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The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Despite of being in different microenvironment, breast cancer cells influence the bone cells and persuade cancer metastasis from breast to bone. Multiple co-culture approaches have been explored to study paracrine signaling between these cells and to study the progression of cancer. However, lack of native tissue microenvironment remains a major bottleneck in existing co-culture technologies. Therefore, in the present study, a tumorigenic and an osteogenic microenvironment have been sutured together to create a multi-cellular environment and has been appraised to study cancer progression in bone tissue. The PCL-polystyrene and PCL-collagen fibrous scaffolds were characterized for tumorigenic and osteogenic potential induction on MDA-MB-231 and MC3T3-E1 cells respectively. Diffusion ability of crystal violet, glucose, and bovine serum albumin across the membrane were used to access the potential paracrine interaction facilitated by device. While in co-cultured condition, MDA-MB-231 cells showed EMT phenotype along with secretion of TNFα and PTHrP which lower down the expression of osteogenic markers including alkaline phosphatase, RUNX2, Osteocalcin and Osteoprotegerin. The cancer progression in bone microenvironment demonstrated the role and necessity of creating multiple tissue microenvironment and its contribution in studying multicellular disease progression and therapeutics., Competing Interests: Declaration of competing interest All the Authors show no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Milk production during lactation places a high demand for calcium that is fulfilled both from maternal bone resorption and diet. While it is known that mammary gland-derived PTHrP drives bone resorption during lactation, the impact of postpartum estrogen loss on bone has been unclear. Methods: We used a case-control study design to test the effect of estrogen loss in lactating mice. Results: In the present study, we show for the first time that estrogen loss during lactation activates memory T-cells (T M ) to produce TNFα and IL-17A to aid in bone resorption and calcium release. Our studies reveal a new mechanism for the release of calcium from bone postpartum. The findings provide several new insights. First, the immune system plays a critical role in milk production postpartum. Second, evolutionarily, the pathway serves the physiological purpose of increasing bone resorption to release calcium for breastmilk production postpartum but becomes maladaptive postmenopause, leading to osteoporosis. Finally, these results highlight the crosstalk between the brain-bone-breast-endocrine axis and the immune system during lactation.

Treating bone-cartilage defects is a fundamental clinical problem. The ability of damaged cartilage to self-repair is limited due to its avascularity. Left untreated, these defects can lead to osteoarthritis. Details of osteochondral defect repair are elusive, but animal models indicate healing occurs via an endochondral ossification-like process, similar to that in the growth plate. In the growth plate, the signalling molecules parathyroid hormone-related protein (PTHrP) and Indian Hedgehog (Ihh) form a feedback loop regulating chondrocyte hypertrophy, with Ihh inducing and PTHrP suppressing hypertrophy. To better understand this repair process and to explore the regulatory role of signalling molecules on the regeneration process, we formulate a reaction-diffusion mathematical model of osteochondral defect regeneration after chondrocyte implantation. The drivers of healing are assumed to be chondrocytes and osteoblasts, and their interaction via signalling molecules. We model cell proliferation, migration and chondrocyte hypertrophy, and matrix production and conversion, spatially and temporally. We further model nutrient and signalling molecule diffusion and their interaction with the cells. We consider the PTHrP-Ihh feedback loop as the backbone mechanisms but the model is flexible to incorporate extra signalling mechanisms if needed. Our mathematical model is able to represent repair of osteochondral defects, starting with cartilage formation throughout the defect. This is followed by chondrocyte hypertrophy, matrix calcification and bone formation deep inside the defect, while cartilage at the surface is maintained and eventually separated from the deeper bone by a thin layer of calcified cartilage. The complete process requires around 48 months. A key highlight of the model demonstrates that the PTHrP-Ihh loop alone is insufficient and an extra mechanism is required to initiate chondrocyte hypertrophy, represented by a critical cartilage density. A parameter sensitivity study reveals that the timing of the repair process crucially depends on parameters, such as the critical cartilage density, and those describing the actions of PTHrP to suppress hypertrophy, such as its diffusion coefficient, threshold concentration and degradation rate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The dental follicle (DF) plays an indispensable role in tooth eruption by regulating bone remodeling through their influence on osteoblast and osteoclast activity. The process of tooth eruption involves a series of intricate regulatory mechanisms and signaling pathways. Disruption of the parathyroid hormone‑related protein (PTHrP) in the PTHrP‑PTHrP receptor signaling pathway inhibits osteoclast differentiation by DF cells (DFCs), thus resulting in obstructed tooth eruption. Furthermore, parathyroid hormone receptor‑1 mutations are linked to primary tooth eruption failure. Additionally, the Wnt/β‑catenin, TGF‑β, bone morphogenetic protein and Hedgehog signaling pathways have crucial roles in DFC involvement in tooth eruption. DFC signal loss or alteration inhibits osteoclast differentiation, affects osteoblast and cementoblast differentiation, and suppresses DFC proliferation, thus resulting in failed tooth eruptions. Abnormal tooth eruption is also associated with a range of systemic syndromes and genetic diseases, predominantly resulting from pathogenic gene mutations. Among these conditions, the following disorders arise due to genetic mutations that disrupt DFCs and impede proper tooth eruption: Cleidocranial dysplasia associated with Runt‑related gene 2 gene mutations; osteosclerosis caused by CLCN7 gene mutations; mucopolysaccharidosis type VI resulting from arylsulfatase B gene mutations; enamel renal syndrome due to FAM20A gene mutations; and dentin dysplasia caused by mutations in the VPS4B gene. In addition, regional odontodysplasia and multiple calcific hyperplastic DFs are involved in tooth eruption failure; however, they are not related to gene mutations. The specific mechanism for this effect requires further investigation. To the best of our knowledge, previous reviews have not comprehensively summarized the syndromes associated with DF abnormalities manifesting as abnormal tooth eruption. Therefore, the present review aims to consolidate the current knowledge on DFC signaling pathways implicated in abnormal tooth eruption, and their association with disorders of tooth eruption in genetic diseases and syndromes, thereby providing a valuable reference for future related research.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 μm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

It is not clear as to whether weight bearing and ambulation may affect bone growth. Our goal was to study the role of mechanical loading (one of the components of ambulation) on endochondral ossification and longitudinal bone growth. Thus, we applied cyclical, biologically relevant strains for a prolonged time period (4 weeks) to one tibia of juvenile mice, while using the contralateral one as an internal control. By the end of the 4-week loading period, the mean tibial growth of the loaded tibiae was significantly greater than that of the unloaded tibiae. The mean height and the mean area of the loaded tibial growth plates were greater than those of the unloaded tibiae. In addition, in female mice we found a greater expression of PTHrP in the loaded tibial growth plates than in the unloaded ones. Lastly, microCT analysis revealed no difference between loaded and unloaded tibiae with respect to the fraction of bone volume relative to the total volume of the region of interest or the tibial trabecular bone volume. Thus, our findings suggest that intermittent compressive forces applied on tibiae at mild-moderate strain magnitude induce a significant and persistent longitudinal bone growth. PTHrP expressed in the growth plate appears to be one growth factor responsible for stimulating endochondral ossification and bone growth in female mice., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The most widely used synthetic glucocorticoid, dexamethasone (DEX), causes stunted growth in children when used excessively or for long periods of time; however, there are still plenty of pediatric patients require long-term treatment with DEX. As an alternative, growth hormone is used in combination, but it has side effects, a high cost, and psychological factors, and it is not satisfactory in terms of effectiveness. It is necessary to develop a safe and affordable treatment that can replace it. The Korean Food and Drug Administration approved HT042, a standardized functional food ingredient, with the claim that it can help height growth of children. In this study, it was found that HT042 activated the Indian hedgehog/parathyroid hormone-related protein signaling pathway and enhanced the number of growth hormone receptors and insulin-like growth factor-1 receptors on the growth plate surface, which were reduced by DEX treatment, and restored growth retardation. In metatarsal bone and primary chondrocyte models, it was found that HT042 can promote the length of growth plate and recover DEX-induced growth retardation. It was also found that HT042 promotes cell proliferation using bromodeoxyuridine and terminal deoxynucleotidyl transferase dUTP nick end labeling assays; moreover, we verified increased expression of GHR/IGF-1R and Ihh/PTHrP pathway activity using qRT-PCR, western blotting, and siRNA analyses to verify its direct action on the growth plate. The anti-apoptotic effect of HT042 was identified by regulating the expression of apoptotic factors such as caspase-3, Bcl2, Bclx, and Bax. These results were identified using both ex vivo and in vitro models. Our study verified that co-administration of HT042 could recover the DEX induced growth retardation.

Purpose of Review: The purpose of this review is to discuss the musculoskeletal consequences of cancer, including those that occur in the absence of bone metastases., Recent Findings: Cancer patients frequently develop cachexia, a debilitating condition reflected by weight loss and skeletal muscle wasting. The negative effects that tumors exert on bone health represents a growing interest amongst cachexia researchers. Recent clinical and pre-clinical evidence demonstrates cancer-induced bone loss, even in the absence of skeletal metastases. Together with muscle wasting, losses in bone demonstrates the impact of cancer on the musculoskeletal system. Identifying therapeutic targets that comprehensively protect musculoskeletal health is essential to improve the quality of life in cancer patients and survivors. IL-6, RANKL, PTHrP, sclerostin, and TGF-β superfamily members represent potential targets to counteract cachexia. However, more research is needed to determine the efficacy of these targets in protecting both skeletal muscle and bone., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma., Methods: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP , cathepsin K ( CTSK ), and nuclear factor of activated T cell cytoplasmic 1 ( NFATc1 ), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis., Results: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P <0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression ( r =0.385, r =0.417, r =-0.316, all P <0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma ( r =0.413, r =0.505, both P <0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group ( P <0.05), with increased mRNA expression levels of TRAP , CTSK , and NFATc1 (all P <0.05). Scanning electron microscopy of bone resorption pits showed that the number ( P <0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group., Conclusions: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca 2+ -activated K + channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca 2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca 2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca 2+ spikes during each burst of spontaneous Ca 2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca 2+ release which prematurely terminates Ca 2+ transient bursts resulting in the attenuation of SPCs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone-related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway-specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein-coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Parathyroid hormone-related peptide (PTHrP) is known to have a pivotal role in the progression of various solid tumors, among which prostate cancer stands out. However, the extent of PTHrP expression and its clinical implications in prostate cancer patients remain shrouded in obscurity. The primary objective of this research endeavor was to shed light on the relevance of PTHrP in the context of prostate cancer patients and to uncover the potential underlying mechanisms., Methods: The expression of PTHrP, E-cadherin, and vimentin in tumor tissues of 88 prostate cancer patients was evaluated by immunohistochemical technique. Subsequently, the associations between PTHrP and clinicopathological parameters and prognosis of patients with prostate cancer were analyzed., Results: Immunohistochemical analysis showed that the expression rates of PTHrP, E-cadherin, and vimentin in prostate cancer tissues were 95.5%, 88.6%, and 84.1%, respectively. Patients with a high level of PTHrP had a decreased expression of E-cadherin (P = .013) and an increased expression of vimentin (P = .010) compared with patients with a low level of PTHrP. Besides, the high expression of PTHrP was significantly correlated with a higher level of initial prostate-specific antigen (P = .026), positive lymph node metastasis (P = .010), osseous metastasis (P = .004), and Gleason score (P = .026). Moreover, patients with a high level of PTHrP had shorter progression-free survival (P = .002) than patients with a low level of PTHrP., Conclusion: The present study indicates that PTHrP is associated with risk factors of poor outcomes in prostate cancer, while epithelial-mesenchymal transition may be involved in this process., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling., (© 2024. The Author(s).)

Background: Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function., Objective: To investigate the effect of local injection of the drug abaloparatide (ABL), an analogue of parathyroid hormone related protein (PTHrP), on promoting lengthening of the mandibular condyle., Methods: Thirty adolescent male Sprague-Dawley rats were randomly divided into two groups, which received the injection of ABL or normal saline (the control) every 3 days in the temporomandibular joint (TMJ) cavity. Cone-beam computed tomography and immunohistochemistry assays were performed at 2, 4 and 6 weeks since the injection. Mandibular condylar chondrocytes (MCC) and pre-osteoblasts were treated with ABL or PBS, followed by the CCK-8 detection, IC50, real-time PCR assay, Western Blot and immunofluorescence staining., Results: In vivo, compared with the control, the ABL group significantly increased the mandibular condylar process length (by 1.34 ± 0.59 mm at 6 weeks), the thickness of the cartilage layer, and enhanced the matrix synthesis. The ABL group had significant up-regulation of SOX 9, COL II, PTHrP and PTH1R, down-regulation of COL X in the cartilage, up-regulation of RUNX 2, and unchanged osteoclastogenesis in the subchondral bone. In vitro, the intra-TMJ injection of ABL promoted the MCC proliferation, with up-regulated expression of chondrogenic genes, and enhanced osteogenic differentiation of the pre-osteoblasts., Conclusions: Intra-TMJ injection of abaloparatide promotes mandibular condyle lengthening in the adolescent rats via enhancing chondrogenesis in the mandibular condylar cartilage and ossification in the subchondral bone., (© 2023 John Wiley & Sons Ltd.)

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca +2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca +2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca +2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies., (© 2023 John Wiley & Sons Ltd.)

Osteocytes express parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptors and respond to the PTHrP analog abaloparatide (ABL) and to the PTH 1-34 fragment teriparatide (TPTD), which are used to treat osteoporosis. Several studies indicate overlapping but distinct skeletal responses to ABL or TPTD, but their effects on cortical bone may differ. Little is known about their differential effects on osteocytes. We compared cortical osteocyte and skeletal responses to ABL and TPTD in sham-operated and ovariectomized mice. Administered 7 weeks after ovariectomy for 4 weeks at a dose of 40 μg/kg/d, TPTD and ABL had similar effects on trabecular bone, but ABL showed stronger effects in cortical bone. In cortical osteocytes, both treatments decreased lacunar area, reflecting altered peri-lacunar remodeling favoring matrix accumulation. Osteocyte RNA-Seq revealed that several genes and pathways were altered by ovariectomy and affected similarly by TPTD and ABL. Notwithstanding, several signaling pathways were uniquely regulated by ABL. Thus, in mice, TPTD and ABL induced a positive osteocyte peri-lacunar remodeling balance, but ABL induced stronger cortical responses and affected the osteocyte transcriptome differently. We concluded that ABL affected the cortical osteocyte transcriptome in a manner subtly different from TPTD, resulting in more beneficial remodeling/modeling changes and homeostasis of the cortex.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl ) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl -/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl . Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (P i ) and pyrophosphate (PP i ) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PP i .

Background: Pseudohypoparathyroidism (PHP) and related disorders newly referred to as inactivating PTH/PTHrP signaling disorders (iPPSD) are rare endocrine diseases. Many clinical features including obesity, neurocognitive impairment, brachydactyly, short stature, parathyroid hormone (PTH) resistance, and resistance to other hormones such as thyroid-stimulating hormone (TSH) have been well described, yet they refer mainly to the full development of the disease during late childhood and adulthood., Objective: A significant delay in diagnosis has been reported; therefore, our objective is to increase awareness on neonatal and early infancy presentation of the diseases. To do so, we analyzed a large cohort of iPPSD/PHP patients., Methods: We included 136 patients diagnosed with iPPSD/PHP. We retrospectively collected data on birth and investigated the rate of neonatal complications occurring in each iPPSD/PHP category within the first month of life., Results: Overall 36% of patients presented at least one neonatal complication, far more than the general population; when considering only the patients with iPPSD2/PHP1A, it reached 47% of the patients. Neonatal hypoglycemia and transient respiratory distress appeared significantly frequent in this latter group, ie, 10.5% and 18.4%, respectively. The presence of neonatal features was associated with earlier resistance to TSH (P < 0.001) and with the development of neurocognitive impairment (P = 0.02) or constipation (P = 0.04) later in life., Conclusion: Our findings suggest that iPPSD/PHP and especially iPPSD2/PHP1A newborns require specific care at birth because of an increased risk of neonatal complications. These complications may predict a more severe course of the disease; however, they are unspecific which likely explains the diagnostic delay., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

A 74-year-old woman was admitted with hypercalcemia and prolonged disturbance of consciousness. The left buttock to the anterior aspect of the left thigh was swollen and erythematous, with a collection of 1.0-cm large, firm, elastic nodules distributed in a zosteriform pattern in the L1-L4 region. Based on autopsy findings, a very rare case of Cobb syndrome was diagnosed due to a spinal vascular malformation at the Th12-L4 level and L5 vertebral hemangioma. Cobb syndrome-associated cutaneous metastasis extending along the same metamere was complicated by immunohistochemically proven parathyroid hormone-related protein-producing advanced bladder carcinoma in this case.

Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP Δ/Δ) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at ∼day 5, are severely stunted in growth, weigh 54% less than control mice at day 1-2 and eventually fail to grow. PTHrP Δ/Δ mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (∼10-20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP Δ/Δ mice were smaller in size but secreted more insulin than littermate controls. PTHrP Δ/Δ and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8-20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP Δ/Δ mice (∼250 µm 2 ) compared to islets from control mice (∼900 µm 2 ), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP Δ/Δ mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP 87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP 87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-Cre ERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP 87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP 87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The PTH-related peptide(1-34) analog, abaloparatide (ABL), is the second anabolic drug available for the treatment of osteoporosis. Previous research demonstrated that ABL had a potent anabolic effect but caused hypercalcemia at a significantly lower rate. However, the mechanism by which ABL maintains the stability of blood calcium levels remains poorly understood. Our in vivo data showed that ABL treatment (40 µg/kg/day for 7 days) significantly increased rat blood level of 1,25-dihydroxyvitamin D [1,25-(OH)2D] without raising the blood calcium value. ABL also significantly augmented the carboxylated osteocalcin (Gla-Ocn) in the blood and bone that is synthesized by osteoblasts, and increased noncarboxylated Ocn, which is released from the bone matrix to the circulation because of osteoclast activation. The in vitro data showed that ABL (10 nM for 24 hours) had little direct effects on 1,25-(OH)2D synthesis and Gla-Ocn formation in nonrenal cells (rat osteoblast-like cells). However, ABL significantly promoted both 1,25-(OH)2D and Gla-Ocn formation when 25-hydroxyvitamin D, the substrate of 1α-hydroxylase, was added to the cells. Thus, the increased 1,25-(OH)2D levels in rats treated by ABL result in high levels of Gla-Ocn and transient calcium increase in the circulation. Gla-Ocn then mediates calcium ions in the extracellular fluid at bone sites to bind to hydroxyapatite at bone surfaces. This regulation by Gla-Ocn at least, in part, maintains the stability of blood calcium levels during ABL treatment. We conclude that the signaling pathway of ABL/1,25-(OH)2D/Gla-Ocn contributes to calcium homeostasis and may help understand the mechanism of ABL for osteoporosis therapy., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Elaborate bioreactor cultivation or expensive growth factor supplementation can enhance extracellular matrix production in engineered neocartilage to provide sufficient mechanical resistance. We here investigated whether raising extracellular calcium levels in chondrogenic cultures to physiologically relevant levels would provide a simple and inexpensive alternative to enhance cartilage neogenesis from human articular chondrocytes (AC) or bone marrow-derived mesenchymal stromal cells (BMSC). Interestingly, AC and BMSC-derived chondrocytes showed an opposite response to a calcium increase from 1.8 mM to 8 mM by which glycosaminoglycan (GAG) and collagen type II production were elevated during BMSC chondrogenesis but depressed in AC, leading to two-fold higher GAG/DNA values in BMSC-based neocartilage compared to the AC group. According to control treatments with Mg 2+ or sucrose, these effects were specific for CaCl 2 rather than divalent cations or osmolarity. Importantly, undesired pro-hypertrophic traits were not stimulated by calcium treatment. Specific induction of PTHrP mRNA and protein by 8.0mM calcium only in AC, along with negative effects of recombinant PTHrP 1-34 on cartilage matrix production, suggested that the PTHrP pathway contributed to the detrimental effects in AC-based neocartilage. Altogether, raising extracellular calcium levels was discovered as a novel, simple and inexpensive stimulator for BMSC-based cartilage neogenesis without the need for special bioreactors, whereas such conditions should be avoided for AC.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor's basal rate of cAMP signaling and decreases its capacity to recruit β-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired β-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with β-arrestin in the mechanism by which the PTH1R regulates bone formation., (© 2023. The Author(s).)

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Bone tissue is a common metastatic site of lung cancer, and bone metastasis is characterized by abnormal differentiation and malfunction of osteoclast, and the roles of exosomes derived from lung cancer have attracted much attention. In our study, we found that the level of HOTAIR expression in A549 and H1299 exosomes was higher than those of normal lung fibrocytes. Overexpression of HOTAIR in A549 and H1299 exosomes promoted osteoclast differentiation. Furthermore, A549-Exos and H1299-Exos targeted bone tissues, and bone formation was significantly inhibited in vivo. Mechanistically, exosomal lncRNA HOTAIR promoted bone resorption by targeting TGF-β/PTHrP/RANKL pathway., (© 2022 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). Published by John Wiley & Sons Ltd.)

Obesity increases the risk of advanced prostate cancer (PCa). The calcium sensing receptor (CaSR) has been shown to be responsive to obesity-mediated cytokines and is upregulated in metastatic PCa. This study used a novel in vitro approach, involving the exposure of PCa cells to sera, from obese or normal weight males, and to CaSR inhibitor NPS-2143. Cell viability was determined using MTT assay. MMP-9 activity and invasion were assessed using zymography and invasion chambers, respectively. Microscopy was used to visualize EMT proteins. qRT-PCR and immunoblot analysis were used to quantify changes in genes and proteins important for tumorigenesis. Exposure to obese sera increased the proliferation, and the invasive capacity of PCa cells and de-localized epithelial-mesenchymal transition markers, which were attenuated with CaSR inhibition. Exposure to obese sera upregulated mRNA expression of PTHrP and protein expression of COX-2, IL-6, and CaSR. Inhibition of CaSR downregulated the mRNA expression of PTHrP and RANK , and protein expression of pERK and TNF-α. Obesity was shown to increase invasion and upregulate the expression of genes and proteins involved in PCa tumorigenesis. CaSR inhibition downregulated the expression of several of these factors. Thus, CaSR is a potentially important protein to target in obesity-mediated PCa progression.

Purpose: This study aims to further explore cartilage development in prenatal ethanol exposure (PEE) offspring at different times to explore the specific time points and mechanism of ethanol-induced fetal cartilage dysplasia., Methods: On gestational day (GD)14, GD17, and GD20, PEE fetal cartilage was evaluated by morphological analysis. RT-qPCR, immunohistochemistry, and immunofluorescence were used to detect the expression of cartilage marker genes and their regulatory factors. Bone marrow mesenchymal stem cells (BMSCs) were used to explore the effect of ethanol on the differentiation of chondrocytes. Additionally, we used inhibitors, overexpression plasmids and a luciferase reporter assay on GD17 chondrocytes to verify the mechanism., Results: PEE significantly reduced cartilage matrix content and the expression of marker genes on GD17 and GD20 but had no effect on GD14. The inhibition of chondrogenic differentiation by PEE mainly occurred on GD14-17. Furthermore, the expression of miR-200b-3p was increased, while that of ERG and PTHrP was markedly reduced in PEE fetal cartilage. In vitro, ethanol (30-120 mM) inhibited the differentiation of BMSCs into chondrocytes in a concentration-dependent manner, accompanied by strong expression of miR-200b-3p and low expression of ERG and PTHrP. Moreover, PTHLH and ERG overexpressed, as well as a miR-200b-3p inhibitor reversed the inhibitory effect of ethanol on the differentiation of fetal chondrocytes. Furthermore, miR-200b-3p could target and negatively regulate ERG., Conclusions: PEE can significantly inhibit the development of articular cartilage, especially during articular cartilage formation. The mechanism is related to the decreased differentiation of fetal cartilage into articular cartilage mediated by the miR-200b-3p/ERG/PTHrP axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) as the bladder fills acts as an endogenous DSM relaxant to facilitate bladder storage function. Here, the effects of exogenous PTHrP on transient pressure rises (TPRs) in the bladder and associated afferent nerve activity during bladder filling were investigated. In anaesthetized rats, changes in the intravesical pressure were measured while the bladder was gradually filled with saline. Afferent nerve activity was simultaneously recorded from their centrally disconnected left pelvic nerves. In DSM strips, spontaneous and nerve-evoked contractions were isometrically recorded. The distribution of PTHrP receptors (PTHrPRs) in the bladder wall was also examined by fluorescence immunostaining. The bladders in which the contralateral pelvic nerve was also centrally disconnected developed nifedipine, an L-type voltage-dependent Ca 2+ channel blocker-sensitive TPRs (< 3 mmHg). Intravenous administration of PTHrP suppressed these TPRs and associated bursts of afferent nerve activity. In the bladders with centrally connected contralateral pelvic nerves, atropine, a muscarinic receptor antagonist-sensitive large TPRs (> 3 mmHg) developed in the late filling phase. PTHrP diminished the large TPRs and corresponding surges of afferent nerve activity. In DSM strips, bath-applied PTHrP (10 nM) suppressed spontaneous phasic contractions, while less affecting nerve-evoked contractions. PTHrPRs were expressed in DSM cells but not in intramural nerve fibers. Thus, PTHrP appears to suppress bladder TPRs and associated afferent nerve activity even under the influence of low degree of parasympathetic neural input during storage phases. Endogenous PTHrP may indirectly attenuate afferent nerve activity by suppressing TPRs to facilitate urinary accommodation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6., (© 2022 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP., Competing Interests: Declaration of interests O.N. is a co-founder and an external director of Curreio Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Implantation of genetically modified chondrogenically competent human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to improve cartilage repair. The goal of this study was to examine the potential benefits of transferring a sequence coding for the bone morphogenetic protein 3 ( BMP-3 ) that modulates bone and cartilage formation, using recombinant adeno-associated virus (rAAV) vectors on the chondroreparative activities of hMSCs. Undifferentiated and chondrogenically induced primary human MSCs were treated with an rAAV-h BMP-3 construct to evaluate its effects on the proliferative, metabolic, and chondrogenic activities of the cells compared with control (reporter rAAV- lacZ vector) condition. Effective BMP-3 expression was noted both in undifferentiated and chondrogenically differentiated cells in the presence of rAAV-h BMP-3 relative to rAAV- lacZ , stimulating cell proliferation and extracellular matrix (proteoglycans, type-II collagen) deposition together with higher levels of chondrogenic sex-determining region Y-type high-mobility group box 9 ( SOX9 ) expression. rAAV-h BMP-3 also advantageously decreased terminal differentiation, hypertrophy, and osteogenesis (type-I/-X collagen and alkaline phosphatase expression), with reduced levels of osteoblast-related runt-related transcription factor 2 ( RUNX-2 ) transcription factor and β-catenin (osteodifferentiation mediator) and enhanced parathyroid hormone-related protein expression (inhibitor of hypertrophic maturation, calcification, and bone formation). This study shows the advantage of modifying hMSCs with rAAV-h BMP-3 to trigger adapted chondroreparative activities as a source of improved cells for transplantation protocols in cartilage defects.

Parathyroid hormone-related protein (PTHrP) C-terminal peptides regulate the metabolism of bone cells. PHTrP [107-111] (osteostatin) promotes bone repair in animal models of bone defects and prevents bone erosion in inflammatory arthritis. In addition to its positive effects on osteoblasts, osteostatin may inhibit bone resorption. The aim of this study was to determine the effects of osteostatin on human osteoclast differentiation and function. We used macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) to induce the osteoclast differentiation of adherent human peripheral blood mononuclear cells. Tartrate-resistant acid phosphatase (TRAP) staining was performed for the detection of the osteoclasts. The function of mature osteoclasts was assessed with a pit resorption assay. Gene expression was evaluated with qRT-PCR, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation was studied by immunofluorescence. We observed that osteostatin (100, 250 and 500 nM) decreased the differentiation of osteoclasts in a concentration-dependent manner, but it did not modify the resorptive ability of mature osteoclasts. In addition, osteostatin decreased the mRNA levels of cathepsin K, osteoclast associated Ig-like receptor (OSCAR) and NFATc1. The nuclear translocation of the master transcription factor in osteoclast differentiation NFATc1 was reduced by osteostatin. Our results suggest that the anti-resorptive effects of osteostatin may be dependent on the inhibition of osteoclastogenesis. This study has shown that osteostatin controls human osteoclast differentiation in vitro through the downregulation of NFATc1.

Cancer-associated hypercalcemia commonly occurs through abnormal secretions of parathyroid hormone-related protein (PTHrP) from cancer cells. Several cases of PTHrP-producing melanoma causing hypercalcemia have been reported; however, effective management of PTHrP-producing BRAF wild-type melanoma causing hypercalcemia after immune checkpoint inhibitor (ICI) failure is unclear. We report a case of PTHrP-producing BRAF wild-type melanoma leading to oncological emergency by hypercalcemia that was successfully treated with dacarbazine after ICI failure. A 65-year-old Japanese woman had advanced BRAF wild-type melanoma that was refractory to ICI, and then led to hypoxia through exacerbated lung metastases and pleural effusion. Moreover, hypercalcemia appeared in parallel with increase of the serum PTHrP level. Dacarbazine was administered, and after the first administration, the pleural effusion was gradually decreased and hypoxia rapidly disappeared, and the serum calcium and PTHrP levels were improved within normal limits. Dacarbazine after ICI failure is potentially a useful option for oncological emergency due to progression of BRAF wild-type melanoma including PTHrP-producing types. Dacarbazine should be reevaluated as a therapeutic option for oncological emergency in patients with BRAF wild-type melanoma after ICI failure., (© 2022 Japanese Dermatological Association.)

PTH-related peptide (PTHrP) improves the bone marrow micro-environment to activate the bone-remodelling, but the coordinated regulation of PTHrP and transforming growth factor-β (TGFβ) signalling in TMJ-OA remains incompletely understood. We used disordered occlusion to establish model animals that recapitulate the ordinary clinical aetiology of TMJ-OA. Immunohistochemical and histological analyses revealed condylar fibrocartilage degeneration in model animals following disordered occlusion. TMJ-OA model animals administered intermittent PTHrP (iPTH) exhibited significantly decreased condylar cartilage degeneration. Micro-CT, histomorphometry, and Western Blot analyses disclosed that iPTH promoted subchondral bone formation in the TMJ-OA model animals. In addition, iPTH increased the number of osterix (OSX)-positive cells and osteocalcin (OCN)-positive cells in the subchondral bone marrow cavity. However, the number of osteoclasts was also increased by iPTH, indicating that subchondral bone volume increase was mainly due to the iPTH-mediated increase in the bone-formation ability of condylar subchondral bone. In vitro, PTHrP treatment increased condylar subchondral bone marrow-derived mesenchymal stem cell (SMSC) osteoblastic differentiation potential and upregulated the gene and protein expression of key regulators of osteogenesis. Furthermore, we found that PTHrP-PTH1R signalling inhibits TGFβ signalling during osteoblastic differentiation. Collectively, these data suggested that iPTH improves OA lesions by enhancing osteoblastic differentiation in subchondral bone and suppressing aberrant active TGFβ signalling. These findings indicated that PTHrP, which targets the TGFβ signalling pathway, may be an effective biological reagent to prevent and treat TMJ-OA in the clinic., (© 2022. The Author(s).)

The cranial base contains a special type of growth plate termed the synchondrosis, which functions as the growth center of the skull. The synchondrosis is composed of bidirectional opposite-facing layers of resting, proliferating, and hypertrophic chondrocytes, and lacks the secondary ossification center. In long bones, the resting zone of the epiphyseal growth plate houses a population of parathyroid hormone-related protein (PTHrP)-expressing chondrocytes that contribute to the formation of columnar chondrocytes. Whether PTHrP + chondrocytes in the synchondrosis possess similar functions remains undefined. Using Pthrp-mCherry knock-in mice, we found that PTHrP + chondrocytes predominantly occupied the lateral wedge-shaped area of the synchondrosis, unlike those in the femoral growth plate that reside in the resting zone within the epiphysis. In vivo cell-lineage analyses using a tamoxifen-inducible Pthrp-creER line revealed that PTHrP + chondrocytes failed to establish columnar chondrocytes in the synchondrosis. Therefore, PTHrP + chondrocytes in the synchondrosis do not possess column-forming capabilities, unlike those in the resting zone of the long bone growth plate. These findings support the importance of the secondary ossification center within the long bone epiphysis in establishing the stem cell niche for PTHrP + chondrocytes, the absence of which may explain the lack of column-forming capabilities of PTHrP + chondrocytes in the cranial base synchondrosis.

Background: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells., Aim: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models., Methods: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10 -8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)&#95;nu mice received daily intratumoral injections of PTHrP (40 μg/kg) in 100 μL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05., Results: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min ( P < 0.01) and at 20 h ( P < 0.01) whereas the levels diminished at 3 min ( P < 0.05), 10 min ( P < 0.01), and 1 h to 5 h ( P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased ( P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis ( P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation ( P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation ( P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation ( P < 0.05), migration ( P < 0.05), and the EMT program ( P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 ( P < 0.001), OXA ( P < 0.01), and DOXO ( P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively ( P < 0.05)., Conclusion: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Homeostasis during lactation is a special case in which the unit for regulation is a dyad comprising the mother and her currently nursing offspring (the mother-offspring dyad). This arrangement is not a trivial. A litter of mice can have a mass greater than the mother and nutrient demands that far exceed her. Homeostasis for milk secretion, appetite, and calcium metabolism must come under integrated control, responding seamlessly to the needs of the mother and the offspring. Serotonin (5-HT) is a primary local regulator of mammary homeostasis. 5-HT synthesis in the mammary epithelium is high during lactation and increases during milk stasis. Two important functions are attributed to the 5-HT system. Firstly, when alveolar spaces are filled with milk 5-HT inhibits milk secretion and opens tight junctions. This feedback induces early phases of involution. Secondly, 5-HT induces synthesis and secretion of parathyroid hormone-related peptide (PTHrP)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Parathyroid hormone-related peptide (PTHrP) overexpression and poor patient outcome have been reported for many human tumors, but no studies are available in laryngeal cancer. Therefore, we studied the expression of PTHrP and its receptor, parathyroid hormone-related peptide receptor type 1 (PTH1R), in primary locally advanced laryngeal squamous cell carcinomas (LALSCC) also in relation to the clinical outcome of patients., Methods: We conducted a retrospective exploratory study, using immunohistochemistry, on PTHrP, PTH1R and HER1 expressions in LALSCC of 66 patients treated with bio-radiotherapy with cetuximab., Results: The expressions of PTHrP and PTH1R in LALSCC were associated with the degree of tumor differentiation (p = 0.01 and 0.04, respectively). Poorly differentiated tumors, with worse prognosis, expressed PTHrP at nuclear level and were PTH1R negative. PTHrP and PTH1R were expressed at cytoplasmic level in normal larynx epithelium and more differentiated laryngeal cancer cells, suggesting an autocrine/paracrine role of PTHrP in squamous cell differentiation of well differentiated tumors with good prognosis. Eighty-one percent HER1 positive tumors expressed PTHrP (p < 0.0001), mainly at nuclear level, consistent with the known up-regulation of PTHrP gene by HER1 signaling. In multivariable analyses, patients with PTHrP positive tumors had a higher relative risk of relapse (HR = 5.49; CI 95% = 1.62-22.24; p = 0.006) and survival (HR = 8.21; CI 95% = 1.19-105.00; p = 0.031) while those with PTH1R positive tumors showed a lower relative risk of relapse (HR = 0.18; CI 95% = 0.04-0.62; p = 0.002) and survival (HR = 0.18; CI 95% = 0.04-0.91; p = 0.029)., Conclusions: In LALSCC nuclear PTHrP and absence of PTH1R expressions could be useful in predicting response and/or resistance to cetuximab in combined therapies, contributing to an aggressive behavior of tumor cells downstream to HER1., (© 2022. The Author(s).)

Background: Lung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour-derived parathyroid hormone-related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia., Methods: Global gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT-PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour-bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA-seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression., Results: Expression of EGFR ligands, EGFR itself, and PTHrP co-clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8-fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65-75% (P < 0.0005) in tumours upon treatment of LLC tumour-bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour-bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8-fold, P < 0.05) and HNSC (17.5-fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P < 0.0001 and R = 0.51, P < 0.0001; HNSC: n = 545, R = 0.34, P < 0.001 and R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log-rank test, P = 0.0451)., Conclusions: Epidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour-bearing mice., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Aims: Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc., Methods: Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h., Results: Pi-gavage elevated plasma Pi and decreased plasma Ca 2+ in both genotypes after 1 h Within 1 h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1 h post-administration in WT and after 4 h in PTH-KO. PTH increased after 1 h in WT animals. After 4 h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4 h after Pi-gavage. Inhibition of IP6-kinases had no effect., Conclusions: (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled., Methods: We developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression., Results: Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling., Conclusions: Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells., (© 2022. The Author(s).)

Background: Triple-negative breast cancer (TNBC) is the most severe type of breast cancer owing to its high heterogeneity, aggressiveness and lack of treatment. Studies have reported that uncarboxylated osteocalcin (GluOC) promotes the development of prostate and other cancers. Studies have also found elevated levels of serum osteocalcin in breast cancer patients with bone metastasis, and serum osteocalcin can be a marker of bone metastasis. However, whether GluOC promotes the development of TNBC and the related mechanisms need to be further clarified., Results: Our results revealed that GluOC is associated with the proliferation and metastasis of MDA-MB-231 cells. GluOC increased the viability and proliferation of MDA-MB-231 cells. In addition, GluOC enhanced the metastatic ability of MDA-MB-231 cells by promoting the expression of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-13 (MMP13), and vascular endothelial growth factor (VEGF) and inducing epithelial-mesenchymal transition (EMT). We also found that GluOC upregulated the expression of interleukin-8 (IL-8) and parathyroid hormone-related protein (PTHrP) genes in MDA-MB-231 breast cancer cells. Moreover, the promoting effect of GluOC was reversed in MDA-MB-231 breast cancer cells treated with specific inhibitor of SMAD3 (SIS3), a SMAD3 phosphorylation inhibitor., Conclusion: Our research proved for the first time that GluOC facilitates the proliferation and metastasis of MDA-MB-231 cells by accelerating the transforming growth factor-β (TGF-β)/SMAD3 signaling pathway. Moreover, GluOC also promotes the gene expression of IL-8 and PTHrP. Both IL-8 and PTHrP can act as osteolytic factors in breast cancer cells. This study indicates that GluOC may be a useful target for preventing TNBC bone metastasis., (© 2022. The Author(s).)

Glucocorticoid (GC)-induced longitudinal bone growth retardation is a common and severe adverse effect in pediatric patients receiving GC immunosuppressive therapy. Molecular mechanisms underlying GC-induced growth inhibition are unclear. GC withdrawal following short-term high-dose use is common, including in the immediate post-transplant period. However, whether skeleton growth recovery is sufficient or whether growth-promoting therapy is required following GC withdrawal is unknown. The aim of this study was to investigate the effect of exogenous growth hormone (GH) on growth plate impairment in GC-induced longitudinal bone growth retardation. Here, apoptotic chondrocytes in the hypertrophic layer of growth plates increased whereas Indian Hedgehog (Ihh) and Parathyroid Hormone Related Peptide (PTHrP) protein levels in the growth plate decreased following GC exposure. The hypertrophic zone of the growth plate expanded following GC withdrawal. Subcutaneously injected GH penetrated the growth plate and modified its organization in rats following GC withdrawal. Ihh and PTHrP expression in GC-induced apoptotic chondrocytes decreased in vitro. GH promoted chondrocyte proliferation by activating Ihh/PTHrP signaling. Downregulating Ihh using specific siRNAs increased chondrocyte apoptosis and inhibited PTHrP, Sox9, and type II collagen (Col2a1) protein expression. GH inhibited apoptosis of Ihh-deficient growth plate chondrocytes by upregulating PTHrP, Sox9, and Col2a1 expression. Thus, reversal of the effect of GC on growth plate impairment following its withdrawal is insufficient, and exogenous GH provides growth plate chondral protection and improved longitudinal growth following GC withdrawal by acting on the Ihh/PTHrP pathway., (Copyright © 2022. Published by Elsevier B.V.)

Breast cancer bone metastases may block normal bone remodeling and promote bone degradation, during which several signaling pathways and small non-coding miRNAs might all play a role. miRNAs and target mRNAs that might be associated with breast cancer bone metastasis were analyzed and selected using bioinformatics analyses based on online data. The 3' untranslated region of key factors associated with breast cancer metastasis were examined for candidate miRNA binding site using Targetscan. The predicted binding was validated. The specific effects of single miRNA and dynamic effects of the miRNA-mRNA axis on breast cancer cell metastasis were investigated. miR-556-5p was downregulated in breast cancer samples according to online datasets and experimental analyses. In breast cancer cells, miR-556-5p overexpression inhibited, whereas miR-556-5p inhibition promoted cancer cell invasion and migration. Among key factors associated with breast cancer bone metastasis, parathyroid hormone related protein (PTHrP) 3'UTR possessed miR-556-5p binding site. Through direct binding, miR-556-5p negatively regulated PTHrP expression. In breast cancer cell lines, miR-556-5p inhibition promoted, whereas PTHrP silencing suppressed cancer cell migration, invasion, and epithelial-mesenchymal transition; the effects of miR-556-5p inhibition were partially reversed by PTHrP silencing. In summary, miR-556-5p targets PTHrP to modulate the cell migration and invasion of breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Obesity, closely related to systematic metabolic disorders, has become a major public health problem in recent decades. Here, we aimed to study the function of Parathyroid hormone-related protein (PTHrP) on high fat diet (HFD) induced murine obesity. Male C57BL/6J mice were transduced with adeno-associated virus vector encoding PTHrP (AAV-PTHrP) or adeno-associated virus control vector (AAV-Vehicle), following with HFD for 8 weeks. In addition, mice without transduction were fed on normal diet or HFD, respectively. Histological, metabolic and biochemical changes were detected. At the endpoint of experiment, body weight of mice treated with AAV-PTHrP did not increase as much as mice with AAV-Vehicle, but similar as mice with normal diet. Food efficiency ratio and weight of interscapular brown adipose tissue and epididymal white adipose tissue in mice overexpressed PTHrP were also lower than mice transducted with AAV-Vehicle. Besides, administration of AAV-PTHrP inhibited HFD-induced adipocyte hypertrophy. Protein level of PKA signaling pathway and thermogenic gene in adipose tissue exhibited a significant raise in HFD + AAV-PTHrP group, whereas transcription of inflammatory gene were decreased. Additionally, PTHrP overexpression ameliorated HFD-induced dyslipidemia, hepatic steatosis and insulin sensitivity. In HFD-induced murine obesity model, PTHrP is crucial to maintain metabolic homeostasis. PTHrP drives white adipose tissue browning and inhibits whitening of brown adipose tissue. Most importantly, PTHrP prevented HFD-induced obesity, hepatic steatosis and insulin resistance.