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The vitamin D receptor (VDR) plays a critical role in the regulation of mineral and bone homeostasis. Upon binding of 1α,25-dihydroxyvitamin D 3 to the VDR, the activation function 2 (AF2) domain repositions and recruits coactivators for the assembly of the transcriptional machinery required for gene transcription. In contrast to coactivator-induced transcriptional activation, the functional effects of coactivator-independent VDR signaling remain unclear. In humans, mutations in the AF2 domain are associated with hereditary vitamin D-resistant rickets, a genetic disorder characterized by impaired bone mineralization and growth. In the present study, we used mice with a systemic or conditional deletion of the VDR-AF2 domain (Vdr ΔAF2 ) to study coactivator-independent VDR signaling. We confirm that ligand-induced transcriptional activation was disabled because the mutant VDR ΔAF2 protein was unable to interact with coactivators. Systemic Vdr ΔAF2 mice developed short, undermineralized bones with dysmorphic growth plates, a bone phenotype that was more pronounced than that of systemic Vdr knockout (Vdr -/- ) mice. Interestingly, a rescue diet that is high in calcium, phosphate, and lactose, normalized this phenotype in Vdr -/- , but not in Vdr ΔAF2 mice. However, osteoblast- and osteoclast-specific Vdr ΔAF2 mice did not recapitulate this bone phenotype indicating coactivator-independent VDR effects are more important in other organs. In addition, RNA-sequencing analysis of duodenum and kidney revealed a decreased expression of VDR target genes in systemic Vdr ΔAF2 mice, which was not observed in Vdr -/- mice. These genes could provide new insights in the compensatory (re)absorption of minerals that are crucial for bone homeostasis. In summary, coactivator-independent VDR effects contribute to mineral and bone homeostasis., (© 2024. The Author(s).)

Objective: In the era of image-guided adaptive radiotherapy, definition of the clinical target volume (CTV) is a challenge in various solid tumors, including esophageal cancer (EC). Many tumor microenvironmental factors, e.g., tumor cell proliferation or cancer stem cells, are hypothesized to be involved in microscopic tumor extension (MTE). Therefore, this study assessed the expression of FAK, ILK, CD44, HIF-1α, and Ki67 in EC patients after neoadjuvant radiochemotherapy followed by tumor resection (NRCHT+R) and correlated these markers with the MTE., Methods: Formalin-fixed paraffin-embedded tumor resection specimens of ten EC patients were analyzed using multiplex immunofluorescence staining. Since gold fiducial markers had been endoscopically implanted at the proximal and distal tumor borders prior to NRCHT+R, correlation of the markers with the MTE was feasible., Results: In tumor resection specimens of EC patients, the overall percentages of FAK + , CD44 + , HIF-1α + , and Ki67 + cells were higher in tumor nests than in the tumor stroma, with the outcome for Ki67 + cells reaching statistical significance (p < 0.001). Conversely, expression of ILK + cells was higher in tumor stroma, albeit not statistically significantly. In three patients, MTE beyond the fiducial markers was found, reaching up to 31 mm., Conclusion: Our findings indicate that the overall expression of FAK, HIF-1α, Ki67, and CD44 was higher in tumor nests, whereas that of ILK was higher in tumor stroma. Differences in the TME between patients with residual tumor cells in the original CTV compared to those without were not found. Thus, there is insufficient evidence that the TME influences the required CTV margin on an individual patient basis., Trial Registration Number and Date: BO-EK-148042017 and BO-EK-177042022 on 20.06.2022, DRKS00011886, https://drks.de/search/de/trial/DRKS00011886 ., (© 2024. The Author(s).)

Transgender youth increasingly present at pediatric gender services. Some of them receive long-term puberty suppression with gonadotropin-releasing hormone analogues (GnRHa) before starting gender-affirming hormones (GAH). The impact of GnRHa use started in early puberty on bone composition and bone mass accrual is unexplored. It is furthermore unclear whether subsequent GAH fully restore GnRHa effects and whether the timing of GAH introduction matters. To answer these questions, we developed a mouse model mimicking the clinical strategy applied in trans boys. Prepubertal 4-week-old female mice were treated with GnRHa alone or with GnRHa supplemented with testosterone (T) from 6 weeks (early puberty) or 8 weeks (late puberty) onward. Outcomes were analyzed at 16 weeks and compared with untreated mice of both sexes. GnRHa markedly increased total body fat mass, decreased lean body mass, and had a modest negative impact on grip strength. Both early and late T administration shaped body composition to adult male levels, whereas grip strength was restored to female values. GnRHa-treated animals showed lower trabecular bone volume and reduced cortical bone mass and strength. These changes were reversed by T to female levels (cortical bone mass and strength) irrespective of the time of administration or even fully up to adult male control values (trabecular parameters) in case of earlier T start. The lower bone mass in GnRHa-treated mice was associated with increased bone marrow adiposity, also reversed by T. In conclusion, prolonged GnRHa use started in prepubertal female mice modifies body composition toward more fat and less lean mass and impairs bone mass acquisition and strength. Subsequent T administration counteracts GnRHa impact on these parameters, shaping body composition and trabecular parameters to male values while restoring cortical bone architecture and strength up to female but not male control levels. These findings could help guide clinical strategies in transgender care. © 2023 American Society for Bone and Mineral Research (ASBMR)., (© 2023 American Society for Bone and Mineral Research (ASBMR).)

Introduction: Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts., Methods: We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D 3 analog WY 1048 or with 1,25(OH) 2 D 3 ., Results and Discussion: We show that Nrp2 expression is induced by 1,25(OH) 2 D 3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D 3 analog WY 1048 (0.4 μg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH) 2 D 3 (0.5 μg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Verlinden, Doms, Janssens, Meyer, Pike, Carmeliet and Verstuyf.)

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis., (© 2022 Matute et al.)

The Vitamin D receptor (VDR) plays a key role in calcium homeostasis, as well as in cell proliferation and differentiation. Among the large number of VDR ligands that have been developed, we have previously shown that BXL-62 and Gemini-72, two C-20-modified vitamin D analogs are highly potent VDR agonists. In this study, we show that both VDR ligands restore the transcriptional activities of VDR variants unresponsive to the natural ligand and identified in patients with rickets. The elucidated mechanisms of action underlying the activities of these C-20-modified analogs emphasize the mutual adaptation of the ligand and the VDR ligand-binding pocket.

Determining the forces that shape diversity in host-associated bacterial communities is critical to understanding the evolution and maintenance of metaorganisms. To gain deeper understanding of the role of host genetics in shaping gut microbial traits, we employed a powerful genetic mapping approach using inbred lines derived from the hybrid zone of two incipient house mouse species. Furthermore, we uniquely performed our analysis on microbial traits measured at the gut mucosal interface, which is in more direct contact with host cells and the immune system. Several mucosa-associated bacterial taxa have high heritability estimates, and interestingly, 16S rRNA transcript-based heritability estimates are positively correlated with cospeciation rate estimates. Genome-wide association mapping identifies 428 loci influencing 120 taxa, with narrow genomic intervals pinpointing promising candidate genes and pathways. Importantly, we identified an enrichment of candidate genes associated with several human diseases, including inflammatory bowel disease, and functional categories including innate immunity and G-protein-coupled receptors. These results highlight key features of the genetic architecture of mammalian host-microbe interactions and how they diverge as new species form., Competing Interests: SD, HF, MR, CC, AK, SI, AF, LT, JB No competing interests declared, (© 2022, Doms et al.)

Active vitamin D 3 , 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH) 2 D 3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D 3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER + ) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER + (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER + BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D 3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER + BC both in vitro and in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vanhevel, Verlinden, Loopmans, Doms, Janssens, Bevers, Stegen, Wildiers and Verstuyf.)

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is primarily known as a key regulator of calcium and phosphate homeostasis. It exerts its biological functions by binding to the vitamin D receptor (VDR), a transcription factor that regulates gene expression in vitamin D-target tissues such as intestine, kidney and bone. Yet, the VDR is expressed in many additional normal and cancerous tissues, where it moderates the antiproliferative, prodifferentiating and immune-modulating effects of 1,25(OH)2D3. Interestingly, several epidemiological studies show that low levels of 25(OH)D, a biological marker for 1,25(OH)2D3 status, are associated with an increased risk of breast cancer (BC) development. Mendelian randomization studies, however, did not find any relationship between single-nucleotide polymorphisms in genes associated with lower serum 25(OH)D and BC risk. Nevertheless, multiple and in vivo preclinical studies illustrate that 1,25(OH)2D3 or its less calcaemic structural analogues influence diverse cellular processes in BC such as proliferation, differentiation, apoptosis, autophagy and the epithelial-mesenchymal transition. Recent insights also demonstrate that 1,25(OH)2D3 treatment impacts on cell metabolism and on the cancer stem cell population. The presence of VDR in the majority of BCs, together with the various anti-tumoural effects of 1,25(OH)2D3, has supported the evaluation of the effects of vitamin D3 supplementation on BC development. However, most randomized controlled clinical trials do not demonstrate a clear decrease in BC incidence with vitamin D3 supplementation. However, 1,25(OH)2D3 or its analogues seem biologically more active and may have more potential anticancer activity in BC upon combination with existing cancer therapies.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)–, receptor-interacting protein kinase 1 (RIPK1)–, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell–derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell–derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

The intestinal microbiome is implicated as an important modulating factor in multiple inflammatory 1,2 , neurologic 3 and neoplastic diseases 4 . Recent genome-wide association studies yielded inconsistent, underpowered and rarely replicated results such that the role of human host genetics as a contributing factor to microbiome assembly and structure remains uncertain 5-11 . Nevertheless, twin studies clearly suggest host genetics as a driver of microbiome composition 11 . In a genome-wide association analysis of 8,956 German individuals, we identified 38 genetic loci to be associated with single bacteria and overall microbiome composition. Further analyses confirm the identified associations of ABO histo-blood groups and FUT2 secretor status with Bacteroides and Faecalibacterium spp. Mendelian randomization analysis suggests causative and protective effects of gut microbes, with clade-specific effects on inflammatory bowel disease. This holistic investigative approach of the host, its genetics and its associated microbial communities as a 'metaorganism' broaden our understanding of disease etiology, and emphasize the potential for implementing microbiota in disease treatment and management.

The long-recognized role of the vitamin D endocrine system is to maintain stable serum calcium concentrations, which are ensured by a complex interplay between parathyroid gland, kidney, intestine, and bone. However, although VDR is expressed in osteoclastogenic cells, the contribution of VDR-mediated signaling to osteoclast differentiation and activity remains undefined. We therefore deleted Vdr expression efficiently and specifically in myeloid cells by use of M lysozyme-driven Cre expression, which targets granulocytes, monocytes, macrophages and osteoclasts (Vdr myel- mice). Bone and calcium homeostasis were investigated under basal conditions and in conditions of increased bone remodeling, by feeding Vdr myel- and Vdr myel+ (wildtype) mice either a normal (1%) or a low (0.02%) calcium diet from weaning onwards. Vdr myel- mice developed normally and were normocalcemic at the age of 8 weeks, both at the normal and the low calcium diet. No differences in trabecular or cortical bone mass were observed between Vdr myel- mice and their wildtype littermates. Dietary calcium restriction resulted in a comparable reduction of trabecular bone mass (40%) and cortical thickness (48%) in Vdr myel- and Vdr myel+ mice, pointing to a massive transfer of calcium from the bone to the serum. In agreement with these results, osteoclastic differentiation of hematopoietic cells of Vdr myel- mice, either induced by M-CSF and RANKL, or cocultured with osteoblasts, occurred as efficiently as osteoclastogenesis from Vdr myel+ mice. In conclusion, our data do not support a role for osteoclastic Vdr signaling in the control of bone homeostasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution, and development of both players. These interdependencies inspired a new view of multicellular organisms as "metaorganisms." The goal of the Collaborative Research Center "Origin and Function of Metaorganisms" is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants., Methods: In order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample., Conclusion: While 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Overall, we recommend single- over multi-step amplification procedures, and although exceptions and trade-offs exist, the V3 V4 over the V1 V2 region of the 16S rRNA gene. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition.

Bisphosphonates like risedronate inhibit osteoclast-mediated bone resorption and are therefore used in the prevention and treatment of osteoporosis. Also vitamin D 3 and calcium supplementation is commonly used in the prevention or treatment of osteoporosis. Combined therapy of risedronate with 1,25(OH) 2 D 3 , the active metabolite of vitamin D 3 , may be advantageous over the use of either monotherapy, but bears a risk of causing hypercalcemia thereby decreasing the therapeutic window for osteoporosis treatment. In this study, we evaluated the effect on bone mass of the combination of risedronate with the 17-methyl 19-nor five-membered D-ring vitamin D 3 analog WY 1048 in a mouse ovariectomy model for postmenopausal osteoporosis. Ovariectomy-induced bone loss was restored by administration of risedronate or a combination of risedronate with 1,25(OH) 2 D 3. However, the combination of WY 1048 with risedronate induced an even higher increase on total body and spine bone mineral density and on trabecular and cortical bone mass. Our data indicate that combination therapy of risedronate with WY 1048 was superior in restoring and improving bone mass over a combination of risedronate with 1,25(OH) 2 D 3 with minimal calcemic side effects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)