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Smad5 (small mothers against decapentaplegic 5) protein is a receptor-regulated member of the Smad family proteins, mainly participating in the bone morphogenetic protein (BMP) signaling pathway in its phosphorylated form. This article will provide a detailed review of Smad5, focusing on its gene characteristics, protein structure, and subcellular localization properties. We will also explore the related signaling pathways and the mechanisms of Smad5 in respiratory diseases, including chronic obstructive pulmonary disease (COPD), bronchial asthma, pulmonary arterial hypertension(PAH), lung cancer, and idiopathic pulmonary fibrosis (IPF). Additionally, the review will cover aspects such as proliferation, differentiation, apoptosis, anti-fibrosis, and mitochondrial function metabolism. In addition, the review will cover aspects of proliferation, differentiation, apoptosis, anti-fibrosis and functional mitochondrial metabolism related to the above topics. Numerous studies suggest that Smad5 may play a unique and important role in the pathogenesis of respiratory system diseases. However, in previous research, Smad5 was mainly used to broadly determine the activation of the BMP signaling pathway, and its own function has not been given much attention. It is worth noting that Smad5 has distinct nuclear-cytoplasmic distribution characteristics different from Smad1 and Smad8. It can undergo significant nuclear-cytoplasmic shuttling when intracellular pH (pHi) changes, playing important roles in both the classical BMP signaling pathway and non-BMP signaling pathways. Given that Smad5 can move intracellularly in response to changes in physicochemical properties, its cellular localization may play a crucial role in the development of respiratory diseases. This article will explore the possibility that its distribution characteristics may be an important factor that is easily overlooked and not adequately considered in disease research., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors read and approved the final version of the manuscript. Otherwise consent not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The proliferation of the endothelium is a highly coordinated process to ensure the emergence, expansion, and homeostasis of the vasculature. While Bone Morphogenetic Protein (BMP) signaling fine-tunes the behaviors of endothelium in health and disease, how BMP signaling influences the proliferation of endothelium and therefore, modulates angiogenesis remains largely unknown. Here, we evaluated the role of Activin A Type I Receptor (ACVR1/ALK2), a key BMP receptor in the endothelium, in modulating the proliferation of endothelial cells. We show that ACVR1/ALK2 is a key modulator for the proliferation of endothelium in the retinal vessels. Loss of endothelial ALK2 leads to a significant reduction in endothelial proliferation and results in fewer branches/endothelial cells in the retinal vessels. Interestingly, venous endothelium appears to be more susceptible to ALK2 deletion. Mechanistically, ACVR1/ALK2 inhibits the expression of CDKN1A/p21, a critical negative regulator of cell cycle progression, in a SMAD1/5-dependent manner, thereby enabling the venous endothelium to undergo active proliferation by suppressing CDKN1A/p21. Taken together, our findings show that BMP signaling mediated by ACVR1/ALK2 provides a critical yet previously underappreciated input to modulate the proliferation of venous endothelium, thereby fine-tuning the context of angiogenesis in health and disease., Competing Interests: Declarations Competing interests None., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective :The aim of our study was to determine the molecular mechanism of BMP4 (bone morphogenetic protein 4) in DR (diabetic retinopathy). Methods : Human retinal endothelial cell (HRECs) induced by high glucose to simulate one of the pathogenesis in the diabetic retinopathy (DR) model. RT-qPCR and western blot were used to detect the mRNA and protein levels of BMP4 in the STZ/HG group. Flow cytometry and TUNEL staining were performed to detect the apoptosis. Angiogenesis was evaluated by tube formation assay. Transwell assay and wound healing assay were used to detect cell migration ability. H&E staining was used to evaluate the pathological changes. Results : BMP4 was significantly upregulated in the STZ/HG group. Sh-BMP4 significantly inhibited the migration and angiogenesis of RVECs induced by HG. In addition, both in vivo and in vitro experiments confirmed that sh-BMP4 could significantly promote RVECs apoptosis in the HG/STZ group. Western blot results showed that sh-BMP4 could down-regulate the expressions of p-smad1, p-smad5 and VEGF. Conclusions : Inhibition of BMP4 could alleviate the damage of diabetic retinopathy by regulating the p-smad1/5/VEGF signaling axis, inhibiting angiogenesis and promoting apoptosis.

Background: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation., Methods: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar 2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays., Results: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells., Conclusion: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells., (© 2024. The Author(s).)

Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from Caenorhabditis elegans and Drosophila , respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on day 5 differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in day 7 differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy. NEW & NOTEWORTHY The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.

Vascular remodeling to match arterial diameter to tissue requirements commonly fails in ischemic disease. Endothelial cells sense fluid shear stress (FSS) from blood flow to maintain FSS within a narrow range in healthy vessels. Thus, high FSS induces vessel outward remodeling, but mechanisms are poorly understood. We previously reported that Smad1/5 is maximally activated at physiological FSS. Smad1/5 limits Akt activation, suggesting that inhibiting Smad1/5 may facilitate outward remodeling. Here we report that high FSS suppresses Smad1/5 by elevating KLF2, which induces the bone morphogenetic protein (BMP) pathway inhibitor, BMP-binding endothelial regulator (BMPER), thereby de-inhibiting Akt. In mice, surgically induced high FSS elevated BMPER expression, inactivated Smad1/5 and induced vessel outward remodeling. Endothelial BMPER deletion impaired blood flow recovery and vascular remodeling. Blocking endothelial cell Smad1/5 activation with BMP9/10 blocking antibodies improved vascular remodeling in mouse models of type 1 and type 2 diabetes. Suppression of Smad1/5 is thus a potential therapeutic approach for ischemic disease., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Aims: Bone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence., Methods and Results: We identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays., Conclusions: This study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction., Competing Interests: Declaration of competing interest P.D.U. and N.W.M. have published US (US10336800) and EU (EP3166628B1) patents entitled: ‘Therapeutic Use of Bone Morphogenetic Proteins’. All other authors declare no conflicts of interests. The funders had no role in the study's design, in the collection, analyses, interpretation of data or writing of the manuscript., (Copyright © 2023. Published by Elsevier Inc.)

Objectives: Promoting the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is a way to regenerate periodontal bone. This study aimed to determine whether lncRNA MALAT1 promotes the osteogenic differentiation of human PDLSCs in vitro., Materials and Methods: Human PDLSCs were extracted from the human periodontal ligament, and after osteogenic differentiation was induced using osteogenic medium, the human PDLSCs were transfected with siRNA-MALAT1, miR-93-5p mimics, and miR-93-5p inhibitors. The expression of osteogenesis-related genes was assessed by RT-qPCR and western blotting, alkaline phosphatase (ALP) activity was assessed by ALP activity assay, and the formation of mineralized nodules was assessed by alizarin red S (ARS) staining. RNA immunoprecipitation (RIP) and luciferase assays were performed to assess the binding of MALAT1, miR-93-5p, and SMAD5., Results: The expression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was upregulated, while that of miR-93-5p was downregulated after PDLSC osteogenic differentiation. Knockdown of MALAT1 inhibited the osteogenic differentiation of PDLSCs, and MALAT1 expression negatively correlated with miR-93-5p expression. miR-93-5p inhibited the osteogenic differentiation of human PDLSCs by specifically binding to SMAD5., Conclusion: MALAT1 regulates human PDLSC differentiation by regulating the miR-93-5p/SMAD5 axis., (© 2023 Wiley Periodicals LLC.)

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci ( Smad1 HA/HA and Smad5 PA/PA ). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers ( IGFBP1, PRL, FOXO1 ) and PR-responsive genes ( RORB , KLF15 ). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy., Competing Interests: ZL, ST, KN, KS, MI, DM, MM No competing interests declared, (© 2023, Liao et al.)

Background: The implication of deregulated circular RNAs in osteoporosis (OP) has gradually been proposed. Herein, we aimed to study the function and mechanism of circ_0001825 in OP using osteogenic-induced human-derived mesenchymal stem cells (hMSCs)., Methods: The content of genes and proteins was tested by quantitative real-time polymerase chain reaction and Western blotting. The osteogenic differentiation in hMSCs were evaluated by ALP activity and Alizarin Red staining, as well as the detection of osteogenesis-related markers. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry. The binding between miR-1270 and circ_0001825 or SMAD5 (SMAD Family Member 5) was confirmed by using dual-luciferase reporter assay and pull-down assay., Results: Circ_0001825 was lowly expressed in OP patients and osteogenic induced hMSCs. Knockdown of circ_0001825 suppressed hMSC viability and osteogenic differentiation, while circ_0001825 overexpression showed the exact opposite effects. Mechanistically, circ_0001825/miR-1270/SMAD5 formed a feedback loop. MiR-1270 was increased and SMAD5 was decreased in OP patients and osteogenic induced hMSCs. MiR-1270 up-regulation suppressed hMSC viability and osteogenic differentiation, which was reversed by SMAD5 overexpression. Moreover, miR-1270 deficiency abolished the effects of circ_0001825 knockdown on hMSCs., Conclusion: Circ_0001825 promoted hMSC viability and osteogenic differentiation via miR-1270/SMAD5 axis, suggesting the potential involvement of circ_0001825 in osteoporosis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we employed multiple laboratory techniques to acknowledge the biological activities and processes of Per2 and Id3 in glioma. We analyzed TCGA and CGGA databases for seeking association among Per2, Id3, and clinical features in glioma. Immunohistochemistry and Western blot were used to detect protein expression levels. CCK-8 assay, colony formation assay, Transwell assay, the wound healing assay, flow cytometric, and Xenograft nude mice were used to acknowledge the impact of Per2 and Id3 on biological behavior of glioma. The results showed that the Per2 mRNA expression was negatively correlated with the WHO grade, while the Id3 mRNA expression was positively correlated with the WHO grade in patients with glioma in TCGA and CGGA databases. Per2 and Id3 maintained separate prognostic abilities and had a negative connection in human glioma. In the clinical sample study, Per2 and Id3 were validated at the protein level with the same results compared to the mRNA expression level in TCGA and CGGA. By using a wide range of functional examples, overexpression of Per2 restrains malignant biological behaviors in glioma cells by many ways, while Id3 promotes malignant biological behaviors in glioma cells. Furthermore, overexpression of Per2 can inhibit Id3 expression via regulating PTEN/AKT/Smad5 signaling pathway and thereby abolish malignant biological behaviors that are caused by Id3 overexpression. These results suggested that Per2 inhibits glioma cell proliferation through regulating PTEN/AKT/Smad5/Id3 signaling pathway, which may be a viable therapeutic target for glioma.

The SMAD1 and SMAD5 genes belong to mothers against decapentaplegic proteins family, which participate in the BMP pathway to control skeletal myogenesis and growth. In the present study, we analyzed the associations between polymorphisms of SMAD1 and SMAD5 genes promoter and important economical traits in Qinchuan cattle. Four SNPs in the SMAD1 gene promoter and three SNPs in the SMAD5 promoter were identified by sequencing of 448 Qinchuan cattles. Allelic and frequency analyses of these SNPs resulted in eight haplotypes both in the promoters of the two genes promoter and identified potential cis-regulatory transcription factor (TF) components. In addition, correlation analysis showed that cattle SMAD1 promoter activity of individuals with Hap4 (P < 0.01) was stronger than that of individuals with Hap2. while the transcriptional activity of individuals with Hap3 within SMAD5 gene promoter was significantly (P < 0.01) higher followed by H2. Uniformly, diplotypes H4-H6 of SMAD1 gene and H1-H3 of SMAD5 gene performed significant (P < 0.01) associations with body measurement and improved carcass quality traits. All these results have indicated that polymorphisms in SMAD1 and SMAD5 genes promoter could impact the transcriptional regulation and then affect muscle content in beef cattle. Moreover, both the SMAD1 and SMAD5 genes were expressed ubiquitously in 10 tissues and had higher expression in the longissimus thoracis tissue from 6-month-old and 12-month-old cattle than in cattle of other ages. We can conclude that SMAD1 and SMAD5 genes may play an important role in muscle growth and development, and the variants mapped within SMAD1 and SMAD5 genes can be utilized in molecular marker-assisted selection for cattle carcass quality and body measurement traits in breed improvement programs of Qinchuan cattle., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Evidence has shown that mesenchymal stem cells' (MSCs) therapy has potential application in treating chronic kidney disease (CKD). In addition, MSCs-derived exosomes can improve the renal function and prevent the progression of CKD. However, the mechanisms by which MSCs-derived exosomes (MSCs-Exo) ameliorate renal fibrosis in CKD remain largely unclear. To mimic an in vitro model of renal fibrosis, rat kidney tubular epithelial cells (NRK52E) were stimulated with transforming growth factor (TGF)-β1. In addition, we established an in vivo model of unilateral ureteric obstruction (UUO)-induced renal fibrosis. Meanwhile, we exploited exosomes derived from MSCs for delivering miR-186-5p agomir into NRK52E cells or kidneys in vitro and in vivo. In this study, we found that level of miR-186-5p was significantly downregulated in TGF-β1-stimulated NRK52E cells and the obstructed kidneys of UUO mice. In addition, miR-186-5p can be transferred from MSCs to NRK52E cells via exosomes. MSCs-delivered miR-186-5p markedly reduced the accumulation of extracellular matrix (ECM) protein, and inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in TGF-β1-stimulated NRK52E cells. Moreover, exosomal miR-186-5p from MSCs attenuated kidney injury and fibrosis in a UUO mouse model via inhibition of the ECM protein accumulation and EMT process. Meanwhile, dual-luciferase assay showed that miR-186-5p downregulated Smad5 expression via direct binding with the 3'-UTR of Smad5. Collectively then, these findings indicated that exosomal miR-186-5p derived from MSCs could attenuate renal fibrosis in vitro and in vivo by downregulation of Smad5. These findings may help to understand the role of MSCs' exosomes in alleviating renal fibrosis in CKD., (© 2021. Japan Human Cell Society.)

Long non-coding RNAs (lncRNAs) are involved in developing hepatocellular carcinoma (HCC). The present study explored the role of lncRNA LINC01194, which is upregulated in HCC tissues and might be a vital regulator in HCC progression. Levels of LINC01194, microRNA (miR)-655-3p, and SMAD family member 5 (SMAD5) were assessed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The bioactivity of Huh-7 cells was assessed using cell counting kit-8 and transwell assays and flow cytometry. Western blotting was conducted to measure the expression of invasion- and apoptosis-related proteins. The relationships between lncRNA LINC01194 and miR-655-3p, and miR-655-3p and SMAD5 were predicted using StarBase and TargetScan, and further verified using a dual-luciferase reporter assay. LINC01194 was overexpressed in HCC cells and in clinical samples. ILINC01194 silencing suppressed proliferation and migration; however, it promoted apoptosis in HCC cell lines. We also confirmed that miR-655-3p could bind to LINC01194, and miR-655-3p was downregulated in HCC. The upregulation of miR-655-3p suppressed HCC cell invasion and migration, and enhanced the number of apoptotic cells. SMAD5, which was overexpressed in HCC cell lines, was directly targeted by miR-655-3p. Therefore, LINC01194 promoted HCC development by decreasing miR-655-3p expression and may serve as a promising therapeutic target for HCC patients.

Accumulating evidence suggests that long non-coding RNAs (lncRNAs) participate in the formation and development of keloids, a benign tumor. In addition, lncRNA H19 has been shown to act on the biological processes of keloids. This study aimed to identify other important mechanisms of the effect of lncRNA H19 on keloid formation. The H19, miR-196b-5p, and SMAD family member 5 (SMAD5) expression levels were detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Subcellular localization of lncRNA H19 was detected using a nuclear-cytoplasmic separation assay. Cell viability and proliferation were measured using counting kit-8 and colony formation assays. Bax and Bcl-2 levels were examined using Western blot analysis. The interaction between H19 and miR-196b-5p or SMAD5 was verified using a dual-luciferase reporter assay. H19 and SMAD5 expression was upregulated in keloid tissue and fibroblasts, whereas miR-196b-5p expression was downregulated. Knockdown of H19, overexpression of miR-196b-5p, or knockdown of SMAD5 inhibited the viability and proliferation of keloid fibroblasts and promoted apoptosis. Overexpression of H19 or SMAD5 and knockdown of miR-196b-5p promoted viability and proliferation and inhibited apoptosis. miR-196b-5p was identified as a H19 sponge, and SMAD5 was identified as a miR-196b-5p target. The combination of lncRNA H19 and miR-196b-5p regulates SMAD5 expression and promotes keloid formation, thus providing a new direction for keloid treatment.

Bladder cancer (BC) is the 9 th most frequent diagnosed tumor and the 2 nd most common urology tumor worldwide. Despite the considerable advancement that BC treatment has made recently, the five-year survival rate of BC remains unsatisfactory. Novel therapeutic strategies for BC clinical intervention are therefore urgently needed now more than ever. circRHOT1 is a newly identified circRNA that plays a crucial role in multiple types of tumorigeneses. However, it remains unclear whether circRHOT1 plays a functional role in BC progression. Our findings suggest that circRHOT1 was highly expressed in BC tumor tissues and cell lines. The results from CCK-8, EDU, Transwell migration, and NK cell-mediated cytotoxicity detection assays suggested that circRHOT1 knockdown could markedly suppress BC cell proliferation and migration level and could aggravate the sensitivity of BC cells to NK cells. Subsequently, we conducted bioinformatics analysis followed by RNA pull-down, ChIP, and luciferase reporter assays, from which we found that circRHOT1 expression in BC cells could be regulated by ZNF652, and circRHOT1 could promote SMAD5 expression to regulate BC cell cellular progression by sponging miR-3666. These results may provide a new direction for developing novel diagnostic or therapeutic targets for BC., Competing Interests: The authors declare no financial or commercial conflict of interest., (Copyright © 2021 Hu Ke et al.)

Gastric cancer (GC) is a common malignant gastrointestinal tumor with high mortality. Previous study has reported that the overexpression of lncRNA HCP5 was observed in gastric cancer tissues. The purpose of this study was to investigate the molecular mechanism underlying the effect of lncRNA HCP5 on the proliferative, migratory, and invasive abilities of GC cells. The relative mRNA expression of HCP5, miR-299-3p, and SMAD5 were determined by RT-qPCR. The expressions of proteins associated with apoptosis and invasion were detected by western blot. The interaction of HCP5 with miR-299-3p and SMAD5 with miR-299-3p was confirmed by luciferase reporter assay. The cellular behaviors of AGS cells were, respectively, detected by CCK-8 assays, colony formation assays, migration and invasion assays, and flow cytometry. In our study, lncRNA HCP5 was highly expressed in GC cell lines compared with normal gastric epithelial cell. LncRNA HCP5 silencing inhibited AGS cells proliferation, migration, and invasion, while promoted cell apoptosis. Moreover, miR-299-3p downregulation could abolish the effect of HCP5 knockdown on cellular behaviors of AGS cells. Interestingly, SMAD5 is identified as the downstream target of miR-299-3p, and its expression was inhibited by miR-299-3p. More importantly, SMAD5 silencing inhibited proliferation, migration, and invasion of GC cells, and promoted cell apoptosis. In a word, lncRNA HCP5 silencing inhibits GC cell proliferation, invasion, and migration while promoting its apoptosis via regulation of miR-299-3p/SMAD5 axis. Hence, lncRNA HCP5 could be a novel and promising target for GC treatment.

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d ​+ ​12 ​h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, TNF-α, NFκB p65, Iκβ, TGF-β, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-β were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68 , Bmpr2 , and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1β, and Cd68 in the lung. In addition, TGF-β, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-β. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1 Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1 G309S -expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis., Competing Interests: Declaration of interests W.W. is a co-founder and consultant for ReKindle Therapeutics. The other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Human dental pulp stem cells (hDPSCs) are the preferable choice of seed cells for craniomaxillofacial bone tissue regeneration. As a member of the miR-17-92 cluster, miR-20a-5p functions as an important regulator during bone remodeling. This study aimed to investigate the roles and mechanisms of miR-20a-5p during osteogenesis of hDPSCs., Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to determine the expression of miR-20a-5p during osteogenesis of hDPSCs. We interfered with the expression of miR-20a-5p in hDPSCs to clarify the function of miR-20a-5p on osteogenesis both in vitro and vivo. Direct bind sites between miR-20a-5p and BAMBI were confirmed by dual-luciferase reporter assay, and the underlying mechanisms were investigated with cell co-transfections., Results: The expression of miR-20a-5p was showed to be upregulated during osteogenesis of hDPSCs. Inhibition of miR-20a-5p could weaken the intensity of ALP/ARS staining and downregulate the expression of mRNAs and proteins of osteogenic markers, while overexpression of miR-20a-5p could enhance the intensity of ALP/ARS staining and the expression of osteogenic markers. Both micro-CT reconstruction images and histological results showed that miR-20a-5p could promote the regeneration of calvarial defects. miR-20a-5p directly targeted bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), and the latter one was an inhibitor of hDPSC osteogenesis. Silencing BAMBI partially reversed the suppression effect of miR-20a-5p knockdown on osteogenesis. Phosphorylation of Smad5 and p38 was decreased when miR-20a-5p was silenced, whereas p-Smad5 and p-p38 were upregulated when miR-20a-5p was overexpressed or BAMBI was silenced., Conclusions: It is demonstrated that miR-20a-5p functioned as a regulator of BAMBI to activate the phosphorylation of Smad5 and p38 during osteogenic differentiation of hDPSCs., (© 2021. The Author(s).)

We studied the effects and mechanisms of action of conophylline in different concentrations in the original in vitro model of myocardial fibrosis (treatment of cardiac fibroblasts isolated form the hearts of newborn rats with angiotensin II). Viability, collagen content, and expression of related protein in cardiac fibroblasts were assessed using the MTT-test, Sircol assay, and Western blotting, respectively. Conophylline markedly protected the cultured cells against the development of angiotensin II-induced fibrosis, which was seen from reduced viability of fibroblasts, decreased collagen content, and down-regulation of the expression of α-smooth muscle actin (α-SMA). Conophylline did not affect the TGF-β pathway altered by angiotensin II, but markedly decreased the level of bone morphogenetic protein-4 (BMP4) enhanced by angiotensin II and BMP4 itself. Conophylline produced no effect on phosphorylation of α-SMA and Smad homologue-1/5/8, the classic BMP4 downstream pathway elements, but reduced the level of c-Jun N-terminal kinase (JNK) elevated by BMP4. Conophylline did not inhibit the development of myocardial fibrosis in the presence of JNK activator anisomycin. Thus, conophylline inhibited angiotensin II-provoked myocardial fibrosis via the BMP4/JNK pathway., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: Long noncoding RNAs have been known to play key roles in myocardial ischemia/reperfusion injury. This study was conducted to investigate whether upregulation of FGD5-AS1 can improve hypoxia/reoxygenation (H/R) injury of cardiomyocytes and its underlying mechanisms. Pc-FGD5-AS1 was used to overexpress FGD5-AS1 in cardiomyocytes. Cholecystokinin octapeptide and flow cytometry assays were performed to detect the effect of FGD5-AS1 on myocardial cell H/R injury. Quantitative real-time polymerase chain reaction and luciferase reporter assay were performed to assess the relationship between FGD5-AS1 and microRNA-106a-5p (miR-106a-5p) or miR-106b-5p. In patients with acute myocardial infarction and in H/R cardiomyocytes and ischemia/reperfusion myocardium, the expression levels of FGD5-AS1 were reduced, whereas the expression levels of miR-106a-5p and miR-106b-5p were increased. Overexpression of FGD5-AS1 increased the viability of H/R-treated cardiomyocytes and reduced the levels of apoptosis and creatine kinase-MB. In addition, FGD5-AS1 could bind to miR-106a-5p or miR-106b-5p and showed a mutual inhibitory effect between them. Furthermore, overexpression of miR-106a-5p or miR-106b-5p inhibited the expression of SMAD5. FGD5-AS1 upregulated the expression of SMAD5. In conclusion, FGD5-AS1 may be a potential therapeutic target for myocardial H/R injury, and its cardioprotective effect may be realized by reducing inflammatory response and cell apoptosis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

BMP10 plays an essential role in regulating cardiac growth, chamber maturation, and maintaining normal expressions of several key cardiogenic factors; however, other functional roles of BMP10 in muscle remain unexplored. This study therefore undertook to investigate the roles of BMP10 in muscle physiology, using mouse-derived C2C12 myoblasts. Bmp10 silencing prevented a number of biological processes such as myogenic differentiation, glucose uptake, and lipid catabolism, whereas exogenous induction of BMP10 in C2C12 cells significantly stimulated the expression of proteins and genes involved in these processes, as well as mitochondrial biogenesis and thermogenesis, resulting in reduced lipid accumulation. A mechanistic study revealed that BMP10 stimulates myogenesis mainly via the Smad 1/5/8 signaling pathway. In conclusion, our data unveiled a previously unknown mechanism in the regulation of lipid metabolisms by BMP10 in muscle cells and identified its significant roles in systemic metabolic homeostasis, shedding light on BMP10 as a pharmacotherapeutic target to treat metabolic disorders.

Maternal nutrition is crucial for the offspring's skeleton development and the onset of osteoporosis later in life. While maternal low protein diet has been shown to regulate bone mass negatively, the effect of a high protein diet (HP) remains unexplored. Here, we found that C57BL/6 mice fed with HP delivered offspring with decreased skeletal mineralization at birth and reduced bone mass throughout their life due to a decline in their osteoblast maturation. A small RNA sequencing study revealed that miR-24-1-5p was highly upregulated in HP group osteoblasts. Target prediction and validation studies identified SMAD-5 as a direct target of miR-24-1-5p. Furthermore, mimic and inhibitor studies showed a negative correlation between miR-24-1-5p expression and osteoblast function. Moreover, ex vivo inhibition of miR-24-1-5p reversed the reduced maturation and SMAD-5 expression in the HP group osteoblasts. Together, we show that maternal HP diminishes the bone mass of the offspring through miR-24-1-5p.

Sertoli cells play vital roles in normal spermatogenesis, and microRNAs (miRNAs) participate in regulating Sertoli cell development. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. Herein, we primarily explored the regulatory roles of miR-130a in immature porcine Sertoli cells using EdU-based high-content screening assay. The results demonstrated that 27 miRNAs have potential roles in the promotion of immature porcine Sertoli cell proliferation, and miR-130a was identified as a promising candidate. miR-130a promoted cell cycle progression and cell proliferation, whereas it impeded cell apoptosis in immature porcine Sertoli cells. It also contributed to Sertoli cell proliferation and testis development in vivo. A TMT-based proteomics approach revealed that miR-130a regulated the expression of 91 proteins and multiple pathways, including the TGF-β and PI3K/AKT signaling. miR-130a did not directly target the 3'-UTR of SMAD5; however, it increased SMAD5 phosphorylation. Moreover, miR-130a enhanced TGF-β signaling by activating SMAD5 protein, and TGF-β signaling further activated the PI3K/AKT signaling pathway to promote cell proliferation and inhibit cell apoptosis in porcine immature Sertoli cells. Collectively, miR-130a promoted immature porcine Sertoli cell growth by activating SMAD5 through the TGF-β-PI3K/AKT signaling pathway. This study, therefore, provides novel insights into the effects of miR-130a on porcine spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

As a stable environmental contaminant, nonylphenol (NP) has been shown to induce some neurological deficits in the cerebellum, although the underlying mechanism is still unknown. In the present study, we aimed to investigate the effects of perinatal exposure to NP on myelination, an important process essential for the intact cerebellar function, in the offspring cerebellum. Exposure to NP delayed the myelination in the offspring cerebellum during perinatal period. The myelination recovered in the cerebellum of offspring exposed to NP over time, and returned to normal in adulthood. In addition, perinatal exposure to NP reduced mature oligodendrocytes (myelin-forming glial cells) and increased astrocytes in the offspring cerebellum. BMP signaling is believed to negatively regulate oligodendrogliogenesis and myelination. In the present study, BMP4, p-Smad1/5, and ID4, key members of BMP signaling, were increased in the cerebellum of offspring exposed to NP. Taken together, these lines of evidence suggest that the activation of BMP signaling may underlie the decreased oligodendrogliogenesis and increased astrogliogenesis, and the consequent delay of myelination in the cerebellum of offspring perinatally exposed to NP., 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 © 2020 Elsevier Inc. All rights reserved.)

Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is downregulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The growth and evolutionary expansion of the cerebral cortex are defined by the spatial-temporal production of neurons, which itself depends on the decision of radial glial cells (RGCs) to self-amplify or to switch to neurogenic divisions. The mechanisms regulating these RGC fate decisions are still incompletely understood. Here, we describe a novel and evolutionarily conserved role of the canonical BMP transcription factors SMAD1/5 in controlling neurogenesis and growth during corticogenesis. Reducing the expression of both SMAD1 and SMAD5 in neural progenitors at early mouse cortical development caused microcephaly and an increased production of early-born cortical neurons at the expense of late-born ones, which correlated with the premature differentiation and depletion of the pool of cortical progenitors. Gain- and loss-of-function experiments performed during early cortical neurogenesis in the chick revealed that SMAD1/5 activity supports self-amplifying RGC divisions and restrains the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signalling effector YAP. We anticipate this SMAD1/5-YAP signalling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: Cholangiocarcinoma (CCA) is generally associated with high incidence and poor prognosis. Nowadays, increasing experimental data demonstrate that long non-coding RNA (lncRNA) plays an indispensable role in tumor occurrence. Nevertheless, the specific mechanism of lncRNA is not clear in CCA., Methods: The relative expressions of lncRNAs, miRNAs, and mRNAs were detected by real-time quantitative PCR (RT-qPCR). CCK8 and colony formation assays were applied to examine cell proliferation ability in CCA. Transwell assay was conducted to measure the migration and invasion capabilities of CCA cells. Nuclear and cytoplasmic separation assay was implemented to figure out the location of LINC01410. Luciferase reporter assay, RIP and RNA pull-down assays were applied to certify the molecular bindings. Western blot was applied to detect the protein level., Results: The high expression of LINC01410 was proved in CCA tissues and CCA cell lines. Also, CCA patients with high LINC01410 level presented poor prognosis. LINC01410 deficiency impeded cell proliferation, migration and invasion in HuCCT1 and RBE cell lines. What's more, LINC01410 interacted with miR-124-3p. Meanwhile, SMAD5 targeted and inhibited by miR-124-3p. SMAD5 expression was enhanced by LINC01410., Conclusion: LINC01410 facilitates cell proliferation, migration and invasion through miR-124-3p/SMAD5 axis., (© 2020 John Wiley & Sons, Ltd.)

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries., (© 2019 Wiley Periodicals, Inc.)

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) is a ubiquitous environmental pollutant and also a strong teratogen for cleft palate (CP). But up to now, the underlying molecular mechanisms of TCDD-induced CP are largely unknown. More recently, accumulating evidences are revealing important roles of long noncoding RNAs (lncRNAs) in all kinds of diseases including CP. However, the role and molecular mechanism of lncRNAs in TCDD-induced CP are still largely unexplored. Thus, identification of differentially expressed lncRNA (DEL) might help figuring out the mechanism of CP induced by TCDD. In this study, a CP offspring model of C57BL/6 female mice was generated by TCDD (64 µg/kg body weight) induce on embryo day 10 (E10). The incidence rate of CP was 100% in the TCDD group (105) after cervical dislocation on E16. Then, the high-throughput RNA sequencing (RNA-seq) was established to search a comprehensive profile of the lncRNAs. In addition, a coexpression network of lncRNA and messenger RNA (mRNA) was performed to discern potential mechanism. The result showed that 26,246 novel lncRNAs and 9635 known lncRNAs were screened out, and 413 lncRNA transcripts and 65 mRNA transcripts were identified as being significantly different between the CP group and control group. Notably, we found that there are seven lncRNAs that can target Smad1 and Smad5, which are key molecules of bone morphogenetic protein (BMP) signaling pathway, which suggested that they may be concerned with BMP signaling in TCDD-induced CP. In addition, some lncRNAs targeted the important molecules of Hippo and Wnt signaling pathways. These results suggested that characteristic lncRNA alterations may play a critical role in TCDD-induced CP, which provided a theoretical basis for further research.

γδT cells have been reported to exert immunosuppressive functions in multiple solid malignant diseases, but their immunosuppressive functional subpopulation in breast cancer (BC) is still undetermined. Here, we collected 40 paired BC and normal tissue samples from Chinese patients for analysis. First, we showed that γδT1 cells comprise the majority of CD3+ T cells in BC; next, we found that CD73+γδT1 cells were the predominant regulatory T-cell (Treg) population in BC, and that their prevalence in peripheral blood was also related to tumour burden. In addition, CD73+γδT1 cells exert an immunosuppressive effect via adenosine generation. We also found that BC could modulate CD73 expression on γδT cells in a non-contact manner. The microarray analysis and functional experiments indicated that breast tumour cell-derived exosomes (TDEs) could transmit lncRNA SNHG16, which upregulates CD73 expression, to Vδ1 T cells. Regarding the mechanism, SNHG16 served as a ceRNA by sponging miR-16-5p, which led to the derepression of its target gene SMAD5 and resulted in potentiation of the TGF-β1/SMAD5 pathway to upregulate CD73 expression in Vδ1 T cells. Our results showed that the BC-derived exosomal SNHG16/miR-16-5p/SMAD5-regulatory axis potentiates TGF-β1/SMAD5 pathway activation, thus inducing CD73 expression in Vδ1 T cells. Our results first identify the significance of CD73+Vδ1 Tregs in BC, and therapy targeting this subpopulation or blocking TDEs might have potential for BC treatment in the future.

Objective: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5 , addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions., Conclusions: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.

Aim: Degenerative calcific aortic valve disease (DCAVD) is a common valve disease characterized by massive calcium deposits in the aortic valve. Osteoblast differentiation of valve interstitial cells (VICs) is responsible for the formation of calcific nodules. This study aims to explore the function and underlying mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 (actin filament-associated protein 1 antisense RNA 1) in the pathogenesis of DCAVD., Methods: AFAP1-AS1, miR-155 and mRNA levels were detected by qRT-PCR. Protein levels were measured by Western blot. Calcification deposition was examined by Alizarin Red staining. The interaction between AFAP1-AS1 and miR-155, as well as miR-155 and SMAD5 was evaluated using luciferase reporter assay., Results: AFAP1-AS1 expression was increased both in calcified aortic valves from DCAVD patients and after osteogenic induction in human VICs. Furthermore, AFAP1-AS1 overexpression promoted osteogenic differentiation of VICs, whereas AFAP1-AS1 knockdown inhibited osteogenic differentiation. Mechanistically, AFAP1-AS1 acted as a sponge for miR-155 to elevate SMAD5 expression. Further functional assays revealed that miR-155 mimic and SMAD5 silencing effectively reversed AFAP1-AS1-promoted osteogenic differentiation of VICs., Conclusion: Collectively, AFAP1-AS1 promotes osteogenic differentiation of VICs, at least in part, by sponging miR-155 to upregulate SMAD5. This study sheds new light on lncRNA-directed therapeutics in DCAVD., Competing Interests: Declaration of competing interest All the authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein-protein action form and plays the role of transcription repressor.

Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5 . We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.

Objective: Osteogenic differentiation plays a crucial role in maintaining general bone homeostasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) act as important regulators during the osteogenesis process. This study aimed to elucidate the mechanism of Potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in osteogenic differentiation., Materials and Methods: Quantitative Real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression of KCNQ1OT1, osteonectin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), miR-320a, mothers against DPP homolog 5 (Smad5). Protein levels of OCN, OPN, RUNX2 and Smad5 were measured by Western blot assay. Alkaline phosphatase (ALP) activity detection assay was performed to examine the ALP activity. The interactions among KCNQ1OT1, miR-320a and Smad5 were determined by dual-luciferase reporter assay., Results: KCNQ1OT1, OCN, OPN and RUNX2 expression were enhanced in human bone marrow-derived mesenchymal stem cells (hBMSCs) treated with osteogenic medium (OM). KCNQ1OT1 positively regulated OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Furthermore, KCNQ1OT1 directly bound to miR-320a, and KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression and ALP activity by suppressing miR-320a expression. Moreover, Smad5 was a target of miR-320a, and miR-320a inhibition abated the effects of Smad5 silencing in OCN, OPN and RUNX2 expression and ALP activity of hBMSCs. Also, KCNQ1OT1 knockdown reduced OCN, OPN and RUNX2 expression by targeting miR-320a/Smad5 axis., Conclusions: KCNQ1OT1 promoted osteogenic differentiation of hBMSCs by regulating Smad5 expression via sponging miR-320a.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1 . Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot., Competing Interests: JD, MV, GC, PM, MP, RN, DO, GR, CM, VR, BC No competing interests declared, (© 2020, Darrigrand et al.)

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs - including in HHT patient blood outgrowth ECs - and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals worldwide. Oxidative stress injury to retinal pigment epithelial (RPE) cells plays a major role in the pathogenesis of AMD. The purpose of this study was to observe the correlation between Hepcidin and neovascular age-related macular degeneration (nAMD) and to further observe whether oxidative stress can inhibit Hepcidin expression through relevant signaling pathways to produce oxidative damage. We compared the concentrations of Hepcidin in the aqueous humor of nAMD patients and a control group and found that the concentration of Hepcidin was lower in nAMD patients. Through PCR and western blotting, we observed that H 2 O 2 can significantly inhibit the expression of Bone morphogenetic protein-6 (BMP-6) and Hepcidin and increase the intracellular iron concentration in RPE cells, while BMP-6 can reverse the inhibition of Hepcidin and the increase in iron concentration caused by H 2 O 2 . In addition, alterations in smad1 and smad5 expression were examined, and pretreatment with BMP-6 was demonstrated to reduce H 2 O 2 -induced activation of smad1 and smad5. The effects of BMP-6 were attenuated by smad1 and smad5 siRNA, further verifying that oxidative stress inhibits the expression of Hepcidin by inhibiting activation of the BMP/SMAD signaling pathway. To some extent, this study verified that oxidative stress injury plays a role in nAMD by affecting the level of hepcidin, which lays a foundation for exploring new methods to treat nAMD., (© 2020 Japanese Society of Developmental Biologists.)

This study demonstrated different effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling on the induction of germ cell formation in chickens. In vitro, BMP4 significantly promoted primordial germ cell (PGC) formation, while RA promoted spermatogonial stem cell (SSC) formation. Hematoxylin-Eosin (HE) staining of reproductive ridge and testicular slices showed that BMP4 signaling was activated during PGC formation but was inhibited during PGC differentiation into SSC. In contrast, RA signaling was significantly activated during PGC differentiation to SSC. Mechanistically, elevated expression of phosphorylated mothers against decapentaplegic homolog 5 (p-Smad5) activated BMP4 signaling, while inhibition of p-Smad5 significantly reduced the PGC formation. Additionally, BMP4 regulated the PGC formation through histone acetylation and DNA methylation in deleted in azoospermia-like (DAZL) gene. Luciferase report showed RA binding to RARα regulated stimulated by RA 8 (Stra8) promoter activity during SSC formation, while mutations in RAR binding sites inhibited the Stra8 expression and SSC formation. Further, both HAT and HDAC regulated the RARα isoform, and HAT binding to RARα activated the Stra8 transcription. RNA-seq of embryonic stem cells (ESC), PGC, and SSC showed inverse expression of genes related to the BMP4 and RA pathways during PGC and SSC formation. Additionally, Smad5 and Smurf were critical for the interactions between the two pathways. Specifically, through Smurf promotion of Smad5 ubiquitination, RA could inhibit the BMP4 signal transduction. In conclusion, the BMP4 and RA signaling pathways play opposing roles in germ cell formation, driven by epigenetic processes such as phosphorylation, ubiquitination, and histone acetylation.

Keloid, characterized by exuberant collagen deposition and invasive growth beyond original wound margins, results from abnormal wound healing. A recent microarray analysis identified homeobox (HOX) A11 antisense (HOXA11-AS) as a keloid-specific long non-coding RNA, although its potential role in keloid formation remains elusive. In this study, hematoxylin-eosin, Masson, and immunohistochemical staining of type I collagen (ColI) revealed abnormal arrangement and hyperplasia of fibers in keloid tissues along with increased ColI level. qRT-PCR and Western blot showed that HOXA11-AS and ColI were significantly upregulated, while miR-124-3p was decreased in both keloid tissues and human keloid fibroblasts (HKFs). Knockdown of HOXA11-AS inhibited cell proliferation (by CCK-8 and immunofluorescence staining of Ki67) and cell migration (by wound healing and transwell assays). Mechanistic experiments verified that HOXA11-AS acted as a sponge of micro-RNA (miR)-124-3p and Smad5 was a target of miR-124-3p. miR-124-3p sufficiently reversed the regulatory effects of HOXA11-AS, and Smad5 was involved in miR-124-3p-mediated biological functions. Furthermore, HOXA11-AS induced ColI synthesis via sponging miR-124-3p-mediated Smad5 signaling, thus promoting keloid formation. Overall, our study implied that HOXA11-AS induces ColI synthesis to promoted keloid formation via sponging miR-124-3p-mediated Smad5 signaling, which might offer a novel target for developing the therapy of keloid formation.

Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 μg·mL -1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) regulate osteogenic differentiation of bone cells, which has applications in orthodontics. Here we evaluated the miRNA expression profile of MC3T3-E1 osteoblasts under cyclic tensile stress with chip technology and found that miR-132-3p was up-regulated by 12% cyclic tensile stress. Alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 cells were decreased under these conditions. Smad2 and Smad5 were identified as potential target genes of miR-132-3p. Native and phosphorylated Smad2 and Smad5 expression was negatively correlated with miR-132-3p levels in the cells under cyclic stretch; however, only Smad5 protein level was reduced upon miR-132-3p overexpression. The luciferase reporter assay confirmed a direct interaction between miR-132-3p and Smad5. Thus, miR-132-3p maybe regulates osteoblast differentiation via Smad5 in response to cyclic tensile stress.