Bone Morphogenetic Protein Receptor Research Articles

ACVR1/ALK2-p21 signaling axis modulates proliferation of the venous endothelium in the retinal vasculature.

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.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Increased bone morphogenetic protein 10 in precapillary pulmonary hypertension patients

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organization for Scientific Research: NWO-VICI num. 918.16.610 and NWO-VIDI num. 917.18.338. Dutch Heart Foundation Dekker senior post-doc grant num. 2018T059 and the Netherlands CardioVascular Research Initiative: CVON-2017-10 DOLPHIN-GENESIS and CVON-2018-29 PHAEDRA-IMPACT. Fundació La Marató de TV3 num.202038. The Spanish Ministry of Science through the Ramón y Cajal grant num.RYC2021-030866-I. Introduction Precapillary pulmonary hypertension (PH) results in increased right atrial (RA) stretch and pressure. The right atrium is the major source of bone morphogenetic protein (BMP) 10 (BMP10) in adults, mostly produced by RA cardiomyocytes. The major predisposing risk factor for PH involves loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene, through which BMP10 and BMP9 transduce signals. Purpose The aim of this study was to investigate if BMP10 expression is altered in PH, and whether BMP10 signaling activity associates with right ventricular (RV) adaptation, RA function and disease progression. Methods We investigated BMP10 gene and protein expressions in RA tissue. We determined BMP10 and BMP9 plasma levels by ELISA. We studied BMP10 signaling activity in PH and CTEPH patients before and after pulmonary endarterectomy (PEA). Results BMP10 mRNA, protein and activity were significantly increased in the PH right atrium. While circulating levels of BMP10 and BMP9 proteins were increased, no significant changes were observed in BMP10 signaling activity between PH and controls. In a cohort of CTEPH patients, BMP10 signaling activity was reduced after PEA which coincided with improved hemodynamics and RV function. Interestingly, high BMP10 activity was associated with reduced RV function and adaptation, increased RA dilation and decreased RA function. No associations were observed with hemodynamics or general disease markers. Conclusions RA BMP10 expression and plasma levels are increased in precapillary PH. High BMP10 transcriptional activity was associated with worse RV function, RV adaptation, RA dilation and RA function. Future studies are needed to further understand the mechanism underlying BMP10 secretion and function in PH.

BMP9 attenuates IL-33 signaling and prevents endothelial-to-mesenchymal transition in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Cardiovascular Alliance - PHAEDRA IMPACT Background Pulmonary Arterial hypertension (PAH) is a rare and progressive disease characterized by increased vascular resistance and remodeling of the pulmonary arteries. Disturbed Transforming growth factor (TGF)-β signaling, often associated with mutations in the gene encoding for the bone morphogenic protein receptor type II (BMPR2) and endothelial-to-mesenchymal transition (EndMT), emerges as a central culprit in the pathogenesis of PAH. Interleukin-33 (IL-33) has been identified as an inducer of EndMT, and its soluble receptor ST2 (sST2) has emerged as a potential biomarker for PAH. Bone morphogenic protein (BMP) 9, a BMPR2 ligand, showing a high affinity for endothelial cells, is being explored as a therapeutic approach. Purpose We hypothesize that a crosstalk between the TGF-β pathway and IL-33, a known promoter of vascular remodeling, occurs in PAH. This study aims to unravel the protective role of BMP9 in PAH, with a specific focus on the involvement of EndMT, a key player in the pathogenesis of vascular diseases. Methods The expression of IL-33 protein levels was assessed in Pulmonary Arterial Endothelial Cells (PAEC) and in lung tissues from experimentally induced PH (Sugen/Hypoxia (SuHx)) or control mice. Signal transduction and EndMT were investigated in PAECs exposed to IL-33, BMP9 and sST2, by gene expression analysis (qPCR), western blotting, ELISA and immunostaining. BMP9 and sST2 levels were measured in plasma from PAH patients by ELISA. Results Elevated IL-33 expression was observed in PAEC from PAH patients and lung tissues from SuHx mice compared to healthy controls. In vitro, BMP9 significantly induced the expression of the IL-33 soluble receptor ST2, while concurrently reducing IL-33 target gene expression. IL-33-induced EndoMT in PAEC was effectively mitigated by pre-incubation with BMP9, proving more efficacious than ST2 neutralizing antibody. Furthermore, a correlation between BMP9 and sST2 levels in plasma from PAH patients was identified. Conclusion Our findings demonstrate that BMP9 offers superior protections against IL-33-induced EndMT in PAECs compared to a ST2 neutralizing antibody. BMP9 induces expression of sST2, leading to IL-33 neutralization and subsequent EndMT inhibition. Therefore, targeting the IL-33 pathway in PAH patients presents a promising therapeutic option for preventing EndMT. This study provides novel insights into the pathogenesis of PAH, revealing a potential therapeutic target for this rare disease.BMP9 inhibits IL-33 downstream signalingBMP9 protects from IL-33-induced EndMT

SEMA3G regulates BMP9 inhibition of VEGF-mediated migration and network formation in pulmonary endothelial cells

AimsBone 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 resultsWe 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. ConclusionsThis 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.

Heritable pulmonary arterial hypertension

Heritable pulmonary arterial hypertension (PAH) can be triggered by at least 18genes. The most frequently altered gene is the bone morphogenetic protein receptor2 (BMPR2). Further genes from the same pathway are also well known PAH-causing genes. Genetic testing can aid to confirm differential diagnoses such as apulmonary veno-occlusive disease. It also enables the testing of healthy family members. In addition to the PAH patient population particularly served by genetic testing, this article touches on the mode of inheritance and provides insights into the first treatments soon on the market that rebalance the BMPR2 signaling pathway.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

O8G Site-Specifically Modified tRF-1-AspGTC: A Novel Therapeutic Target and Biomarker for Pulmonary Hypertension.

Hypoxia and oxidative stress contribute to the development of pulmonary hypertension (PH). tRNA-derived fragments play important roles in RNA interference and cell proliferation, but their epitranscriptional roles in PH development have not been investigated. We aimed to gain insight into the mechanistic contribution of oxidative stress-induced 8-oxoguanine in pulmonary vascular remodeling. Through small RNA modification array analysis and quantitative polymerase chain reaction, a significant upregulation of the 8-oxoguanine -modified tRF-1-AspGTC was found in the lung tissues and the serum of patients with PH. This modification occurs at the position 5 of the tRF-1-AspGTC (5o8G tRF). Inhibition of the 5o8G tRF reversed hypoxia-induced proliferation and apoptosis resistance in pulmonary artery smooth muscle cells. Further investigation unveiled that the 5o8G tRF retargeted mRNA of WNT5A (Wingless-type MMTV integration site family, member 5A) and CASP3 (Caspase3) and inhibited their expression. Ultimately, BMPR2 (Bone morphogenetic protein receptor 2) -reactive oxygen species/5o8G tRF/WNT5A signaling pathway exacerbated the progression of PH. Our study highlights the role of site-specific 8-oxoguanine-modified tRF in promoting the development of PH. Our findings present a promising therapeutic avenue for managing PH and propose 5o8G tRF as a potential innovative marker for diagnosing this disease.

Blood flow regulates acvrl1 transcription via ligand-dependent Alk1 activity.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by the development of arteriovenous malformations (AVMs) that can result in significant morbidity and mortality. HHT is caused primarily by mutations in bone morphogenetic protein receptors ACVRL1/ALK1, a signaling receptor, or endoglin (ENG), an accessory receptor. Because overexpression of Acvrl1 prevents AVM development in both Acvrl1 and Eng null mice, enhancing ACVRL1 expression may be a promising approach to development of targeted therapies for HHT. Therefore, we sought to understand the molecular mechanism of ACVRL1 regulation. We previously demonstrated in zebrafish embryos that acvrl1 is predominantly expressed in arterial endothelial cells and that expression requires blood flow. Here, we document that flow dependence exhibits regional heterogeneity and that acvrl1 expression is rapidly restored after reinitiation of flow. Furthermore, we find that acvrl1 expression is significantly decreased in mutants that lack the circulating Alk1 ligand, Bmp10, and that, in the absence of flow, intravascular injection of BMP10 or the related ligand, BMP9, restores acvrl1 expression in an Alk1-dependent manner. Using a transgenic acvrl1:egfp reporter line, we find that flow and Bmp10 regulate acvrl1 at the level of transcription. Finally, we observe similar ALK1 ligand-dependent increases in ACVRL1 in human endothelial cells subjected to shear stress. These data suggest that ligand-dependent Alk1 activity acts downstream of blood flow to maintain or enhance acvrl1 expression via a positive feedback mechanism, and that ALK1 activating therapeutics may have dual functionality by increasing both ALK1 signaling flux and ACVRL1 expression.

MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

Ethanol-induced lung and cardiac right ventricular inflammation and remodeling underlie progression to pulmonary arterial hypertension.

Current research on ethanol-induced cardiovascular anomalies has focused on left ventricular (LV) function and blood pressure. To extend this area of research, we sought to determine whether ethanol-induced alterations in the structure and function of the right cardiac ventricle (RV) and pulmonary artery (PA) lead to pulmonary arterial hypertension (PAH). Two groups of male Sprague-Dawley rats received a balanced liquid diet containing 5% ethanol (w/v) or a pair-fed isocaloric liquid diet for 8 weeks. Weekly echocardiography was conducted to evaluate cardiopulmonary function, and lung and RV tissues were collected for exvivo histological and molecular studies. The ethanol-treated rats exhibited: (1) Elevated mean pulmonary arterial pressure and decreased pulmonary artery acceleration time/ejection time; (2) Pulmonary vascular remodeling comprising intrapulmonary artery medial layer thickening; and (3) RV hypertrophy along with increased RV/LV + septum, RV diameter, RV cardiomyocyte cross-sectional area, and LV mass/body weight ratio. These responses were associated with increased lung and RV pro-inflammatory markers, endothelin-1 (ET-1), TNF-α, and IL-6 levels and higher ET-1,ET-1 type A/B receptor ratio, and downregulation of the cytoprotective protein, bone morphogenetic protein receptor 2 (BMPR2), in the lungs. These findings show that moderate ethanol-induced cardiopulmonary changes underlie progression to PAH via an upregulated proinflammatory ET1-TNFα-IL6 pathway and suppression of the anti-inflammatory BMPR2.

Capitella teleta gets left out: possible evolutionary shift causes loss of left tissues rather than increased neural tissue from dominant-negative BMPR1

BackgroundThe evolution of central nervous systems (CNSs) is a fascinating and complex topic; further work is needed to understand the genetic and developmental homology between organisms with a CNS. Research into a limited number of species suggests that CNSs may be homologous across Bilateria. This hypothesis is based in part on similar functions of BMP signaling in establishing fates along the dorsal-ventral (D-V) axis, including limiting neural specification to one ectodermal region. From an evolutionary-developmental perspective, the best way to understand a system is to explore it in a wide range of organisms to create a full picture.MethodsHere, we expand our understanding of BMP signaling in Spiralia, the third major clade of bilaterians, by examining phenotypes after expression of a dominant-negative BMP Receptor 1 and after knock-down of the putative BMP antagonist Chordin-like using CRISPR/Cas9 gene editing in the annelid Capitella teleta (Pleistoannelida).ResultsEctopic expression of the dominant-negative Ct-BMPR1 did not increase CNS tissue or alter overall D-V axis formation in the trunk. Instead, we observed a unique asymmetrical phenotype: a distinct loss of left tissues, including the left eye, brain, foregut, and trunk mesoderm. Adding ectopic BMP4 early during cleavage stages reversed the dominant-negative Ct-BMPR1 phenotype, leading to a similar loss or reduction of right tissues instead. Surprisingly, a similar asymmetrical loss of left tissues was evident from CRISPR knock-down of Ct-Chordin-like but concentrated in the trunk rather than the episphere.ConclusionsOur data highlight a novel asymmetrical phenotype, giving us further insight into the complicated story of BMP’s developmental role. We further solidify the hypothesis that the function of BMP signaling during the establishment of the D-V axis and CNS is fundamentally different in at least Pleistoannelida, possibly in Spiralia, and is not required for nervous system delimitation in this group.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling

BackgroundBone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive.MethodsTo assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients.ResultsIn the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis.ConclusionsBMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Mechanistic Insights into Nonylphenol Stress on BMP2 and BMP4 Gene Expression in Red Crucian Carp (Carassius auratus Red var.)

Nonylphenol (NP) is a known endocrine-disrupting chemical (EDC) that has been shown to affect bone development in mammals. However, the detrimental impacts of NP on the skeletal growth and development of aquatic species, especially bony fish, remain poorly understood. Bone morphogenic proteins (BMPs), essential for bone formation and osteoblast differentiation, act through the BMP-Smad signaling pathway. In this study, two BMP genes, BMP2 and BMP4, were cloned and characterized in the red crucian carp (Carassius auratus red var.). The full-length cDNAs of BMP2 and BMP4 were 2029 bp and 2095 bp, respectively, encoding polypeptides of 411 and 433 amino acids, and share a typical TGF-β domain with other BMPs. The tissue expression patterns of both genes were identified, showing ubiquitous expression across all studied tissues. Additionally, the exposure of embryos or adult fish to NP stress resulted in a downregulation of BMP2, BMP4, and other genes associated with the BMP-Smad signaling pathway. Moreover, the combined treatment of adult fish with NP and the specific BMP receptor inhibitor significantly reduced these genes’ expression. These findings elucidate the mechanism of NP stress on BMP2 and BMP4, suggesting a role for the BMP-Smad signaling pathway in the response to endocrine-disrupting chemicals in fish.

Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 μM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

Association of polymorphism in the promotor area of the caprine BMPR1B gene with litter size and body measurement traits in Damani goats.

This study aimed to explore polymorphisms in the promoter region of the caprine BMPR1B (Bone morphogenetic protein receptor 1 beta) gene and its association with body measurement and litter size traits in Damani does. A total of 53 blood samples were collected to analyze the association between the BMPR1B gene polymorphism and 11 phenotypic traits in Damani female goats. The results revealed that three novel SNPs were identified in the promoter region of the caprine BMPR1B gene, including g.67 A > C (SNP1), g.170 G > A(SNP2), and g.501A > T (SNP3), among which the SNP1 and SNP2 were significantly (p < 0.05) associated with litter size and body measurement traits in Damani goats. In SNP1 the AC genotype could be used as a marker for litter size, and the CC genotype for body weight in Damani goats. In SNP2, the genotype GG was significantly (p < 0.05) associated with ear and head length. Therefore, we can conclude from the present study, that genetic variants AC and CC of the caprine BMPR1B gene could be used as genetic markers for economic traits through marker-assisted selection for the breed improvement program of the Damani goat.

Effects of the ferroptosis inducer erastin on osteogenic differentiation and biological pathways of primary osteoblasts

ABSTRACT Background Periodontitis is a chronic destructive inflammatory disease exacerbated by osteoblast dysfunction. Ferroptosis has emerged as a significant factor that could contribute to the pathological changes observed in periodontitis. However, the impact of ferroptosis on osteogenic differentiation and gene expression patterns of primary osteoblasts remain elusive. Methods In this study, osteoblasts were osteogenically induced for specific durations with and without the ferroptosis inducer erastin. Subsequently, cell proliferation, ferroptosis-related molecules, and osteogenic differentiation capacity were assessed. Furthermore, the differences in transcriptome expression following erastin treatment were analyzed by RNA sequencing. Results The results demonstrated that erastin treatment induced ferroptosis, resulting in suppressed cell proliferation and impaired osteogenic differentiation. Transcriptomic analysis revealed significant alterations in processes such as hydrogen peroxide catabolism, response to lipid peroxidation, and metal iron binding, as well as BMP receptor activity and collagen type XI trimer. Conclusion The ferroptosis inducer erastin inhibited osteoblast proliferation and differentiation. Our study provides novel insights into the effect of ferroptosis on osteogenesis, suggesting that targeting ferroptosis may present a promising approach in the treatment of periodontitis.

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept

BackgroundIn vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-β is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-β can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors – compound A (CompA) and LDN-212854 (LDN21) – in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-β receptors during MSC in vitro chondrogenesis.MethodsStandard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-β and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1–34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice.ResultsAll groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups.ConclusionsOverall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-β that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-β-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

Coexistence of pulmonary arterial hypertension and straight back syndrome in a patient with a novel BMPR2 variant affecting cytoplasmic tail domain

BackgroundPathologic variants in the bone morphogenetic protein receptor-2 (BMPR2) gene cause a pulmonary arterial hypertension phenotype in an autosomal-dominant pattern with incomplete penetrance. Straight back syndrome is one of the causes of pseudo-heart diseases. To date, no cases of idiopathic or heritable pulmonary arterial hypertension with straight back syndrome have been reported.Case presentationA 30-year-old female was diagnosed with pulmonary arterial hypertension by right heart catheterization. Computed tomography revealed a decreased anteroposterior thoracic space with heart compression, indicating a straight back syndrome. Genetic analysis by whole exome sequencing identified a novel c.2423_2424delGT (p.G808Gfs*4) germline frameshift variant within BMPR2 affecting the cytoplasmic tail domain.ConclusionsThis is the first report of different straight back characteristics in heritable pulmonary arterial hypertension with a novel germline BMPR2 variant. This finding may provide a new perspective on the variable penetrance of the pulmonary arterial hypertension phenotype.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.