Bone Morphogenetic Protein Signaling Pathway Research Articles

Toxicity mechanisms regulating bone differentiation and development defects following abnormal expressions of miR-30c targeted by triclosan in zebrafish

As a ubiquitous environmental estrogen-disrupting chemical, triclosan (TCS) can induce severe osteotoxicity; however, the underlying molecular mechanisms remain uncertain. Herein, we evaluated the toxic effects of TCS on the development of cartilage and osteogenesis in 5-dpf zebrafish. Under TCS exposure from 62.5 to 250 μg/L, several osteodevelopmental malformations were observed, such as defect of craniofacial cartilage, pharyngeal arch cartilage dysplasia, and impairments on skeletal mineralization. Further, the morphology of mature chondrocytes became swollen and deformed, their number decreased, nucleus displacement occurred, and most immature chondrocytes were crowded at both ends of ceratobranchial. SEM observation of larval caudal fin revealed that, the layer of collagen fibers and the mineralized calcium nodules were significantly decreased, with the collagen fibers becoming shorter upon TCS exposure. The activity of bone-derived alkaline phosphatase significantly reduced, and marker functional genes related to cartilage and osteoblast development were abnormally expressed. RNA-seq and bioinformatics analysis indicated, that changes in marker genes intimately related to the negative regulation of miR-30c-5p overexpression targeted by TCS, and the up-regulation of miR-30c induced bone developmental defects by inhibiting the bone morphogenetic protein (BMP) signaling pathway. These findings were confirmed by artificially intervening the expression of miR-30c and using BMP pathway agonists in vivo. In sum, TCS induced osteototoxicity by targeting miR-30c up-regulation and interfering in the BMP signaling pathway. These findings enhance mechanistic understanding of TCS-induced spontaneous bone disorders and bone metastatic diseases. Further research is necessary to monitor chronic TCS-exposure levels in surrounding environments and develop relevant safety precautions based on TCS environmental risk.

Context-dependent tumor-suppressive BMP signaling in diffuse intrinsic pontine glioma regulates stemness through epigenetic regulation of CXXC5.

The most lethal subtype of diffuse intrinsic pontine glioma (DIPG) is H3K27M. Although ACVR1 mutations have been implicated in the pathogenesis of this currently incurable disease, the impacts of bone morphogenetic protein (BMP) signaling on more than 60% of H3K27M DIPG carrying ACVR1 wild-type remain unknown. Here we show that BMP ligands exert potent tumor-suppressive effects against H3.3K27M and ACVR1 WT DIPG in a SMAD-dependent manner. Specifically, clinical data revealed that many DIPG tumors have exploited the capacity of CHRDL1 to hijack BMP ligands. We discovered that activation of BMP signaling promotes the exit of DIPG tumor cells from 'prolonged stem-cell-like' state to differentiation by epigenetically regulating CXXC5, which acts as a tumor suppressor and positive regulator of BMP signaling. Beyond showing how BMP signaling impacts DIPG, our study also identified the potent antitumor efficacy of Dacinostat for DIPG. Thus, our study delineates context-dependent features of the BMP signaling pathway in a DIPG subtype.

Selective targeting BMP2 and 4 in SMAD4 negative esophageal adenocarcinoma inhibits tumor growth and aggressiveness in preclinical models

PurposeAbnormalities within the Sonic Hedgehog (SHH), Bone Morphogenetic Protein (BMP) and SMAD4 signalling pathways have been associated with the malignant behavior of esophageal adenocarcinoma (EAC). We recently developed two specific llama-derived antibodies (VHHs), C4C4 and C8C8, which target BMP4 and BMP2/4, respectively. Here we aimed to demonstrate the feasibility of the VHHs for the treatment of EAC and to elucidate its underlying mechanism.MethodsGene Set Enrichment Analysis (GSEA) was performed on a TCGA dataset, while expression of SHH, BMP2/4 and SMAD4 was validated in a cohort of EAC patients. The effects of the VHHs were tested on the recently established SMAD4(-) ISO76A primary EAC cell line and its counterpart SMAD4(+) ISO76A. In a patient-derived xenograft (PDX) model, the VHHs were evaluated for their ability to selectively target tumor cells and for their effects on tumor growth and survival.ResultsHigh expression of BMP2/4 was detected in all SMAD4 negative EACs. SHH upregulated BMP2/4 expression and induced p38 MAPK signaling in the SMAD4(-) ISO76A cells. Inhibition of BMP2/4 by VHHs decreased the aggressive and chemo-resistant phenotype of the SMAD4(-) ISO76A but not of the SMAD4(+) ISO76A cells. In the PDX model, in vivo imaging indicated that VHHs effectively targeted tumor cells. Both VHHs significantly inhibited tumor growth and acted synergistically with cisplatin. Furthermore, we found that C8C8 significantly improved survival of the mice.ConclusionsOur data indicate that increased BMP2/4 expression triggers aggressive non-canonical BMP signaling in SMAD4 negative EAC. Inhibiting BMP2/4 decreases malignant behavior and improves survival. Therefore, VHHs directed against BMP2/4 hold promise for the treatment of SMAD4 negative EAC.

The role of BMP4 signaling in trophoblast emergence from pluripotency.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

Functions of the bone morphogenetic protein signaling pathway through non-coding RNAs.

Bone morphogenetic proteins (BMPs) are proteins of the transforming growth factor-β (TGF-β) family, which plays an important role in the formation of skeletal and cartilage tissue and their regeneration. BMPs play a key role in the formation of new blood vessels and promote the migration, proliferation, and differentiation of mesenchymal stem cells (MSCs) into chondroblasts and osteoblasts. It is known that malfunction of BMPs signaling can cause a disease state. Epigenetic regulation of expression plays a key role in the control of many cellular processes. Important participants in this regulation are non-coding RNAs (ncRNAs), which are RNA molecules that are not translated into proteins. The best known of these are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). In addition, the results of many studies make it possible to establish an unambiguous functional relationship between these ncRNAs. Being involved in the regulation of a large number of target genes responsible for the life of the cell, miRNAs, lncRNAs, and circRNAs are essential for the normal development and functioning of the body, and the violation of their functions accompanies the development of many pathophysiological processes including oncogenesis. In the present review, we discuss different insights into the regulation of BMPs signaling pathway by miRNAs, lncRNAs and circRNAs governed.

A Process for the Design and Development of Novel Bone Morphogenetic Protein-7 (BMP-7) Mimetics With an Example: THR-184.

Growth Factors have been evaluated as therapeutic targets for the treatment of a broad spectrum of diseases. Because they are proteins with pleiotropic effects, the quest to harness their beneficial effects has presented challenges. Most Growth Factors operate at the extracellular-receptor level and have natural feedback mechanisms that modulate their effects. As proteins, they are difficult and expensive to manufacture. Frequently proteins must be administered parenterally, may invoke an immune response, and may be neutralized by naturally occurring inhibitors. To circumvent these limitations, we have undertaken an effort to develop mimetics for the Bone Morphogenetic Protein (BMP) signaling pathway effects that incorporate the beneficial effects, eliminate the deleterious effects, and thereby create effective drug-like compounds.To this end, we have designed and tested a family of small peptide BMP mimetics. The design used the three-dimensional structure of BMP-7 to identify likely active surface regions. Lead sequences were then optimized based on in vitro assays that examine the selective binding to BMP receptors, demonstrate the phosphorylation of Smad-1,5,8, detect anti-apoptosis and anti-inflammation, and block the epithelial to mesenchymal transition (EMT) in renal tubular epithelial cells. These sequences were further optimized using in vivo assays of the attenuation of acute kidney injury in a rat-model of unilateral clamp ischemic reperfusion. This process uses a Structure Variance Analysis algorithm (SVA) to identify structure/activity relationships. One member of this family, THR-184, is an agonist of BMP signaling and a potent antagonist of TGFβ signaling. This small peptide mimetic inhibits inflammation, apoptosis, fibrosis and reverses epithelial to mesenchymal transition (EMT) by regulating multiple signaling pathways involved in the cellular injury of multiple organs. Its effects have been shown to control Acute Kidney Injury (AKI). THR-184 has progressed through phase I and II clinical trials for the prevention of Cardio-Vascular Surgery (CVS) associated AKI. This work provides a roadmap for the development of other growth factor mimetics and demonstrates how we might harness their therapeutic potential.

ZNF750 facilitates carcinogenesis via promoting the expression of long non-coding RNA CYTOR and influences pharmacotherapy response in colon adenocarcinoma.

The epidermal cell differentiation regulator zinc finger protein 750 (ZNF750) is a transcription factor containing the Cys2His2 (C2H2) domain, the zinc finger structure of which is located at the N-terminal 25‍‍-‍46 amino acids of ZNF750. It can promote the expression of differentiation-related factors while inhibiting the expression of progenitor cell-related genes. ZNF750 is directly regulated by p63 (encoded by the TP63 gene, belonging to the TP53 superfamily). The Krüppel-like factor 4 (KLF4), repressor element-1 (RE-1)‍-silencing transcription factor (REST) corepressor 1 (RCOR1), lysine demethylase 1A (KDM1A), and C-terminal-binding protein 1/2 (CTBP1/2) chromatin regulators cooperate with ZNF750 to repress epidermal progenitor genes and activate the expression of epidermal terminal differentiation genes (Sen et al., 2012; Boxer et al., 2014). Besides, ZNF750 and the regulatory network composed of bone morphogenetic protein (BMP) signaling pathway, long non-coding RNAs (lncRNAs) (anti-differentiation non-coding RNA (ANCR) and tissue differentiation-inducing non-protein coding RNA (TINCR)), musculoaponeurotic fibrosarcoma oncogene (MAF)/MAF family B (MAFB), grainy head-like 3 (GRHL3), and positive regulatory domain zinc finger protein 1 (PRDM1) jointly promote epidermal cell differentiation (Sen et al., 2012).

Cistanche deserticola polysaccharides extracted from Cistanche deserticola Y.C. Ma promote the differentiation of mouse female germline stem cells in vitro

Ethnopharmacological relevanceTraditional Chinese herbal medicine Cistanche deserticola Y.C. Ma has been recorded and treatment for infertility and impotence since ancient times, which is widely distributed in northwest China, and is mainly composed of phenylethanol glycosides, iridoids, lignans, polysaccharides, alkaloids, etc. C. deserticola polysaccharides (CDPs) is one of its main active ingredients, studies of its effect on germline stem cells are limited so far.Aim of the study: The aim of this study was to clarify that CDPs promoted the differentiation of FGSCs in vitro, and to initially clarify its possible cell signaling pathways. Material and methodsThe cells were randomly divided into two groups. Normal FGSCs culture medium and the optimal concentration of CDPs (0.5 μg/mL) were added for culture, which was the selected treatment concentration that could promote cell differentiation on the basis of maintaining cell viability. After treatment for different time periods (12 h, 24 h, 36 h, 48 h), the cell proliferation and differentiation were evaluated by CCK-8, real-time PCR (qPCR), cell immunofluorescence and Western blot. Subsequently, RNA-Seq and data analysis were used to preliminarily analyze and verify the different genes and possible signal pathways. ResultsUnder the treatment of CDPs, cell viability was relatively better, and the expression of meiotic markers stimulated by retinoic acid gene 8 protein (Stra8) and synaptonemal complex protein 3 (Sycp3) significantly increased. In addition, their cell morphology was more similar to oocytes. Comparison of gene expression in FGSCs identified key differential expression genes (DEGs) by RNA-Seq that consisted of 549 upregulated and 465 downregulated genes. The DEGs enriched in the functional categories of germline cell development and relevant signaling pathways, which jointly regulate self-renewal and differentiation of FGSCs. The transforming growth factor β (TGF-β) signaling pathway and bone morphogenetic protein (BMP) signaling pathway might be activated to synergistically influence cell differentiation during the CDPs treatment of FGSCs. ConclusionThese findings indicated that CDPs could promote the differentiation of FGSCs in vitro and could be regulated by different DEGs and signal transduction. Preliminary mechanism studies have shown that CDPs can exert their biological activities by regulating the TGF-β and BMP signaling pathways.

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An invitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Bone Morphogenetic Protein Signaling in Cancer; Some Topics in the Recent 10 Years.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β (TGF-β) family, are multifunctional cytokines. BMPs have a broad range of functions, and abnormalities in BMP signaling pathways are involved in cancer progression. BMPs activate the proliferation of certain cancer cells. Malignant phenotypes of cancer cells, such as increased motility, invasiveness, and stemness, are enhanced by BMPs. Simultaneously, BMPs act on various cellular components and regulate angiogenesis in the tumor microenvironment. Thus, BMPs function as pro-tumorigenic factors in various types of cancer. However, similar to TGF-β, which shows both positive and negative effects on tumorigenesis, BMPs also act as tumor suppressors in other types of cancers. In this article, we review important findings published in the recent decade and summarize the pro-oncogenic functions of BMPs and their underlying mechanisms. The current status of BMP-targeted therapies for cancers is also discussed.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

IntroductionEfficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.MethodsWe differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.ResultsWe demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.ConclusionsWe demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Potential Nephroprotective Effect of Dorsomorphin Homolog 1 (DMH1) in a rat model of diabetic nephropathy.

Diabetic nephropathy (DN) represents the most common cause of end-stage renal disease. On the other hand, Bone Morphogenetic Protein signaling pathway (BMP/Smad) is one of the most interesting prophylactic targets, since inhibition of this pathway may preserve kidney functions. Therefore, a BMP pharmacological inhibitor, Dorsomorphin Homolog 1 (DMH1) was used to assess the potential nephroprotective effect in an animal model of DN. STZ-induced diabetic rat was the selected model to assess the nephroprotective effect of DMH1(5 mg/kg) for eight weeks. Rats were divided into normal control (C=10), diabetic control (DC=10), diabetic+vehicle (DV=10) and diabetic DMH1-treated rats (DT=10). Fasting blood glucose (FBG) level was measured on a weekly basis. Then, glycated hemoglobin (HbA1c), serum Creatinine (sCr), Cystatin-C (Cys-C) and Blood Urea Nitrogen (BUN) were measured by the end of the experiment. Furthermore, Tumor Necrosis Factor (TNF-α), Interleukin-6 (IL-6) and Malondialdehyde (MDA) levels were determined in kidney tissues. The histopathological study was also performed using Hematoxylin and Eosin (H&E), Periodic acid Schiff (PAS) and Masson's trichrome stains. DMH1 treatment has significantly reduced HbA1c along with sCr, Cys-C and BUN vs. the diabetic non-treated groups (p< 0.001). Furthermore, TNF-α, IL-6 and MDA levels were also significantly decreased in the DT group compared to the diabetic non-treated groups (p< 0.001). This improvement was further confirmed and found in correspondence with histopathological findings. The present findings revealed a nephroprotective activity of DMH1 against STZ-induced DN in rats. DMH1 also showed anti-inflammatory and antioxidant activities, which may explain part of the nephroprotective mechanism. This can shed light on the importance of DMH1 and BMP/Smad pathway for further experimental studies.

Dissecting Human Gonadal Cell Lineage Specification and Sex Determination Using A Single-cell RNA-seq Approach

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series single-cell RNA sequencing (scRNA-seq) strategy, we analyzed fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that the bone morphogenetic protein (BMP) signaling pathway plays cell type-specific and developmental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition (GST) in late-stage testicular mitotic arrest FGCs. Intriguingly, testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner. In our study, potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development of and the interactions among human FGCs and gonadal somatic cells.

Premature Vertebral Mineralization in hmx1-Mutant Zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development, and mutations in HMX1 are linked to an ocular defect termed oculoauricular syndrome of Schorderet–Munier–Franceschetti (OAS) (MIM #612109). Recently, additional altered orofacial features have been reported, including short mandibular rami, asymmetry of the jaws, and altered premaxilla. We found that in two mutant zebrafish lines termed hmx1mut10 and hmx1mut150, precocious mineralization of the proximal vertebrae occurred. Zebrafish hmx1mut10 and hmx1mut150 report mutations in the SD1 and HD domains, which are essential for dimerization and activity of hmx1. In hmx1mut10, the bone morphogenetic protein (BMP) antagonists chordin and noggin1 were downregulated, while bmp2b and bmp4 were highly expressed and specifically localized to the dorsal region prior to the initiation of the osteogenic process. The osteogenic promoters runx2b and spp1 were also upregulated. Supplementation with DMH1—an inhibitor of the BMP signaling pathway—at the specific stage in which bmp2b and bmp4 are highly expressed resulted in reduced vertebral mineralization, resembling the wildtype mineralization progress of the axial skeleton. These results point to a possible role of hmx1 as part of a complex gene network that inhibits bmp2b and bmp4 in the dorsal region, thus regulating early axial skeleton development.

Protein kinase A is activated during bone morphogenetic protein 2-induced osteogenic differentiation of dental follicle stem cells via endogenous parathyroid hormone-related protein

ObjectiveThe aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. DesignHuman dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. ResultsPKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7−34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. ConclusionsBMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop.

The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation.

The bone morphogenetic proteins (BMPs) are a group of potent morphogens which are critical for the patterning, development, and function of the central nervous system. The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within in vitro tissue engineering approaches and pre-clinical practices and develop futuristic therapeutic strategies for patients with neurological disease.

Operation of the Atypical Canonical Bone Morphogenetic Protein Signaling Pathway During Early Human Odontogenesis.

Bone morphogenetic protein (BMP) signaling plays essential roles in the regulation of early tooth development. It is well acknowledged that extracellular BMP ligands bind to the type I and type II transmembrane serine/threonine kinase receptor complexes to trigger the BMP signaling pathway. Then, the receptor-activated Smad1/5/8 in cytoplasm binds to Smad4, the central mediator of the canonical BMP signaling pathway, to form transfer complexes for entering the nucleus and regulating target gene expression. However, a recent study revealed the functional operation of a novel BMP-mediated signaling pathway named the atypical BMP canonical signaling pathway in mouse developing tooth, which is Smad1/5/8 dependent but Smad4 independent. In this study, we investigated whether this atypical BMP canonical signaling is conserved in human odontogenesis. We showed that pSMAD1/5/8 is required for the expression of Msh homeobox 1 (MSX1), a well-defined BMP signaling target gene, in human dental mesenchyme, but the typical BMP canonical signaling is in fact not operating in the early human developing tooth, as evidenced by the absence of pSMAD1/5/8-SMAD4 complexes in the dental mesenchyme and translocation of pSMAD1/5/8, and the expression of MSX1 induced by BMP4 is mothers against decapentaplegic homolog 4 (SMAD4)-independent in human dental mesenchymal cells. Moreover, integrative analysis of RNA-Seq data sets comparing the transcriptome profiles of human dental mesenchymal cells with and without SMAD4 knockdown by siRNA displays unchanged expression profiles of pSMAD1/5/8 downstream target genes, further affirming the functional operation of the atypical canonical BMP signaling pathway in a SMAD1/5/8-dependent but SMAD4-independent manner in the dental mesenchyme during early odontogenesis in humans.

RGMa regulates CCL5 expression via the BMP receptor in experimental autoimmune encephalomyelitis mice and endothelial cells.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Repulsive guidance molecule a (RGMa) has been indicated to act as a bone morphogenetic protein (BMP) co-receptor, enhancing BMP signalling activity. However, the role and downstream pathways of the BMP signalling pathway mediated by RGMa have yet to be fully elucidated. A recent study revealed that C-C motif chemokine ligand 5 (CCL5) has a major role in the pathogenesis of MS via the recruitment of macrophages and T-lymphocytes into the CNS. The present study aimed to evaluate whether RGMa regulates CCL5 via the BMP pathway in MS. The results demonstrated that RGMa regulated CCL5 expression in a BMP ligand-dependent manner in experimental autoimmune encephalomyelitis (EAE) mice in vivo and in endothelial cells in vitro. First, specific inhibition of the expression of RGMa via RNA interference led to a significant reduction of the expression of RGMa and this was associated with a significant delay of EAE, an alleviated disease course and downregulation of CCL5 expression at both the protein and mRNA levels. Furthermore, exogenous noggin, an extracellular antagonist of BMP ligand, abolished the induction effect of RGMa on CCL5 in endothelial cells. Taken together, these results suggested that RGMa is an important regulator of MS and inflammatory mediators such as CCL5, and the present results should prove to be useful in terms of further elucidating the RGMa-BMP receptor signalling pathway and the pathogenesis of RGMa on MS as far as the involvement of blood-brain barrier permeability is concerned.

Regulation of bone phosphorus retention and bone development possibly by BMP and MAPK signaling pathways in broilers

The bone morphogenetic protein (BMP) and mitogen-activated protein kinase (MAPK) signaling pathways play an important role in regulation of bone formation and development, however, it remains unclear that the effect of dietary different levels of non-phytate phosphorus (NPP) on these signaling pathways and their correlations with bone phosphorus (P) retention and bone development in broilers. Therefore, this experiment was conducted to investigate the effect of dietary P supplementation on BMP and MAPK signaling pathways and their correlations with bone P retention and bone development in broilers. A total of 800 one-day-old Arbor Acres male broilers were randomly allotted to 1 of 5 treatments with 8 replicates in a completely randomized design. The 5 treatments of dietary NPP levels were 0.15, 0.25, 0.35, 0.45 and 0.55% or 0.15, 0.22, 0.29, 0.36 and 0.43% for broilers from 1 to 21 days of age or 22 to 42 days of age, respectively. The results showed that extracellular signal-regulated kinase 1 (ERK1) mRNA expression in the tibia of broilers on days 14 and 28, phosphorylated-ERK1 (p-ERK1) on day 14, and BMP2 protein expression on days 28 and 42 decreased linearly (P<0.04), while c-Jun N-terminal kinase 1 (JNK1) mRNA expression on day 42 increased linearly (P<0.02) with the increase of dietary NPP level. At 14 days of age, total P accumulation in tibia ash (TPTA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS) and tibia ash were negatively correlated (r=–0.726 to –0.359, P<0.05) with ERK1 and JNK1 mRNA as well as p-ERK1; tibia alkaline phosphatase (ALP) and bone gal protein (BGP) were positively correlated (r=0.405 to 0.665, P<0.01) with ERK1 mRNA and p-ERK1. At 28 days of age, TPTA, BMC, BMD, BBS and tibia ash were negatively correlated (r=–0.518 to –0.370, P<0.05) with ERK1 mRNA and BMP2 protein, while tibia ALP was positively correlated (r=0.382 to 0.648, P<0.05) with them. The results indicated that TPTA, BMC, BMD, BBS or tibia ash had negative correlations, while tibia ALP and BGP had positive correlations with ERK1 and JNK1 mRNAs, BMP2 protein and p-ERK1, suggesting that bone P retention and bone development might be regulated by BMP and MAPK signaling pathways in broiler chickens.

The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

Background & AimsHepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis.MethodsExperimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means.ResultsIn this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation.ConclusionsHepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.