Long noncoding RNA KCNMA1-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the SMAD9 signaling pathway.

The human bone marrow mesenchymal stem cells (hBMSCs) undergo intense osteogenic differentiation, a crucial bone formation mechanism. Evidence from prior studies suggested an association between long noncoding RNAs (lncRNAs) and the osteogenic differentiation of hBMSCs. However, precise roles and molecular mechanisms are still largely unknown. In this work, we report for the first time that lncRNA KCNMA1 antisense RNA 1 (KCNMA1-AS1) plays a vital role in regulating hBMSCs' osteogenic differentiation. Here, it was observed that the KCNMA1-AS1 expression levels were significantly upregulated during osteogenic differentiation. In addition, KCNMA1-AS1 overexpression enhanced in vitro osteogenic differentiation of hBMSCs and in vivo bone formation, whereas knockdown of KCNMA1-AS1 resulted in the opposite result. Additionally, the interaction between KCNMA1-AS1 and mothers against decapentaplegic homolog 9 (SMAD9) was confirmed by an RNA pull-down experiment, mass spectrometry, and RIP assay. This interaction regulated the activation of the SMAD9 signaling pathway. Moreover, rescue assays demonstrated that the inhibitor of the SMAD9 signaling pathway reversed the stimulative effects on osteogenic differentiation of hBMSCs by KCNMA1-AS1 overexpression. Altogether, our results stipulate that KCNMA1-AS1 promotes osteogenic differentiation of hBMSCs via activating the SMAD9 signaling pathway and can serve as a biomarker and therapeutic target in treating bone defects.

SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor occurring during childhood and high-risk NB patients have a poor prognosis. The amplified MYCN gene serves as an important determinant of a high risk of NB. METHODS: We performed an integrative screen using public NB tissue and cell line data, and identified that SMAD9 played an important role in high-risk NB. An investigation of the super-enhancers database (SEdb) and chromatin immunoprecipitation sequencing (ChIP-seq) dataset along with biological experiments of incorporating gene knockdown and CRISPR interference (CRISPRi) were performed to identify upstream regulatory mechanism of SMAD9. Gene knockdown and rescue, quantitative real-time PCR (Q-RT-PCR), cell titer Glo assays, colony formation assays, a subcutaneous xenograft model and immunohistochemistry were used to determine the functional role of SMAD9 in NB. An integrative analysis of ChIP-seq data with the validation of CRISPRi and dual-luciferase reporter assays and RNA sequencing (RNA-seq) data with Q-RT-PCR validation was conducted to analyze the downstream regulatory mechanism of SMAD9. RESULTS: High expression of SMAD9 was specifically induced by the transcription factors including MYCN, PHOX2B, GATA3 and HAND2 at the enhancer region. Genetic suppression of SMAD9 inhibited MYCN-amplified NB cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that SMAD9 bound to the MYCN promoter and transcriptionally regulate MYCN expression, with MYCN reciprocally binding to the SMAD9 enhancer and transactivating SMAD9, thus forming a positive feedback loop along with the MYCN-associated cancer cell cycle. CONCLUSION: This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5.

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.

Smad8 Is Increased in Duchenne Muscular Dystrophy and Suppresses miR-1, miR-133a, and miR-133b.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor ß (TGFß) signaling. In this report, we investigated the major transducers of TGFß signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.

ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis.

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin-proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

Uptake and Patient Perspectives on Additional Testing for Novel Disease-Associated Genes: Lessons from a PAH Cohort.

BACKGROUND: Pulmonary arterial hypertension (PAH) has an identifiable genetic cause in 5% of all PAH cases. Due to health benefits conferred by the early detection of PAH and the recent identification of additional PAH-associated genes, we decided to offer (extended) genetic testing to all incident and prevalent idiopathic PAH (iPAH) and pulmonary veno-occlusive disease (PVOD) patients in our clinic. Here, we report the lessons learned from (re-)contacting iPAH/PVOD patients concerning the uptake and analysis of identified PAH-associated genes and patient perspectives of the approach. METHODS: Between January 2018 and April 2020, all iPAH/PVOD patients who were not previously genetically tested (contact group) and those who tested negative on prior analysis of BMPR2 and SMAD9 variants (re-contact group) were (re-)contacted for (additional) genetic testing. RESULTS: With our approach, 58% of patients (84 out of 165) opted for genetic counselling, and a pathogenic variant was found in 12% of cases (n = 10) (re-contact group, 11%, and contact group, 13%). Eighty-six percent of participants of the survey study appreciated being (re-)contacted for genetic testing. Mild psychosocial impacts were observed. CONCLUSIONS: Our report shows the importance of (re-)contact and interest of patients (as indicated by the uptake, mild psychosocial impact and appreciation) in PAH.

Thymopentin treatment of murine premature ovarian failure via attenuation of immune cell activity and promotion of the BMP4/Smad9 signalling pathway.

Premature ovarian failure (POF) is a typical form of pathological aging with complex pathogenesis and no effective treatment. Meanwhile, recent studies have reported that a high-fat and high-sugar (HFHS) diet adversely affects ovarian function and ovum quality. Here, we investigated the therapeutic effect of thymopentin (TP-5) as a treatment for murine POF derived from HFHS and its target. Pathological examination and hormone assays confirmed that TP-5 significantly improved murine POF symptoms. And, TP-5 could reduce oxidative stress injury and blood lipids in the murine POF derived from HFHS. Flow cytometry and qPCR results suggested that TP-5 attenuated activation of CD3+ T cells and type I macrophages. RNA-Seq results indicated somedifferences in gene transcription between the TP-5 intervention group and the control group. KEGG analysis indicated that the expression of genes involved in the mTOR signaling pathway was the most significantly different between the two groups. Additionally, compared with the control groups, the expression levels of interleukin, NFκB, and TNF families of genes were significantly downregulated in the POF+TP-5 group, whereas expression of the TGFß/Smad9 genes was significantly upregulated. Finally, immunofluorescence staining and qPCR confirmed that TP-5 promoted the polarization of Mø2 cells in the ovary by activating the expression of the BMP4/Smad9 signalling pathway. Thus, our study confirmed that TP-5 has a significant therapeutic effect on POF by upregulating BMP4/Smad9 signalling pathway so as to promote the balance and polarization of immune cell and reducing the release of inflammatory factors and reduce lipid oxidative stress injury.

Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis.

Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-ß family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-ß family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-ß families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors.

Role of BMP signaling during early development of the annelid Capitella teleta.

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 â€‹+ â€‹12 â€‹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.

Conophylline Suppresses Angiotensin II-Induced Myocardial Fibrosis In Vitro via the BMP4/JNK Pathway.

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-ß 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.

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3).

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations.

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

Changes in Smad1/5/9 expression and phosphorylation in astrocytes of the rat hippocampus after transient global cerebral ischemia.

Smad proteins are known to transduce the actions of the transforming growth factor-ß (TGF-ß) family including TGF-ßs, activins, and bone morphogenetic proteins (BMPs). We previously reported that Smad1/5/9 immunoreactivity was observed in astrocytes of various rat brain regions including the hippocampus, suggesting that Smad1/5/9 may be associated with the physiology of astrocytes. However, the Smad1/5/9 expression and activation in the hippocampal astrocytes after global cerebral ischemia has not been yet elucidated. In this study, we examined temporal changes in the expression and phosphorylation of Smad1/5/9 in the hippocampus using a rat model of global cerebral ischemia. Furthermore, we examined the candidate ligand involved in the phosphorylation of Smad1/5/9 in the hippocampus after ischemia. Pyramidal neuronal cell death in the CA1 regions was visible at 3 days, and maximum death occurred within 7 days after ischemia. At 7 days after ischemia, astrocytes that showed strong immunoreactivity for Smad1/5/9 were frequently observed in the CA1 region. Additionally, there was an increase in phosphorylated Smad1/5/9 (phospho-Smad1/5/9) -immunopositive astrocytes in the CA1 region 7 days after ischemia. Real-time PCR analysis showed an increase in the expression level of TGF-ß1 mRNA in the hippocampus after ischemia. Intracerebroventricular injection of SB525334, an inhibitor of TGF-ß/Smad signaling, reduced immunoreactivity for phospho-Smad1/5/9 in astrocytes. These results suggest that TGF-ß1 may be a key molecule for ischemia-induced Smad1/5/9 phosphorylation in astrocytes, and TGF-ß1-Smad1/5/9 signaling may play a role in post-ischemic events, including brain inflammation or tissue repair rather than neuroprotection of the hippocampus.

Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

Reduction of miR-744 delivered by NSCLC cell-derived extracellular vesicles upregulates SUV39H1 to promote NSCLC progression via activation of the Smad9/BMP9 axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a common type of lung cancer. Extracellular vehicles (EVs) are nano-sized particles containing proteins, lipids, and miRNAs secreted by various cells, which play important roles in the development of lung cancer by regulating a wide range of signaling pathways. This study focused on elucidating a potential mechanism by which EVs promote the development of NSCLC. METHODS: Expression levels of miR-744, SUV39H1, Smad9, and BMP4 in clinical tissue samples of NSCLC patients and cell lines were quantified by RT-qPCR and/or western blot analysis. The interaction between SUV39H1 and miR-744 was identified by dual-luciferase reporter assay. miR-744, SUV39H1, and BMP4 expression levels were modulated in A549 and H460 cells, in order to evaluate their effects on cell proliferation, colony formation and cell cycle. A NSCLC xenograft mouse model was used to verify the in vitro findings. NSCLC cell-derived EVs and normal bronchial epithelial cell-derived EVs were isolated and their roles in NSCLC development were evaluated in vivo and in vitro. RESULTS: miR-744 expression was lower in cancer cell-derived derived EVs than in normal lung epithelial cell-derived EVs. Reduced miR-744 expression in EVs upregulated SUV39H1 in NSCLC cells and further increased BMP4 levels to promote NSCLC development. BMP4 was upregulated in NSCLC cells upon suppression of Smad9 mediated by SUV39H1. Reduced miR-744 expression transferred from cancer cell-derived EVs into NSCLC cells enhanced cancer development. CONCLUSION: Overall, our findings unveiled a mechanism whereby miR-744 delivered by NSCLC-derived EVs upregulated SUV39H1, activating the Smad9/BMP9 axis and thus promoted cancer development.

BMP10 positively regulates myogenic differentiation in C2C12 myoblasts via the Smad 1/5/8 signaling pathway.

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.

RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR), currently considered as a neurovascular disease, has become the major cause of blindness. More and more scholars believe that DR is no longer just a kind of microvascular disease, but accompanied by retinal neurodegenerative changes. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is a classic treatment for DR; however, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. The aim of this study was to explore the pathogenesis of DR and identified new treatments that can provide dual-target intervention for angiogenesis and fibrosis. METHODS: We explored changes in gene expression in high glucose-induced vascular endothelial cells using RNA sequencing (RNA-seq) technology. We identified bone morphogenetic protein 4 (BMP4) and SMAD family member 9 (SMAD9) among 449 differentially expressed genes from RNA-seq data and confirmed the expression of these two genes in the blood of diabetes patients by RT-PCR and in streptozotocin-induced rat retinas by RT-PCR, immunofluorescence, and western blot. Moreover, considering that DR is a multifactorial and multicellular disease, we used hydrogen peroxide (H2O2), advanced glycation end products (AGEs), CoCl2, 4-hydroxynonenal (4-HNE), and hypoxia to induce three human retinal cell types (Müller, retinal pigment epithelium, and human retinal capillary endothelial cells) to simulate the pathogenesis of DR, and MTT experiment, scratch experiment, Transwell experiment, and lumen formation experiment were used to test whether the model was successfully established. Then, we verified the overexpression of these two genes in the cell models by RT-PCR, immunofluorescence, and western blot. We further tested the effects of BMP4 on retinal cells. We use BMP4 to stimulate retinal cells and observe the effect of BMP4 on retinal cells by MTT experiment, scratch experiment, and RT-PCR. RESULTS: The results demonstrated that BMP4 and SMAD9 were highly expressed in both in vivo and in vitro models, while BMP4 could significantly upregulate the expression of SMAD9 and promote the expression of VEGF and fibrosis factors. CONCLUSIONS: This study is the first to analyze the mechanism by which high glucose levels affect retinal vascular endothelial cells through RNA transcriptome sequencing and indicates that BMP4 may be a potential target for the dual-target treatment (anti-VEGF and anti-fibrosis) of DR. KEY MESSAGES: • High-glucose effect on vascular endothelial cell was analyzed by RNA-seq. • KEGG analysis revealed enrichment of TGF-beta signaling pathway. • SMAD9 and BMP4 expression was upregulated in all samples. • Dual-target therapy of PDR by antagonizing BMP4.

Whole Exome Sequence Analysis Provides Novel Insights into the Genetic Framework of Childhood-Onset Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) describes a rare, progressive vascular disease caused by the obstruction of pulmonary arterioles, typically resulting in right heart failure. Whilst PAH most often manifests in adulthood, paediatric disease is considered to be a distinct entity with increased morbidity and often an unexplained resistance to current therapies. Recent genetic studies have substantially increased our understanding of PAH pathogenesis, providing opportunities for molecular diagnosis and presymptomatic genetic testing in families. However, the genetic architecture of childhood-onset PAH remains relatively poorly characterised. We sought to investigate a previously unsolved paediatric cohort (n = 18) using whole exome sequencing to improve the molecular diagnosis of childhood-onset PAH. Through a targeted investigation of 26 candidate genes, we applied a rigorous variant filtering methodology to enrich for rare, likely pathogenic variants. This analysis led to the detection of novel PAH risk alleles in five genes, including the first identification of a heterozygous ATP13A3 mutation in childhood-onset disease. In addition, we provide the first independent validation of BMP10 and PDGFD as genetic risk factors for PAH. These data provide a molecular diagnosis in 28% of paediatric cases, reflecting the increased genetic burden in childhood-onset disease and highlighting the importance of next-generation sequencing approaches to diagnostic surveillance.

Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Prunella vulgaris L protects glucocorticoids-induced osteogenesis inhibition in bone marrow mesenchymal stem cells through activating the Smad pathway.

OBJECTIVE: To elucidate the role of Prunella vulgaris L (PVL) in protecting glucocorticoids (GC)-induced osteogenesis inhibition, thereafter, protecting the deterioration of osteoporosis (OP). MATERIALS AND METHODS: Cell Counting Kit-8 (CCK-8) assay was conducted to assess the influence of PVL treatment on MSCs viability. Osteogenesis in MSCs was induced by Dexamethasone (DEX) stimulation. Regulatory effects of PVL on osteogenesis-related gene expressions, ALP activity, and mineralization ability in DEX-induced MSCs were determined. At last, protein levels of p-Smad1/5/9 and total-Smad1/5/9 influenced by DEX and PVL were measured by Western blot. RESULTS: PVL treatment did not pose a time- or dose-dependent influence on MSCs viability. DEX induction in MSCs downregulated ALP, RUNX2, Bglap, and Osterix. ALP activity and mineralization in DEX-induced MSCs were suppressed. Downregulated osteogenesis-related genes decreased ALP activity and mineralization in MSCs undergoing DEX stimulation were partially reversed by PVL treatment. Moreover, the downregulated p-Smad1/5/9 level in DEX-induced MSCs was elevated by PVL treatment, while total-Smad1/5/9 was not affected. CONCLUSIONS: PVL alleviated GC-induced suppression in MSCs osteogenesis by activating the Smad pathway, thereafter, protecting the deterioration of OP.