The SPI1/SMAD5 cascade in the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells: a mechanism study.

BACKGROUND: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation. METHODS: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays. RESULTS: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells. CONCLUSION: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells.

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

AIMS: Bone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence. METHODS AND RESULTS: We identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays. CONCLUSIONS: This study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.

mTORC1 and BMP-Smad1/5 regulation of serum-stimulated myotube hypertrophy: a role for autophagy.

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

Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HA and Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.

Circ_0001825 promotes osteogenic differentiation in human-derived mesenchymal stem cells via miR-1270/SMAD5 axis.

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

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.

Circadian Period 2 (Per2) downregulate inhibitor of differentiation 3 (Id3) expression via PTEN/AKT/Smad5 axis to inhibits glioma cell proliferation.

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

Long non-coding RNA H19 aggravates keloid progression by upregulating SMAD family member 5 expression via miR-196b-5p.

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

Role of lncRNA LINC01194 in hepatocellular carcinoma via the miR-655-3p/SMAD family member 5 axis.

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

Effect of genetic variants in the SMAD1 and SMAD5 genes promoter on growth and beef quality traits in cattle.

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

Exosomes derived from mesenchymal stem cells ameliorate renal fibrosis via delivery of miR-186-5p.

Evidence has shown that mesenchymal stem cells' (MSCs) therapy has potential application in treating chronic kidney disease (CKD). In addition, MSCs-derived exosomes can improve the renal function and prevent the progression of CKD. However, the mechanisms by which MSCs-derived exosomes (MSCs-Exo) ameliorate renal fibrosis in CKD remain largely unclear. To mimic an in vitro model of renal fibrosis, rat kidney tubular epithelial cells (NRK52E) were stimulated with transforming growth factor (TGF)-ß1. In addition, we established an in vivo model of unilateral ureteric obstruction (UUO)-induced renal fibrosis. Meanwhile, we exploited exosomes derived from MSCs for delivering miR-186-5p agomir into NRK52E cells or kidneys in vitro and in vivo. In this study, we found that level of miR-186-5p was significantly downregulated in TGF-ß1-stimulated NRK52E cells and the obstructed kidneys of UUO mice. In addition, miR-186-5p can be transferred from MSCs to NRK52E cells via exosomes. MSCs-delivered miR-186-5p markedly reduced the accumulation of extracellular matrix (ECM) protein, and inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in TGF-ß1-stimulated NRK52E cells. Moreover, exosomal miR-186-5p from MSCs attenuated kidney injury and fibrosis in a UUO mouse model via inhibition of the ECM protein accumulation and EMT process. Meanwhile, dual-luciferase assay showed that miR-186-5p downregulated Smad5 expression via direct binding with the 3'-UTR of Smad5. Collectively then, these findings indicated that exosomal miR-186-5p derived from MSCs could attenuate renal fibrosis in vitro and in vivo by downregulation of Smad5. These findings may help to understand the role of MSCs' exosomes in alleviating renal fibrosis in CKD.

ZNF652-Induced circRHOT1 Promotes SMAD5 Expression to Modulate Tumorigenic Properties and Nature Killer Cell-Mediated Toxicity in Bladder Cancer via Targeting miR-3666.

Bladder cancer (BC) is the 9th most frequent diagnosed tumor and the 2nd most common urology tumor worldwide. Despite the considerable advancement that BC treatment has made recently, the five-year survival rate of BC remains unsatisfactory. Novel therapeutic strategies for BC clinical intervention are therefore urgently needed now more than ever. circRHOT1 is a newly identified circRNA that plays a crucial role in multiple types of tumorigeneses. However, it remains unclear whether circRHOT1 plays a functional role in BC progression. Our findings suggest that circRHOT1 was highly expressed in BC tumor tissues and cell lines. The results from CCK-8, EDU, Transwell migration, and NK cell-mediated cytotoxicity detection assays suggested that circRHOT1 knockdown could markedly suppress BC cell proliferation and migration level and could aggravate the sensitivity of BC cells to NK cells. Subsequently, we conducted bioinformatics analysis followed by RNA pull-down, ChIP, and luciferase reporter assays, from which we found that circRHOT1 expression in BC cells could be regulated by ZNF652, and circRHOT1 could promote SMAD5 expression to regulate BC cell cellular progression by sponging miR-3666. These results may provide a new direction for developing novel diagnostic or therapeutic targets for BC.

Litopenaeus vannamei Sma and Mad related protein 5 gene is involved in stress response and white spot syndrome virus infection.

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

Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension.

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

miR-20a-5p contributes to osteogenic differentiation of human dental pulp stem cells by regulating BAMBI and activating the phosphorylation of Smad5 and p38.

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

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.

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.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

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

Upregulation of Long Noncoding RNA FGD5-AS1 Ameliorates Myocardial Ischemia/Reperfusion Injury via MicroRNA-106a-5p and MicroRNA-106b-5p.

ABSTRACT: Long noncoding RNAs have been known to play key roles in myocardial ischemia/reperfusion injury. This study was conducted to investigate whether upregulation of FGD5-AS1 can improve hypoxia/reoxygenation (H/R) injury of cardiomyocytes and its underlying mechanisms. Pc-FGD5-AS1 was used to overexpress FGD5-AS1 in cardiomyocytes. Cholecystokinin octapeptide and flow cytometry assays were performed to detect the effect of FGD5-AS1 on myocardial cell H/R injury. Quantitative real-time polymerase chain reaction and luciferase reporter assay were performed to assess the relationship between FGD5-AS1 and microRNA-106a-5p (miR-106a-5p) or miR-106b-5p. In patients with acute myocardial infarction and in H/R cardiomyocytes and ischemia/reperfusion myocardium, the expression levels of FGD5-AS1 were reduced, whereas the expression levels of miR-106a-5p and miR-106b-5p were increased. Overexpression of FGD5-AS1 increased the viability of H/R-treated cardiomyocytes and reduced the levels of apoptosis and creatine kinase-MB. In addition, FGD5-AS1 could bind to miR-106a-5p or miR-106b-5p and showed a mutual inhibitory effect between them. Furthermore, overexpression of miR-106a-5p or miR-106b-5p inhibited the expression of SMAD5. FGD5-AS1 upregulated the expression of SMAD5. In conclusion, FGD5-AS1 may be a potential therapeutic target for myocardial H/R injury, and its cardioprotective effect may be realized by reducing inflammatory response and cell apoptosis.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.