Xbra modulates the activity of linker region phosphorylated Smad1 during Xenopus development.

The Bmp/Smad1 pathway plays a crucial role in developmental processes and tissue homeostasis. Mitogen-activated protein kinase (Mapk)/Erk mediated phosphorylation of Smad1 in the linker region leads to Smad1 degradation, cytoplasmic retention and inhibition of Bmp/Smad1 signaling. While Fgf/Erk pathway has been documented to inhibit Bmp/Smad1 signaling, several studies also suggests the cooperative interaction between these two pathways in different context. However, the precise role and molecular pathway of this collaborative interaction remain obscure. Here, we identified Xbra induced by Fgf/Erk signaling as a factor in a protective mechanism for Smad1. Xbra physically interacted with the linker region phosphorylated Smad1 to make Xbra/Smad1/Smad4 trimeric complex, leading to Smad1 nuclear localization and protecting it from ubiquitin-mediated proteasomal degradation. This interaction of Xbra/Smad1/Smad4 led to sustained nuclear localization of Smad1 and the upregulation of lateral mesoderm genes, while concurrently suppression of neural and blood forming genes. Taken together, the results suggests Xbra-dependent cooperative interplays between Fgf/Erk and Bmp/Smad1 signaling during lateral mesoderm specification in Xenopus embryos.

Oxidative-stress induced Bmp2-Smad1/5/8 signaling dependent differentiation of early cardiomyocytes from embryonic and adult epicardial cells.

Heart failure has become a major life-threatening cause affecting millions globally, characterized by the permanent loss of adult functional cardiomyocytes leading to fibrosis which ultimately deprives the heart of its functional efficacy. Here we investigated the reparative property of embryonic and adult epicardial cells towards cardiomyocyte differentiation under oxidative stress-induced conditions along with the identification of a possible molecular signaling pathway. Isolated epicardial cells from embryonic chick hearts subjected to oxidative stress and hypoxia induction. Initial assessment of successful injury induction reveals hypertrophy of isolated epicardial cells. Detailed marker gene expression analyses and inhibitor studies reveal Bone morphogenic protein (Bmp)2-Smad1/5/8 signaling dependent cardiomyocyte lineage specification via epithelial to mesenchymal transition (EMT) post-injury. EMT is further confirmed by increased proliferation, migration, and differentiation towards cardiomyocyte lineage. We have also established an in-vivo model in adult male rats using Isoproterenol. Successful oxidative stress-mediated injury induction in adult heart was marked by increased activated fibroblasts followed by apoptosis of adult cardiomyocytes. The detailed characterization of adult epicardial cells reveals similar findings to our avian in-vitro data. Both in-vitro and in-vivo results show a significant increase in the expression of cardiomyocyte specific markers indicative of lineage specificity and activation of epicardial cells post oxidative stress mediated injury. Our findings suggest an EMT-induced reactivation of epicardial cells and early cardiomyocyte lineage specification following oxidative stress in a Bmp2- Smad1/5/8 dependent manner. Overall, this regulatory mechanism of cardiomyocyte differentiation induced by oxidative stress may contribute to the field of cardiac repair and regenerative therapeutics.

Downregulated BMP-Smad1/5/8 signaling causes emphysema via dysfunction of alveolar type II epithelial cells.

Bone morphogenetic protein (BMP)-Smad1/5/8 signaling plays a crucial regulatory role in lung development and adult lung homeostasis. However, it remains elusive whether BMP-Smad1/5/8 signaling is involved in the pathogenesis of emphysema. In this study, we downregulated BMP-Smad1/5/8 signaling by overexpressing its antagonist Noggin in adult mouse alveolar type II epithelial cells (AT2s), resulting in an emphysematous phenotype mimicking the typical pathological features of human emphysema, including distal airspace enlargement, pulmonary inflammation, extracellular matrix remodeling, and impaired lung function. Dysregulation of BMP-Smad1/5/8 signaling in AT2s leads to inflammatory destruction dominated by macrophage infiltration, associated with reduced secretion of surfactant proteins and inhibition of AT2 proliferation and differentiation. Reactivation of BMP-Smad1/5/8 signaling by genetics or chemotherapy significantly attenuated the morphology and pathophysiology of emphysema and improved the lung function in Noggin-overexpressing lungs. We also found that BMP-Smad1/5/8 signaling was downregulated in cigarette smoke-induced emphysema, and that enhancing its activity in AT2s prevented or even reversed emphysema in the mouse model. Our data suggest that BMP-Smad1/5/8 signaling, located at the top of the signaling cascade that regulates lung homeostasis, represents a key molecular regulator of alveolar stem cell secretory and regenerative function, and could serve as a potential target for future prevention and treatment of pulmonary emphysema. © 2023 The Pathological Society of Great Britain and Ireland.

MiR-20 promotes cartilage repair in knee osteoarthritis rats via BMP2/Smad1 signaling pathway.

To explore the effect of micro ribonucleic acid (miR)-20b on knee osteoarthritis rats by regulating the bone morphogenetic protein 2 (BMP2)/Smad1 pathway, a total of 36 SD rats were randomly divided into normal group (n=12), model group (n=12) and miR-20b mimics group (n=12). The rats in normal group were fed normally, while those in model group and miR-20b mimics group were used to establish knee osteoarthritis models. After modeling, model group was not given any intervention, but miR-20b mimics group received intra-articular injection of miR-20b mimics once a day for 2 weeks. Basso, Beattie and Bresnahan (BBB) limb motor function scoring was performed at 1, 5, 7 and 14 days after the modeling, and samples were obtained after 2 weeks of intervention. Next, hematoxylin and eosin (H&E) staining was applied to observe tissue morphology, Markin's scoring was utilized to evaluate articular cartilage degeneration, and immunohistochemistry was employed to detect the expressions of BMP2 and Smad1. Thereafter, the expression of miR-20b was detected via qPCR, the content of cartilage oligomeric matrix protein (COMP) and C-telopeptide of type II collagen (CTX-II) was measured via enzyme-linked immunosorbent assay (ELISA), and the expressions of BMP2 and Smad1 proteins were examined via Western blotting (WB). BBB limb motor function scoring showed that compared with that in normal group, the BBB limb motor function score of rats in the other two groups was reduced (P<0.05). In comparison with that in model group, the BBB limb motor function score in miR-20b mimics group was increased from the 7th day after intervention (P<0.05). In addition, H&E staining results manifested that the articular surface in normal group was smooth and flat, with normal morphology, clear structure and no obvious damage. In model group, the articular surface was not smooth and uneven, and more articular cartilage fractures, morphological disorders and structural damages could be observed. Moreover, the articular surface in miR-20b mimics group was slightly damaged and smoother, and its morphology and structure were markedly improved in contrast to that in model group. The Markin's score in normal group was lower than that in model group and miR-20b mimics group (P<0.05), and it was overtly decreased in miR-20b mimics group in comparison with that in model group (P<0.05). Next, immunohistochemistry demonstrated that compared with normal group, the other two groups had lowered positive expressions of BMP2 and Smad1 (P<0.05). In comparison with model group, miR-20b mimics group exhibited notably raised positive expressions of BMP2 and Smad1 (P<0.05). Then it was found from qPCR results that the expression level of miR-20b in the other two groups was overtly reduced compared with that in normal group (P<0.05), and it was prominently elevated in miR-20b mimics group in contrast to that in model group (P<0.05). Besides, ELISA illustrated that the content of COMP and CTX-II in the cartilage tissues in the other two groups was evidently reduced compared with that in normal group (P<0.05), and it was increased prominently in miR-20b mimics group compared with that in model group (P<0.05). Finally, it was revealed by WB examination that the relative expression levels of BMP2 and Smad1 proteins in the other two groups markedly declined in comparison with those in normal group (P<0.05), and they were elevated in contrast to those in model group (P<0.05). MiR-20b can promote cartilage repair and improve articular function in knee osteoarthritis rats by up-regulating the BMP2/Smad1 signaling pathway.

Hyaluronan and Proteoglycan Link Protein 1 Activates the BMP4/Smad1/5/8 Signaling Pathway to Promote Osteogenic Differentiation: an Implication in Fracture Healing.

Osteoblast regeneration, characterized by osteoblast differentiation, is the basis of fracture healing and accelerates fracture repair. It has been reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) is overexpressed during osteoblast differentiation and regulates cartilage regeneration, but its function in fracture healing remains unclear. To elucidate this issue, we collected clinical blood samples of fracture healing, established a femoral fracture rat model, and induced an osteoblast differentiation cell model. We found that HAPLN1 was overexpressed in the serum of patients with fracture healing and the bone tissues of rats with fracture healing. Furthermore, the expression of HAPLN1 was increased time dependently during the osteogenic differentiation of MC3T3-E1 cells. HAPLN1 silencing prevented osteoblast differentiation and mineralization in MC3T3-E1 cells as evidenced by decreased osteoblast differentiation-related factors, suppressed alkaline phosphatase activities, and reduced alizarin red positive staining. Mechanically, the bone morphogenic protein 4 (BMP4)/Smad1/5/8 pathway, a facilitator of osteoblastic differentiation, was found to be inhibited by HAPLN1 knockdown, and inhibition of BMP4/Smad1/5/8 signaling enhanced the effects caused by HAPLN1 silencing. These findings demonstrated that HAPLN1 might promote fracture healing by facilitating osteogenic differentiation through the BMP4/Smad1/5/8 pathway, indicating that targeting HAPLN1 may be a feasible therapeutic candidate for fracture repair.

BMP/Smad Pathway Is Involved in Lithium Carbonate-Induced Neural-Tube Defects in Mice and Neural Stem Cells.

Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.

Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

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.

BMP/SMAD1/5 Signaling in the Endometrial Epithelium Is Essential for Receptivity and Early Pregnancy.

The biological processes that control endometrial receptivity and embryo implantation are critical for the successful outcome of pregnancy. The endometrium is the complex inner lining of the uterine wall that is under the cyclical control of estrogen and progesterone and is a site of intimate contact between mother and blastocyst. The bone morphogenetic signaling (BMP) pathway is a highly conserved signaling pathway that controls key cellular processes throughout pregnancy and exerts intracellular effects via the SMAD1/5 transcription factors. To delineate the endometrial compartment-specific roles of BMP signaling, we generated mice with epithelial-specific conditional deletion of SMAD1/5 using Lactoferrin-icre (Smad1flox/flox;Smad5flox/flox;Lactoferrin-cre, "Smad1/5 cKO"). Histological analysis of the reproductive tracts showed that Smad1/5 cKO mice were developmentally normal and displayed no defects in glandular morphology. In fertility analyses, single SMAD1 or SMAD5 deletion had no effect on fertility; however, double-conditional deletion of SMAD1 and SMAD5 resulted in severe subfertility. Timed mating analyses revealed endometrial receptivity defects in the Smad1/5 cKO mice beginning at 3.5 days post coitum (dpc) that perturbed embryo implantation at 4.5 dpc, as demonstrated by the detection of unattached blastocysts in the uterus, decreased COX2 expression, and FOXO1 cytoplasmic mislocalization. We also found that defects that arose during peri-implantation adversely affected embryonic and decidual development at 5.5 and 6.5 dpc. Thus, uterine epithelial BMP/SMAD1/5 signaling is essential during early pregnancy and SMAD1/5 epithelial-specific deletion has detrimental effects on stromal cell decidualization and pregnancy development.

Sodium tanshinone IIA sulfonate enhances the BMP9-BMPR2-Smad1/5/9 signaling pathway in rat pulmonary microvascular endothelial cells and human embryonic stem cell-derived endothelial cells.

BACKGROUND: Recent studies have demonstrated the beneficial effects of STS in treating pulmonary hypertension by inhibiting the pulmonary vascular remodeling and suppressing the abnormally elevated proliferation and migration of PASMCs. However, the roles of STS on pulmonary vascular endothelium remain largely known. METHODS: In this study, we investigated the effects and mechanisms of STS on pulmonary vascular endothelial dysfunction by using a chronic hypoxia-induced pulmonary hypertension (HPH) rat model, as well as in primarily cultured rat PMVECs and human ESC-ECs cell models. RESULTS: Firstly, a 21-day treatment of STS significantly prevents the disease development of HPH by normalizing the right ventricular systolic pressure and right ventricular hypertrophy, improving the cardiac output. Then, STS treatment markedly inhibits the hypoxia-induced medial wall thickening of the distal intrapulmonary arteries. Notably, STS significantly inhibits the hypoxia-induced apoptosis in both the pulmonary endothelium of HPH rats and primarily cultured PMVECs, through the stabilization of BMPR2 protein and protection of the diminished BMP9-BMPR2-Smad1/5/9 signaling pathway. In mechanism, STS treatment retrieves the hypoxic downregulation of BMPR2 by stabilizing the BMPR2 protein, inhibiting the BMPR2 protein degradation via lysosome system, and promoting the plasma membrane localization of BMPR2, all of which together reinforcing the BMP9-induced signaling transduction in both PMVECs and human ESC-ECs. However, these effects are absent in hESC-ECs expressing heterozygous dysfunctional BMPR2 protein (BMPR2+/R899X). CONCLUSION: STS may exert anti-apoptotic roles, at least partially, via induction of the BMP9-BMPR2-Smad1/5/9 signaling transduction in pulmonary endothelium and PMVECs.

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.

Exosomal miR-100-5p inhibits osteogenesis of hBMSCs and angiogenesis of HUVECs by suppressing the BMPR2/Smad1/5/9 signalling pathway.

BACKGROUND: Nontraumatic osteonecrosis of the femoral head (NONFH) is a common, progressive, and refractory orthopaedic disease. Decreased osteogenesis and angiogenesis are considered the main factors in the pathogenesis of NONFH. We aimed to figure out whether exosomes and exosomal miRNA from necrotic bone tissues of patients with NONFH are involved in the pathogenesis of NONFH and reveal the underlying mechanisms. METHODS: RT-PCR and western blotting (WB) were used to detect the expression of osteogenic, adipogenic, and angiogenic markers. ALP staining and Alizarin Red S (ARS) staining were used to evaluate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to assess the adipocyte deposition. A tube formation assay was used to study angiogenesis of human umbilical vascular endothelial cells (HUVECs). H&E staining and immunohistochemistry (IHC) staining were used to detect the effect of the NONFH exosomes in vivo. MicroRNA sequencing was conducted to identify potential regulators in the NONFH exosomes. The target relationship between miR-100-5p and BMPR2 was predicted and confirmed by a dual luciferase reporter assay and WB. RESULTS: The NONFH exosomes reduced the osteogenic differentiation of hBMSCs and angiogenesis of HUVECs. In addition, the injection of the NONFH exosomes caused thinning and disruption of bone trabeculae in the femoral heads of rats. MiR-100-5p expression was upregulated in the NONFH exosomes and inhibited the osteogenesis of hBMSCs and angiogenesis of HUVECs by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway. Silencing miR-100-5p expression rescued the reduction in osteogenesis and angiogenesis caused by the NONFH exosomes by activating the BMPR2/SMAD1/5/9 signalling pathway. CONCLUSION: The NONFH exosomal miR-100-5p can lead to NONFH-like damage by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway, which may be involved in the pathophysiological mechanisms of nontraumatic osteonecrosis of the femoral head (NONFH).

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.

DUSP5 promotes osteogenic differentiation through SCP1/2-dependent phosphorylation of SMAD1.

Dual-specificity phosphatases (DUSPs) are defined by their capability to dephosphorylate both phosphoserine/phosphothreonine (pSer/pThr) and phosphotyrosine (pTyr). DUSP5, a member of DUSPs superfamily, is located in the nucleus and plays crucially regulatory roles in the signaling pathway transduction. In our present study, we discover that DUSP5 significantly promotes osteogenic differentiation of mesenchymal stromal cells (MSCs) by activating SMAD1 signaling pathway. Mechanistically, DUSP5 physically interacts with the phosphatase domain of small C-terminal phosphatase 1/2 (SCP1/2, SMAD1 phosphatases) by the linker region. In addition, we further confirm that DUSP5 activates SMAD1 signaling through a SCP1/2-dependent manner. Specifically, DUSP5 attenuates the SCP1/2-SMAD1 interaction by competitively binding to SCP1/2, which is responsible for the SMAD1 dephosphorylation, and thus results in the activation of SMAD1 signaling. Importantly, DUSP5 expression in mouse bone marrow MSCs is significantly reduced in ovariectomized (OVX) mice in which osteogenesis is highly passive, and overexpression of Dusp5 via tail vein injection reverses the bone loss of OVX mice efficiently. Collectively, this work demonstrates that the linker region of DUSP5 maybe a novel chemically modifiable target for controlling MSCs fate choices and for osteoporosis treatment.

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.

Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

Rationale: Cardiac fibrosis is an important feature of cardiac remodeling and is a hallmark of heart failure. Recent studies indicate that elevated IgE plays a causal role in pathological cardiac remodeling. However, the underlying mechanism of how IgE promotes cardiac fibrosis has not been fully elucidated. Methods and Results: To explore the function of IgE in cardiac fibrosis, we stimulated mouse primary cardiac fibroblasts (CFs) with IgE and found that both IgE receptor (FcεR1) and fibrosis related proteins were increased after IgE stimulation. Specific deletion of FcεR1 in CFs alleviated angiotensin II (Ang II)-induced cardiac fibrosis in mice. To investigate the mechanisms underlying the IgE-mediated cardiac fibrosis, deep miRNA-seq was performed. Bioinformatics and signaling pathway analysis revealed that IgE upregulated Col1a1 and Col3a1 expression in CFs by repressing miR-486a-5p, with Smad1 participating downstream of miR-486a-5p in this process. Lentivirus-mediated overexpression of miR-486a-5p was found to alleviate Ang II-induced myocardial interstitial fibrosis in mice. Moreover, miR-486-5p serum levels were lower in patients with heart failure than in healthy controls, and were negatively correlated with NT-proBNP levels. Conclusions: Our study demonstrates that elevated IgE promotes pathological cardiac fibrosis by modulating miR-486a-5p and downstream factors, such as Smad1. These findings suggest new targets for pathological cardiac fibrosis intervention.

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.

Adipose-derived mesenchymal stromal cell-derived exosomes promote tendon healing by activating both SMAD1/5/9 and SMAD2/3.

BACKGROUND: The use of adipose-derived mesenchymal stromal cell-derived exosomes (ADSC-Exos) may become a new therapeutic method in biomedicine owing to their important role in regenerative medicine. However, the role of ADSC-Exos in tendon repair has not yet been evaluated. Therefore, we aimed to clarify the healing effects of ADSC-Exos on tendon injury. METHODS: The adipose-derived mesenchymal stromal cells (ADSCs) and tendon stem cells (TSCs) were isolated from the subcutaneous fat and tendon tissues of Sprague-Dawley rats, respectively, and exosomes were isolated from ADSCs. The proliferation and migration of TSCs induced by ADSC-Exos were analyzed by EdU, cell scratch, and transwell assays. We used western blot to analyze the tenogenic differentiation of TSCs and the role of the SMAD signaling pathways. Then, we explored a new treatment method for tendon injury, combining exosome therapy with local targeting using a biohydrogel. Immunofluorescence and immunohistochemistry were used to detect the expression of inflammatory and tenogenic differentiation after tendon injury, respectively. The quality of tendon healing was evaluated by hematoxylin-eosin (H&E) staining and biomechanical testing. RESULTS: ADSC-Exos could be absorbed by TSCs and promoted the proliferation, migration, and tenogenic differentiation of these cells. This effect may have depended on the activation of the SMAD2/3 and SMAD1/5/9 pathways. Furthermore, ADSC-Exos inhibited the early inflammatory reaction and promoted tendon healing in vivo. CONCLUSIONS: Overall, we demonstrated that ADSC-Exos contributed to tendon regeneration and provided proof of concept of a new approach for treating tendon injuries.

Identification, molecular evolution, and expression analysis of the transcription factor Smad gene family in lamprey.

Transcription factor small mothers against decapentaplegic (Smad) family SMAD proteins are the essential intracellular signal mediators and transcription factors for transforming growth factor ß (TGF-ß) signal transduction pathway, which usually exert pleiotropic actions on cell physiology, including immune response, cell migration and differentiation. In this study, the Smad family was identified in the most primitive vertebrates through the investigation of the transcriptome data of lampreys. The topology of phylogenetic tree showed that the four Smads (Smad1, Smad3, Smad4 and Smad6) in lampreys were subdivided into four different groups. Meanwhile, homology analysis indicated that most Smads were conserved with typical Mad Homology (MH) 1 and MH2 domains. In addition, Lethenteron reissneri Smads (Lr-Smads) adopted general Smads folding structure and had high tertiary structural similarity with human Smads (H-Smads). Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey Smads presented dramatic differences compared with jawed vertebrates. Importantly, quantitative real-time PCR analysis demonstrated that Smads were widely expressed in lamprey, and the expression level of Lr-Smads mRNA was up-regulated with different pathogenic stimulations. Moreover, depending on the weighted gene co-expression network analysis (WGCNA), four Lr-Smads were identified as two meaningful modules (green and gray). The functional analysis of these two modules showed that they might have a correlation with ployI:C. And these genes presented strong positive correlation during the immune response from the results of Pearson's correlation analysis. In conclusion, our results would not only enrich the information of Smad family in jawless vertebrates, but also lay the foundation for immunity in further study.