HMCES modulates the transcriptional regulation of nodal/activin and BMP signaling in mESCs.

Despite the fundamental roles of TGF-ß family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-ß signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-ß family signaling.

MicroRNAs involved in the TGF-ß signaling pathway in atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease with a multifactorial pathogenesis. It becomes a global health concern, especially causing an array of fatal consequences among the elderly. However, the mechanisms of AS remain unexplained. The transforming growth factor-ß (TGF-ß) signaling pathway is widely involved in the inflammation, immune function, proliferation, differentiation，and apoptosis in vivo. Based on previous researches, it has not been confirmed whether the TGF-ß pathway promotes or inhibits atherosclerosis. Furthermore, more and more studies have found that microRNAs can regulate atherosclerosis through the TGF-ß signaling pathway. In this review, we summarize and discuss the role of microRNAs in the pathogenesis of atherosclerosis via the TGF-ß signaling pathway.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial-mesenchymal transition via the Smad1/5 signaling pathway.

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

Cissus quadrangularis extract attenuates diabetic nephropathy by altering SIRT1/DNMT1 axis.

OBJECTIVES: Hyperglycemia-induced SIRT1, DNMT1, SODs, as well as oxidative stress, play a pivotal role in the progression of diabetic nephropathy. Cissus quadrangularis, holds antioxidant and hypoglycemic activity; however, a direct link between its activity and prevention of diabetic nephropathy has not been ascertained yet. Accordingly, we aimed to delineate the protective effect of ethanolic extract of Cissus quadrangularis (EECQ) against high-fat diet/streptozotocin (HFD/STZ) induced diabetic nephropathy rats. METHODS: The control group was fed with a normal chow diet. Rats kept on an HFD for 12 weeks with a single low dose of STZ manifested the features of diabetic nephropathy. The treatment was done by the oral administration of EECQ (200 mg/kg) for six weeks (six rats in each group). KEY FINDINGS: Treatment with EECQ demonstrated substantial attenuation of elevated insulin resistance, lipid profile and creatinine level. Additionally, EECQ restored albuminuria, glomerular filtration rate and creatinine clearance in diabetic nephropathy rats. Furthermore, HFD consumption in rats culminated in reduced SIRT1 and enhanced DNMT1 expression, nonetheless, rescued by EECQ. Moreover, EECQ augmented the SOD 1 and 3 levels, thereby safeguarded from oxidative damage and renal inflammation. Besides, treatment protected from renal fibrosis by downregulating TGFß, Smad2/3 and col1/3 expression in diseased rats. CONCLUSIONS: Thus, based on the above findings, we conclude that EECQ shows a protective effect against diabetic nephropathy.

Pretreatment with valproic acid alleviates pulmonary fibrosis through epithelial-mesenchymal transition inhibition in vitro and in vivo.

Epithelial-mesenchymal transition (EMT) plays a crucial role in the development of pulmonary fibrosis. This study aims to investigate the effects of valproic acid (VPA) on EMT in vitro and in vivo. In vitro, EMT was induced by the administration of transforming growth factor-ß1 (TGF-ß1) in a human alveolar epithelial cell line (A549). The dose effects of VPA (0.1-3 mM) on EMT were subsequently evaluated at different timepoints. VPA (1 mM) was applied prior to the administration of TGF-ß1 and the expression of E-cadherin, vimentin, p-Smad2/3 and p-Akt was assessed. In addition, the effects of a TGF-ß type I receptor inhibitor (A8301) and PI3K-Akt inhibitor (LY294002) on EMT were evaluated. In vivo, the effects of VPA on bleomycin-induced lung fibrosis were evaluated by assessing variables such as survival rate, body weight and histopathological changes, whilst the expression of E-cadherin and vimentin in lung tissue was also evaluated. A8301 and LY294002 were used to ascertain the cellular signaling pathways involved in this model. The administration of VPA prior to TGF-ß1 in A549 cells prevented EMT in both a time- and concentration-dependent manner. Pretreatment with VPA downregulated the expression of both p-Smad2/3 and p-Akt. A8301 administration increased the expression of E-cadherin and reduced the expression of vimentin. LY294002 inhibited Akt phosphorylation induced by TGF-ß1 but failed to prevent EMT. Pretreatment with VPA both increased the survival rate and prevented the loss of body weight in mice with pulmonary fibrosis. Interestingly, both VPA and A8301 prevented EMT and facilitated an improvement in lung structure. Overall, pretreatment with VPA attenuated the development of pulmonary fibrosis by inhibiting EMT in mice, which was associated with Smad2/3 deactivation but without Akt cellular signal involvement.

lncRNA CDKN2A-AS1 facilitates tumorigenesis and progression of epithelial ovarian cancer via modulating the SOSTDC1-mediated BMP-SMAD signaling pathway.

Ovarian cancer (OC) is the fifth most common female malignant tumor and the leading cause of cancer-related death in women worldwide. Epithelial ovarian cancer (EOC) is the predominant type of OC. Investigating the mechanism underlying tumorigenesis and progression of EOC is urgent. Our previous research has shown that long non-coding RNAs (lncRNAs) CDKN2A-AS1 is upregulated in EOC tissues and cells. Furthermore, we have predicted that CDKN2A-AS1 is associated with the bone morphogenetic protein (BMP)-SMAD signaling pathway, which is negatively regulated by the sclerostin domain containing 1 (SOSTDC1). Therefore, we conjecture that the CDKN2A-AS1 regulate BMP-SMAD signaling pathway via interacting with SOSTDC1, which need more investigation. Moreover, the functions of the BMP-SMAD signaling pathway and the SOSTDC1 on EOC are still unclear. Herein, we unearthed that CDKN2A-AS1, BMP2/4/7, SMAD1/5/9 and phosphorylation of SMAD1/5/9 (p-SMAD1/5/9) were upregulated in EOC tissues and cells, whereas SOSTDC1 was downregulated in EOC tissues and cells. We firstly demonstrated that CDKN2A-AS1 bound directly with the SOSTDC1. CDKN2A-AS1 downregulated the expression of SOSTDC1, but upregulated the expression of BMP2/4/7, SMAD1/5/9, and p-SMAD1/5/9. CDKN2A-AS1 promoted the proliferation, migration, invasion of EOC cells and tumor growth in vivo, whereas SOSTDC1 inhibited the proliferation, migration, invasion of EOC cells. Knockdown SOSTDC1 rescued the inhibitory effect of si-lncRNA CDKN2A-AS1 on the EOC cells proliferation, migration and invasion. These results demonstrated that CDKN2A-AS1activated the BMP-SMAD signaling pathway by directly bind with SOSTDC1 to promote EOC tumor growth. CDKN2A-AS1/SOSTDC1 axis may provide a novel therapeutic strategy for EOC treatment.

Exosomes-Mediated LncRNA ZEB1-AS1 Facilitates Cell Injuries by miR-590-5p/ETS1 Axis Through the TGF-ß/Smad Pathway in Oxidized Low-density Lipoprotein-induced Human Umbilical Vein Endothelial Cells.

ABSTRACT: Atherosclerosis is a chronic lipid-induced inflammation of the vessel wall. Oxidized low-density lipoprotein was confirmed to drive the onset of atherogenesis. Zinc finger e-box-binding homeobox 1 antisense 1 (ZEB1-AS1) is a long noncoding RNA that is involved in human diseases, including atherosclerosis. In this study, the role of exosomes-mediated ZEB1-AS1 and its underlying mechanisms in atherosclerosis were explored in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). Exosomes were extracted from HUVECs. Quantitative real-time polymerase chain reaction was conducted to measure the expression of ZEB1-AS1, microRNA-590-5p (miR-590-5p), or erythroblastosis virus E26 oncogene homolog 1 (ETS1) in cells or exosomes. Cell proliferation and apoptosis were assessed by MTT assay and flow cytometry analysis, respectively. Western blot was performed to detect apoptosis-related factors, ETS1, and TGF-ß/Smad pathway protein levels. The secretion of inflammatory factors in supernatant was detected by ELISA assay. Oxidative stress damage indicators were used to assess cellular damage. Relationship between miR-590-5p and ZEB1-AS1 or ETS1 was analyzed. Our data indicated that ox-LDL-induced exosomes-mediated ZEB1-AS1 in HUVECs. Ox-LDL treatment resulted in limited proliferation, proapoptosis, inflammation, and oxidative stress damage, whereas knockdown of ZEB1-AS1 could reverse these effects. Mechanically, ZEB1-AS1 sponged miR-590-5p to regulate ETS1 expression. MiR-590-5p knockdown inverted effects above of si-ZEB1-AS1 on HUVECs under ox-LDL exposure. Moreover, ETS1 reversed miR-590-5p-induced effects and activated the TGF-ß/Smad pathway in ox-LDL-treated HUVECs. Taken together, our findings demonstrated that exosomes-mediated ZEB1-AS1 enhanced cell injuries by miR-590-5p/ETS1 axis through the TGF-ß/Smad pathway in ox-LDL-induced HUVECs, suggesting that inhibiting ZEB1-AS1 might be an effective way for atherosclerosis treatment.

Transcriptional regulation of the basic helix-loop-helix factor AmeloD during tooth development.

The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.

Long non-coding RNA H19 deficiency ameliorates bleomycin-induced pulmonary inflammation and fibrosis.

BACKGROUND: The poor understanding of pathogenesis in idiopathic pulmonary fibrosis (IPF) impaired development of effective therapeutic strategies. The aim of the current study is to investigate the roles of long non-coding RNA H19 (lncRNA H19) in the pulmonary inflammation and fibrosis of IPF. METHODS: Bleomycin was used to induce pulmonary inflammation and fibrosis in mice. The mRNAs and proteins expression in lung tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. H19 knockout (H19-/-) mice were generated by CRISPR/Cas9. RESULTS: The expression of H19 mRNA was up-regulated in fibrotic lungs patients with IPF as well as in lungs tissues that obtained from bleomycin-treated mice. H19-/- mice suppressed bleomycin-mediated pulmonary inflammation and inhibited the Il6/Stat3 signaling. H19 deficiency ameliorated bleomycin-induced pulmonary fibrosis and repressed the activation of TGF-ß/Smad and S1pr2/Sphk2 in the lungs of bleomycin-treated mice. CONCLUSIONS: Our data suggests that H19 is a profibrotic lncRNA and a potential therapeutic target for IPF.

Enhanced asthma-related fibroblast to myofibroblast transition is the result of profibrotic TGF-ß/Smad2/3 pathway intensification and antifibrotic TGF-ß/Smad1/5/(8)9 pathway impairment.

Airway remodelling with subepithelial fibrosis, which abolishes the physiological functions of the bronchial wall, is a major issue in bronchial asthma. Human bronchial fibroblasts (HBFs) derived from patients diagnosed with asthma display in vitro predestination towards TGF-ß1-induced fibroblast-to-myofibroblast transition (FMT), a key event in subepithelial fibrosis. As commonly used anti-asthmatic drugs do not reverse the structural changes of the airways, and the molecular mechanism of enhanced asthma-related TGF-ß1-induced FMT is poorly understood, we investigated the balance between the profibrotic TGF-ß/Smad2/3 and the antifibrotic TGF-ß/Smad1/5/9 signalling pathways and its role in the myofibroblast formation of HBF populations derived from asthmatic and non-asthmatic donors. Our findings showed for the first time that TGF-ß-induced activation of the profibrotic Smad2/3 signalling pathway was enhanced, but the activation of the antifibrotic Smad1/5/(8)9 pathway by TGF-ß1 was significantly diminished in fibroblasts from asthmatic donors compared to those from their healthy counterparts. The impairment of the antifibrotic TGF-ß/Smad1/5/(8)9 pathway in HBFs derived from asthmatic donors was correlated with enhanced FMT. Furthermore, we showed that Smad1 silencing in HBFs from non-asthmatic donors increased the FMT potential in these cells. Additionally, we demonstrated that activation of antifibrotic Smad signalling via BMP7 or isoliquiritigenin [a small-molecule activator of the TGF-ß/Smad1/5/(8)9 pathway] administration prevents FMT in HBFs from asthmatic donors through downregulation of profibrotic genes, e.g., α-SMA and fibronectin. Our data suggest that influencing the balance between the antifibrotic and profibrotic TGF-ß/Smad signalling pathways using BMP7-mimetic compounds presents an unprecedented opportunity to inhibit subepithelial fibrosis during airway remodelling in asthma.

Ablation of Fam20c causes amelogenesis imperfecta via inhibiting Smad dependent BMP signaling pathway.

BACKGROUND: Amelogenesis imperfecta (AI) is a type of hereditary diseases that manifest defects in the formation or mineralization of enamel. Recently, it is reported that inactivation of FAM20C, a well-known Golgi casein kinase, caused AI. However, the mechanism of it is still unknown. The aim of this study was to explore the molecular mechanism of AI, which caused by ablation of FAM20C. RESULTS: In the Sox2-Cre;Fam20Cfl/fl (cKO) mouse, we found abnormal differentiation of ameloblasts, improper formation and mineralization of enamel, and downregulation of both mRNA and protein level of enamel matrix proteins, including amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). The levels of BMP2, BMP4 and BMP7, the ligands of BMP signaling pathway, and phosphorylation of Smad1/5/8, the key regulators of BMP signaling pathway, were all decreased in the enamel matrix and the ameloblast of the cKO mice, respectively. The expression of cyclin-dependent kinase inhibitor (P21), muscle segment homeobox genes 2 (Msx2), which are the target genes of the BMP signaling pathway, and laminin 3, the downstream factor of Msx2, were all significantly decreased in the ameloblasts of the cKO mice compared to the control mice. CONCLUSION: the results of our study suggest that ablation of FAM20C leads to AI through inhibiting the Smad dependent BMP signaling pathway in the process of amelogenesis.

Green Tomato Extract Prevents Bone Loss in Ovariectomized Rats, a Model of Osteoporosis.

Although drug therapies are available for postmenopausal osteoporosis, these drugs are not free of side effects and long-term adherence to them are low. A safe and effective nutritional approach to counter postmenopausal osteoporosis is an important research goal. We fed ovariectomized (OVX) Sprague-Dawley rats a diet supplemented with 1% or 2% green tomato extract (GTE). After 12 weeks, micro-computed tomography scans revealed that GTE supplementation effectively prevented distal femur bone loss. This prevention was due to improved bone formation and suppressed bone resorption as observed by the regulation of osteoblast and osteoclast activities. GTE supplementation also improved bone formation through Bmp2-Smad 1/5/8-Runx2 signaling, while bone resorption was regulated by the receptor activator of nuclear factor kappa-B (RANKL)/osteoprogeterin (OPG) pathway. These results suggest that GTE supplementation prevents severe postmenopausal bone loss by maintaining the regulation of bone homeostasis in OVX rats. GTE as a diet supplement might be a potential novel alternative for the prevention of postmenopausal osteoporosis.

NLRC5 deficiency ameliorates cardiac fibrosis in diabetic cardiomyopathy by regulating EndMT through Smad2/3 signaling pathway.

Diabetic cardiomyopathy (DCM) is one of the main causes of heart failure in patients with diabetes. Cardiac fibrosis caused by endothelial mesenchymal transformation (EndMT) plays an important role in the pathogenesis of DCM. NLRC5 is a recently discovered immune and inflammatory regulatory molecule in the NOD-like receptor family, and is involved in organ fibrosis. In this study, we found that the expression of NLRC5 was up-regulated in endothelial cells (ECs) and cardiac fibroblasts (CFs) in diabetes models both in vivo and in vitro. NLRC5 knockdown significantly inhibited high glucose-induced EndMT. In addition, NLRC5 deficiency inhibited the expression of phosphorylated Smad2/3 and the activation of EndMT-related transcription factors in ECs induced by high glucose. However, the effect of NLRC5 deficiency on CFs was not obvious. In summary, our results suggest that NLRC5 deficiency ameliorates cardiac fibrosis in DCM by inhibiting EndMT through Smad2/3 signaling pathway and related transcription factors. NLRC5 is likely to be a biomarker and therapeutic target of cardiac fibrosis in diabetic cardiomyopathy.

Astragalus polysaccharides protect renal function and affect the TGF-ß/Smad signaling pathway in streptozotocin-induced diabetic rats.

OBJECTIVES: The objective was to observe the effects of Astragalus polysaccharides on diabetes and on regulation of the TGF-ß/Smad signaling pathway. METHODS: A type 2 diabetic rat model was established with a high-fat diet in combination with low-dose streptozotocin (35 mg/kg). Astragalus polysaccharides were applied as treatment intervention and changes in blood glucose and kidney morphology and function were assessed. RESULTS: Eight weeks after model establishment, kidney weight as a proportion of total weight (KW/TW) in the high-, medium-, and low-dose Astragalus polysaccharide groups was significantly lower than that in the model group, and the KW/TW value gradually decreased with increasing dose of polysaccharides in each treatment group. Fasting blood glucose in the low- and medium-dose Astragalus polysaccharide groups was numerically lower than that in the model group and fasting blood glucose in rats in the high-dose group was significantly lower than that in the model group. Levels of 24-hour urinary microalbumin, creatinine, blood urea nitrogen, collagens I, III, and IV, α-smooth muscle actin, transforming growth factor-ß1, and Smad3 in Astragalus polysaccharide groups (all doses) were significantly lower than those in the model group. CONCLUSIONS: Astragalus polysaccharide significantly improved blood glucose and protected kidney function in a rat diabetes model.

Kaempferol Protects Renal Fibrosis through Activating the BMP-7-Smad1/5 Signaling Pathway.

Renal interstitial fibrosis (RIF) is a common pathological characteristic associated with end-stage renal disease. However, treatment strategies for RIF are still very limited. In this study, we reported that kaempferol, a classic flavonoid, exhibited strong and widely inhibitory effect on the expression of fibrosis related genes in transforming growth factor beta 1 (TGF-ß1) treated NRK-52E cells. Further studies revealed that kaempferol inhibited TGF-ß1 induced epithelial-mesenchymal transition (EMT) process of NRK-52E cells and improved renal function deterioration and RIF in unilateral ureteral obstruction (UUO) rats. After exploring the underlying mechanisms, we found that kaempferol was able to activate the BMP-7-Smad1/5 pathway, rather than the TGF-ß1-Smad2/3 pathway. To further validate these results, DMH1 and BMP-7 knockdown were utilized at the cellular level and the results showed that both methods were able to antagonize the effects of kaempferol on the EMT process of NRK-52E cells induced by TGF-ß1. In UUO rats, inhibition of BMP-7 signaling by DMH1 also reversed the effects of kaempferol on renal function decline and RIF. Taken together, our findings demonstrated that kaempferol could be a good candidate for renal fibrosis treatment.

The Phosphodiesterase-5 Inhibitor Vardenafil Improves the Activation of BMP Signaling in Response to Hydrogen Peroxide.

PURPOSE: The pleiotropic roles of phosphodiesterase-5 inhibitors (PDE5is) in cardiovascular diseases have attracted attention. The effect of vardenafil (a PDE5i) is partly mediated through reduced oxidative stress, but it is unclear whether vardenafil protects against hydrogen peroxide (H2O2)-induced endothelial cell injury, and the molecular mechanisms that are involved remain unknown. We determined the protective role of vardenafil on H2O2-induced endothelial cell injury in cultured human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Vardenafil decreased the number of TUNEL-positive cells, increased the Bcl2/Bax ratio, and ameliorated the numbers of BrdU-positive cells in H2O2-treated HUVECs. The bone morphogenetic protein receptor (BMPR)/p-Smad/MSX2 pathway was enhanced in response to H2O2, and vardenafil treatment could normalize this pathway. To determine whether the BMP pathway is involved, we blocked the BMP pathway using dorsomorphin, which abolished the protective effects of vardenafil. We found that vardenafil improved the H2O2-induced downregulation of BMP-binding endothelial regulator protein (BMPER), which possibly intersects with the BMP pathway in the regulation of endothelial cell injury in response to oxidative stress. CONCLUSIONS: We demonstrated for the first time that exogenous H2O2 activates BMPR expression and promotes Smad1/5/8 phosphorylation. Additionally, vardenafil can attenuate H2O2-induced endothelial cell injury in HUVECs. Vardenafil decreases apoptosis through an improved Bcl-2/Bax ratio and increases cell proliferation. Vardenafil protects against endothelial cell injury through ameliorating the intracellular oxidative stress level and BMPER expression. The protective role of vardenafil on H2O2-induced endothelial cell injury is mediated through BMPR/p-Smad/MSX2 in HUVECs.

Inhibition of bone morphogenetic protein 6 receptors ameliorates Sjögren's syndrome in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease, with only palliative treatments available. Recent work has suggested that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction. We examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models. Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.

Estrogen inhibits vascular calcification in rats via hypoxia-induced factor-1α signaling.

OBJECTIVE: Calcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway. METHODS: Eight-week-old healthy female Sprague-Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17ß-estradiol) and bone morphogenetic protein 2 siRNA intervention. RESULTS: The estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification (p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro (p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein (p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 (p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells (p < 0.01). CONCLUSION: Estrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.

Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesis.

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.

GdCl3 attenuates the glomerular sclerosis of streptozotocin (STZ) induced diabetic rats via inhibiting TGF-ß/Smads signal pathway.

Diabetic nephropathy (DN) is the most serious end-stage renal disease which characterized by renal glomerular sclerosis including glomerular hypertrophy, glomerular basement membrane (GBM) thickening, mesangial expansion and renal fibrosis. TGF-ß/Smads signal pathway plays a crucial role in the development of renal fibrosis. In this study, we found that GdCl3 which was an agonist of Calcium-sensing receptor (CaSR) could repress the activation of TGF-ß/Smads signal pathway induced by TGF-ß1 or high glucose and then alleviated the accumulation of extracellular matrix (ECM) in mesangial cells and the kidney of type1 diabetic rats. Further study indicated that GdCl3 could induce the binding of CaSR and TßR II and then both of these two receptors translocated from cell membrane to cytoplasm, in this case, TßR II on the cell membrane was decreased and then desensitized to the stimulation of its ligand TGF-ß1, so that the activation of its downstream factors such as Smad2 and Smad3 were blocked, finally, ECM expression in mesangial cells were inhibited. We concluded that GdCl3 could alleviate the accumulation of ECM in mesangial cells via antagonizing TGF-ß/Smads signal pathway in diabetes mellitus.