Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4.

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-ß signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets Smad2, Smad3 and Smad4 mRNA 3'UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-ß, B cells downregulated miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline Iα-Cα transcription. Deletion of miR-146a in miR-146a-/- mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a-/- mice, the elevated systemic IgA levels were associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline Iα-Cα transcription and CSR to IgA in miR-146a-/- B cells in vitro proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed in vivo by construction and OVA immunization of mixed bone marrow µMT/miR-146a-/- chimeric mice. Thus, by inhibiting Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.

SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Transforming Growth Factor-ß1/Smad Signaling in Glomerulonephritis and Its Association with Progression to Chronic Kidney Disease.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine, with diverse roles in fibrosis and inflammation, which acts through Smad signaling in renal pathology. We intended to investigate the expression of TGF-ß/Smad signaling in glomerulonephritis (GN) and to assess its role as risk factor for progression to chronic kidney disease (CKD). METHODS: We evaluated the immunohistochemical expression of TGF-ß1, phosphorylated Smad3 (pSmad3), and Smad7 semiquantitatively and quantitatively using computerized image analysis program in different compartments of 50 renal biopsies with GN, and the results were statistically analyzed with clinicopathological parameters. We also examined the associations among their expressions, the impact of their co-expression, and their role in progression to CKD. RESULTS: TGF-ß1 expression correlated positively with segmental glomerulosclerosis (p= 0.025) and creatinine level at diagnosis (p = 0.002), while pSmad3 expression with interstitial inflammation (p = 0.024). In glomerulus, concomitant expressions of high Smad7 and medium pSmad3 were observed to be correlated with renal inflammation, such as cellular crescent (p = 0.011), intense interstitial inflammation (p = 0.029), and lower serum complement (C) 3 (p = 0.028) and C4 (p = 0.029). We also reported a significant association between pSmad3 expression in glomerular endothelial cells of proliferative GN (p = 0.045) and in podocytes of nonproliferative GN (p = 0.005). Finally, on multivariate Cox-regression analysis, TGF-ß1 expression (hazard ratio = 6.078; 95% confidence interval: 1.168-31.627; p = 0.032) was emerged as independent predictor for CKD. DISCUSSION/CONCLUSION: TGF-ß1/Smad signaling is upregulated with specific characteristics in different forms of GN. TGF-ß1 expression is indicated as independent risk factor for progression to CKD, while specific co-expression pattern of pSmad3 and Smad7 in glomerulus is correlated with renal inflammation.

TGF­ß inhibitor RepSox suppresses osteosarcoma via the JNK/Smad3 signaling pathway.

Osteosarcoma (OS) is the most common malignant bone tumor and the long­term survival rates remain unsatisfactory. Transforming growth factor­ß (TGF­ß) has been revealed to play a crucial role in OS progression, and RepSox is an effective TGF­ß inhibitor. In the present study, the effect of RepSox on the proliferation of the OS cell lines (HOS and 143B) was detected. The results revealed that RepSox effectively inhibited the proliferation of OS cells by inducing S­phase arrest and apoptosis. Moreover, the inhibitory effect of RepSox on cell migration and invasion was confirmed by wound­healing and Transwell assays. Furthermore, western blotting revealed that the protein levels of molecules associated with the epithelial­mesenchymal transition (EMT) phenotype, including E­cadherin, N­cadherin, Vimentin, matrix metalloproteinase (MMP)­2 and MMP­9, were reduced by RepSox treatment. Concurrently, it was also revealed that the JNK and Smad3 signaling pathway was inhibited. Our in vivo findings using a xenograft model also revealed that RepSox markedly inhibited the growth of tumors. In general, our data demonstrated that RepSox suppressed OS proliferation, EMT and promoted apoptosis by inhibiting the JNK/Smad3 signaling pathway. Thus, RepSox may be a potential anti­OS drug.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

Deletion of Smad3 protects against diabetic myocardiopathy in db/db mice.

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF-ß/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT-db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO-db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2-mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF-kB-driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3-dependent miRNAs by up-regulating cardiac miR-29b while suppressing miR-21 to exhibit the cardioprotective effect on Smad3KO-db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.

Nupr1 mediates renal fibrosis via activating fibroblast and promoting epithelial-mesenchymal transition.

Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

A review for natural polysaccharides with anti-pulmonary fibrosis properties, which may benefit to patients infected by 2019-nCoV.

Pulmonary fibrosis (PF) is a lung disease with highly heterogeneous and mortality rate, but its therapeutic options are now still limited. Corona virus disease 2019 (COVID-19) has been characterized by WHO as a pandemic, and the global number of confirmed COVID-19 cases has been more than 8.0 million. It is strongly supported for that PF should be one of the major complications in COVID-19 patients by the evidences of epidemiology, viral immunology and current clinical researches. The anti-PF properties of naturally occurring polysaccharides have attracted increasing attention in last two decades, but is still lack of a comprehensively understanding. In present review, the resources, structural features, anti-PF activities, and underlying mechanisms of these polysaccharides are summarized and analyzed, which was expected to provide a scientific evidence supporting the application of polysaccharides for preventing or treating PF in COVID-19 patients.

CO2 lattice laser reverses skin aging caused by UVB.

The carbon dioxide (CO2) lattice laser has been successfully used to treat facial skin photoaging induced by UV light. In this study, we analyzed the effect of CO2 lattice laser irradiation on skin photoaging, and investigated the underlying mechanisms. Our results demonstrate that the laser promoted collagen synthesis and proliferation of primary human skin fibroblasts, inhibited cell senescence, and induced expression of superoxide dismutase (SOD) and the signaling protein SMAD3. In addition, this laser reversed cell cycle arrest and fibroblast apoptosis induced by UVB irradiation, and restored fibroblast proliferation inhibited by SMAD3 silencing. Using a rat model of photoaging, our results show that the laser increased collagen expression and dermal thickness, demonstrating that the CO2 lattice laser has a profound therapeutic effect on photoaged skin. Together, our in vitro and in vivo data show that the CO2 lattice laser can reverse the skin aging caused by UVB, and indicate that this effect is mediated through SMAD3.

Angiocrine FSTL1 (Follistatin-Like Protein 1) Insufficiency Leads to Atrial and Venous Wall Fibrosis via SMAD3 Activation.

OBJECTIVE: Angiocrine factors, mediating the endothelial-mural cell interaction in vascular wall construction as well as maintenance, are incompletely characterized. This study aims to investigate the role of endothelial cell-derived FSTL1 (follistatin-like protein 1) in vascular homeostasis. Approach and Results: Using conditional knockout mouse models, we show that loss of FSTL1 in endothelial cells (Fstl1ECKO) led to an increase of pulmonary vascular resistance, resulting in the heart regurgitation especially with tricuspid valves. However, this abnormality was not detected in mutant mice with Fstl1 knockout in smooth muscle cells or hematopoietic cells. We further showed that there was excessive αSMA (α-smooth muscle actin) associated with atrial endocardia, heart valves, veins, and microvessels after the endothelial FSTL1 deletion. There was also an increase in collagen deposition, as demonstrated in livers of Fstl1ECKO mutants. The SMAD3 (mothers against decapentaplegic homolog 3) phosphorylation (pSMAD3) was significantly enhanced, and pSMAD3 staining was colocalized with αSMA in vein walls, suggesting the activation of TGFß (transforming growth factor ß) signaling in vascular mural cells of Fstl1ECKO mice. Consistently, treatment with a TGFß pathway inhibitor reduced the abnormal association of αSMA with the atria and blood vessels in Fstl1ECKO mutant mice. CONCLUSIONS: The findings imply that endothelial FSTL1 is critical for the homeostasis of vascular walls, and its insufficiency may favor cardiovascular fibrosis leading to heart failure.

Function and characterization of the promoter region of perilipin 1 (PLIN1): Roles of E2F1, PLAG1, C/EBPß, and SMAD3 in bovine adipocytes.

Perilipin 1 (PLIN1) protein, also known as lipid droplet-associated protein, is encoded by the PLIN1 gene and is able to anchor itself to the membranes of lipid droplets. The phosphorylation of PLIN1 is critical for the mobilization of fat in adipose tissue and plays an important role in regulating lipolysis and lipid storage in adipocytes. However, research on the synthesis and lipid metabolism of lipid droplets by PLIN1 in bovine adipocytes is limited. In the present study, we found that bovine PLIN1 was highly expressed in subcutaneous adipose tissue. The highest level of PLIN1 mRNA expression in bovine adipocytes was observed on day 6 of differentiation. Moreover, the cytoplasmic subcellular localization of PLIN1 was observed in bovine preadipocytes. To elucidate the molecular mechanism of bovine PLIN1 transcriptional regulation, we cloned eight fragments containing the 5' regulatory region of the PLIN1 gene. The results showed that the -209/-17 bp region of the bovine PLIN1 gene was the core promoter region. Based on the transcriptional activities of the promoter vector fragments, the luciferase activity of the mutated fragment, the siRNA interference, and the results of the electrophoretic mobility shift assay (EMSA), we identified the binding sites of E2F transcription factor 1 (E2F1), pleiomorphic adenoma gene 1 (PLAG1), CCAAT enhancer binding protein beta (C/EBPß), and SMAD family member 3 (SMAD3) as the transcriptional activators or repressors of the core promoter region. Further experiments confirmed that the knockdown of the PLIN1 gene affected the ability of these transcription factors to regulate the lipid metabolism in bovine adipocytes. In conclusion, our results reveal a potential mechanism for the transcriptional regulation of PLIN1 in bovine adipocytes.

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

TGF-ßs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-ß1, -ß2 and -ß3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Effects of Acupoint Application Therapy with TianGui Powder on Osteoporosis in Ovariectomized Rats through TGF-ß1 and Smad2/3 Signaling Pathway.

OBJECTIVES: To explore the effects of acupoint application therapy (AAT) with TianGui Powder (TGP) on the expressions of the transforming growth factor ß1 (TGF-ß1) and Smad-2/3 signaling pathway in ovariectomized osteoporosis rats. METHODS: Sixty rats were randomly divided into four groups: normal group (group A), model group (group B), TGP group (group C), and Western medicine group (group D). Group A had only the corresponding amount of adipose tissue around the ovary removed; rats in the other groups had bilateral ovariectomies. After 1 week, groups A and B were given 1 mL/100 mg normal saline solution by gavage, group C was treated with AAT with TGP on ShenQue acupoint (0.2 piece/rat, 6 h/time, 1 time/d) and group D was given calcium carbonate vitamin D3 (36 mg/kg/d) and alfacalcidol (0.05 µg/kg/d) tablet suspension. In this study, the bone mineral density (BMD) , the levels of BALP, TRAP-5b, and BGP in serum and the changes in bone histomorphology was detected. For acquiring lumbar experimental data, the expression of TGF-ß1, Smad-2/3 proteins and mRNA of TGF-ß1and Smad-2/3 were assessed. After 12 weeks, the data were collected for analysis. RESULTS: Compared with group A, the bone trabecula was thinner and significantly reduced in other groups. The result of BMD improved significantly in both groups C and D compared to group B after intervention (P < 0.05). In contrast, compared to group B, the levels of BALP, TRAP-5b, and BGP significantly declined in both groups C and D. In group C, the results of protein expressions in TGF-ß1, Smad-2/3 were 2.870 ± 0.270, 1.552 ± 0.111, and 1.420 ± 0.079, respectively. In groups C and D, those indications significantly declined compared to group B (P < 0.01). In group C, the level of mRNA expressions of TGF-ß1, Smad-2/3 were 1.872 ± 0.177, 1.672 ± 0.086, and 1.790 ± 0.136, respectively. Compared with group B, those indications had significant difference in groups C and D (P < 0.05). CONCLUSION: Acupoint application therapy with TGP could significantly improve the BMD. The TGF-ß1 and Smad-2/3 signaling pathway could be a therapeutic target of TGP in postmenopausal osteoporosis rats.

Fibroblast Primary Cilia Are Required for Cardiac Fibrosis.

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Genome-Wide mRNA-Seq Profiling Reveals that LEF1 and SMAD3 Regulate Epithelial-Mesenchymal Transition Through the Hippo Signaling Pathway During Palatal Fusion.

BACKGROUND: Epithelial-mesenchymal transition (EMT) of the medial edge epithelium (MEE) occurs through fusion of the palatal shelves and is a crucial step in palatogenesis. The key genes, however, and the related signaling pathway of EMT are not yet fully understood. Therefore, the aim of this study was to reveal the key genes and the related signaling pathway of EMT during palatal fusion. MATERIALS AND METHODS: C57BL/6J mice at embryonic gestation day 14.5 (E14.5; n = 6) were used to establish the cleft palate model for mRNA-Seq (HiSeq X Ten). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for functional annotations of the differentially expressed genes. Quantitative polymerase chain reaction (qPCR) assays were used to validate the RNAseq data. RESULTS: A total of 936 differentially expressed genes, including 558 upregulated and 378 downregulated genes were identified in cases versus controls, respectively. Among these genes, the GO analysis showed that Lymphoid Enhancer-Binding Factor 1 (LEF1) and SMAD Family Member 3 (SMAD3) significantly enriched biological processes, which were EMT related. The KEGG analysis showed that these genes regulated EMT through the Hippo signaling pathway. LEF1 and SMAD3 were downregulated, and the qPCR results corroborated the RNA-seq data. CONCLUSIONS: These results demonstrate that LEF1 and SMAD3 inhibits EMT at the MEE through the Hippo signaling pathway; and that this could contribute to cleft palate formation in embryonic palatal fusion at E 14.5.

Long noncoding RNA Crnde attenuates cardiac fibrosis via Smad3-Crnde negative feedback in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM)-ventricular dysfunction in the absence of underlying heart disease-is a common complication of diabetes and a leading cause of mortality associated with the disease. In DCM, cardiac fibrosis is the main cause of heart failure. Although it is well-established that the transforming growth factor-beta signaling pathway plays a part in inducing cardiac fibrosis in DCM, details of the molecular mechanism involved remain elusive. Therefore, it is crucial to study the gene reg;ulation of key signaling effectors in DCM-associated cardiac fibrosis. A recently emerged hotspot in the field of gene regulation is the role of long noncoding RNAs (lncRNAs). Recent evidence indicates that lncRNAs play a critical role in cardiac fibrosis; however, in DCM, the function of these regulatory RNAs have not been studied in depth. In this study, we identified a conserved cardiac-specific lncRNA named colorectal neoplasia differentially expressed (Crnde). By analyzing 376 human heart tissues, it was found that Crnde expression is negatively correlated with that of cardiac fibrosis marker genes. Moreover, Crnde expression was shown to be enriched in cardiac fibroblasts (CFs). Overexpression of Crnde attenuated cardiac fibrosis and enhanced cardiac function in mice with DCM. Further, in vitro experiments showed that Crnde negatively regulates the myofibroblast differentiation of CFs. The expression of Crnde was activated by SMAD family member 3 (Smad3), shedding light on the underlying molecular mechanism. Interestingly, Crnde also inhibited the transcriptional activation of Smad3 on target genes, thereby inhibiting the expression of myofibroblastic marker genes in CFs. Overall, our data provide valuable insights into the development of potential anti-cardiac fibrosis strategies centered on lncRNAs, for the treatment of DCM.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1.

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Hydrogen sulfide attenuates paraquat-induced epithelial-mesenchymal transition of human alveolar epithelial cells through regulating transforming growth factor-ß1/Smad2/3 signaling pathway.

Exogenous H2 S donor, sodium hydrosulfide (NaHS), can influence the bleomycin-induced pulmonary fibrosis by attenuating the epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, but whether NaHS affects paraquat (PQ)-induced EMT and the molecular mechanisms remain unclarified. The aim of the present study is to examine the effect of exogenous NaHS on PQ-induced EMT in human alveolar epithelial cells (A549 cells) and assess if this effect occurs through regulating transforming growth factor (TGF)-ß1/Smad2/3 signaling pathway. The expressions of endogenous H2 S producing enzymes, namely cystathionine ß-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfur transferase, were detected by reverse transcription-polymerase chain reaction and western blotting. The induced EMT was assessed by morphological and phenotypic characterizations, and the protein level of E-cadherin and vimentin were detected by western blotting. To investigate the effect of NaHS on PQ-induced EMT and potential mechanism, A549 cells were pretreated with NaHS before incubating with PQ and then evaluated by morphological changes, cell migration ability, the expression of EMT markers and TGF-ß1/Smad2/3 signaling pathway related proteins. PQ significantly downregulated the expression levels of cystathionine ß-synthase and cystathionine γ-lyase, but not 3-mercaptopyruvate sulfur transferase, in a time-dependent manner in A549 cells. Exogenous NaHS could significantly retard PQ-induced morphological changes and cell migration ability. Furthermore, exogenous NaHS significantly upregulated the expression of E-cadherin, whereas it downregulated the expression of vimentin. In addition, exogenous NaHS could also significantly attenuates PQ-induced TGF-ß1, phosphorylated Smad2/3 proteins expression, which induced by PQ in a time-dependent manner. This study provides the first evidence that exogenous NaHS attenuates PQ-induced EMT and migration of human alveolar epithelial cells through regulating the TGF-ß1/Smad2/3 signaling pathway.

Downregulation of TGF-ß1 suppressed proliferation and increased chemosensitivity of ovarian cancer cells by promoting BRCA1/Smad3 signaling.

BACKGROUND: Studies have demonstrated that transforming growth factor beta-1 (TGF-ß1) exhibits oncogenic activity in different types of cancer, including ovarian cancer (OC). However, its regulatory mechanism in OC and whether TGF-ß1 is involved in chemosensitivity regulation remains unclear. Thus, the aim of this study was to investigate the role of TGF-ß1 in OC. METHODS: The OC cell line SKOV3 was employed, and TGF-ß1 overexpression or knockdown vectors were constructed. The cell proliferation of SKOV3 was evaluated with the cell counting kit (CCK8) kit after treatment with different concentrations of cis-platinum. Western blot and protein immunoprecipitation were employed to detect changes in BRCA1 and Smad3 expression and their interactions. Tumor growth in nude mice was evaluated. RESULTS: The results showed that TGF-ß1 knockdown increased chemosensitivity by promoting BRCA1 expression and Smad3 phosphorylation. In vivo studies showed that TGF-ß1 knockdown significantly inhibited the growth of tumors, also by upregulating BRCA1 expression and Smad3 phosphorylation. CONCLUSION: Taken together, our results suggest that TGF-ß1 knockdown inhibits tumor growth and increases chemosensitivity by promotion of BRCA1/Smad3 signaling.