Induced Smad2 Phosphorylation Research Articles

Retraction: GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF- β -Induced Smad3 Phosphorylation.

Retraction: GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF- β -Induced Smad3 Phosphorylation.

The IL-1β-p65 axis stimulates quiescent odontogenic epithelial cell rests via TGF-β signalling to promote cell proliferation of the lining epithelia in radicular cysts: A laboratory investigation.

Cyst formation of the jaws is frequently accompanied by the proliferation of odontogenic epithelial cells located in the periodontal ligament (PDL), which consists of heterozygous cells and includes the most fibroblasts. The lining epithelium of radicular cyst, an odontogenic cyst of inflammatory origin, is derived from the proliferation of the remnants of the Hertwig epithelial root sheath (odontogenic epithelial cell rests of Malassez; ERMs) in the PDL. ERMs are maintained at a lower proliferative state under physiological conditions, but the regulatory mechanisms underlying the inflammation-dependent enhanced-proliferative capabilities of ERMs are not fully understood. The aim of this study was to evaluate the effects of cytokine pathway association between TGF-β signalling and IL-1β signalling on the regulation of odontogenic epithelial cell proliferation using radicular cyst pathological specimens and odontogenic epithelial cell lines. Immunofluorescence analyses were performed to clarify the expression levels of Smad2/3 and Ki-67 in ERMs of 8-week-old mouse molar specimens. In radicular cyst (n = 52) and dentigerous cysts (n = 6) specimens from human patients, the expression of p65 (a main subunit of NF-κB), Smad2/3 and Ki-67 were investigated using immunohistochemical analyses. Odontogenic epithelial cells and PDL fibroblastic cells were co-cultured with or without an inhibitor or siRNAs. Odontogenic epithelial cells were cultured with or without TGF-β1 and IL-1β. The proliferative capabilities and Smad2 phosphorylation levels of odontogenic epithelial cells were examined. Immunohistochemically, Smad2/3-positivity was increased, and p65-positivity and Ki-67-positivity were decreased both in ERMs and in the epithelial cells in dentigerous cysts, a non-inflammatory developmental cyst. In contrast, p65-positive cells, along with the expression of Ki-67, were increased and Smad2/3-positive cells were decreased in the lining epithelia of radicular cysts. Co-culture experiments with odontogenic epithelial cells and PDL fibroblastic cells revealed that PDL cells-derived TGF-β1/2 and their downstream signalling suppressed odontogenic epithelial cell proliferation. Moreover, TGF-β1 stimulation induced Smad2 phosphorylation and suppressed odontogenic epithelial cell proliferation, while IL-1β stimulation reversed these phenotypes through p65 transactivation. These results suggest that IL-1β-p65 signalling promotes odontogenic epithelial cell proliferation through suppressing TGF-β-Smad2 signalling, which would be involved in the pathogenesis of radicular cysts.

A gut microbial metabolite of linoleic acid ameliorates liver fibrosis by inhibiting TGF-β signaling in hepatic stellate cells

The antidiabetic drug pioglitazone ameliorates insulin resistance by activating the transcription factor PPARγ. In addition to its blood glucose–lowering action, pioglitazone exerts pleiotropic effects including amelioration of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The mechanism by which pioglitazone achieves this latter effect has remained unclear, however. We here show that pioglitazone administration increases the amount of linoleic acid (LA) metabolites in adipose tissue of KK-Ay mice. These metabolites are produced by lactic acid bacteria in the gut, and pioglitazone also increased the fraction of Lactobacillus in the gut microbiota. Administration of the LA metabolite HYA (10-hydroxy-cis-12-octadecenoic acid) to C57BL/6 J mice fed a high-fat diet improved liver histology including steatosis, inflammatory cell infiltration, and fibrosis. Gene ontology analysis of RNA-sequencing data for the liver revealed that the top category for genes downregulated by HYA treatment was related to extracellular matrix, and the expression of individual genes related to fibrosis was confirmed to be attenuated by HYA treatment. Mechanistically, HYA suppressed TGF-β–induced Smad3 phosphorylation and fibrosis-related gene expression in human hepatic stellate cells (LX-2). Our results implicate LA metabolites in the mechanism by which pioglitazone ameliorates liver fibrosis, and they suggest that HYA is a potential therapeutic for NAFLD/NASH.

Crosstalk between protein kinase C α and transforming growth factor β signaling mediated by Runx2 in intestinal epithelial cells

Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor β (TGFβ) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFβ signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFβ receptor 1 (TGFβR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFβR1 mRNA and protein expression in these cells. Effects on TGFβR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFβR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFβR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFβR1 signaling axis. In further support of a link between PKCα and TGFβ signaling, PKCα knockdown reduced the ability of TGFβ to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFβR1 decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by The Cancer Genome Atlas data showing correlation between PKCα, Runx2, and TGFβR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFβR1 in endometrial cancer cells, and PKCα, Runx2, and TGFβR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFβ signaling may be a common mechanism in diverse epithelial tissues.

Long noncoding TSI attenuates aortic valve calcification by suppressing TGF-β1-induced osteoblastic differentiation of valve interstitial cells

IntroductionCalcific aortic valve disease (CAVD) is an active and cellular-driven fibrocalcific process characterised by differentiation of valve interstitial cells (VICs) towards an osteogenic-like phenotype. A recently identified lncRNA, lncTSI, has been reported to inhibit fibrogenesis through transforming growth factor (TGF)-β/Smad3 pathway. Here, the present study aimed to investigate the role of lncTSI in CAVD. MethodsThe effect of TGF-β1 on lncTSI of VICs was measured. TGF-β1, RUNX2 and collagen I expression between calcified aortic valve tissue and normal samples by immunohistochemistry and western blotting. Human VICs were cultured and treated with TGF-β1. SiRNA and pcDNA3.1-lncTSI plasmid transfection were used to silence and overexpress lncTSI in VICs for 48 h, Smads phosphorylation, RUNX2 and collagen I expression were then verified by western blotting. In ApoE−/− mice fed with 0.25 % high-cholesterol diet, AAV2-lncTSI were injected intravenously to observe their effect on the formation of aortic valve calcification. ResultslncTSI was highly expressed in VICs treated with TGF-β1. lncTSI was transcriptionally regulated by Smad3 and reversely inhibited TGF-β1-induced Smad3 phosphorylation and downregulated profibrotic gene expression. Silencing lncTSI increased TGF-β1–induced Smad3 phosphorylation, and subsequently, upregulated RUNX2 and collagen I expressions in VICs. While overexpression of lncTSI reversed the production of RUNX2 and collagen I in VICs. In a mouse CAVD model of 24 week 0.25 % high-cholesterol diet feeding, overexpression of lncTSI significantly reduced calcium deposition, RUNX2, pSmad3, and collagen I expression in aortic valve leaflets, with less aortic valve stenosis. ConclusionsThe novel findings of present study suggested that lncTSI alleviated aortic valve calcification through negative regulation of the TGF-β/Smad3 pathway. The results may help elucidate new diagnostic and therapeutic targets to prevent CAVD progression.

Linderanoids A–O, dimeric sesquiterpenoids from the roots of Lindera aggregata (Sims) Kosterm

Fifteen undescribed dimeric sesquiterpenoids, linderanoids A–O along with one known lindenane-type sesquiterpenoid dimer, lindenaneolide F, were isolated and identified from the roots of Lindera aggregata. Their structures and absolute configurations were elucidated using spectroscopy and electronic circular dichroism (ECD) analysis. All the isolated compounds were screened for transforming growth factor (TGF)-β inhibitory activity, and the results showed that linderanoid E significantly inhibited the TGF- β induced smad2 phosphorylation at a concentration of 25 μM.

Crosstalk between 17β-Estradiol and TGF-β Signaling Modulates Glioblastoma Progression.

Epithelial–mesenchymal transition (EMT) is an essential mechanism contributing to glioblastoma multiforme (GBM) progression, the most common and malignant brain tumor. EMT is induced by signaling pathways that crosstalk and regulate an intricate regulatory network of transcription factors. It has been shown that downstream components of 17β-estradiol (E2) and transforming growth factor β (TGF-β) signaling pathways crosstalk in estrogen-sensitive tumors. However, little is known about the interaction between the E2 and TGF-β signaling components in brain tumors. We have investigated the relationship between E2 and TGF-β signaling pathways and their effects on EMT induction in human GBM-derived cells. Here, we showed that E2 and TGF-β negatively regulated the expression of estrogen receptor α (ER-α) and Smad2/3. TGF-β induced Smad2 phosphorylation and its subsequent nuclear translocation, which E2 inhibited. Both TGF-β and E2 induced cellular processes related to EMT, such as morphological changes, actin filament reorganization, and mesenchymal markers (N-cadherin and vimentin) expression. Interestingly, we found that the co-treatment of E2 and TGF-β blocked EMT activation. Our results suggest that E2 and TGF-β signaling pathways interact through ER-α and Smad2/3 mediators in cells derived from human GBM and inhibit EMT activation induced by both factors alone.

Differential Responses to Targeting Matrix Metalloproteinase 9 in Idiopathic Pulmonary Fibrosis.

Rationale: Aberrant lung remodeling in idiopathic pulmonary fibrosis (IPF) is characterized by elevated MMP9 (matrix metalloproteinase 9) expression, but the precise role of this matrix metalloproteinase in this disease has yet to be fully elucidated.Objectives: To evaluate antifibrotic effects of MMP9 inhibition on IPF.Methods: Quantitative genomic, proteomic, and functional analyses both in vitro and in vivo were used to determine MMP9 expression in IPF cells and the effects of MMP9 inhibition on profibrotic mechanisms.Measurements and Main Results: In the present study, we demonstrate that MMP9 expression was increased in airway basal cell (ABC)-like cells from IPF lungs compared with ABC cells from normal lungs. The inhibition of MMP9 activity with an anti-MMP9 antibody, andecaliximab, blocked TGF-β1 (transforming growth factor β1)-induced Smad2 phosphorylation. However, in a subset of cells from patients with IPF, TGF-β1 activation in their ABC-like cells was unaffected or enhanced by MMP9 blockade (i.e., nonresponders). Further analysis of nonresponder ABC-like cells treated with andecaliximab revealed an association with type 1 IFN expression, and the addition of IFNα to these cells modulated both MMP9 expression and TGF-β1 activation. Finally, the inhibition of MMP9 ameliorated pulmonary fibrosis induced by responder lung cells but not a nonresponder in a humanized immunodeficient mouse model of IPF.Conclusions: Together, these data demonstrate that MMP9 regulates the activation of ABC-like cells in IPF and that targeting this MMP might be beneficial to a subset of patients with IPF who show sufficient expression of type 1 IFNs.

Full-length IL-33 regulates Smad3 phosphorylation and gene transcription in a distinctive AP2-dependent manner

IL-33 has emerged as a central mediator of immune, inflammatory, and fibrotic responses. Many studies have focused on mature IL-33, but elevated expression of the precursor, full-length IL-33 (FLIL33), has also been implicated in a spectrum of diseases, including tissue fibrosis. We previously reported and now confirmed that overexpression of FLIL33 induced phosphorylation of the key profibrotic signaling mediator of TGF-β, Smad3, in primary human lung fibroblasts from healthy donors and idiopathic pulmonary fibrosis patients. Presently, we demonstrate that FLIL33-induced Smad3 phosphorylation was not abrogated by anti-TGF-β antibody but was abrogated by ALK5/TGFBR1-specific and Smad3-specific inhibition, indicating that FLIL33 effect was independent of TGF-β but dependent on its receptor, TGFBR. Western blotting analyses revealed that FLIL33 overexpression increased levels, but did not affect subcellular distribution, of the AP2A1 and AP2B1 subunits of the adaptor protein complex 2 (AP2), a known TGFBR binding partner. siRNA-mediated inhibition of these subunits blocked FLIL33-induced Smad3 phosphorylation, whereas AP2 subunit overexpression induced Smad3 phosphorylation even in the absence of FLIL33. RNA-Seq transcriptomic analyses revealed that fibroblast stimulation with TGF-β induced major changes in expression levels of numerous genes, whereas overexpression of FLIL33 induced modest expression changes in a small number of genes. Furthermore, qRT-PCR tests demonstrated that despite inducing Smad3 phosphorylation, FLIL33 did not induce collagen gene transcription and even mildly attenuated TGF-β-induced levels of collagen I and III mRNAs. We conclude that FLIL33 induces Smad3 phosphorylation through a TGF-β-independent but TGF-β receptor- and AP2- dependent mechanism and has limited downstream transcriptomic consequences.

TGF-β1-induced miR-424 promotes pulmonary myofibroblast differentiation by targeting Slit2 protein expression

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. Myofibroblasts in the lung are key cellular mediators of IPF progression. Transforming growth factor (TGF)-β1, a major profibrogenic cytokine, induces pulmonary myofibroblast differentiation, and emerging evidence has established the importance of microRNAs (miRs) in the development of IPF. The objective of this study was to define the pro-fibrotic roles and mechanisms of miRs in TGF-β1-induced pulmonary myofibroblast differentiation. Using RNA sequencing, we identified miR-424 as an important TGF-β1-induced miR in human lung fibroblasts (HLFs). Quantitative RT-PCR confirmed that miR-424 expression was increased by 2.6-fold in HLFs in response to TGF-β1 and was 1.7-fold higher in human fibrotic lung tissues as compared to non-fibrotic lung tissues. TGF-β1-induced upregulation of miR-424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of this canonical TGF-β1 signaling pathway. Transfection of a miR-424 hairpin inhibitor into HLFs reduced TGF-β1-induced expression of classic myofibroblast differentiation markers including ɑ-smooth muscle actin (ɑ-SMA) and connective tissue growth factor (CTGF), whereas a miR-424 mimic significantly enhanced TGF-β1-induced myofibroblast differentiation. In addition, TGF-β1 induced Smad3 phosphorylation in HLFs, and this response was reduced by the miR-424 inhibitor. In silico analysis identified Slit2, a protein that inhibits TGF-β1 profibrogenic signaling, as a putative target of regulation by miR-424. Slit2 is less highly expressed in human fibrotic lung tissues than in non-fibrotic lung tissues, and knockdown of Slit2 by its siRNA enhanced TGF-β1-induced HLF differentiation. Overexpression of a miR-424 mimic down-regulated expression of Slit2 but not the Slit2 major receptor ROBO1 in HLFs. Luciferase reporter assays showed that the miR-424 mimic represses Slit2 3′ untranslated region (3′-UTR) reporter activity, and mutations at the seeding regions in the 3′-UTR of Slit2 abolish this inhibition. Together, these data demonstrate a pro-fibrotic role of miR-424 in TGF-β1-induced HLF differentiation. It functions as a positive feed-back regulator of the TGF-β1 signaling pathway by reducing expression of the negative regulator Slit2. Thus, targeting miR-424 may provide a new therapeutic strategy to prevent myofibroblast differentiation and IPF progression.

Activin A Signaling Regulates IL13Rα2 Expression to Promote Breast Cancer Metastasis.

Metastatic dissemination of cancer cells to distal organs is the major cause of death for patients suffering from the aggressive basal-like breast cancer (BLBC) subtype. Recently, we have shown that interleukin 13 receptor alpha 2 (IL13Rα2) is a critical gene that is overexpressed in a subset of BLBC primary tumors associated with poor distant metastasis-free survival (DMFS) and can promote extravasation and metastasis of breast cancer cells to the lungs. However, the upstream signaling mechanisms that promote aberrant IL13Rα2 expression during tumor progression remain unknown. Driven by our previously published gene expression microarray data derived from a well-characterized cell line model for BLBC progression, we show that both Inhibin βA (INHBA) and IL13Rα2 genes exhibit similarly higher expression levels in metastatic compared to non-metastatic cells and that overexpression of both genes predicts worse metastasis-free survival of patients with high grade tumors. Activin A, a member of the TGFβ superfamily comprising two INHBA subunits, has been shown to play context-depended roles in cancer progression. Here, we demonstrate that INHBA depletion downregulates IL13Rα2 expression in metastatic breast cancer cells, whereas treatment with Activin A in non-metastatic cells increases its expression levels. We also find that Activin A predominantly induces Smad2 phosphorylation and to a lesser extent activates Smad3 and Akt. Interestingly, we also show that Activin A-mediated upregulation of IL13Rα2 is Smad2-dependent since knocking down Smad2 or using the ALK4/ALK5 inhibitors EW-7197 and SB-505124 abolishes this effect. Most importantly, our data indicate that knocking down INHBA levels in breast cancer cells delays primary tumor growth, suppresses migration in vitro and inhibits the formation of lung metastases in vivo. Conclusively, our findings presented here suggest that the development of therapeutic interventions employing small molecule inhibitors against Activin receptors or neutralizing antibodies targeting Activin A ligand, could serve as alternative approaches against breast tumors overexpressing INHBA and/or IL13Rα2.

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.

Notch signaling inhibits cardiac fibroblast to myofibroblast transformation by antagonizing TGF‐β1/Smad3 signaling

During myocardial infarction (MI), cardiac fibroblasts (CFs) transform into myofibroblast (CMT). This study aimed to investigate the crosstalk of Notch1 and transforming growth factor-β1 (TGF-β1)/Smad3 signaling in the regulation of CMT and myocardial fibrosis. Primary CFs were isolated from young rats and treated with TGF-β1 or adenovirus to overexpress or knockdown Notch1 intracellular domain (N1ICD) or Smad3. TGF-β1 decreased the expression of fibroblast markers but increased the expression of myofibroblast markers in rat CFs. TGF-β1 increased the proliferation, invasion, and adhesion, and the secretion of collagen I of CFs, and these effects were inhibited by N1ICD overexpression. Moreover, endogenous Smad3 phosphorylation in CFs was enhanced by N1ICD knockdown, whereas TGF-β1 induced Smad3 phosphorylation was antagonized by the N1ICD overexpression. Conversely, endogenous N1ICD activation in CFs was antagonized by Smad3, whereas TGF-β1 induced N1ICD inactivation was antagonized by Smad3 knockdown. Coimmunoprecipitation showed that N1ICD interacted with Smad3 and immunostaining revealed the colocalization of N1ICD and Smad3 in the nuclei of CFs. Moreover, we demonstrated the functional antagonism of N1ICD and Smad3 on the phenotypes of CFs. Finally, TGF-β1/Smad3 signaling promoted whereas Notch signaling inhibited myocardial fibrosis in rat MI model. Notch signaling inhibits CMT by antagonizing TGF-β1/Smad3 signaling. Notch signaling activators and TGF-β1/Smad3 signaling inhibitors could be exploited for therapeutic intervention to inhibit myocardial fibrosis after MI.

Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1.

Functional cytokine networks have been poorly characterized in systemic sclerosis (SSc). While interleukin-17A (IL-17A) is increased in SSc skin and other organs, its role is still debated, particularly considering fibrogenesis. We uncover here a dual function of IL-17A in the presence of transforming growth factor-β 1 (TGF-β), the master pro-fibrotic cytokine. In the one hand, we report an unexpected synergic activity resulting in enhanced production of IL-6 by dermal fibroblasts; in the other hand, a substantial inhibition of type I collagen (col-I) production. IL-17A or TGF-β enhanced the production of IL-6 by 8- to 16-folds when compared to control in healthy donors (HD) and SSc cultures. However, the joint presence of IL-17A and TGF-β resulted in robustly exuberant responses with levels of IL-6 up to 100-folds higher than those observed in untreated cells. Inhibition of NFκB signaling pathway preferentially inhibited the production of IL-6 driven by IL-17A in HD fibroblasts, while inhibition of PI3K preferentially inhibited the production of IL-6 driven by TGF-β. Interestingly, when p38 MAPK was inhibited, substantial reduction of IL-6 production was observed for both IL-17A and TGF-β. Consistently with the inhibition experiments, the combined stimulation of fibroblasts by IL-17A and TGF-β resulted in 1.8-fold increase in p38 MAPK phosphorylation (P = 0.025), when compared to levels of phosphorylated p38 MAPK induced by IL-17A alone. Furthermore, the enhanced phosphorylation of p38 MAPK in the joint presence of IL-17A and TGF-β was unique among the signaling molecules we examined. As expected, TGF-β induced SMAD2 phosphorylation and col-I production. However, in fibroblasts cultured in the joint presence of TGF-β and IL-17A, SMAD2 phosphorylation was decreased by 0.6-folds (P = 0.022) when compared to that induced by TGF-β alone. Remarkably, in this condition, the production of col-I and fibronectin was significantly decreased in both HD and SSc. Thus, IL-17A and TGF-β reciprocally influence each other effector functions in fibroblasts. Intracellular molecular switches may favor synergic or antagonistic activities, which are revealed by specific readouts. The implications of these data in the context of SSc are far reaching, particularly in terms of therapeutic approaches since IL-6, IL-17A, and TGF-β are all putative targets of treatment.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-β1 signaling

Transforming growth factor-β (TGF-β) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-β1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-β1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-β1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-β and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-β1-stimulated α-SMA and fibronectin expression, and reversed the TGF-β1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-β1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.The authors identified BMP-2 downregulation in human and experimental fibrotic livers, and found that adenovirus-mediated BMP-2 gene delivery ameliorates liver fibrosis and biliary injury in mice. Mechanistically, BMP-2 suppresses proliferation and migration of hepatic stellate cells by antagonizing TGF-β signaling and epithelial-to-mesenchymal transition. BMP-2 may therefore constitute a therapeutic target for liver fibrosis treatment.

TGF-β induces Smad2 Phosphorylation, ARE Induction, and Trophoblast Differentiation

BackgroundTransforming growth factor beta (TGF-β) signaling has been shown to control a large number of critical cellular actions such as cell death, differentiation, and development and has been implicated as a major regulator of placental function. SM10 cells are a mouse placental progenitor cell line, which has been previously shown to differentiate into nutrient transporting, labyrinthine-like cells upon treatment with TGF-β. However, the signal transduction pathway activated by TGF-β to induce SM10 progenitor differentiation has yet to be fully investigated.Materials and MethodsIn this study the SM10 labyrinthine progenitor cell line was used to investigate TGF-β induced differentiation. Activation of the TGF-β pathway and the ability of TGF-β to induce differentiation were investigated by light microscopy, luciferase assays, and Western blot analysis.Results and ConclusionsIn this report, we show that three isoforms of TGF-β have the ability to terminally differentiate SM10 cells, whereas other predominant members of the TGF-β superfamily, Nodal and Activin A, do not. Additionally, we have determined that TGF-β induced Smad2 phosphorylation can be mediated via the ALK-5 receptor with subsequent transactivation of the Activin response element. Our studies identify an important regulatory signaling pathway in SM10 progenitor cells that is involved in labyrinthine trophoblast differentiation.

FGFR1 is critical for the anti-endothelial mesenchymal transition effect of N-acetyl-seryl-aspartyl-lysyl-proline via induction of the MAP4K4 pathway

Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to organ fibrogenesis, and we have reported that the anti-EndMT effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is associated with restoring expression of diabetes-suppressed fibroblast growth factor receptor (FGFR), the key anti-EndMT molecule. FGFR1 is the key inhibitor of EndMT via the suppression of the transforming growth factor β (TGFβ) signaling pathway, and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) inhibits integrin β1, a key factor in activating TGFβ signaling and EndMT. Here, we showed that the close proximity between AcSDKP and FGFR1 was essential for the suppression of TGFβ/smad signaling and EndMT associated with MAP4K4 phosphorylation (P-MAP4K4) in endothelial cells. In cultured human dermal microvascular endothelial cells (HMVECs), the anti-EndMT and anti-TGFβ/smad effects of AcSDKP were lost following treatment with a neutralizing FGFR1 antibody (N-FGFR1) or transfection of FRS2 siRNA. The physical interaction between FGFR1 and P-MAP4K4 in HMVECs was confirmed by proximity ligation analysis and an immunoprecipitation assay. AcSDKP induced P-MAP4K4 in HMVECs, which was significantly inhibited by treatment with either N-FGFR1 or FRS2 siRNA. Furthermore, MAP4K4 knockdown using specific siRNAs induced smad3 phosphorylation and EndMT in HMVECs, which was not suppressed by AcSDKP. Streptozotocin-induced diabetic CD-1 mice exhibited suppression of both FGFR1 and P-MAP4K4 expression levels associated with the induction of TGFβ/smad3 signaling and EndMT in their hearts and kidneys; those were restored by AcSDKP treatment. These data demonstrate that the AcSDKP–FGFR1–MAP4K4 axis has an important role in combating EndMT-associated fibrotic disorders.

Lipopolysaccharides induce Smad2 phosphorylation through PI3K/Akt and MAPK cascades in HSC-T6 hepatic stellate cells

AimsEndotoxemia and its pro-fibrogenic signaling play a significant role in the development of hepatic fibrosis. This study investigated whether lipopolysaccharide (LPS) directly activate cultured HSC-T6 hepatic stellate cells (HSCs) through triggering Smad-dependent pro-fibrogenic signaling pathway. Main methodsDirect cell counting and assays for cell proliferation and migration were used to measure the effects of LPS on HSC behaviors. Quantitative PCR, Western blot, and gelatin zymography were used to quantify the molecular effects of LPS on expression of HSC activation markers and signaling activity. Key findingsLong-term exposure to LPS exhibited moderately stimulatory effect on HSC cell growth. A wound-healing cell migration assay showed that LPS suppressed HSC-T6 cell migration. qPCR and Western blotting detection indicated that LPS treatment induced upregulation of type I and IV collagens, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-9 (MMP-9). Gelatin zymography confirmed that LPS elevated MMP-9, but not MMP-2 gelatinolytic activity. Moreover, LPS immediately stimulated Akt, EKR1/2, JNK, p38 MAPK, and Smad2 hyperphosphorylation, supporting that LPS directly triggers pro-fibrogenic Smad signaling cascade without TGF-β1 stimulation. Kinase blockade experiments demonstrated the involvement of PI3K/Akt, JNK, p38 MAPK, but not ERK1/2 signaling activation in the LPS-elicited Smad2 phosphorylation as well as the overexpression of type I collagen and α-SMA in HSC-T6 cells. SignificanceThese findings demonstrate that LPS exerts pro-fibrogenic effect through activation and transformation of HSCs. The tissue-remodeling effect of LPS may be attributable to its ability to activate non-canonical Smad pathway through PI3K/Akt and MAPK signaling cascades.

210 Syndecan-4/fgf-2/pkca signalling regulates vascular smooth muscle cell calcification via cross-talk with tgfb

Vascular smooth muscle cells (VSMCs) were induced to mineralise with β-glycerophosphate (β-GP). Controls were cultured without β-GP. FGF-2 mRNA (~40-fold increase, P<0.001) and protein (~2-fold increase, P<0.05) expression are significantly...

Relatively Low Sensitivity of CD109(-) Hematopoietic Stem/Progenitor Cells (HSPCs) to TGF-β: A Possible Mechanism Responsible for the Preferential Commitment of Piga Mutant HSPCs in Immune-Mediated Bone Marrow Failure

[Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ.[Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells.[Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2).[Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. [Display omitted] [Display omitted] DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.