Smad-independent Signaling Pathways Research Articles

Transforming Growth Factor β Signaling Pathway as a Potential Drug Target in Treating Aortic Diseases

Review Transforming Growth Factor β Signaling Pathway as a Potential Drug Target in Treating Aortic Diseases Zijie Liu 1,2, Tianyu Song 3, and Liping Xie 1,2,3, * 1 Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China 2 School of Basic Medical Sciences, Nanjing Medical University, Nanjing 211166, China 3 ‍Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Nanjing Medical University, Nanjing 211166, China * Correspondence: lipingxie@njmu.edu.cn Received: 16 October 2023 Accepted: 20 November 2023 Published: 6 March 2024 Abstract: The transforming growth factor β (TGF-‍β) signaling pathway is crucial for preserving the structural homeostasis of the aorta and promoting aortic development. This pathway encompasses both SMAD-dependent canonical pathway and SMAD-independent non-canonical signaling pathway. Heritable thoracic aortic aneurysms and dissection are highly correlated with genetic alterations in TGF-‍β canonical signaling-related genes. However, depending on the stage of the disease, the TGF-‍β signaling pathway can have either inhibitory or aggravation effects, making its roles in aortic disease complex and occasionally contradictory. This review aims to elucidate the biological mechanisms underlying the TGF-‍β signaling pathway in the most common aortic diseases, namely acute aortic syndromes and aortic aneurysms, and to evaluate the potential clinical application of TGF-β-targeting therapies in aortic diseases.

Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-β) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

Enhanced BMP signaling leads to enlarged nasal cartilage formation in mice

Bone morphogenetic proteins (BMPs) are required for craniofacial bone development. However, it remains elusive how BMP signaling regulates craniofacial cartilage development. To address this question, we utilized a genetic system to enhance BMP signaling via one of BMP type I receptors ALK2 in a chondrocyte-specific manner (hereafter Ca-Alk2:Col2-Cre) in mice. Ca-Alk2:Col2-Cre mice died shortly after birth due to severe craniofacial abnormalities including cleft palate, defective tongue, and shorter mandible formation. Histological analysis revealed that these phenotypes were attributed to the extensive chondrogenesis. Compared with controls, enhanced SOX9 and RUNX2 production were observed in nasal cartilage of Ca-Alk2:Col2-Cre mice. To reveal the mechanisms responsible for enlarged nasal cartilage, we examined Smad-dependent and Smad-independent BMP signaling pathways. While the Smad-independent BMP signaling pathway including p38, ERK, and JNK remained silent, the Smad1/5/9 was highly phosphorylated in Ca-Alk2:Col2-Cre mice. Interestingly, Ca-Alk2:Col2-Cre mice showed enhanced S6 kinase phosphorylation, a readout of mammalian target of rapamycin complex 1 (mTORC1). These findings may suggest that enhanced Smad-dependent BMP signaling positively regulates the mTOR pathway and stimulates chondrocytes toward hypertrophic differentiation, thereby leading to enlarged nasal cartilage formation in mice.

Discovery of a chalcone derivative as an anti-fibrotic agent targeting transforming growth factor-β1 signaling: Potential therapy of renal fibrosis

As a final common pathway of renal injuries, renal fibrosis leads to chronic kidney disease (CKD). Currently, there is no safe and effective therapy to prevent the progression of renal fibrosis to CKD. Inhibition of transforming growth factor-β1 (TGF-β1) pathway is proposed as one of the most promising approaches for anti-renal fibrosis therapies. This study aimed to identify novel anti-fibrotic agents using the TGF-β1-induced fibrosis in renal proximal tubule epithelial cells (RPTEC) and characterize their mechanism of action as well as in vivo efficacy. By screening 362 natural product-based compounds for their ability to reduce collagen accumulation assessed by picro-sirius red (PSR) staining in RPTEC cells, a chalcone derivative AD-021 was identified as an anti-fibrotic agent with IC50 of 14.93 μM. AD-021 suppressed TGF-β1-induced collagen production, expression of pro-fibrotic proteins (fibronectin and α-smooth muscle actin (αSMA)), and Smad-dependent and Smad-independent signaling pathways via suppression of TGF-β receptor II (TGFβRII) phosphorylation in RPTEC cells. Furthermore, TGF-β1-induced mitochondrial fission in RPTEC cells was ameliorated by AD-021 via mechanisms involving inhibition of Drp1 phosphorylation. In a mouse model of unilateral ureteral obstruction (UUO)-induced renal fibrosis, AD-021 reduced plasma TGF-β1, ameliorated renal fibrosis and improved renal function. Collectively, AD-021 represents a novel class of natural product-based anti-fibrotic agent that has therapeutic potential in the prevention of fibrosis-associated renal disorders including CKD.

Bone morphogenetic protein 4 inhibits rat stem/progenitor Leydig cell development and regeneration via SMAD-dependent and SMAD-independent signaling

Bone morphogenetic protein 4 (BMP4) is an important member of the transforming growth factor-β superfamily. BMP4 is expressed in the Leydig cell lineage. We hypothesized that BMP4 might regulate the development of stem/progenitor Leydig cells. The BMP4 receptors, BMPR1A, BMPR1B, and BMPR2 were found to be expressed in progenitor Leydig cells of prepubertal testis and isolated cells. BMP4 at 1 and 10 ng/mL significantly reduced androgen production and down-regulated steroidogenesis-related gene and protein expression possibly by activating the SMAD signaling pathway (increasing SMAD1/5 phosphorylation and SMAD4) at 24 h treatment. BMP4 at 0.1 ng/mL and higher concentrations markedly reduced the EdU labeling index of CD90+ stem Leydig cells after 24 h treatment and significantly reduced the number of EdU+ stem Leydig cells on the surface of seminiferous tubules after 7 days of culture. BMP4 at 0.01 ng/mL and higher concentrations significantly blocked the differentiation of stem Leydig cells into adult cells, as shown by the reduction of testosterone secretion and the downregulation of Lhcgr, Scarb1, Cyp11a1, Hsd11b1, and Insl3 and their function after 3D seminiferous tubule culture for 3 weeks, and this effect was reversed by co-treatment with the BMP4 antagonists noggin and doxomorphine. In addition, BMP4 also blocked stem Leydig cell differentiation through SMAD-independent signaling pathways (ERK1/2 and AMPK). Ethanedimethane sulfonate (EDS) single injection can result in reduction of testosterone, restoration can happen post treatment. In an in vivo model of Leydig cell regeneration following EDS treatment, intratesticular injection of BMP4 from day 14 to day 28 post-elimination significantly reduced serum testosterone levels and down-regulated the expression of Scarb1, Star, Hsd11b1, and Insl3 and its proteins, possibly through SMAD-dependent and SMAD-independent (ERK1/2 and AMPK) signaling pathways. In conclusion, BMP4 is expressed in cells of the Leydig cell lineage and blocks entry of stem/progenitor Leydig cells into adult Leydig cells through SMAD-dependent and SMAD-independent signaling pathways.

The type II TGF-β receptor phosphorylates Tyr182 in the type I receptor to activate downstream Src signaling.

Transforming growth factor-β (TGF-β) signaling has important roles during embryonic development and in tissue homeostasis. TGF-β ligands exert cellular effects by binding to type I (TβRI) and type II (TβRII) receptors and inducing both SMAD-dependent and SMAD-independent intracellular signaling pathways, the latter of which includes the activation of the tyrosine kinase Src. We investigated the mechanism by which TGF-β stimulation activates Src in human and mouse cells. Before TGF-β stimulation, inactive Src was complexed with TβRII. Upon TGF-β1 stimulation, TβRII associated with and phosphorylated TβRI at Tyr182. Binding of Src to TβRI involved the interaction of the Src SH2 domain with phosphorylated Tyr182 and the interaction of the Src SH3 domain with a proline-rich region in TβRI and led to the activation of Src kinase activity and Src autophosphorylation. TGF-β1-induced Src activation required the kinase activities of TβRII and Src but not that of TβRI. Activated Src also phosphorylated TβRI on several tyrosine residues, which may stabilize the binding of Src to the receptor. Src activation was required for the ability of TGF-β to induce fibronectin production and migration in human breast carcinoma cells and to induce α-smooth muscle actin and actin reorganization in mouse fibroblasts. Thus, TGF-β induces Src activation by stimulating a direct interaction with TβRI that depends on tyrosine phosphorylation of TβRI by TβRII.

Cinnamomum bejolghota Extract Inhibits Colorectal Cancer Cell Metastasis and TGF-β1-Induced Epithelial-Mesenchymal Transition via Smad and Non-Smad Signaling Pathway

Cinnamomum bejolghota, used in Thai traditional medicine remedies, has several biological activities including antimicrobial, antifungal, and anticancer. In colorectal cancer, epithelial-mesenchymal transition (EMT) is an initial step of cancer metastasis. Thus, this study investigated the effects of C. bejolghota bark extract (CBE) on colorectal cancer cell metastasis and transforming growth factor-β1 (TGF-β1) induced EMT in LoVo cells. The results showed that CBE could reduce cell migration, invasion, and adhesion of LoVo cells in a dose-dependent manner. In addition, our studies also showed that CBE could reverse TGF-β1-induced morphological changes as well as increase an epithelial marker, E-cadherin, while the expression of the mesenchymal marker, N-cadherin, was decreased in TGF-β1-treated LoVo cells. MMP-2 expression was effectively decreased but TIMP-1 and TIMP-2 expression was increased by the CBE treatment in LoVo cells. CBE also inhibited Smad2/3 phosphorylation and nuclear translocation as well as decreased the expression of Snail, Slug, and TCF8/ZEB1 transcription factors in LoVo cells. Moreover, CBE could inhibit TGF-β1-induced Smad-independent signaling pathway by decreased phosphorylation of ERK1/2, p38, and Akt. These findings suggest that CBE inhibited TGF-β1-induced EMT in LoVo cells via both Smad-dependent and Smad-independent pathways. Therefore, CBE may function as an alternative therapeutic treatment for colorectal cancer metastasis.

Enhanced Intestinal TGF-β/SMAD-Dependent Signaling in Simian Immunodeficiency Virus Infected Rhesus Macaques.

Transforming growth factor-β signaling (TGF-β) maintains a balanced physiological function including cell growth, differentiation, and proliferation and regulation of immune system by modulating either SMAD2/3 and SMAD7 (SMAD-dependent) or SMAD-independent signaling pathways under normal conditions. Increased production of TGF-β promotes immunosuppression in Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection. However, the cellular source and downstream events of increased TGF-β production that attributes to its pathological manifestations remain unknown. Here, we have shown increased production of TGF-β in a majority of intestinal CD3−CD20−CD68+ cells from acute and chronically SIV infected rhesus macaques, which negatively correlated with the frequency of jejunum CD4+ T cells. No significant changes in intestinal TGF-β receptor II expression were observed but increased production of the pSMAD2/3 protein and SMAD3 gene expression in jejunum tissues that were accompanied by a downregulation of SMAD7 protein and gene expression. Enhanced TGF-β production by intestinal CD3−CD20−CD68+ cells and increased TGF-β/SMAD-dependent signaling might be due to a disruption of a negative feedback loop mediated by SMAD7. This suggests that SIV infection impacts the SMAD-dependent signaling pathway of TGF-β and provides a potential framework for further study to understand the role of viral factor(s) in modulating TGF-β production and downregulating SMAD7 expression in SIV. Regulation of mucosal TGF-β expression by therapeutic TGF-β blockers may help to create effective antiviral mucosal immune responses.

Anti-fibrotic effects of branched-chain amino acids on hepatic stellate cells.

Background/AimsPatients with liver cirrhosis (LC) have low levels of branched-chain amino acids (BCAAs). There is accumulating evidence that BCAAs have anti-fibrotic effects in cirrhosis. This study is aimed to evaluate the effect of BCAAs on the function and phenotype of activated hepatic stellate cells (HSCs).MethodsLX-2, an immortalized human stellate cell line, was used in in vitro experiments. LX-2 cells were exposed to transforming growth factor β1 (TGF-β1) and BCAAs or to valine, leucine, and isoleucine, which are components of BCAAs. Activation of the TGF-β signaling pathway in LX-2 cells was observed using real-time quantitative polymerase chain reaction and Western blotting.ResultsThe increased expression of snail family transcriptional repressor 1 (SNAI1) was observed in LX-2 cells activated by TGF-β1. After BCAA treatment, its expression was significantly decreased at the mRNA level. The increased expression of Col1α1 and TIMP2 at the mRNA level and alpha smooth muscle actin at the protein level in activated LX-2 cells decreased after BCAA treatment. Among the BCAA components, leucine and valine significantly abrogated TGF-β-induced activation of LX-2 cells. BCAA treatment led to the decreased phosphorylation of Smad2 and p38 proteins, which are markers for Smad and Smad-independent p38 mitogen-activated protein kinase signaling pathways, respectively.ConclusionsBCAA treatment can improve hepatic fibrosis by directly affecting the activated state of hepatic stellate cells through inhibition of the TGF-β signaling pathway. Among BCAA components, leucine and valine mainly abrogated TGF-β-induced activation of HSCs. Our results suggest that BCAA may be used to attenuate the progression of liver fibrosis.

Trophoblast cell influence on peripheral blood natural killer cell proliferation and phenotype in non-pregnant women and women in early pregnancy.

Natural killer (NK) cells are the main population of leukocytes in decidua during the first trimester of pregnancy. NK cells can have contact with trophoblast cells during pregnancy, which raises the possibility of mutual influence. This research aimed to evaluate the proliferation and phenotype of peripheral blood NK cells in the presence of trophoblast cells of the JEG-3 cell line. We showed that trophoblast cells of the JEG-3 cell line (American Type Culture Collection (ATCC), USA) produced TGFβ. However, co-culturing of NK and trophoblast cells did not change the SMAD2/3 to pSMAD2/3 ratio within NK cells. These data indicate that the canonical signaling pathway from TGFβ is not activated, but do not preclude activation of SMAD-independent signaling pathways through the effect of TGFβ and/or other cytokines. We established that trophoblast cells inhibited both constitutive and IL-2-induced expression of Ki-67 proliferation marker by NK cells in vitro in both pregnant and non-pregnant women. Constitutive and induced Ki-67 expression by peripheral blood NK cells was increased in pregnant women compared with non-pregnant women. The influence of trophoblast cells on Ki-67 expression by NK cells was more pronounced in the presence of other mononuclear cells than in their absence. In the presence of trophoblast cells and IL-2, the number of NK cells with the CD16+CD57- phenotype in peripheral blood mononuclear cells (PBMCs) was increased in pregnant and non-pregnant women, compared with culturing with IL-2 only. This might reflect a decrease in the number of NK cells at the terminal stage of differentiation. We also revealed the increased content of NK cells with the CD16-CD56bright phenotype in PBMCs of pregnant women when incubated with trophoblast cells and IL-2, compared with culturing with trophoblast cells only. Our results suggest that NK cells need contact interactions with trophoblast cells and additional cytokine stimulation (IL-2, cytokines of other mononuclear cells) to acquire the CD56bright phenotype.

Role of NADPH oxidase and its therapeutic intervention in TGF-\u03b2-mediated EMT progression: an in vitro analysis on HeLa cervical cancer cells

Epithelial to mesenchymal transition (EMT) is a complex biological event, wherein polarized epithelial cells lose their integrity resulting in a mesenchymal phenotype with enhanced motility, a phenomenon known as metastasis. However, the underlying mechanisms of EMT are still poorly understood in cervical carcinomas. In this study, we investigated the molecular signalling events responsible for the effect of TGF-β, a potent inducer of EMT, on HeLa cervical cancer cells. We observed that TGF-β treatment (5 ng/mL) upregulates the expression of EMT-associated transcription factors such as Snail and Slug and downregulates the expression of epithelial markers such as ZO-1 and E-cadherin. Furthermore, treatment with TGF-β activates both Smad-dependent and Smad-independent signaling pathways, which subsides upon addition of Diphenyleneiodonium (DPI), a potent ROS inhibitor that inhibits NADPHoxidase (NOX). TGF-β treatment enhanced cellular migration and invasion ability was diminished in the presence of ROS inhibitors. In addition, we also observed that ROS-mediated, TGF-β-induced EMT progression was inhibited using therapeutic candidates that target the key signal transduction mediators, including PI3K/AKT, ERK, and P38/MAPK. Accordingly, we demonstrated the involvement of redox biology (NOX2 and NOX4 mediate migration and invasion) in TGF-β-mediated EMT advancement and explored suitable therapeutic interventions.

Role of hypoxia in skeletal muscle fibrosis: Synergism between hypoxia and TGF-β signaling upregulates CCN2/CTGF expression specifically in muscle fibers

Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-β (TGF-β) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle.We evaluated the role of hypoxia and TGF-β on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-β1 under hypoxic conditions. Hypoxia and TGF-β1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-β signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-β1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-β signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.

Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways.

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

Combination of TWEAK and TGF-β1 induces the production of TSLP, RANTES, and TARC in BEAS-2B human bronchial epithelial cells during epithelial-mesenchymal transition

Aim of the study: In patients with asthma, chronic inflammatory processes and the subsequent remodeling of the airways contribute to the symptoms and the pathophysiological changes. Epithelial-mesenchymal transition (EMT) is thought to play an important role in tissue remodeling. Previous reports show that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF superfamily, exerts pro-inflammatory effects, and enhances transforming growth factor (TGF)-β-induced EMT in bronchial epithelial cells. In this study, we investigated the TWEAK-induced cytokine and chemokine production in the human bronchial epithelial cell line BEAS-2B during EMT. Materials and Methods: Quantitative real-time RT-PCR, enzyme-linked immunosorbent assays, western blotting, and immunohistochemistry were used to define the production of cytokines and chemokines. Results: We found that TWEAK increases mRNA and protein levels of thymic stromal lymphopoietin (TSLP), monocyte chemoattractant protein -1 (MCP-1), regulated upon activation normal T cell express sequence (RANTES), and IL-8 in BEAS-2B bronchial epithelial cells. Moreover, co-treatment with TWEAK and TGF-β1 induces not only features of EMT but also enhances the production of TSLP and RANTES. Thymus- and activation-regulated chemokines (TARC) production is induced by the co-treatment of TWEAK and TGF-β1 but not by TWEAK or TGF-β1 stimulation alone. Furthermore, the increased mRNA expression of TSLP and RANTES after co-treatment with TWEAK and TGF-β1 is prevented by inhibitors of Smad-independent signaling pathways. Conclusions: In the present study, we have revealed a novel mechanism for the production of asthma-related cytokines and chemokines in EMT driven by the co-stimulation with TWEAK and TGF-β1. We conclude that cellular EMT processes caused by TWEAK and TGF-β1 may contribute to chronic airway inflammation and remodeling.

Abstract 2685: Targeting TGFβ signaling pathways in bone metastatic breast cancer cells to limit metastatic progression using curcuminoids, a turmeric-derived natural product

Abstract Tumor-cell TGFβ signaling has been demonstrated to drive osteolytic breast cancer bone metastases (BrCa BMET) in mouse models. This established an important link between the microenvironment and tumor cells in bone, wherein osteoclast-mediated bone resorption stimulates the release of TGFβ from bone matrix, which induces tumoral secretion of osteolytic factors, such as parathyroid-related protein (PTHrP) that drive further osteoclast-mediated bone destruction in a positive feedback loop. These findings have prompted interest in the use of pharmacologic TGFβ inhibitors, currently under development, for the treatment of BrCa BMET. Interestingly, studies have demonstrated antagonistic effects of a natural product, turmeric-derived polyphenols called curcuminoids (CURC), on BrCa BMET progression and TGFβ-inducible, Smad-dependent signaling in a TGFβ-dependent human xenograft model (human MDA-MB-231 [MDA-SA]).The studies described here were undertaken to determine whether this inhibitory effect is generalizable to other BrCa cells that form TGFβ-dependent BMETs (human MDA-1833 and MDA-2287; murine 4T1) and to begin to elucidate the mechanism by which CURC block BrCa cell TGFβ signaling. Analogous to prior findings using MDA-SA cells forming BMET that are dependent on TGFβ-inducible tumoral secretion of PTHrP via Smad and non-Smad (specifically, p38) dependent pathways, CURC inhibited TGFβ inducible activation (phosphorylation) of receptor Smad2 (Western) in all TGFβ-dependent BrCa cell lines tested.In MDA-SA cells, CURC inhibition of TGFβ-stimulated Smad2 phosphorylation was time- and dose-dependent (Western), and associated with decreased expression of a Smad-dependent luciferase reporter gene in transiently transfected cells. In both MDA-SA and 4T1 cells, these changes were accompanied by decreased TGFβ-stimulated PTHrP secretion (RIA). These effects in MDA-SA cells were not associated with changes in the expression of canonical TGFβ signaling proteins, such as Smad anchor for receptor activation (SARA), which is involved in Smad phosphorylation, or Smad4, a required cofactor for Smad-mediated gene transcription. Also unchanged were noncanonical TGFβ-stimulated, Smad-independent signaling pathways (e.g., p38, ERK1/2 or JNK). Of note, however, in all cell lines tested, CURC inhibition of Smad2 phosphorylation was accompanied by a decrease in constitutive Smad2 levels, with variable decreases in the ratio of phosphorylated vs. total Smad levels. These results suggest that CURC can specifically and uniformly inhibit Smad-regulated TGFβ signaling in bone metastatic BCa cells forming TGFβ-dependent osteolytic lesions, an effect that may be mediated, at least in part, by changes in constitutive levels of receptor-regulated Smad expression. Citation Format: Andrew Kunihiro, Julia A. Brickey, Jen B. Frye, Janet L. Funk. Targeting TGFβ signaling pathways in bone metastatic breast cancer cells to limit metastatic progression using curcuminoids, a turmeric-derived natural product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2685.

The potential role of aquaporin 1 on aristolochic acid I induced epithelial mesenchymal transition on HK-2 cells.

Aristolochic acid I (AA-I), one of the main active components in Aristolochaia herbs, may induce aristolochic acid nephropathy (AAN). Renal interstitial fibrosis is one of the most typical features of AAN. To investigate the mechanism of Aristolochic acid I (AA-I) -induced renal epithelial-mesenchymal transition (EMT) and determine the role of aquaporin-1 (AQP1) in this process, we established an AA-I-induced EMT model in human proximal tubular epithelial cells (HK-2 cells). Morphological examination, MTT assay, and Western blot analysis were performed. Aquaporin 1 (AQP1) and several EMT-related proteins were detected, thereby suggesting the occurrence of AA-I-induced EMT. Two main pathways of transforming growth factor-β (TGF-β) signaling, namely, Smad-dependent and Smad-independent signaling pathways, were also detected. The results showed that the TGF-β / Smad-independent signaling pathways (β-catenin, Ras-Raf-Erk1/2 signaling pathways) were activated, and AQP1 expression was decreased during the AA-I induced EMT on HK-2 cells. With the presence of TGF-β1 receptor inhibitor (LY364947) and Erk1/2 inhibitor (PD98059), AQP1 expression was altered by PD98059, suggested that AQP1 could be adjusted by Erk1/2 signaling. Moreover, the inhibitory effect of AA-I on AQP1 was stronger than that of TGF-β1, suggested that AQP1 may be an important target on AAN clinical therapy.

Transforming growth factor β: A potential biomarker and therapeutic target of ventricular remodeling

Transforming growth factor β (TGF-β) is a multifunctional cytokine that is synthesized by many types of cells and regulates the cell cycle. Increasing evidence has led to TGF-β receiving increased and deserved attention in recent years because it may play a potentially novel and critical role in the development and progression of myocardial fibrosis and the subsequent progress of ventricular remodeling (VR). Numerous studies have highlighted a crucial role of TGF-β in VR and suggest potential therapeutic targets of the TGF-β signaling pathways for VR. Changes in TGF-β activity may elicit anti-VR activity and may serve as a novel therapeutic target for VR therapy. This review we discusses the smad-dependent signaling pathway, such as TGF-β/Smads, TGF-β/Sirtuins, TGF-β/BMP, TGF-β/miRNAs, TGF-β/MAPK, and Smad-independent signaling pathway of TGF-β, such as TGF-β/PI3K/Akt, TGF-β/Rho/ROCK,TGF-β/Wnt/β-catenin in the cardiac fibrosis and subsequent progression of VR. Furthermore, agonists and antagonists of TGF-β as potential therapeutic targets in VR are also described.

Characterization of TGF-β expression and signaling profile in the adipose tissue of rats fed with high-fat and energy-restricted diets

Transforming growth factor beta (TGF-β) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-β and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-β, TGF-β receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-β and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-β and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-β mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-β receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-β expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

Response of Hepatic Stellate Cells to TGFB1 Differs from the Response of Myofibroblasts. Decorin Protects against the Action of Growth Factor.

Regardless to the exact nature of damage, hepatic stellate cells (HSCs) and other non-parenchymal liver cells transform to activated myofibroblasts, synthesizing the accumulating extracellular matrix (ECM) proteins, and transforming growth factor-β1 (TGF-β1) plays a crucial role in this process. Later it was discovered that decorin, member of the small leucin rich proteoglycan family is able to inhibit this action of TGF-β1. The aim of our present study was to clarify whether HSCs and activated myofibroblasts of portal region exert identical or different response to TGF-β1 exposure, and the inhibitory action of decorin against the growth factor is a generalized phenomenon on myofibroblast of different origin? To this end we measured mRNA expression and production of major collagen components (collagen type I, III and IV) of the liver after stimulation and co-stimulation with TGF-β1 and decorin in primary cell cultures of HSCs and myofibroblasts (MFs). Production of matrix proteins, decorin and members of the TGF-β1 signaling pathways were assessed on Western blots. Messenger RNA expression of collagens and TIEG was quantified by real-time RT-PCR. HSCs and MFs responded differently to TGF-β1 exposure. In contrast to HSCs in which TGF-β1 stimulated the synthesis of collagen type I, type III, and type IV, only the increase of collagen type IV was detected in portal MFs. However, in a combined treatment, decorin seemed to interfere with TGF-β1 and its stimulatory effect was abolished. The different mode of TGF-β1 action is mirrored by the different activation of signaling pathways in activated HSCs and portal fibroblasts. In HSCs the activation of pSMAD2 whereas in myofibroblasts the activation of MAPK pathway was detected. The inhibitory effect of decorin was neither related to the Smad-dependent nor to the Smad-independent signaling pathways.

Molecular mechanisms of suberoylanilide hydroxamic acid in the inhibition of TGF-β1-mediated canine corneal fibrosis.

To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase (MMP) activity. Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-β1 (5 ng/mL) and SAHA (2.5 μm) for 24 h. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2, and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8, and MMP9 mRNA expressions and MMP2 and MMP9 protein activities, respectively. TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho-JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expressions but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression, but SAHA treatment +/-TGF-β1 significantly increased MMP2 and MMP9 protein expressions. The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity.