Divergent effects of canonical and non‐canonical TGF‐β signalling on mixed contractile‐synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.     

Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.     

microRNA let‐7g suppresses PDGF‐induced conversion of vascular smooth muscle cell into the synthetic phenotype

Platelet‐derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let‐7g in phenotypic switching. Bioinformatics prediction was used to find let‐7g target genes in the PDGF/mitogen‐activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal‐regulated kinase (ERK)/Krüppel‐like factor‐4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let‐7g transfection were used to confirm let‐7g target genes. Two contractile proteins alpha‐smooth muscle actin (α‐SMA) and calponin were VSMC‐specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let‐7g gene was injected to apolipoprotein E knockout (apoE^?/?) mice to confirm let‐7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF‐BB can inhibit α‐SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let‐7g binding sites, which were confirmed by our reporter assays. Transfection of let‐7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let‐7g decreased phosphorylated‐ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC‐specific gene expression by preventing myocardin–serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let‐7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let‐7g can increase α‐SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let‐7g gene increased let‐7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE^?/? mice. We demonstrated that let‐7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.     

Rotavirus‐induced miR‐142‐5p elicits proviral milieu by targeting non‐canonical transforming growth factor beta signalling and apoptosis in cells

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa‐miR‐142‐5p was up‐regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR‐142‐5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR‐142‐5p. Functional studies of hsa‐miR‐142‐5p identified its role in transforming growth factor beta (TGFβ) signalling as TGFβ receptor 2 and SMAD3 were degraded during both hsa‐miR‐142‐5p overexpression and rotavirus infection. TGFβ is induced during rotavirus infection, which may promote apoptosis by activation of non‐canonical pathways in HT29 cells. However, up‐regulated miR‐142‐5p resulted in the inhibition of TGFβ‐induced apoptosis suggesting its anti‐apoptotic function. Rotavirus NSP5 was identified as a regulator of miR‐142‐5p expression. Concurrently, NSP5‐HT29 cells showed inhibition of TGFβ‐induced apoptosis and epithelial to mesenchymal transition by blocking non‐canonical pathways. Overall, the study identified proviral function of hsa‐miR‐142‐5p during rotavirus infection. In addition, modulation of TGFβ‐induced non‐canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.     

The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways

The members of the transforming growth factor beta (TGFβ) family of cytokines, including bone morphogenetic proteins (BMP), play fundamental roles in development and tissue homeostasis. Hence, aberrant TGFβ/BMP signalling is associated with several human diseases such as fibrosis, bone and immune disorders, cancer progression and metastasis. Consequently, targeting TGFβ signalling for intervention potentially offers therapeutic opportunities against these diseases. Many investigations have focussed on understanding the molecular mechanisms underpinning the regulation of TGFβ signalling. One of the key areas has been to investigate the regulation of the protein components of the TGFβ/BMP signal transduction pathways by ubiquitylation and deubiquitylation. In the last 15 years, extensive research has led to the discovery and characterisation of several E3 ubiquitin ligases that influence the TGFβ pathway. However, the research on DUBs regulating the TGFβ pathway has received prominence only recently and is still an emerging field. This review will provide a concise summary of our current understanding of how DUBs regulate TGFβ signalling.     

Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF‐Rβ

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet‐derived growth factor (PDGF)‐BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF‐Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF‐BB. CSMC were enzymatically isolated from Sprague–Dawley rats, and the effect of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, transforming growth factor (TGF), IL‐17A and IL‐2 on CSMC growth and responsiveness to PDGF‐BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid‐induced colitis. Neither CM alone nor cytokines caused proliferation of early‐passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF‐BB. IL‐1β, TNF‐α and IL‐17A, but not other cytokines, increased the effect of PDGF‐BB because of up‐regulation of mRNA and protein for PDGF‐Rβ without change in receptor phosphorylation. PDGF‐BB was identified in adult rat serum (RS) and RS‐induced CSMC proliferation was inhibited by imatinib, suggesting that blood‐derived PDGF‐BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL‐1β and TNF‐α induced the early expression of PDGF‐Rβ, while imatinib blocked subsequent RS‐induced cell proliferation. Thus, pro‐inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF‐Rβ and serum‐derived PDGF‐BB, and control of PDGF‐Rβ expression may be beneficial in chronic intestinal inflammation.     

Tissue transglutaminase promotes PDGF/PDGFR‐mediated signaling and responses in vascular smooth muscle cells

Although the pivotal role of platelet derived growth factor (PDGF)‐mediated signaling in vascular diseases was demonstrated, the pathophysiological mechanisms driving its over‐activation remain incompletely understood. Tissue transglutaminase (tTG) is a multifunctional protein expressed in the vasculature, including smooth muscle cells (SMCs), and implicated in several vascular pathologies. The goal of this study is to define the regulation of PDGF‐BB/PDGFRβ‐induced signaling pathways and cell responses by tTG in vascular SMCs. We find that in human aortic SMCs, shRNA‐mediated depletion and over‐expression of tTG reveals its ability to down‐regulate PDGFRβ levels and induce receptor clustering. In these cells, tTG specifically amplifies the activation of PDGFRβ and its multiple downstream signaling targets in response to PDGF‐BB. Furthermore, tTG promotes dedifferentiation and increases survival, proliferation, and migration of human aortic SMCs mediated by this growth factor. Finally, PDGF‐BB stimulates tTG expression in human aortic SMCs in culture and in the blood vessels in response to injury. Together, our results show that tTG in vascular SMCs acts as a principal enhancer within the PDGF‐BB/PDGFRβ signaling axis involved in phenotypic modulation of these cells, thereby suggesting a novel role for this protein in the progression of vascular diseases. J. Cell. Physiol. 227: 2089–2096, 2012. © 2011 Wiley Periodicals, Inc.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.     

PDGF-induced proliferation in human arterial and venous smooth muscle cells: molecular basis for differential effects of PDGF isoforms

Platelet‐derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum‐starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF‐AA, venous SMCs showed no proliferative response to PDGF‐AA, but instead demonstrated a significantly greater proliferative response to PDGF‐BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5‐fold higher (P < 0.05) density of PDGF receptor‐α (PDGF‐Rα) and a 6.6‐fold lower (P < 0.05) density of PDGF‐Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF‐AA in arterial SMCs was a marked PDGF‐Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c‐Jun NH2‐terminal kinase (JNK), and a significant reduction in expression of the cell‐cycle inhibitor p27^kip1. This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF‐Rα was detected after venous SMC exposure to PDGF‐AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF‐BB stimulation of venous SMC resulted in PDGF‐Rβ activation as well as transactivation of epidermal growth factor receptor (EGF‐R); transactivation of EGF‐R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins. J. Cell. Biochem. 112: 289–298, 2011. © 2010 Wiley‐Liss, Inc.     

CTRP3/cartducin is induced by transforming growth factor‐β1 and promotes vascular smooth muscle cell proliferation

CTRP3 (C1q and tumour necrosis factor‐related protein 3)/cartducin, a novel serum protein, is a member of the CTRP superfamily. Although the CTRP3/cartducin gene is markedly up‐regulated in rat carotid arteries after balloon injury, little is known about its biological roles in arterial remodelling and neointima formation in injured blood vessels. We have investigated the mechanisms underlying CTRP3/cartducin up‐regulation and the in vitro effects of CTRP3/cartducin on vascular smooth muscle cells. CTRP3/cartducin expression in cultured p53LMAC01 vascular smooth muscle cells was induced by TGF‐β1 (transforming growth factor‐β1), but not by bFGF (basic fibroblast growth factor) or PDGF‐BB (platelet‐derived growth factor‐BB). Exogenous CTRP3/cartducin promoted the proliferation of p53LMAC01 cells in a dose‐dependent manner via ERK1/2 (extracellular signal‐regulated kinase 1/2)‐ and MAPK (p38 mitogen‐activated protein kinase)‐signalling pathways. In contrast, CTRP3/cartducin exhibited no effect on the migration of p53LMAC01 cells. Taken together, the results of the present study demonstrate a novel biological role of CTRP3/cartducin in promoting vascular smooth muscle cell proliferation in blood vessel walls after injury.     

Interaction of TGFβ and BMP signaling pathways during chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.     

Interferon‐γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)‐1 and transforming growth factor (TGF) β2

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1, and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF, and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. 228: 1774–1783, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.