Down-regulation of Id-1 expression is associated with TGFβ1-induced growth arrest in prostate epithelial cells

Transforming growth factor β1 (TGFβ1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGFβ1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGFβ1 was able to suppress the expression of Id-1, a helix–loop–helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGFβ1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGFβ1 signaling pathway. The fact that up-regulation of p21^WAF1, one of the downstream effectors of Id-1, was observed after exposure to TGFβ1 further indicates the involvement of Id-1 in the TGFβ1-induced growth arrest in HPr-1 cells. However, increased expression of p27^KIP1 was also observed in the TGFβ1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGFβ1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGFβ1 may be one of the upstream regulators of Id-1.     

Ectopic Expression of eIF-4E in Human Colon Cancer Cells Promotes the Stimulation of Adhesion Molecules by Transforming Growth Factorβ

Transforming growth factor β1 (TGFβ) inhibits cellular proliferation, promotes differentiation, and stimulates the expression and secretion of the extracellular matrix adhesion molecules fibronectin and laminin and the colon-associated intercellular adhesion molecule carcinoembryonic antigen. This is collectively called the TGFβ-mediated adhesion response and occurs in the human colon cancer cell line Moser while the cell line KM12SM is relatively unresponsive to TGFβ. We have previously shown that TGFβ rapidly stimulates protein kinase C (PKC) phosphotransferase activity in the Moser cells and that the induction of the adhesion response (but not antiproliferation) by TGFβ is dependent on PKC. Because resistance to growth factors may be due to translational suppression and the translation initiation factor eIF-4E may alleviate translational suppression, we determined the effect of eIF-4E expression on the responses of Moser and KM12SM cells to TGFβ. Ectopic expression of eIF-4E in the TGFβ-responsive Moser cells enhanced the activation of PKC by TGFβ and the induction of the adhesion response, especially the secretion of adhesion molecules, but not the antiproliferative response. Ectopic expression of eIF-4E in the TGFβ-resistant KM12SM cells increased TGFβ stimulation of PKC and the TGFβ-mediated adhesion response (but not antiproliferation). The secretion of adhesion molecules was significantly increased by TGFβ. These results showed in these cells that eIF-4E promotes TGFβ-regulated adhesion but not antiproliferation in a PKC-dependent manner.     

Increase in AMPK brought about by cocoa is renoprotective in experimental diabetes mellitus by reducing NOX4/TGFβ-1 signaling

The aims of the present study were to investigate, in diabetes mellitus (DM), the mechanism of NOX4 up-regulation, its link with 5′ adenosine monophosphate-activated protein kinase (AMPK) inactivation and transforming growth factor (TGF) ß-1 signaling in determining the accumulation of kidney extracellular matrix (ECM), and the possible action of cocoa enriched with polyphenols (CH) in these events. After 16 weeks of DM, spontaneously hypertensive rats showed increased kidney TGFβ-1 levels and expression of phosphorylated smad2, collagen IV and fibronectin in parallel with elevated NOX4 expression and reduced phosphorylated AMPK. CH treatment in diabetic rats prevented all of these abnormalities. In immortalized human mesangial cells exposed to high glucose (HG), or TGFβ-1, CH, nicotinamide adenine dinucleotide phosphate blocker, or silencing NOX4 ameliorated enhanced phosphorylated smad2 and collagen IV. Reduction in phosphorylated AMPK induced by HG or TGFβ-1 was ameliorated by CH or activation of AMPK, which reduced phosphorylation of smad2 and collagen IV via reduction in NOX4 expression. The effects of CH were abolished by AMPK blockade. These results suggest that inactivation in AMPK leads to NOX4 up-regulation, activation of TGFβ-1 signaling and increased ECM accumulation. Additionally, increased TGF-ß1 per se leads to the amplification of ECM production by reducing AMPK and promoting the activation of NOX4. It is suggested that the activation of AMPK by CH followed by reduction in NOX4/TGFβ-1 signaling may have a therapeutic potential in diabetic nephropathy.     

MicroRNA-24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

Cyclic mechanical stress (CMS) leads to alterations of cellular functions in the trabecular meshwork (TM), including the up-regulation of transforming growth factor beta 1 (TGFβ1), that can potentially contribute to the pathogenesis of glaucoma. Although microRNAs (miRNAs) are known to play important roles in many biological functions, little is known about their potential involvement in the cellular responses elicited by mechanical stress. Here we analyzed changes in miRNA expression induced by CMS, and examined the possible role of miR-24 in the response of human TM cells to CMS. CMS induced the expression of miR-24 that led to the down regulation of the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1. FURIN was confirmed as a novel target of miR-24 by 3' UTR luciferase assay and western blot. Overexpression of miR-24 resulted in a significant decrease in activated TGFβ1. This effect was mimicked by down regulation of FURIN by siRNA. Conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. Furthermore, the increase in active TGFβ1 induced by CMS in HTM cells was prevented by miR-24. Altogether, our results suggest that miRNAs might contribute to the regulation of responses to CMS in TM cells. Specifically, miR-24 might play an important role in modulating the induction of TGFβ1 mediated by CMS through direct targeting of FURIN.     

Uniaxial mechanical strain modulates the differentiation of neural crest stem cells into smooth muscle lineage on micropatterned surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.     

Sphingosine-1-phosphate as a mediator involved in development of fibrotic diseases

Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-β (TGFβ), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFβ signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFβ, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Evaluation of the prognostic value of TGF-β superfamily type I receptor and TGF-β type II receptor expression in nasopharyngeal carcinoma using high-throughput tissue microarrays

Gene expression profiling had revealed that TGF-β superfamily type I receptor (also known as activin receptor-like kinase-1, ALK1) and TGFβR2 (TGF-β type II receptor) were down-regulated in nasopharyngeal carcinoma (NPC) (P < 0.05, respectively). However, no study with significantly large clinical samples to address the relevance of ALK1 and TGFβR2 in NPC progression or in patient outcomes has been reported. This study aims to assess the possible correlations of ALK1 and TGFβR2 expression with NPC progression and their potential prognostic predictive ability in NPC outcomes. ALK1 and TGFβR2 mRNA and protein levels were detected by qRT-PCR and NPC tissue microarray (TMA), which included 742 tissue cores. Both mRNA and protein levels of ALK1 and TGFβR2 were significantly lower in the cancer tissues compared with the non-cancerous tissues (P < 0.05). Epstein-Barr virus small RNA (EBER-1) hybridization signals in NPC showed significant associations with ALK1 and TGFβR2 proteins (P = 0.000 and 0.003, respectively). In the final logistic regression analysis model, the abnormal expression of ALK1 and TGFβR2 were found to be independent contributors to nasopharyngeal carcinogenesis (P = 0.000 and 0.000, respectively). A survival analysis revealed that ALK1 (Disease Free Survival (DFS): P = 0.002, Overall Survival (OS): P = 0.007) and TGFβR2 (DFS: P = 0.072, OS: P = 0.045) could predict the prognosis of NPC patients. The positive expression of ALK1 and TGFβR2 were independent risk factors for DFS and OS in multivariate analyses (DFS: P = 0.001 and 0.420, respectively; OS: P = 0.018 and 0.047, respectively). These results suggest that ALK1 and TGFβR2 may be useful prognostic biomarkers in NPC.     

GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.     

Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.