Smad4 is essential for down-regulation of E-cadherin induced by TGF-beta in pancreatic cancer cell line PANC-1

Smad4 is a tumour suppressor gene frequently deleted in pancreatic cancer. To investigate the roles of Smad4 deficiency in invasive and matastatic capabilities of pancreatic cancer, we examined the effects of Smad4 deficiency on regulation of the invasion suppressor E-cadherin in pancreatic cancer cell line PANC-1. TGF-beta decreased expression of E-cadherin and beta-catenin proteins at the plasma membrane, increased Snail and Slug mRNA expression, and induced fibroblastoid morphology in PANC-1 cells. These effects of TGF-beta were abrogated in Smad4-knocked-down PANC-1 cells. We also found that TGF-beta-induced down-regulation of E-cadherin expression was partially inhibited in Snail- and Slug-knocked-down PANC-1 cells. Thus, Smad4 mediates down-regulation of E-cadherin induced by TGF-beta in PANC-1 cells, at least in part, through Snail and Slug induction. These results suggest that Smad4 deficiency observed in invasive and metastatic pancreatic cancer might not be linked to the loss of E-cadherin.     

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.     

MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer

MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. DPC4/Smad4 is a critical tumor suppressor involved in the progression of pancreatic cancer, but few studies have been conducted to determine its relationship with miRNAs. In this study, we identify miR-421 as a potential regulator of DPC4/Smad4. We find that in human clinical specimens of pancreatic cancer miR-421 is aberrantly upregulated while DPC4/Smad4 is strongly repressed, and their levels of expression are inversely correlated. Moreover, ectopic expression of miR-421 significantly decreases DPC4/Smad4 protein level in pancreatic cancer cell lines and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-421 as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for treatment of DPC4/Smad4-driven pancreatic cancer.     

MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer

Both deregulation of tumor-suppressor genes and misexpression of microRNAs (miRNAs) have been implicated in the development of pancreatic cancer, but their relationship during this process remains less clear. Here, we report that the expression of miR-483-3p is strongly enhanced in pancreatic cancer tissues compared to side normal tissues using a miRNA-array differential analysis. Furthermore, DPC4/Smad4 is identified as a target of miR-483-3p and their expression levels are inversely correlated in human clinical specimens. Ectopic expression of miR-483-3p significantly represses DPC4/Smad4 protein levels in pancreatic cancer cell lines, and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-483-3p as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for the treatment of DPC4/Smad4-driven pancreatic cancer.     

Transforming growth factor-beta regulates stearoyl coenzyme A desaturase expression through a Smad signaling pathway.

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.     

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

Enhancement of tenogenic differentiation of rat tendon-derived stem cells by biglycan

Biglycan (BGN) has been identified as one of the critical components of the tendon-derived stem cells (TDSCs) niche and may be related to tendon formation. However, so far, no study has demonstrated whether the soluble BGN could induce the tenogenic differentiation of TDSCs in vitro. The aim of this study was to investigate the effect of BGN on the tenogenic differentiation of TDSCs. The proliferation and tenogenic differentiation of TDSCs exposed to different concentrations of BGN (0, 50, 100, and 500 ng/ml) were determined by the live/dead cell staining assay, CCK-8 assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. The BGN signaling pathway of TDSCs (with and without 50 ng/ml of BGN) was determined by western blot analysis and qRT-PCR analysis. At a concentration of 50 ng/ml, BGN increased the expression of the tenogenic markers THBS-4 and TNMD at both the messenger RNA (mRNA) and protein levels. Meanwhile, 50 ng/ml of BGN inhibited the expression of the chondrogenic and osteogenic markers SOX9, ACN, and RUNX2 at both the mRNA and protein levels. Moreover, BGN (50 ng/ml) affected the expression of the components of the extracellular matrix of TDSCs. Additionally, BGN activated the Smad1/5/8 pathway as indicated by an increase in phosphorylation and demonstrated by inhibition experiments. Upregulation in the gene expression of BMP-associated receptors (BMPRII, ActR-IIa, and BMPR-Ib) and Smad pathway components (Smad4 and 8) was observed. Taken together, BGN regulates tenogenic differentiation of TDSCs via BMP7/Smad1/5/8 pathway and this regulation may provide a basic insight into treating tendon injury.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

Smad4 dependency defines two classes of transforming growth factor {beta} (TGF-{beta}) target genes and distinguishes TGF-{beta}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.     

Glypican-1 antisense transfection modulates TGF-beta-dependent signaling in Colo-357 pancreatic cancer cells

The heparan sulfate proteoglycan glypican-1 is essential as a co-receptor for heparin binding growth factors, such as HB-EGF and FGF-2, in pancreatic cancer cells. In the present study, the role of glypican-1 in the regulation of TGF-beta signaling was investigated. Colo-357 pancreatic cancer cells were stably transfected with a full-length glypican-1 antisense construct. Cell growth was determined by MTT and soft agar assays. TGF-beta1 induced p21 expression and Smad2 phosphorylation were analyzed by immunoblotting. PAI-1 promoter activity was determined by luciferase assays. Down-regulation of glypican-1 expression by stable transfection of a full-length glypican-1 antisense construct resulted in decreased anchorage-dependent and -independent cell growth in Colo-357 pancreatic cancer cells and attenuated TGF-beta1 induced cell growth inhibition, Smad2 phosphorylation, and PAI-1 promoter activity. There was, however, no significant difference in TGF-beta1 induced p21 expression and Smad2 nuclear translocation. In conclusion, glypican-1 is required for efficient TGF-beta1 signaling in pancreatic cancer cells.     

Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-beta signaling in pancreatic acinar cells

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-beta signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-beta-responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH terminal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. Acinar cells expressed dominant-negative Smad4 within 8 h after infection, and expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protein, and TGF-beta was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and [(3)H]thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-beta inhibition of [(3)H]thymidine incorporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-beta-mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-beta-induced expression of the cell cycle regulatory proteins p21(Cip1) and p27(Kip1). TGF-beta induced upregulation of p21(Cip1), which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-beta-induced expression of the TGF-beta-responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-beta-mediated signaling in pancreatic acinar cells.