Expression Profiling Of Genes Regulated By TGF-β : Role Of Multiple Signaling Pathways
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Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.