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Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3' untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β's antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.
Expression of claudin-1, a tight junction protein, is highly upregulated in colon cancer. We have reported that claudin-1 expression in colon cancer cells is epigenetically regulated as histone deacetylase (HDAC) inhibitors decrease claudin-1 messenger RNA (mRNA) stability and thus expression. In this regard, our data suggested a role of the 3'-untranslated region (UTR) in the regulation of HDAC-dependent regulation of claudin-1 mRNA stability. In the current study, we demonstrate, based on our continued investigation, that the ELAV-like RNA-binding proteins (RBPs), human antigen R (HuR) and tristetraprolin (TTP) associate with the 3'-UTR of claudin-1 mRNA to modulate the latter's stability. Ribonomic and site-directed mutagenesis approaches were used to confirm the binding of HuR and TTP to the 3'-UTR of claudin-1. We further confirmed their roles in the stabilization of claudin-1 mRNA, under conditions of HDAC inhibition. In summary, we report that HuR and TTP are the critical regulators of the posttranscriptional regulation of claudin-1 expression in colon cancer cells. We also demonstrate that inhibition of HDACs by trichostatin treatment decreased the binding of HuR while increasing the binding of TTP to the 3'-UTR of claudin-1. Additionally, we provide data showing transcriptional regulation of claudin-1 expression, through the regulation of transcription factor Sp1. Taken together, we demonstrate epigenetic regulation of claudin-1 expression in colon cancer cells at the transcriptional and posttranscriptional levels.
In human colorectal adenocarcinoma cell lines, we found two major transcripts of cyclooxygenase-2, the full-length mRNA and a short polyadenylation variant (2577 kb) lacking the distal segment of the 3'-untranslated region. Tristetraprolin, an mRNA-binding protein that promotes message instability, was shown to bind the cyclooxygenase-2 mRNA in the region of the 3'-untranslated region between nucleotides 3125 and 3432 and to reduce levels of the full-length mRNA. During cell growth and confluence, the expression of tristetraprolin mRNA was inversely correlated with that of the full-length cyclooxygenase-2 transcript, and transfection of tristetraprolin into HCA-7 cells reduced the level of full-length cyclooxygenase-2 mRNA. However, the truncated transcript escaped tristetraprolin binding and downregulation.
The epithelium lining the intestine undergoes rapid and continuous renewal. Growth factors play a role in intestinal epithelial growth regulation in vitro and in vivo. In this study, transforming growth factor alpha (TGF alpha) is shown to act as a mitogen and induce the expression of two zinc finger-containing immediate early genes [Zif268 (zinc finger protein 268) and Nup475 (nuclear protein 475)] in rat intestinal epithelial (RIE-1) cells in culture. These two gene products were initially isolated from serum-treated fibroblasts and represent growth-stimulated transcription factors. In TGF alpha-treated RIE-1 cells, nuclear run-on experiments demonstrate that TGF alpha induction of these two genes is regulated predominantly at the level of gene transcription. Utilizing in situ hybridization techniques, we show that systemic administration of TGF alpha induces expression of these two genes in the rat intestine. The predominant expression of zif268 is observed in the proliferative crypt compartment, whereas nup475 expression is concentrated in the postmitotic luminal compartment. These studies demonstrate that two immediate early genes, Nup475 and Zif268, are induced in intestinal epithelium in vitro and in vivo and thus may play a role in intestinal epithelial growth and/or differentiation.