Abstract 3975: CLIC4 induces a Smad7 variant to enhance TGF-β signaling in keratinocytes

Abstract

Abstract CLIC4 (Chloride intracellular channel 4) is a member of a family of intracellular chloride channel proteins that are ubiquitously expressed in multiple tissue types. It is a dimorphic protein that exists in both soluble and membrane-bound forms. CLIC4 is multifunctional, traffics between the cytoplasm and nucleus, and participates in cell cycle control and differentiation in keratinocytes and other cell types. CLIC4 expression is lost in multiple human epithelial cancers. We have recently shown that nuclear CLIC4 enhances transforming growth factor beta (TGF-β) signaling through stabilization of phospho-Smad2/3. The TGF-β signaling pathway is a major growth regulator of multiple cell types, and its regulation is altered in many human cancers suggesting that CLIC4 could participate in altering TGF-β regulation in tumor cells. Here we show that in addition to stabilizing phospho-Smads 2/3, CLIC4 alters Smad7, a feedback inhibitor of the TGF-β pathway. Elevated levels of CLIC4 in primary keratinocytes lead to the expression of a truncated form of Smad7 (Smad7Δ), which is detectable as a PCR product. The Smad7Δ variant contains a missing region of 94bp in exon 4 of Smad7 and appears to be an alternately spliced form. Absence of these 94bp leads to a frameshift mutation and an early protein truncation causing the Smad7Δ variant to lack the MH2 domain of Smad7, the domain responsible for inhibition of TGF-β signaling. While wild-type Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ variant does not inhibit TGF-β signaling, based upon analysis of TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Together, these data suggest that CLIC4 regulates TGF-β signaling by multiple mechanisms. In light of this, CLIC4 may serve as a target for modifying TGF-β function in cancer and other conditions where this pathway is involved in pathological changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3975.