Restricted high level expression of Tcf-4 protein in intestinal and mammary gland epithelium

Abstract

Tcf-4 is a member of the Tcf/Lef family of transcription factors that interact functionally with beta-catenin to mediate Wnt signaling in vertebrates. We have previously demonstrated that the tumor suppressor function of APC in the small intestine is mediated via regulation of Tcf-4/beta-catenin transcriptional activity. To gain further insight into the role of Tcf-4 in development and carcinogenesis we have generated several mouse monoclonal antibodies, one of which is specific for Tcf-4 and another of which recognizes both Tcf-3 and Tcf-4. Immunohistochemistry performed with the Tcf 4- specific monoclonal antibody revealed high levels of expression in normal intestinal and mammary epithelium and carcinomas derived therefrom. Additional sites of Tcf-3 expression, as revealed by staining with the Tcf-3/-4 antibody, occurred only within the stomach epithelium, hair follicles, and keratinocytes of the skin. A temporal Tcf-4 expression gradient was observed along the crypt-villus axis of human small intestinal epithelium: strong Tcf-4 expression was present within the crypts of early (week 16) human fetal small intestine, with the villi showing barely detectable Tcf-4 protein levels. Tcf-4 expression levels increased dramatically on the villi of more highly developed (week 22) fetal small intestine. We conclude that Tcf-4 exhibits a highly restricted expression pattern related to the developmental stage of the intestinal epithelium. The high levels of Tcf-4 expression in mammary epithelium and mammary carcinomas may also indicate a role in the development of this tissue and breast carcinoma.

Figure 1.

Immunohistochemical staining of hTcf-4 and mTcf-3 in COS cells using the 6H5 and 6F12 mAbs. a: Staining of COS cells expressing hTcf-4 (arrows) by the 6H5 mAb. b: Absence of staining on COS cells expressing mTcf-3 by the 6H5 mAb. Magnification, ×200. c,d: Staining of COS cells expressing hTcf-4 or mTcf-3 (arrows) by the 6F12 mAb. Magnification, ×200.

Figure 2.

Immunohistochemical analysis of Tcf-4 and Tcf-3 expression in human and mouse tissues. a-h: Immunohistochemical stainings generated using the Tcf-4 specific mAb (6H5). i-j: Immunohistochemical stainings generated using the Tcf-3/−4 mAb (6F12). a,b: Tcf-4 is highly expressed at the tops of the crypts (arrows) of human adult colonic epithelium (a) and human colon carcinoma (b). Magnification, ×150. c,d: Tcf-4 is expressed at high levels in the fibrous tissue (open arrows) and crypts (shaded arrows) of human (c) and mouse (d) adult small intestinal epithelium. Magnification, ×100. e,f: Tcf-4 expression increases along the crypt-villus axis (arrows) during week 16 (e) and week 22 (f) of the development of human small intestinal epithelium. Magnification, ×50. g,h: Tcf-4 is expressed at high levels in the epithelium (shaded arrows) and fibrous tissue (open arrows) of human mammary gland (g) and mammary carcinoma (h). Magnification, ×100. i-j: Tcf-3 is expressed in hair follicles (i) and keratinocytes of skin (j). Magnification, ×100.

Figure 3.

a: Gel retardation of Tcf-4 complexes from HT-29 colon carcinoma cells. Tcf-4 complexed to an optimal Tcf binding site probe can be supershifted by addition of the Tcf-4-specific mAb 6H5. Lane 1: Nonspecific (NS) bands generated using a probe comprising a disrupted Tcf binding site. Lane 2 : Specific Tcf-4/probe complex. Lane 3: Supershift of the Tcf-4/probe complex by addition of 6H5 mAb. Lane 4: Supershift of the Tcf-4/probe complex on addition of 6F12 mAb. Lanes 5 and 6: No supershift of the Tcf-4/probe complex induced on addition of control antibodies (anti-APC and anti-Plakoglobin). b: Co-immunoprecipitation of Tcf-4 and β-catenin from SW620 nuclear extracts. Lane 1: A 92-kd band (arrow) visualized by Western blot using an anti-β-catenin mAb after Tcf-4 immunoprecipitation from SW620 nuclear extracts using the 6H5 mAb. H, antibody heavy chain; L, antibody light chain. Lane 2: Western blot analysis of total β-catenin (arrow) in SW620 total cell extracts.