Fig. 3.
Smad phosphoisoform signaling can determine specific cellular responses to TGF-β in normal enterocytes, a colonic adenoma and an invasive carcinoma metastasized from colon. Phospho-Smad3 signaling and the biology of colonic crypt (A) and adenoma (B). Formalin-fixed paraffin-embedded sections of normal colonic crypt (A) and adenoma (B) were stained with anti-pSmad3L (Ser208/213) Ab [α pSmad3L (Ser208/213) column], anti-c-Myc Ab (α c-Myc column), anti-pSmad3C Ab (α pSmad3C column) or anti-p21WAF1 Ab (α p21WAF1 column). Sections stained for pSmad3L (Ser208/213) or pSmad3C were paired with adjacent sections stained with anti-c-Myc Ab or anti-p21WAF1 Ab, respectively. All sections were counterstained with hemotoxylin (blue). Brown product indicates specific Ab reactivity. Scale bars = 100 μm. (A) Physiologic roles of pSmad3L and pSmad3C in normal colonic crypts. Immunoreactivity for pSmad3L shows a striking distribution, localized to the nuclei of c-Myc-immunoreactive progenitor cells at the bottom of mucosal crypts [proliferative compartments: lower portions of crypts in α pSmad3L (Ser208/213) and α c-Myc columns]. In the colonic progenitor cells, linker phosphorylation of Smad3 prevents the cytostatic p21WAF1 effect by shutting down phosphorylation at Smad3C. As enterocytes migrate upward from the crypt base to the lumen, the growth-stimulatory signal via pSmad3L ceases in the differentiated enterocytes (differentiated compartments). TGF-β can then activate the promoter of p21WAF1 via pSmad3C to arrest the growth of enterocytes (upper portions in α pSmad3C and α p21WAF1 columns). (B) pSmad3L/c-Myc-immunoreactive adenomas accumulate within entire crypts and ultimately form an adenomatous polyp. pSmad3L/c-Myc-immunoreactive colonic adenoma carrying K-Ras mutation [α pSmad3L (Ser208/213) and α c-Myc columns] becomes able to divide indefinitely (red arrow), instead of undergoing physiologic pSmad3C/p21WAF1-mediated differentiation and death. At the edges of advanced carcinomas invading adjacent tissues, pSmad2L/C and pSmad3L/C transmit pro-tumorigenic TGF-β signal (C and D). (C) The upper panels show formalin-fixed, paraffin-embedded sections of invasive carcinomas carrying K-Ras mutation, which had metastasized from the colon, as well as uninvolved liver. Tissues were stained with anti-pSmad2/3L (Thr) Ab [α pSmad2/3L (Thr) column] or anti-pSmad2/3C Ab (α pSmad2/3C column). Sections stained for pSmad2/3L (Thr) were paired with adjacent sections stained with pSmad2/3C. All sections were counterstained with hemotoxylin (blue). Brown product indicates specific Ab reactivity. Scale bars = 100 μm. Fibroblasts, macrophages and hepatocytes in the uninvolved liver tissue (lower portions) show little phosphorylation of Smad2/3 at the linker regions [α pSmad2/3L (Thr) column], but moderate phosphorylation at their C-tail regions (α pSmad2/3C column). In the carcinomas (upper portions) invading adjacent liver tissue, nuclear Smad2 and Smad3 are highly phosphorylated at both their linker and C-tail regions [α pSmad2/3L (Thr) and α pSmad2/3C columns]. The lower panels show sections of invasive tumor tissue stained for immunofluorescence to simultaneously detect pSmad2/3L (Thr) (red) and pSmad2/3C (green). Yellow color indicates the presence of both pSmad2L/C and pSmad3L/C. Scale bars = 100 μm. At invasion fronts of the carcinomas, nuclear pSmad2/3L (Thr) co-localizes with pSmad2/3C. (D) upper right: Tumor cells (pink) take on an invasive phenotype at the edge of carcinomas (red) invading adjacent liver tissue. Balloon, left: pSmad2L/C and pSmad3L/C transmit the protumorigenic TGF-β signal in combination with oncogenic Ras. Balloon, lower right: Invasive carcinomas carrying K-Ras mutation gain access to abundant TGF-β from either autocrine or microenvironmental sources. Macrophages and fibroblasts release TGF-β in the reactive stroma.