Volume 115, Issue 1, Pages 75-85 (July 1998) Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes  Gordana Verstovsek*, Andrew Byrd*, Mark R. Frey*, Nicholas J. Petrelli‡, Jennifer D. Black*  Gastroenterology  Volume 115, Issue 1, Pages 75-85 (July 1998) DOI: 10.1016/S0016-5085(98)70367-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Structural organization of the colonic epithelium. (A) Diagrammatic representation of a colonic crypt. Proliferating cells are confined to the lower two thirds of the crypt. Cells undergo growth arrest in the upper crypt region (arrow), and mature cells are found on the flat surface mucosa. (B) Section of mouse colon immunostained to detect BrdU incorporation. Cells synthesizing DNA are confined to the lower and midcrypt region. (C) Corresponding phase-contrast micrograph. (D) Section of rat colon immunostained for the Ki67 nuclear antigen to identify cycling cells. Stained nuclei are found in the lower two thirds of the crypt. (E) Corresponding phase-contrast micrograph. S, surface mucosa. The arrows in B, C, D, and E indicate the end of the proliferation zone (bars = 10 μm). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Structural organization of the colonic epithelium. (A) Diagrammatic representation of a colonic crypt. Proliferating cells are confined to the lower two thirds of the crypt. Cells undergo growth arrest in the upper crypt region (arrow), and mature cells are found on the flat surface mucosa. (B) Section of mouse colon immunostained to detect BrdU incorporation. Cells synthesizing DNA are confined to the lower and midcrypt region. (C) Corresponding phase-contrast micrograph. (D) Section of rat colon immunostained for the Ki67 nuclear antigen to identify cycling cells. Stained nuclei are found in the lower two thirds of the crypt. (E) Corresponding phase-contrast micrograph. S, surface mucosa. The arrows in B, C, D, and E indicate the end of the proliferation zone (bars = 10 μm). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunofluorescence localization of (A and B) PKC α, (C–E) PKC βII, (F and G) PKC δ, (H–J) PKC ϵ, and (K–M) PKC ζ in mouse colonic epithelium. The large arrows indicate the crypt base. Note the marked increase in levels of PKC isozymes in cells of the upper crypts (arrowheads) and the membrane localization of PKC isozymes in postmitotic cells of the surface mucosa (small arrows). Although not always detectable in the micrographs shown, proliferating cells of the lower crypts generally expressed lower levels of PKC isozymes in a diffuse cytoplasmic pattern (e.g., I and L). The intensity of the fluorescence signal in mature cells of the surface mucosa precluded the use of optimal exposure times to show clearly the weaker, diffuse signal in lower crypt cells. B, E, G, J, and M are phase-contrast images corresponding to A, D, F, I, and L, respectively. The data presented are representative of more than three independent experiments. In each case, the fluorescence staining shown was completely blocked by preincubation of the PKC antibody with the appropriate inhibitory peptide (bars = 10 μm). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunofluorescence localization of PKC isozymes in rat colonic epithelium. (A), Colonic crypt (C) and surface mucosa (S) stained for PKC α. PKC α is diffusely distributed throughout the cytoplasm of proliferating crypt cells (C). Note that nuclei are devoid of staining (small arrows). Higher levels are detected in cells of the surface mucosa, particularly in the apical domain. (B) High magnification of postmitotic cells of the mucosal surface showing PKC α in the cytoplasm, in the lateral membrane domains (arrowhead) and in the brush border (arrow). (C) PKC βII is expressed at low levels in proliferating cells of the lower crypts (C). Levels markedly increase in the cytoplasm and at the periphery of cells of the upper crypt and surface mucosa (S). The large arrows indicate the crypt base and the small arrows indicate the point at which the pattern of staining changes. (D) Expression of PKC δ is markedly increased (small arrow) in cells of the upper crypts and surface mucosa. (E) Corresponding phase-contrast micrograph. (F) PKC δ is found in the cytoplasm and in the lateral and apical membrane domains (small arrows) of postmitotic colonocytes of the surface mucosa. The large arrow indicates the crypt base. (G) PKC ϵ is expressed in the cytoplasm and in the lateral membrane domains (arrow) of cells of the surface mucosa. Lower levels of diffuse staining were detected in proliferating crypt cells (not shown). (H) Corresponding phase-contrast micrograph. (I) Levels of PKC ζ increase in postmitotic cells (arrowhead). Note the cytoplasmic and membrane distribution (small arrow) of this PKC isozyme in cells of the mucosal surface. The large arrow indicates the crypt base. The data presented are representative of more than three independent experiments. In each case, the fluorescence staining shown was completely blocked by preincubation of the PKC antibody with the appropriate inhibitory peptide (bars = 10 μm; B = 5 μm). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Postmitotic colonocytes of the surface epithelium show prominent localized PKC isozyme staining of lateral and/or apical membrane domains. Higher magnification immunofluorescence micrographs of (A and B) rat and (C and D) mouse surface colonocytes stained for (A) PKC βII, (B) δ, (C) ϵ, and (D) ζ. (A) PKC βII staining is detected at the lateral membranes (arrow) and in the cytoplasm (in a filamentous pattern) of cells of the surface mucosa. (B) PKC δ is localized both in the brush border microvilli and at the lateral membranes, particularly in the upper lateral membrane domains. Note the absence of PKC δ from the junctional complexes between cells (arrow). (C) PKC ϵ is found only at the lateral membranes (arrow). (D) PKC ζ is detected at the lateral membranes of postmitotic colonocytes and is accumulated in the junctional complexes between cells (arrow) (bar = 5 μm). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Verification of the composition of the epithelial fractions obtained by sequential scraping of rat colonic mucosa. Whole-cell lysates were prepared from colonic epithelial fractions I and IV obtained, as described in Materials and Methods, and examined for Rb phosphorylation state and p21waf1/cip1 expression by Western blot analysis. Cells in fraction IV express predominantly hyperphosphorylated, growth-permissive Rb, and low levels of the CDK inhibitor p21waf1/cip1, whereas cells in fraction I express hypophosphorylated, growth-suppressive Rb and high levels of p21waf1/cip1. Rb-p, hyperphosphorylated Rb. Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Western blot analysis of the expression, subcellular distribution, and membrane-association of PKC isozymes in isolated proliferating and functionally mature colonocytes. (A) Enriched populations of proliferating cells from the crypt base (fraction IV) and differentiated colonocytes from the surface mucosa (fraction I), isolated by sequential scraping, were partitioned into soluble (S) and membrane/cytoskeletal particulate (P) fractions and examined for PKC isozyme expression by Western blot analysis. Levels of PKC α, βII, δ, ϵ, and ζ are significantly higher, both in the soluble and particulate fractions, in mature cells relative to proliferating cells of the crypt base. The faster migrating bands detected on several of the immunoblots represent specific PKC isozyme degradation products that were also observed in samples of small intestine.26 Data are representative of three independent experiments. (B) Membrane association of PKC isozymes in isolated proliferating (fraction IV) and postmitotic (fraction I) colonocytes. Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Expression of PKC α is markedly reduced in human colonic adenocarcinoma tissue relative to normal adjacent colonic mucosa. Extracts (30 μg) of normal human colonic epithelium (N) and colonic tumor tissue (T) from 16 patients were examined for PKC α expression by Western blot analysis. The specificity of the immunoreactive band was verified by preincubation of the anti–PKC α antibody with the appropriate antigenic peptide (not shown). Gastroenterology 1998 115, 75-85DOI: (10.1016/S0016-5085(98)70367-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions