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, 286 (20), 18104-17

Protein Kinase Cα Signaling Regulates Inhibitor of DNA Binding 1 in the Intestinal Epithelium

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Protein Kinase Cα Signaling Regulates Inhibitor of DNA Binding 1 in the Intestinal Epithelium

Fang Hao et al. J Biol Chem.

Abstract

Increasing evidence supports a role for PKCα in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKCα signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKCα and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKCα activation, and (b) loss of PKCα in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKCα activity in IEC-18 nontransformed intestinal crypt cells determined that PKCα suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKCα, but not PKCδ, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKCα. Notably, Id1 expression was elevated in the intestinal epithelium of PKCα-knock-out mice, confirming that PKCα regulates Id1 in vivo. A wider role for PKCα in control of inhibitor of DNA binding factors is supported by its ability to down-regulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKCα → Erk ⊣ Id1 → cyclin D1 signaling axis in the maintenance of intestinal homeostasis.

Figures

FIGURE 1.
FIGURE 1.
Id1 expression inversely correlates with PKCα activation in the intestinal epithelium. A, immunohistochemical analysis of Id1, cyclin D1, and PKCα expression in the mouse small intestine. Panels i and ii, detection of Id1 using a highly specific rabbit monoclonal anti-Id1 antibody. Id1 staining is seen in nuclei of proliferating crypt epithelial cells. Down-regulation of Id1 occurs at the crypt-villus junction (J), and the protein is absent from villus cells (arrowheads) and Paneth cells (P). Open arrows point to the presumptive stem cells at the crypt base, and block arrows indicate Id1-positive stromal cells. Panels iii and iv, staining for the proliferation marker, cyclin D1, parallels that of Id1 (arrows as in panels i and ii). Panels v and vi, PKCα is diffusely distributed/inactive in the proliferating crypt cells (solid arrows) and becomes membrane-associated in nondividing cells of the villus (open arrows) and in Paneth cells (P). Brackets, Crypt compartment; V, villus; C, crypt; J, crypt-villus junction. Panel vii, villus and crypt fractions were isolated by sequential washing of intestinal segments in chelating buffer and subjected to Western blot analysis for the indicated proteins. As confirmed by the crypt marker cyclin D1, fractions 1 and 2 predominantly contain villus cells, and fractions 4 and 5 contain crypt cells. Varying low levels of crypt cell marker were detected in fraction 3. B, immunohistochemical analysis of Id1, cyclin D1, and PKCα expression in the mouse colon. Panels i and ii, Id1 and cyclin D1 staining is seen in nuclei of crypt epithelial cells (arrows) but is absent from the nuclei of the surface mucosa (arrowhead). Block arrow, stromal Id1 staining. Panel iii, PKCα membrane association/activity is only evident in epithelial cells of the surface mucosa (open arrows). Solid arrows indicate the presence of diffuse cytosolic/inactive PKCα in proliferating crypt cells. SM, surface mucosa; C, crypt. Bars, 50 μm. Data are representative of >3 independent experiments.
FIGURE 2.
FIGURE 2.
Id1 is overexpressed in intestinal tumors. A, immunostaining for Id1 and PKCα in small intestinal tissue from APCmin/+ mice (panels i–iii) and colonic tissue from azoxymethane (AOM)-treated mice (panels iv–viii). Panels i and ii, iv and v, vii and viii show serial sections from the same tissue stained for Id1 and PKCα. Id1 is generally overexpressed in the nuclei of tumor (T) cells relative to nuclei of normal crypt (NC) cells (panels i, iv, and vi; arrows), whereas PKCα is lost from the tumors (panels ii, v, and viii). The dashed line in panels iv and v demarcates the boundary between normal and tumor tissue. Higher magnification images in panels iii and vii show nuclear localization of Id1 in tumor cells. Bars, 50 μm. Data are representative of >3 independent experiments. B, immunohistochemical analysis of normal and neoplastic colonic tissue from human patients. Panel i, Id1 expression in normal colonic mucosa and adjacent adenocarcinoma. Tumor (T) and normal tissue are from the same section, and images were processed identically. Nuclear staining for Id1 is seen in the normal crypt (NC) epithelium (solid arrows) but is absent from the surface mucosa (SM; arrowhead), whereas more intense nuclear staining can be seen in tumor cells. Data are representative of analysis of samples from two patients. Panel ii, inverse correlation of PKCα and Id1 expression in colon tumors from human patients. Sections of tumor tissue from four patients were stained for PKCα or Id1. Note the high nuclear expression of Id1 in tumors that lack PKCα and the absence of Id1 in a relatively rare tumor that retains expression of the enzyme (bottom panels). Bars, 50 μm.
FIGURE 3.
FIGURE 3.
PKC signaling down-regulates Id1 in IEC-18 cells. A, PKC agonist treatment leads to down-regulation of Id1 in IEC-18 cells. Protein extracts from cells treated with PMA (100 nm), bryostatin (Bryo; 100 nm), or DiC8 (20 μg/ml) for 2 h were subjected to immunoblot analysis for Id1 and actin (loading control). B, effects of PKC agonists on Id1 are PKC-dependent. Panel i, PKC agonist-responsive isozymes (PKCα, -δ, and -ϵ) were depleted from IEC-18 cells by treatment with 1 μm PDBu for 24 h. PDBu was removed, and cells were treated with vehicle (C) or 100 nm PMA (P) for 2 h prior to protein extraction and immunoblot analysis for the indicated proteins. Panel ii, cells were pretreated with 1 μm Gö6983 for 30 min prior to addition of vehicle or 100 nm PMA for 2 h. C, PKC activation down-regulates Id1 mRNA. Panel i, cells were treated with PKC agonists or vehicle for 3 h as above, and total cellular RNA was subjected to real time RT-PCR analysis. Levels of Id1 mRNA were normalized to 18 S rRNA and are displayed as relative to vehicle control. Panel ii, cells were depleted of agonist-responsive PKC isozymes by prolonged treatment with PDBU or treated with Gö6983 prior to addition of vehicle (V) or PMA for 3 h. RNA was extracted and analyzed by real time RT-PCR. Data are representative (A and B) or averages ± S.E. (C) of at least three independent experiments. Asterisks signify statistically different (p < 0.05) from vehicle-only control (*) or PMA-treated control (**).
FIGURE 4.
FIGURE 4.
PKCα down-regulates Id1 in intestinal epithelial cells. A, loss of PKCα correlates with restoration of Id1 levels in PKC agonist-treated IEC-18 cells. Panel i, cells were treated with 100 nm PMA, 100 nm bryostatin (Bryo), or 20 μg/ml DiC8 for the indicated times and subjected to Western blot analysis. Each panel shows proteins from a single blot; vertical solid lines indicate where the position of lanes has been changed for clarity. Panel ii, Id1 and PKCα expression in cells treated with DiC8 or vehicle for 8 and 12 h. B, Id1 down-regulation is prolonged in PKCα-overexpressing cells. IEC-18 cells were infected with adenovirus expressing lacZ or PKCα (m.o.i. of 10). After 48 h, cells were treated with PMA and analyzed as in A. The vertical dashed line is included for clarity. Endogenous PKCα is not apparent in lacZ-expressing cells due to lower antibody concentrations and shorter exposures used to detect the exogenous protein. C, inhibition of PKCα activity abrogates PKC agonist-induced Id1 down-regulation. IEC-18 cells were pretreated with vehicle (C) or Gö6976 for 30 min prior to addition of PMA (P) or vehicle (C). After 2 h, protein was extracted and subjected to Western analysis for the indicated proteins. Note that Gö6976 is specific for PKCα in these cells as indicated by its ability to block down-regulation of this isozyme but not that of PKCδ or PKCϵ (enzyme down-regulation is dependent on catalytic activity). Data are representative of at least three independent experiments.
FIGURE 5.
FIGURE 5.
Erk signaling mediates the effects of PKC activation on Id1 expression. A and B, IEC-18 cells were pretreated with vehicle (C), LY294002 (50 μm), U0126 (10 μm), or PD98059 (50 μm) prior to addition of PMA (P) or vehicle (C). After 2 h, protein was extracted and subjected to Western blotting for the indicated proteins. Each panel shows data from a single blot; vertical solid lines indicate where lanes have been realigned for clarity. Dashed lines are included for clarity. C, IEC-18 cells, infected with adenovirus expressing lacZ (control) or PKCα, were treated with PMA (P) or vehicle (ethanol, E) for the indicated times and subjected to Western blotting for the indicated proteins. Prolonged suppression of Id1 in PKCα-overexpressing cells is associated with sustained Erk activation. Data are representative of at least three independent experiments.
FIGURE 6.
FIGURE 6.
PKCα induces Erk activation and down-regulates Id1 in colon cancer cells. A and B, indicated colon cancer cells were infected with adenovirus expressing lacZ, PKCα, or PKCδ at an m.o.i. of 10. After 48 h, protein was extracted and subjected to Western blot analysis for the indicated proteins. Each panel shows data from a single blot; the vertical line on the FET pERK blot indicates where lanes have been rearranged for consistency. Data are representative of at least three independent experiments. C, HCT116 cells were pretreated with vehicle (DMSO) or 10 μm U0126 (in DMSO) for 30 min prior to addition of vehicle (ethanol, E) or 100 nm PMA (P) for 30 min (left panel) or 2 h (right panel). Cells were than processed for Western blot analysis for the indicated proteins. D, HCT116 and DLD1 cells were infected with adenovirus expressing lacZ or PKCα as above and total cellular RNA was subjected to real time RT-PCR analysis. Levels of Id1 mRNA were normalized to 18 S rRNA and are displayed as relative to lacZ-transduced cells. Data are averages (±S.E.) of two (DLD1) or three (HCT116) independent experiments. *, significantly different from lacZ control (p < 0.05).
FIGURE 7.
FIGURE 7.
Id1 regulates cyclin D1 in intestinal epithelial cells. A, knockdown of Id1 results in down-regulation of cyclin D1. Indicated cells were transfected with nontargeting siRNA (NS) or siRNA targeting rat (IEC-18) or human (HCT-116 and DLD1) Id1. #1, #2, and #3 designate IEC-18 cells transfected with one of three independent siRNAs targeting rat Id1. 48 h after transfection, cells were harvested and analyzed by Western blotting for the indicated proteins. B, panel i, IEC-18 cells expressing an EGFP-Id1 fusion protein or EGFP (C) were analyzed by Western blotting for the indicated proteins. Panel ii, cells expressing EGFP-Id1 fusion protein were treated with vehicle (C) or 100 nm PMA (P) for 2 h prior to extraction and Western blot analysis. Data are representative of >3 independent experiments.
FIGURE 8.
FIGURE 8.
Id1 is deregulated in the intestine and colon of PKCα−/− mice. A, immunohistochemical analysis of Id1 expression in small intestine of age-matched wild-type and PKCα−/− mice. To allow direct comparison, sections were stained simultaneously on the same slide, and images were processed identically. Arrows indicate Id1 staining on the villus (V) of PKCα−/− mice. C, crypt. Bars, 50 μm. B, Id1 expression in isolated small intestinal epithelial fractions. Fractions, isolated as in Fig. 1A, were subjected to Western blot analysis for the indicated proteins. Cyclin D1 staining confirmed that fractions 1 and 2 contain only villus cells, whereas fractions 4 and 5 contain crypt cells. Dashed lines are included for clarity. C, immunohistochemical analysis of Id1 expression in wild-type and PKCα−/− colon. The arrows in panels ii and iv indicate Id1 staining on the surface mucosa (SM) of PKCα−/− mice. Bars, 50 μm. Data are representative of >3 experiments.
FIGURE 9.
FIGURE 9.
PKCα down-regulates Id2 and Id3. IEC-18 cells were treated with vehicle, PMA, or DiC8 for 2 h (left panels) or pretreated with vehicle (control), Gö6983, or Gö6976 for 30 min prior to addition of PMA or vehicle for 2 h (right panels). RNA was extracted and subjected to real time RT-PCR analysis of Id2 (A) or Id3 (B) mRNA. Data, which are normalized to 18 S rRNA levels and expressed relative to vehicle control, are averages of three independent experiments ±S.E. Asterisks signify statistically different (p < 0.05) from control (*), from control and PMA alone (**) or from PMA alone (***).

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