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Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD - J. Cell Biol. (2000)

Bottom Line: PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes.These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6.Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. jennifer.black@roswellpark.org

ABSTRACT
Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

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Developmental regulation of CKI expression in the small intestinal epithelium. (a–d) Immunofluorescence localization of CKIs in rat duodenum. (ai and aii) Expression of p21waf1/cip1 is weak in the nuclei of proliferating crypt cells (P), increases markedly coincident with growth arrest (arrow in ai), and declines in functional cells (F in aii) of the villus. D, differentiation zone. (b) p27kip1 staining is undetectable in cells of the lower crypt (P) and increases markedly with growth arrest, remaining high along the length of the villus. (ci and cii) p15 staining is readily detectable in nuclei of cells throughout the crypt and increases at the crypt–villus junction (J). V, villus; arrow, point of growth arrest. (di, dii) p16 staining is low in crypt cells (C) and increases gradually as cells migrate towards the villus tip. In dii, arrow points to a villus cell nucleus expressing p16. (e) Whole cell lysates (30 μg protein) of crypt (C), lower villus (LV), and upper villus (UV) populations were examined by Western blot analysis for expression of p21waf1/cip1, p27kip1, p15, and p16. Note that the crypt fraction includes some postmitotic cells of the upper crypt region which account for the p21waf1/cip1 and p27kip1 detected in this sample. Data are representative of at least three independent experiments. Bars, 10 μm.
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Figure 8: Developmental regulation of CKI expression in the small intestinal epithelium. (a–d) Immunofluorescence localization of CKIs in rat duodenum. (ai and aii) Expression of p21waf1/cip1 is weak in the nuclei of proliferating crypt cells (P), increases markedly coincident with growth arrest (arrow in ai), and declines in functional cells (F in aii) of the villus. D, differentiation zone. (b) p27kip1 staining is undetectable in cells of the lower crypt (P) and increases markedly with growth arrest, remaining high along the length of the villus. (ci and cii) p15 staining is readily detectable in nuclei of cells throughout the crypt and increases at the crypt–villus junction (J). V, villus; arrow, point of growth arrest. (di, dii) p16 staining is low in crypt cells (C) and increases gradually as cells migrate towards the villus tip. In dii, arrow points to a villus cell nucleus expressing p16. (e) Whole cell lysates (30 μg protein) of crypt (C), lower villus (LV), and upper villus (UV) populations were examined by Western blot analysis for expression of p21waf1/cip1, p27kip1, p15, and p16. Note that the crypt fraction includes some postmitotic cells of the upper crypt region which account for the p21waf1/cip1 and p27kip1 detected in this sample. Data are representative of at least three independent experiments. Bars, 10 μm.

Mentions: Immunofluorescence analysis revealed that PKC-mediated control of CKI expression in IEC-18 cells parallels CKI regulation along the crypt–villus axis in situ. Relatively low levels of p21waf1/cip1 were observed in the nuclei of proliferating lower crypt cells, whereas p27kip1 was undetectable in these cells (Fig. 8, a and b). Coincident with growth arrest, upregulation/activation of PKC, and alterations in pocket protein expression/phosphorylation, levels of both Cip/Kip CKIs increased markedly. Upregulation of p21waf1/cip1 was transient; expression remained high in the actively differentiating cells of the upper crypt and lower villus and decreased to barely detectable levels in functional cells of the upper villus. p27kip1 expression, on the other hand, remained relatively high in the entire postmitotic compartment. In contrast, changes in p15 and p16 expression did not correlate with growth arrest and PKC activation in the midcrypt region (Fig. 8c and Fig. d). Moderate levels of p15 and low amounts of p16 were detected in cells throughout the entire crypt. Increased p15 expression became apparent at the crypt–villus junction, which is well into the postmitotic compartment (Fig. 8 c), and levels of this molecule remained high along the length of the villus. Levels of p16, on the other hand, increased gradually with cell migration up the villus (Fig. 8 d). These patterns of Cip/Kip and Ink4 family CKI regulation along the crypt–villus unit were consistent with those observed by Western blot analysis of isolated crypt, lower villus, and upper villus cells (Fig. 8 e). Thus, upregulation of p21waf1/cip1 and p27kip1 expression in the midcrypt region coincides precisely with changes in PKC expression/activation, alterations in pocket protein expression/phosphorylation, and growth arrest. Changes in p15 and p16 do not correlate with PKC activation in this system.


Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD - J. Cell Biol. (2000)

Developmental regulation of CKI expression in the small intestinal epithelium. (a–d) Immunofluorescence localization of CKIs in rat duodenum. (ai and aii) Expression of p21waf1/cip1 is weak in the nuclei of proliferating crypt cells (P), increases markedly coincident with growth arrest (arrow in ai), and declines in functional cells (F in aii) of the villus. D, differentiation zone. (b) p27kip1 staining is undetectable in cells of the lower crypt (P) and increases markedly with growth arrest, remaining high along the length of the villus. (ci and cii) p15 staining is readily detectable in nuclei of cells throughout the crypt and increases at the crypt–villus junction (J). V, villus; arrow, point of growth arrest. (di, dii) p16 staining is low in crypt cells (C) and increases gradually as cells migrate towards the villus tip. In dii, arrow points to a villus cell nucleus expressing p16. (e) Whole cell lysates (30 μg protein) of crypt (C), lower villus (LV), and upper villus (UV) populations were examined by Western blot analysis for expression of p21waf1/cip1, p27kip1, p15, and p16. Note that the crypt fraction includes some postmitotic cells of the upper crypt region which account for the p21waf1/cip1 and p27kip1 detected in this sample. Data are representative of at least three independent experiments. Bars, 10 μm.
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Figure 8: Developmental regulation of CKI expression in the small intestinal epithelium. (a–d) Immunofluorescence localization of CKIs in rat duodenum. (ai and aii) Expression of p21waf1/cip1 is weak in the nuclei of proliferating crypt cells (P), increases markedly coincident with growth arrest (arrow in ai), and declines in functional cells (F in aii) of the villus. D, differentiation zone. (b) p27kip1 staining is undetectable in cells of the lower crypt (P) and increases markedly with growth arrest, remaining high along the length of the villus. (ci and cii) p15 staining is readily detectable in nuclei of cells throughout the crypt and increases at the crypt–villus junction (J). V, villus; arrow, point of growth arrest. (di, dii) p16 staining is low in crypt cells (C) and increases gradually as cells migrate towards the villus tip. In dii, arrow points to a villus cell nucleus expressing p16. (e) Whole cell lysates (30 μg protein) of crypt (C), lower villus (LV), and upper villus (UV) populations were examined by Western blot analysis for expression of p21waf1/cip1, p27kip1, p15, and p16. Note that the crypt fraction includes some postmitotic cells of the upper crypt region which account for the p21waf1/cip1 and p27kip1 detected in this sample. Data are representative of at least three independent experiments. Bars, 10 μm.
Mentions: Immunofluorescence analysis revealed that PKC-mediated control of CKI expression in IEC-18 cells parallels CKI regulation along the crypt–villus axis in situ. Relatively low levels of p21waf1/cip1 were observed in the nuclei of proliferating lower crypt cells, whereas p27kip1 was undetectable in these cells (Fig. 8, a and b). Coincident with growth arrest, upregulation/activation of PKC, and alterations in pocket protein expression/phosphorylation, levels of both Cip/Kip CKIs increased markedly. Upregulation of p21waf1/cip1 was transient; expression remained high in the actively differentiating cells of the upper crypt and lower villus and decreased to barely detectable levels in functional cells of the upper villus. p27kip1 expression, on the other hand, remained relatively high in the entire postmitotic compartment. In contrast, changes in p15 and p16 expression did not correlate with growth arrest and PKC activation in the midcrypt region (Fig. 8c and Fig. d). Moderate levels of p15 and low amounts of p16 were detected in cells throughout the entire crypt. Increased p15 expression became apparent at the crypt–villus junction, which is well into the postmitotic compartment (Fig. 8 c), and levels of this molecule remained high along the length of the villus. Levels of p16, on the other hand, increased gradually with cell migration up the villus (Fig. 8 d). These patterns of Cip/Kip and Ink4 family CKI regulation along the crypt–villus unit were consistent with those observed by Western blot analysis of isolated crypt, lower villus, and upper villus cells (Fig. 8 e). Thus, upregulation of p21waf1/cip1 and p27kip1 expression in the midcrypt region coincides precisely with changes in PKC expression/activation, alterations in pocket protein expression/phosphorylation, and growth arrest. Changes in p15 and p16 do not correlate with PKC activation in this system.

Bottom Line: PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes.These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6.Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. jennifer.black@roswellpark.org

ABSTRACT
Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Show MeSH
Related in: MedlinePlus