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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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PKC activation in IEC-18 cells leads to loss of cdc6 expression. Cells were treated with 100 nM PMA for the indicated times and subjected to Western blot analysis for cdc6 (U, untreated). Data are representative of three independent experiments.
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Figure 9: PKC activation in IEC-18 cells leads to loss of cdc6 expression. Cells were treated with 100 nM PMA for the indicated times and subjected to Western blot analysis for cdc6 (U, untreated). Data are representative of three independent experiments.

Mentions: The depletion of D-type cyclins and coordinated alterations in pocket protein expression/phosphorylation observed after PMA treatment of IEC-18 cells suggested that PKC activation in this system signals cell cycle withdrawal into G0. To further investigate this possibility, control and PMA-treated IEC-18 cells were analyzed for expression of the cdc6 DNA licensing factor, a molecule that is strictly associated with actively cycling populations and whose downregulation is indicative of cell cycle exit (Stillman 1996; Fujita 1999). PKC activation in IEC-18 cells resulted in marked downregulation of cdc6 by 2 h of treatment (Fig. 9), confirming cell cycle withdrawal into G0 or a G0-like state. Levels of this molecule recovered by 10–16 h, reflecting downregulation of PKC isozymes and reentry of IEC-18 cells into the cell cycle.


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)

PKC activation in IEC-18 cells leads to loss of cdc6 expression. Cells were treated with 100 nM PMA for the indicated times and subjected to Western blot analysis for cdc6 (U, untreated). Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169440&req=5

Figure 9: PKC activation in IEC-18 cells leads to loss of cdc6 expression. Cells were treated with 100 nM PMA for the indicated times and subjected to Western blot analysis for cdc6 (U, untreated). Data are representative of three independent experiments.
Mentions: The depletion of D-type cyclins and coordinated alterations in pocket protein expression/phosphorylation observed after PMA treatment of IEC-18 cells suggested that PKC activation in this system signals cell cycle withdrawal into G0. To further investigate this possibility, control and PMA-treated IEC-18 cells were analyzed for expression of the cdc6 DNA licensing factor, a molecule that is strictly associated with actively cycling populations and whose downregulation is indicative of cell cycle exit (Stillman 1996; Fujita 1999). PKC activation in IEC-18 cells resulted in marked downregulation of cdc6 by 2 h of treatment (Fig. 9), confirming cell cycle withdrawal into G0 or a G0-like state. Levels of this molecule recovered by 10–16 h, reflecting downregulation of PKC isozymes and reentry of IEC-18 cells into the cell cycle.

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