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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 pocket protein expression and phosphorylation state in the intestinal epithelium in situ. (a–c) Immunofluorescence localization of pocket proteins. P, proliferation zone; D, differentiation zone; V, villus/functional zone. (a) p107 is readily detected in the nuclei of proliferating lower crypt cells (P). Coincident with growth arrest (arrow), p107 expression decreases to barely detectable levels. (bi and bii) pRb staining is evident in both nuclear and cytosolic compartments of proliferating crypt cells (P), and becomes predominantly nuclear in postmitotic cells of the upper crypt and villus (V). The arrow indicates the point of growth arrest. (c) p130 staining is low in proliferating crypt cells (P), but increases markedly coincident with growth arrest (arrow). (d) Whole cell lysates (30 μg protein) of isolated crypt (C), lower villus (LV), and upper villus (UV) cells were subjected to Western blot analysis for expression and migration/phosphorylation state of pocket proteins. Note that the crypt fraction includes some postmitotic cells of the upper crypt region. p130 form 3, which is only found in cycling cells, is only detected in the crypt fraction; the presence of forms 1 and 2 in this fraction reflects the postmitotic cells in this sample. Data are representative of at least three independent experiments. Bar, 10 μm.
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Figure 3: Developmental regulation of pocket protein expression and phosphorylation state in the intestinal epithelium in situ. (a–c) Immunofluorescence localization of pocket proteins. P, proliferation zone; D, differentiation zone; V, villus/functional zone. (a) p107 is readily detected in the nuclei of proliferating lower crypt cells (P). Coincident with growth arrest (arrow), p107 expression decreases to barely detectable levels. (bi and bii) pRb staining is evident in both nuclear and cytosolic compartments of proliferating crypt cells (P), and becomes predominantly nuclear in postmitotic cells of the upper crypt and villus (V). The arrow indicates the point of growth arrest. (c) p130 staining is low in proliferating crypt cells (P), but increases markedly coincident with growth arrest (arrow). (d) Whole cell lysates (30 μg protein) of isolated crypt (C), lower villus (LV), and upper villus (UV) cells were subjected to Western blot analysis for expression and migration/phosphorylation state of pocket proteins. Note that the crypt fraction includes some postmitotic cells of the upper crypt region. p130 form 3, which is only found in cycling cells, is only detected in the crypt fraction; the presence of forms 1 and 2 in this fraction reflects the postmitotic cells in this sample. Data are representative of at least three independent experiments. Bar, 10 μm.

Mentions: To examine the physiological relevance of these data, a combined biochemical and morphological approach (described previously in Saxon et al. 1994) was used to compare the developmental regulation of pocket protein expression, subcellular distribution, and phosphorylation state along the crypt–villus axis in situ with changes in PKC activation/expression. Immunofluorescence analysis revealed that p107, pRb, and low levels of p130 are expressed in proliferating crypt cells (Fig. 3, a–c): p107 staining was predominantly nuclear, whereas pRb staining was detected both in the cytoplasm and the nucleus. Coincident with cell growth arrest and PKC activation in the midcrypt region (cell position 14–18 from the crypt base; Fig. 1b and Fig. c), all three pocket proteins underwent changes in expression and/or subcellular compartmentalization. p107 became barely detectable in postmitotic cells, whereas staining for pRb became restricted to the nucleus. Levels of p130, on the other hand, increased markedly with growth arrest and remained elevated on the villus, decreasing somewhat towards the villus tip. Consistent with these immunofluorescence data, Western blot analysis of pocket protein expression in isolated crypt, lower villus, and upper villus cells demonstrated marked decreases in levels of p107 and pRb in villus cells relative to crypt cells (Fig. 3 d). Similar levels of p130 were detected in the crypt and lower villus fractions, likely reflecting the presence of postmitotic upper crypt cells in the crypt sample and of some midvillus cells in the lower villus fraction (see Materials and Methods). Western blot analysis also demonstrated that pRb and p130 are primarily expressed in their underphosphorylated forms in postmitotic cells of the villus. Comparison of the data obtained from in vitro and in situ studies (see Fig. 2 above) demonstrates that direct activation of PKC isozymes in IEC-18 cells leads to alterations in pocket protein expression and phosphorylation state that closely parallel those seen coincident with PKC activation within intestinal crypts in situ. Notably, the data demonstrate that PKC activation in this system can initiate a coordinated program of pocket protein regulation that has been associated with cell cycle exit in several systems (Garriga et al. 1998).


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 pocket protein expression and phosphorylation state in the intestinal epithelium in situ. (a–c) Immunofluorescence localization of pocket proteins. P, proliferation zone; D, differentiation zone; V, villus/functional zone. (a) p107 is readily detected in the nuclei of proliferating lower crypt cells (P). Coincident with growth arrest (arrow), p107 expression decreases to barely detectable levels. (bi and bii) pRb staining is evident in both nuclear and cytosolic compartments of proliferating crypt cells (P), and becomes predominantly nuclear in postmitotic cells of the upper crypt and villus (V). The arrow indicates the point of growth arrest. (c) p130 staining is low in proliferating crypt cells (P), but increases markedly coincident with growth arrest (arrow). (d) Whole cell lysates (30 μg protein) of isolated crypt (C), lower villus (LV), and upper villus (UV) cells were subjected to Western blot analysis for expression and migration/phosphorylation state of pocket proteins. Note that the crypt fraction includes some postmitotic cells of the upper crypt region. p130 form 3, which is only found in cycling cells, is only detected in the crypt fraction; the presence of forms 1 and 2 in this fraction reflects the postmitotic cells in this sample. Data are representative of at least three independent experiments. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Developmental regulation of pocket protein expression and phosphorylation state in the intestinal epithelium in situ. (a–c) Immunofluorescence localization of pocket proteins. P, proliferation zone; D, differentiation zone; V, villus/functional zone. (a) p107 is readily detected in the nuclei of proliferating lower crypt cells (P). Coincident with growth arrest (arrow), p107 expression decreases to barely detectable levels. (bi and bii) pRb staining is evident in both nuclear and cytosolic compartments of proliferating crypt cells (P), and becomes predominantly nuclear in postmitotic cells of the upper crypt and villus (V). The arrow indicates the point of growth arrest. (c) p130 staining is low in proliferating crypt cells (P), but increases markedly coincident with growth arrest (arrow). (d) Whole cell lysates (30 μg protein) of isolated crypt (C), lower villus (LV), and upper villus (UV) cells were subjected to Western blot analysis for expression and migration/phosphorylation state of pocket proteins. Note that the crypt fraction includes some postmitotic cells of the upper crypt region. p130 form 3, which is only found in cycling cells, is only detected in the crypt fraction; the presence of forms 1 and 2 in this fraction reflects the postmitotic cells in this sample. Data are representative of at least three independent experiments. Bar, 10 μm.
Mentions: To examine the physiological relevance of these data, a combined biochemical and morphological approach (described previously in Saxon et al. 1994) was used to compare the developmental regulation of pocket protein expression, subcellular distribution, and phosphorylation state along the crypt–villus axis in situ with changes in PKC activation/expression. Immunofluorescence analysis revealed that p107, pRb, and low levels of p130 are expressed in proliferating crypt cells (Fig. 3, a–c): p107 staining was predominantly nuclear, whereas pRb staining was detected both in the cytoplasm and the nucleus. Coincident with cell growth arrest and PKC activation in the midcrypt region (cell position 14–18 from the crypt base; Fig. 1b and Fig. c), all three pocket proteins underwent changes in expression and/or subcellular compartmentalization. p107 became barely detectable in postmitotic cells, whereas staining for pRb became restricted to the nucleus. Levels of p130, on the other hand, increased markedly with growth arrest and remained elevated on the villus, decreasing somewhat towards the villus tip. Consistent with these immunofluorescence data, Western blot analysis of pocket protein expression in isolated crypt, lower villus, and upper villus cells demonstrated marked decreases in levels of p107 and pRb in villus cells relative to crypt cells (Fig. 3 d). Similar levels of p130 were detected in the crypt and lower villus fractions, likely reflecting the presence of postmitotic upper crypt cells in the crypt sample and of some midvillus cells in the lower villus fraction (see Materials and Methods). Western blot analysis also demonstrated that pRb and p130 are primarily expressed in their underphosphorylated forms in postmitotic cells of the villus. Comparison of the data obtained from in vitro and in situ studies (see Fig. 2 above) demonstrates that direct activation of PKC isozymes in IEC-18 cells leads to alterations in pocket protein expression and phosphorylation state that closely parallel those seen coincident with PKC activation within intestinal crypts in situ. Notably, the data demonstrate that PKC activation in this system can initiate a coordinated program of pocket protein regulation that has been associated with cell cycle exit in several systems (Garriga et al. 1998).

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