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1.
J Biol Chem ; 286(20): 18104-17, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454537

RESUMO

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.


Assuntos
Neoplasias do Colo/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais , Animais , Neoplasias do Colo/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Homeostase/genética , Proteína 1 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteína Quinase C-alfa/genética , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Exp Cell Res ; 315(8): 1415-28, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19232344

RESUMO

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCalpha in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCalpha or PKCdelta in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCalpha and delta levels were generally reduced in colon carcinoma cell lines, PKCbetaII was elevated and PKCepsilon showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCalpha downregulated cyclin D1 by two independent mechanisms. PKCalpha expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCdelta had modest and variable effects on cyclin D1 steady-state levels and failed to restore responsiveness to PKC agonists. Notably, PKCalpha expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCdelta had only minor effects. Loss of PKCalpha and effects of its re-expression were independent of the status of the APC/beta-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCalpha is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.


Assuntos
Ciclina D1/metabolismo , Neoplasias Intestinais/fisiopatologia , Proteína Quinase C/metabolismo , Animais , Linhagem Celular Tumoral , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas , Proteína Quinase C/farmacologia , Ratos , Transdução de Sinais , Transcrição Gênica
3.
J Biol Chem ; 282(19): 14213-25, 2007 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-17360714

RESUMO

We reported previously that protein kinase Calpha (PKCalpha), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCalpha-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr(45) and Ser(64) on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCalpha-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an approximately 2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H(2)O(2) and ceramide, two naturally occurring PKCalpha agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCalpha-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCalpha-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.


Assuntos
Proteínas de Transporte/metabolismo , Ciclinas/metabolismo , Mucosa Intestinal/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Ciclina D , Regulação para Baixo , Células Epiteliais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteína Fosfatase 2 , Ratos , Transdução de Sinais
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