RESUMO
We previously reported that ras and polyoma middle T (PyMT), a constitutive activator of the src protooncogene product, up-regulated Caco-2 cell proliferation along with protein kinase C (PKC) alpha expression and PKC activity. We aimed to investigate whether oncogene-induced up-regulation of Caco-2 cell proliferation involved stimulation of the autocrine IGF-II/IGF-I receptor (IGFIR) loop described in these cells and if so, to analyse the role of overexpressed and activated PKC. Compared with control vector transfected Caco-2 cells, ras- and PyMT-transfected cells exhibited increased expression of the 6.0 and 4.8 kb IGF-II transcripts. This was due to increased activity of the P3 and P4 promoters of the IGF-II gene which correlated with increased expression and DNA-binding activity of Sp1, a transcription factor interacting with several specific sites in P3 and P4 promoters. Oncogene-transfected cells displayed enhanced autocrine IGF-II production, which was fully responsible for the oncogene-induced increase in their proliferation since this increase was blunted by anti-human IGF-II and IGF1R (alphaIR3) antibodies. PKC mediated oncogene activation of the IGF-II gene presumably through action on Sp1 since (i) PKC activation by phorbol 12-myristate 13-acetate increased Sp1 expression, P3 and P4 activity and IGF-II mRNA in control but not in oncogene-transfected cells; and (ii) PKC inhibition by the PKC inhibitor Gö6976 reduced Sp1, P3 and P4 activity and IGF-II mRNA in all three cell lines. This is the first evidence that ras- and PyMT/src oncogenes up-regulate Caco-2 cell proliferation through a PKC-mediated pathway which stimulates IGF-II gene transcription and thereby increases autocrine IGF-II production. The mechanisms underlying IGF-II gene activation by PKC most probably involve action on Sp1.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Divisão Celular/genética , Regulação Neoplásica da Expressão Gênica , Genes ras , Fator de Crescimento Insulin-Like II/genética , Oncogenes , Proteína Quinase C/metabolismo , Transcrição Gênica , Anticorpos Monoclonais/farmacologia , Antígenos Transformantes de Poliomavirus/biossíntese , Células CACO-2 , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/biossíntese , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like II/biossíntese , Fator de Crescimento Insulin-Like II/fisiologia , Receptor IGF Tipo 1/fisiologia , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional , TransfecçãoRESUMO
In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-beta, serum withdrawal, loss of cell adhesion (anoikis) and TNF-alpha. Smad7 increased TGF-beta-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-kappaB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-kappaB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-alpha stimulation of NF-kappaB and increased TNF-alpha-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-kappaB factor, providing a potential mechanism whereby Smad7 potentiates cell death.