RESUMEN
Members of the C/EBP (CCAAT/enhancer binding protein) family of transcription factors play important roles in mediating the acute-phase response (APR), an inflammatory process resulting from infection and/or tissue damage. Among the C/EBP family of proteins, C/EBPbeta and -delta were thought to be the primary mediators of the APR. The function of C/EBPalpha in the APR has not been fully characterized to date. Here, we investigate the role of C/EBPalpha in the APR by using neonatal mice that lack C/EBPalpha expression. Northern blot analysis of acute-phase protein gene expression in neonatal mice treated with purified bacterial lipopolysaccharide or recombinant interleukin 1beta as an inflammation stimulus showed a strong APR in wild-type mice, but a response in C/EBPalpha null animals was completely lacking. The C/EBPalpha knockout and wild-type mice demonstrated elevations in C/EBPbeta and -delta mRNA expression and DNA binding as well as increased DNA binding of NF-kappaB, all of which are known to be important in the APR. Null mice, however, failed to activate STAT3 binding in response to lipopolysaccharide. Our results provide the first evidence that C/EBPalpha is absolutely required for the APR in neonatal mice, is involved in STAT3 regulation, and cannot be compensated for by other C/EBP family members.
Asunto(s)
Reacción de Fase Aguda/genética , Proteínas de Unión al ADN/genética , Inflamación/genética , Proteínas Nucleares/genética , Proteínas de Fase Aguda/genética , Reacción de Fase Aguda/inducido químicamente , Animales , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Genotipo , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Hígado/metabolismo , Pulmón/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , ARN Mensajero/genética , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels.
Asunto(s)
Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Hígado/citología , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Animales , Animales Recién Nacidos , Proteínas Potenciadoras de Unión a CCAAT , División Celular/genética , División Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , ADN/biosíntesis , Proteínas de Unión al ADN/genética , Técnicas In Vitro , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Both CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta are intronless, yet can create various N-terminally truncated protein products with distinct DNA binding and transactivation potentials. These proteins can be generated via two distinct mechanisms, one translational and the other post-translational. In the translational mechanism, there is alternative translational start site selection of the different AUG codons present in the single messenger RNA (mRNA) species via a process of leaky ribosome scanning. Additionally, a post-translational method of isoform formation, through specific proteolytic cleavage of the full length protein has also been described. In this manuscript, we present evidence that the production of C/EBPbeta protein isoforms in the neonatal mouse liver is regulated by C/EBPalpha. In C/EBPalpha knockout mice, the predominant C/EBPbeta proteins are the larger 38- and 35-kd isoforms, whereas wild-type animals primarily possess the smaller 21- and 14-kd isoforms. These C/EBPalpha-dependent differences are liver specific, not present in lung or adipose tissues, and present at day 18 of development. Additionally, we show that induction of C/EBPalpha expression leads to an increase in the production of the 21-kd C/EBPbeta isoform in cell culture studies. As the various C/EBPbeta protein isoforms have different transcriptional capabilities, it is important to understand the regulation of the production of these isoforms. Our observations suggest a novel role for the C/EBPalpha transcription factor in this process.