RESUMO
Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25-dihydroxyvitamin D3 (calcitriol) and all-trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger-1 (MZF-1), a hematopoietic transcription factor. We show the interaction of MZF-1 with the CTGF and NOV promoters in several cell types. Up-regulation of MZF-1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF-1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/biossíntese , Hematopoese/genética , Fatores de Transcrição Kruppel-Like/genética , Proteína Sobre-Expressa em Nefroblastoma/biossíntese , Medula Óssea/metabolismo , Calcitriol/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Tretinoína/metabolismoRESUMO
Recently, new tissue-specific functions for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been discovered, aside from its archetypal function in glycolysis. This casts doubt on the legitimacy of using GAPDH as a normalization control for gene expression analysis. We report the binding of the myeloid zinc finger-1 (MZF-1) transcription factor to the human GAPDH promoter. Furthermore, we show that up-regulation of MZF-1 by 1,25-dihydroxyvitamin D3 (calcitriol) induces GAPDH in HS-5 stromal fibroblasts, while knockdown of MZF1 by shRNA leads to a concomitant reduction in GAPDH expression. This argues that MZF-1 regulates GAPDH, indicating a role for GAPDH in calcitriol-mediated signaling.
Assuntos
Calcitriol/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Sequência de Bases , Calcitriol/metabolismo , Linhagem Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Transdução de Sinais , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Intestinal epithelial cells (IEC) play a role in mucosal inflammation by producing pro-inflammatory chemokines that may initiate or amplify local responses. IL-1 is a potent activator of IEC and its receptor localizes to focal adhesions. Since the Rho-associated kinase, ROCK, also localizes to focal adhesions, we examined the role of ROCK in IL-1-induced chemokine responses in IEC cell lines. Suppressing ROCK with the Y27632 inhibitor suppressed IL-1-stimulated Caco-2 cell CXCL8/IL-8 and IEC-6 cell CCL2/MCP-1 secretion and mRNA levels. ROCK inhibition also suppressed IL-1-induced JNK phosphorylation in both cell lines, but high levels of the inhibitor had no significant effect on IL-1-stimulated Caco-2 IκBα phosphorylation and degradation or IKK phosphorylation and kinase activity. Therefore, ROCK may exert an effect on IL-1-stimulated JNK signaling to AP-1 activation, with little effect on IKK/IκBα signaling, defining a potentially important mechanism for regulating IL-1 signaling in IEC that may be essential for optimal cytokine responses.
Assuntos
Células Epiteliais/efeitos dos fármacos , Interleucina-1/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Quinases Associadas a rho/fisiologia , Células CACO-2 , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/fisiologia , Fosforilação , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Intestinal epithelial cells express the alpha3beta1 integrin which binds to laminin-5. We have previously shown that activation of the alpha3 integrin through laminin-5 binding or a cross-linking antibody results in a suppression of IL-1 induced cytokine secretion and intracellular signaling through IKK to NF-kappaB and JNK to AP-1 in Caco-2 cells. In the present study, the effects of alpha3 integrin activation on the proximal events of IL-1 induced signaling were examined. Monoclonal antibody activation of the alpha3 integrin on Caco-2 cells prior to IL-1 stimulation had no effect on the association of the adapter protein TAB2 with TAK1. However, the association of TRAF6 with TAK1, and TRAF6 with the IL-1 receptor I was significantly suppressed. Activation of the alpha3 integrin had no effect on total levels of TRAF6. Finally, the IL-1 induced formation of higher molecular weight, presumably phosphorylated, forms of IRAK-1 were not altered by alpha3 integrin activation, suggesting that signaling events leading up to IRAK-1 were unaffected. These results suggest that the suppressive effects of alpha3 integrin activation on IL-1 signaling may be due to an effect on the function of TRAF6, preventing the transmission of the signal from the IL-1RI complex to the TAK1 complex.