RESUMO
BACKGROUND AND AIMS: Fibronectin (FN) is an essential factor for the induction of migration of primary colonic lamina propria fibroblasts (CLPF). The FN isoform ED-A is an important inducer of migration. Recently, we have shown that CLPF isolated from inflamed Crohn's disease (CD) mucosa migrated significantly less than control CLPF. We, therefore, investigated changes in FN or integrin expression that could be relevant for CLPF migration. MATERIALS AND METHODS: mRNA of control-CLPF and CLPF isolated from fibrotic mucosa of CD patients was subtractively hybridized. Expression of FN, ED-A, and ED-B in frozen sections from intestinal mucosa was determined by immunohistochemistry. The mRNA expression of the FN isoforms in control, CD, and fibrosis biopsies was quantified by real-time polymerase chain reaction (PCR). Integrin alpha5beta1 protein and mRNA expression was analyzed by fluorescence activated cell sorting (FACS) and PCR, respectively. RESULTS: Subtractive hybridization indicated differential regulation of FN isoform expression in CD. The immunohistochemical analysis of FN protein revealed a reduction of FN isoforms in inflamed CD mucosa compared to control mucosa. In CD fistulae, the ED-A and ED-B isoforms were virtually absent. In fibrotic mucosa, both proteins were increased. Real-time PCR showed a decrease of FN and ED-A expression during mucosal inflammation in CD in contrast to UC and a significant increase of FN and isoforms in CD fibrosis. No difference was found for protein and mRNA of integrin alpha5beta1 in control, CD, and fibrosis CLPF by FACS and PCR. CONCLUSION: Downregulated expression of migration-inducing FN-isoforms in contrast to unchanged FN receptor expression may contribute to the observed alterations of CD CLPF migration.
Assuntos
Processamento Alternativo/genética , Doença de Crohn/genética , Fibronectinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Adulto , Idoso , Estudos de Casos e Controles , DNA Complementar , Feminino , Fibroblastos/patologia , Fibronectinas/química , Fibronectinas/metabolismo , Citometria de Fluxo , Humanos , Integrina alfa5beta1/metabolismo , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Interleukin-11 (IL-11) has been evaluated as an anti-inflammatory and mucosa-protective therapeutic agent in inflammatory bowel diseases (IBDs). Activity of IL-11 requires binding to the alpha receptor subunit (IL-11Ralpha) that provides ligand specificity. Recently, we showed that in the intestinal mucosa, IL-11Ralpha is mainly present on epithelial cells mediating antiapoptotic effects. The aim of this study was to investigate the expression profiling of IL-11Ralpha and its downstream signaling cascade in colonic adenoma and carcinoma. MATERIALS AND METHODS: The expression of IL-11Ralpha in normal colonic mucosa, 11 colonic adenomas, and 10 carcinomas was analyzed by immunohistochemistry. In addition, IL-11Ralpha-expression and IL-11Ralpha-induced phosphorylation of signal transducer and activator of transcription (STAT)3 were investigated by Western blot analysis. RESULTS: Immunohistochemistry revealed significant IL-11-Ralpha expression in epithelial cells of normal colonic mucosa. In contrast, the expression of IL-11-Ralpha in colon adenomas and carcinomas was either absent or only detectable in very few scattered epithelial cells. Densitometric analysis of Western blots confirmed these results, showing a decrease of IL-11Ralpha-protein in cells isolated from adenomas or carcinomas. Reduced STAT3-phosphorylation in carcinoma cells indicated functional consequences of decreased IL-11Ralpha-protein expression on signal transduction. CONCLUSION: This study demonstrates a decrease of IL-11-Ralpha-protein expression in epithelial cells isolated from colon carcinomas and adenomas compared to normal colonic mucosa and a reduced STAT3 signaling. Because of reduced binding and signal transduction, it is unlikely that therapeutically administered IL-11 would contribute to colorectal carcinoma induction and growth.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias do Colo/metabolismo , Subunidade alfa de Receptor de Interleucina-11/biossíntese , Mucosa Intestinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Neoplasias do Colo/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/fisiologiaRESUMO
A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Ralpha monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Ralpha selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Ralpha mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Ralpha restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity.