Regulation of Cytokines and G-protein gene expression by Cholera toxin

Zafar Nawaz1, Bukhtiar H Shah2, University of karachi; 2, Aga khan University

Infection with Vibrio cholera is a major cause of secretory diarrhoea in under-developed as well as in developed countries. V. cholera produces cholera toxin (CTX), responsible for symptoms of diarrhoea. In the present study, the molecular mechanism of CTX action was studied in mice intestines. Results showed that CTX (1mg/ml) increased intracellular cAMP levels as measured by radio immuno assay. However, immunoblot results revealed that CTX caused a decrease in Gas but increase membrane Gaq without any change in Gai proteins. cAMP analogue, dbcAMP, treatment did not mimic the effects of CTX, indicating that the effects of CTX on G-proteins were cAMP-independent. The CTX-induced reduction in Gas levels was not reflected by change in its transcripts, however, Gaq increase was accompanied by increase in Gaq mRNA levels as detected by RT/PCR. Lack of any effect on Gas mRNA levels revealed that CTX down regulated Gs-a from membrane through some proteolytic pathway. Proinflammatory cytokines play an important role in the pathogenesis of infectious diseases. To examine if cytokines, IL-6 and TNF-a, were involved in CTX action, their levels were measured by ELISA in intestinal fluid. The results showed that treatment with CTX increased both IL-6 and TNF-a and their mRNA levels in intestinal epithelial cells. The mechanism of CTX-mediated increase in cytokines was examined using pharmacological inhibitors of different signalling pathways. It was found that CTX-mediated regulation of IL-6 and TNF-a involved PKC, MAP kinase, tyrosine kinase and transcription factor NF-kB at transcriptional level as reflected by changes in cytokine mRNA levels. These results showed that stimulation of G-proteins with CTX led to induction of diverse signaling pathways which increased the expression of IL-6 and TNF-a genes. Nitric Oxide (NO) is involved in many physiological and pathophysiological conditions in the body. NO has a short half-life and is rapidly converted into nitrite, which can be measured by Griess reaction. It was found that CTX treatment increased NO as well as iNOS mRNA levels in intestines. dbcAMP and forskolin had no significant changes in NO levels indicating that the NO production was independent of cAMP/PKA pathway. Likewise CTX-mediated NO production was not effected by PKC inhibitors. However, CTX-induced NO formtion and iNOS expression were inhibited by selective inhibitors of protein tyrosine kinases, MEK and NF-kB suggesting the involvement of tyrosine kinae/MAP kinase linked pathways in the cascade. These studies demonstrate that CTX exerts its effects through activation of both G-proteins and tyrosine kinase linked signalling pathways in mice intestine.