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Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells.

ABSTRACT: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells.Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA.CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release.Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

SUBMITTER: Gkoumassi E

PROVIDER: S-EPMC2042924 | biostudies-other | 2007 Aug

REPOSITORIES: biostudies-other

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Project description:MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.

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Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells.

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