Pathogenic inflammation of the colonic mucosa is accompanied by suppression of circular muscle contractility, which makes important contributions to the symptom of diarrhea. Previous studies have established that the suppression of contractility results from specific changes in the expression of cell signaling molecules that mediate excitation-contraction coupling. In particular, a decrease in the expression of the pore-forming alpha1c subunit of L-type calcium channels, which reduces calcium influx, is a key factor in this effect. The alterations in the expression of cell signaling molecules are mediated by inflammatory response mediators, such as TNFalpha released from the immunocytes in the muscularis externa. Our hypothesis is that TNFalpha suppresses contractility by down-regulating the gene expression of the alpha1c subunit of L-type calcium channels through the activation of transcription factor NF-kappaB and by modulating alpha1c mRNA stability. This hypothesis will be tested in studies performed on primary cultures of human colonic circular smooth muscle cells (HCCSMC).
The specific aims of the proposal are to investigate: 1) whether NF-kappaB activation in HCCSMC muscle strips down-regulates the expression of alpha1c subunit of L-type calcium channels, 2) the molecular mechanisms of down-regulation of (alpha1c by p50 and p65 subunits of NF-KappaB in response to TNFalpha, 3) which signaling molecules regulate the activation of NF-kappaB subunits in HCCSMC in response to TNFalpha, 4) whether TNFalpha alters the stability of a1c mRNA and, if so, is this effect mediated by mitogen-activated protein kinases (MAPKs), and 5) whether TNFalpha treatment of circular muscle strips suppresses their contractility and, if so, is this effect mediated by p50, p65, PKCzeta and MAPKs. The experiments will utilize complimentary pharmacologic and molecular approaches. The findings will suggest new cellular targets for therapeutic interventions to modulate physiologically and pathophysiologically important contractions of colonic smooth muscle. The inhibition of NF-kappaB activation is an emerging therapy in treating inflammatory disorders.
Showing the most recent 10 out of 87 publications
