Regulation of attachment and proliferation of interstitial cells of Cajal by c-Kit
Gibbons, Simon J Roeder, Jaime L Wouters, Mira Farrugia, Gianrico #
Neurogastroenterology and motility pages:23-24
ISNM location:Korea date:September 2007
Background: Interstitial cells of Cajal (ICC) are required for normal gastrointestinal motility. The signaling pathways that regulate ICC proliferation are not well understood. 5-HT2B receptors are expressed on ICC and activation of the 5-HT2B receptor on ICC results in proliferation. 5-HT2B receptor activation is known to induce proliferation through phospholipase C (PLC) and phosphatidyl inositol 3’-kinase (PI3K) in other cell types. The aim of this study was to identify the signaling cascades involved in the effect of 5-HT2B receptors on proliferation of ICC.
Methods: Experiments were carried out on primary cell cultures from small intestinal muscle strips obtained from 2-3 day old mice. Enzymatically dissociated cells were plated in serum containing medium on a feeder layer of Steel factor producing fibroblasts to enrich for ICC. LY294002, a PI3K inhibitor and U73122, a PLC inhibitor, were used to determine if the increase in proliferation induced by the 5-HT2B receptor agonist BW723C86 was through PI3K or PLC. Compounds were added every 20h and cell cultures were fixed at 48 hours. ACK-2 and Ki67 antisera were used to label for ICC and proliferating cells respectively. Data were compared using a Wilcoxon matched-pairs signed-ranks test and data are presented as mean ± s.e.m. Every n-value represents a separate experiment.
Results: As previously reported, the 5-HT2B receptor agonist, BW723C86 (50 nM) increased proliferation of ICC in primary cell culture in all experiments. In the presence of the PI3K inhibitor LY294002 (1 µM), proliferation of ICC was reduced (control; 17.2±2.6%, LY; 11.0±2.2%, n=6, p=0.03). However, activation of 5-HT2B receptor still resulted in increased proliferation in the presence of 1 µM LY294002 (LY; 11.0±2.0%, LY + BW; 16.8±3.2%, n=6; p=0.03). The PLC inhibitor, U73122, had no significant effect on proliferation of ICC (control; 19.8±1.5%, 1 M U; 24.3±5.5%, 10 µM U; 16.6±1.4%, n=6, p>0.05). In contrast to the lack of effect of PI3K inhibition, the proliferative response of ICC to 5HT2B receptor activation was blocked by the PLC inhibitor U73122 (1 µM U; 24.3±5.5%, 1 µM U + BW; 21.7±5.9%, 10 µM U; 16.6±1.4%, 10 µM U + BW 18.6±2.2%; n=6 respectively). Conclusion: Increased proliferation due to activation of the 5-HT2B receptor on ICC is PLC dependent and does not appear to require activation of PI3K.
Supported by NIH grant DK57061.