Calcium Mobilization through L-type Channels in Hepatic Stellate Cell Is Essential for TGF-b-mediated CTGF = Calcium Mobilization through L-type Channels in Hepatic Stellate Cell Is Essential for TGF-b-mediated CTGF

다국어 초록 (Multilingual Abstract)

Aims: In hepatic fibrogenesis hepatic stellate cell (HSC) is a major cell type responsible for producing a major profibrogenic cytokine TGF-b, and connective tissue growth factor (CTGF), a major fibrogenic mediator in several organs. The multi-functional nature of TGF-b signaling in hepatic fibrogenesis is still elusive. At the previous The Liver Week(2015) we reported that Pyk2 is essential for TGF-b-mediated, Smad-independent CTGF induction. Pyk2 is known to be calcium- sensitive, and TGF-b was reported to increase intracellular calcium level. Therefore, we investigated the relation between intracellular calcium levels and pro-fibrogenic TGF-b/Pyk2 axis in hepatic stellate cell. Methods: Immortalized human stellate cell line, LX-2, has been cultured. After TGF-b treatment, expression of CTGF and a-SMA were assessed with RT-PCR and western blot. Pharmacological inhibitor and siRNA-mediated knockdown were used to modulate the activities and expression levels of protein. Intracelllular calcium mobilization was measured with Fura-4/AM. Activation of Pyk2 was addressed in western blot using different phosphorylation site-specific antibodies. Results: CTGF expression was up-regulated within 1hr in TGF-b stimulated LX-2. This up-regulation was greatly suppressed by siRNA-mediated knockdown and pharmacological inhibitor of Pyk2. TGF-b treatment increased phosphorylation of Pyk2 on tyrosine 402, 579/580, and 881. Consistent with the previous reports, TGF-b increased intracellular calcium concentration in Fura-4-preloaded LX-2. CTGF induction by TGF-b was blocked in dose dependent manner by pre-treatment with BAPTA-AM, an intracellular calcium chelator, while A23187, a calcium ionopore, increased CTGF induction even in the absence of TGF-b, suggesting that increase of intracellular calcium level is enough to induce CTGF expression. In addition, A23187 increased phosphorylation of Pyk2, and CTGF induction by A23187 is also greatly reduced by siRNA of Pyk2. Pre-treament of LX-2 with Nifedipine (an L-type calcium channel blocker) suppressed CTGF induction by TGF-b in dose-dependent manner, while FPL64176 (L-type channel activator) increased CTGF expression without TGF-b. The CTGF up-regulations by TGF-b, A23187, and FPL64176 were all suppressed by siRNA-mediated knockdown and pharmacological inhibitors of Pyk2. Conclusions: In hepatic stellate cell, TGF-b increases the intracellular calcium level through L-type calcium channel, leading to downstream Pyk2 signaling for CTGF induction.