Ablation of DGK facilitates -smooth muscle actin expression via the Smad and PKC signaling pathways during the acute phase of CCl₄-induced hepatic injury

Expression of alpha-smooth muscle actin (alpha SMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by alpha SMA expression, which is regulated by the TGF beta-induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate alpha SMA expression. Diacylglycerol kinase (DGK) metabolizes a second-messenger DG, thereby controlling components of DG-mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGK alpha in alpha SMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes alpha SMA expression and PKC delta phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGF beta stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGF beta stimulation engenders alpha SMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGF beta stimulation significantly induces alpha SMA incorporation in stress fibers. Through animal model experiments, we observed that DGK alpha-knockout mice exhibit increased expression of alpha SMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKC delta. Together, these findings suggest that DGK alpha negatively regulates alpha SMA expression by acting on the Smad and PKC delta signaling pathways, which differentially regulate stress fiber incorporation and protein expression of alpha SMA, respectively.