Effects Of EPAC On The Proliferation And Activation Of Rat Hepatic Stellate Cells Induced By Acetaldehyde

Alcoholic liver disease (ALD) is one of the most common etiologies of liver disease and has become a main cause of morbidity and mortality worldwide. ALD encompasses a broad spectrum of disorders, ranging from fatty liver to more severe forms of liver injury, including alcoholic hepatitis, fibrosis, cirrhosis, and superimposed hepatocellular carcinoma. Alcoholic liver fibrosis (ALF) is identified as a breaking point in ALD because it can lead to cirrhosis. Mounting evidence suggests that the principal metabolite of ethanol, acetaldehyde, plays a critical role in the onset and maintenance of the fibrogenetic process. Many studies have demonstrated that acetaldehyde stimulates the deposition of extracellular matrix (ECM) by activating the hepatic stellate cells (HSCs), the major cell type involved in ALF. As response to acetaldehyde-induced stimulation, HSCs change from quiescent vitamin A-storing cells to activated myofibroblast-like cells, which proliferate and become the primary source of ECM.The cyclic adenosine 3’,5’-monophosphate (cAMP) is a prominent multifunctional intracellular second messenger. Most of the effects of cAMP are dependent upon the activation of its downstream effectors protein kinase A (PKA). In our previous study, we found that the activation of cAMP/PKA signaling pathway promotes the activation and proliferation of HSCs.Exchange protein activated by cAMP (EPAC) regulate many cellular processes, including migration, proliferation and apoptosis, via a PKA-independent pathway. There are two main EPAC isoforms (EPAC1 and EPAC2, also known as RapGEF3 and RapGEF4, respectively) that are expressed from different genes in a variety of tissues. EPAC proteins respond to cAMP and act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 which were activated by exchanging bound GDP for GTP, while GTPase activating proteins return them to inactive GDP-bound states. Based on these findings, we hypothesized that cAMP may influence acetaldehyde-induced HSCs activation by regulating the activation of both PKA and EPAC signaling pathways.In this study, the rat HSC line were cultured and treated with 200 μM acetaldehyde for 48 h, which was proposed to mimic the activation process in vivo. We then tried to identify the role of cAMP/EPAC/Rapl signaling pathway in the regulation of HSCs activation and proliferation induced by acetaldehyde. The main contents can be summarized as follows:1. Quantification of the expression of EPAC1 and EPAC2 in acetaldehyde-induced HSCsTo investigate whether the expression of EPAC1 and EPAC2 were altered in acetaldehyde-induced HSCs, the QRT-PCR and western blotting were applied to quantify the levels of the mRNA and protein for EPAC1 and EPAC2. The HSCs were treated with 200 μM acetaldehyde for 48 h. Our results showed that the expression of EPAC2 at both protein and mRNA levels were significantly increased as compared with that of control group, whereas the expression of EPAC 1 was decreased markedly by the addition of acetaldehyde.2. Effect of EPAC on proliferation and activation of acetaldehyde-induced HSCsThe HSCs were pretreated with acetaldehyde (200 μM) for 24 h, and co-cultured with Me-cAMP (50 μM) for 24 h. Results showed that Me-cAMP restored the expression of EPAC 1, and the expression EPAC2 was slightly increased. MTT assay showed that Me-cAMP displayed remarkable inhibition of the proliferation in acetaldehyde-induced HSCs. The results of cell cycle analysis showed that Me-cAMP increased the ratio of cells in the G0/G1 phase and decreased those in the S and G2/M phase. There were significant differences in the ratio of cells in G0/G1 phase and S plus G2/M phase between Me-cAMP-treated groups and model group. Furthermore, addition of Me-cAMP potently decreased the mRNA and protein expression of a-SMA, collagen type I and III in acetaldehyde-induced HSCs. To gain insight into the potential signaling pathways mediated by EPAC, the protein levels of both Rapl-GTP and total Rapl were determined. The expression of Rapl-GTP were significantly downregulated by acetaldehyde but upregulated by Me-cAMP, whereas total Rapl protein levels remained unchanged, indicating that EPAC activation by Me-cAMP enhanced the expression of GTP-bound (activated) Rapl.3. Effect of EPAC siRNA on proliferation and activation of acetaldehyde-induced HSCsTo further dissect the functional role of EPAC1 and EPAC2, the small interfering RNA was used. After 48 h following the transfection, the cells were photographed at x920 magnification by fluorescence microscope. Transfection efficiency detected by western blot and QRT-PCR. EPAC2 siRNA significantly inhibited the proliferation of HSCs, whereas EPAC1 siRNA and double knockdown of EPAC 1 and 2 remarkably increased the rate of HSC proliferation. In accordance with the findings from previous studies, the results of cell cycle analysis showed that only EPAC2 siRNA enhanced the ratio of cells in the G0/G1 phase and decreased those in the S and G2/M phase apparently. In addition, double knockdown of EPAC 1 and 2 had a profound promotion of the expression of a-SMA and collagen type Ⅰ and type Ⅲ.QRT-PCR and western blot results indicated that knockdown of EPAC1 further enhanced acetaldehyde-induced a-SMA and collagen type Ⅰ and Ⅲ mRNA and protein levels. However, EPAC2 depletion prevented the increase of both collagen type Ⅰ and Ⅲ, and inhibited acetaldehyde-induced a-SMA expression strongly. Taken together, these data suggested that the activation and proliferation of HSCs could be suppressed by EPAC1 and promoted by EPAC2.4. EPAC and PKA activation had opposite effects on synthesis of collagen and a-SMA in acetaldehyde-induced HSCsTo better understand the roles of individual cAMP effectors EPAC and PKA in the regulation of HSCs activation induced by acetaldehyde, we applied the selective PKA activator Phe-cAMP and the selective EPAC activator Me-cAMP. Consistent with our previous findings, treatment of acetaldehyde-induced HSCs with Me-cAMP significantly decreased the protein expression of markers of HSC activation, including α-SMA, collagen type Ⅰ and Ⅲ. In contrast, the activation of PKA obviously enhanced the expression of the HSCs activation marker. In addition, we also assessed the role of Rap 1, a downstream target of EPAC, on EPAC-regulated activation of acetaldehyde-induced HSCs. EPAC activation by Me-cAMP increased the expression of GTP-bound Rapl, while PKA activation by Phe-cAMP had negligible effects on such binding. These data further demonstrated that EPAC could play a protective role against acetaldehyde-induced HSCs activation via Rap1.