MOLECULAR MEDICINE REPORTS 18: 3997-4003, 2018 Hypoxia‑inducible factor 1α and ROCK1 regulate proliferation and collagen synthesis in hepatic stellate cells under hypoxia YIBING HU, DANPING HU, HUANHUAN YU, WANGWANG XU and RONGQUAN FU Department of Infectious Diseases, The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325200, P.R. China Received May 16, 2017; Accepted February 27, 2018 DOI: 10.3892/mmr.2018.9397 Abstract. Hypoxia serves a critical role in the pathogenesis of be a critical factor that regulates cell proliferation and collagen liver fibrosis. Hypoxia‑inducible factor α1 (HIF1-α) is induced synthesis in rat HSCs under hypoxia. when cells are exposed to low O2 concentrations. Recently, it has been suggested that Rho‑associated coiled‑coil‑forming Introduction kinase 1 (ROCK1) may be an important HIF1‑α regulator. In the present study, it was analyzed whether crosstalk between Hepatic fibrosis is a common consequence of chronic liver HIF1-α and ROCK1 regulates cell proliferation and collagen diseases of various etiologies, such as hepatitis virus, alcoholic synthesis in hepatic stellate cells (HSCs) under hypoxic liver disease (ALD), non‑alcoholic steatohepatitis (NASH), conditions. For this purpose, a rat hepatic HSC line (HSC‑T6) and others (1). The development of liver fibrosis encompasses was cultured under hypoxic or normoxic conditions, and activation of hepatic stellate cells (HSCs) and their crosstalk HIF1-α and ROCK1 expression was measured at different with liver parenchymal cells, such as hepatocytes and biliary time points. Additionally, HSC‑T6 cells were transfected with epithelial cells (2). HSCs become activated and differentiate HIF1-α small interfering RNA (siHIF1‑α), and measured into myofibroblasts, thus playing a critical role in the produc- protein expression and mRNA transcript levels of α‑smooth tion of collagen and excessive accumulation of extracellular muscle actin, collagen 1A1 and ROCK1. Collagen 3A1 matrix (ECM), which are two main features of hepatic fibro- secretion was also measured by ELISA. Cell proliferation was genesis (3). Liver fibrosis is a form of chronic liver injury that assessed by the MTT assay under these hypoxic conditions. is strongly associated with hypoxia. Moreover, exposure of The results indicated that a specific ROCK inhibitor, Y‑27632, HSCs to hypoxia affects development of hepatic fibrosis (4,5). increased HIF1‑α and ROCK1 expression over time in During the past decades, increasing evidence has suggested HSC‑T6 cells in response to hypoxia. In addition, knockdown that overexpression of hypoxia‑inducible factor 1‑α (HIF1-α) of HIF1-α inhibited HSC‑T6 proliferation, suppressed is closely associated with liver fibrogenesis (6-8). Indeed, over- collagen 1A1 expression, decreased collagen 3A1 secretion expression of HIF1-α in vitro results in HSC activation and and attenuated ROCK1 expression. Notably, ROCK1 inhibition upregulation of genes associated with matrix deposition (9). caused HSC‑T6 quiescence, suppressed collagen secretion and HIF1-α and collagen 1A1 (Col1A1) expression increase in downregulated HIF1‑α expression. Collectively, these findings HSCs under hypoxia (4,10). Transfection with short inter- indicated that the interplay between HIF1‑α and ROCK1 may fering RNA against HIF1-α (siHIF1-α) prevents human HSC migration (7), while upregulation of HIF1‑α is associated with viral hepatitis‑derived fibrosis, HSC activation, and mitogen activated protein kinase (MAPK) activity (11). Additionally, Correspondence to: Dr Rongquan Fu, Department of Infectious expression of fibrogenic mediators is decreased in bile duct Diseases, The Third Affiliated Hospital of Wenzhou Medical ligated HIF1‑α‑knockout mice compared to controls (6). University, 108 Wansong Road, Rui'an, Wenzhou, Zhejiang 325200, Altogether, this strong evidence indicates that HIF1‑α is P.R. China highly involved in hepatic fibrosis; however, the underlying E‑mail: [email protected] regulatory mechanism is still not fully understood. Previously, it was reported that the Rho/Rho‑associated Abbreviations: HSCs, hepatic stellate cells; α-SMA, coiled‑coil‑forming kinase 1 (ROCK1) pathway is involved α‑smooth muscle actin; COL1A1, collagen type 1 α1 chain; COL3A1, collagen type 3 α1 chain; RT‑qPCR, reverse in HSC activation (12). Furthermore, ROCK inhibition with transcription‑quantitative polymerase chain reaction; DMEM, Y‑27632 was demonstrated to suppress HSC proliferation Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; and type I collagen production (13). Several studies have MTT, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide clearly indicated that there is crosstalk between ROCK1 and HIF1-α (14-16). However, the relationship between HIF1‑α Key words: hypoxia inducible factor 1‑α, Rho‑associated and ROCK1 in HSCs remains unexplored. coiled‑coil‑forming kinase‑1, HSCs, hypoxia, collagen In the present study, we measured changes in HIF1‑α and ROCK1 expression in HSCs under hypoxia at different 3998 HU et al: HIF1-α AND ROCK‑1 CROSSTALK IN HYPOXIC HSC time points. In order to functionally address the role of was loaded into 8‑10% gradient SDS‑PAGE gels, and HIF1-α in HSC proliferation and collagen synthesis, and transferred to a polyvinylidene difluoride membrane the interplay between HIF1‑α and ROCK1 signaling, we (EMD Millipore, Billerica, MA, USA). Membranes were performed knockdown experiments using siRNA transfection. blocked with 5% skimmed milk for 1 h and then incubated Finally, we investigated whether ROCK1 inhibition mediates with primary antibodies (β-actin, HIF1-α, ROCK1, α-SMA, HIF1-α‑derived HSC activation, thus providing an attractive Col1A1, and Col3A1) at 4˚C overnight. Goat anti‑mouse IgG target for the treatment of hepatic fibrosis. or anti‑rabbit IgG (Jackson, USA) conjugated to horseradish peroxidase was then added for 1 h at 37˚C. Finally, antibody Materials and methods binding was developed using an Odyssey infrared scanner and Western Bright ECL reagents (Thermo Fisher Scientific, Inc.). Cell culture. The HSC‑T6 cell line was maintained in room Samples were washed three times with 1X Tris buffered saline air or in hypoxic conditions (1% O2) in an incubator at 37˚C. and 0.1% Tween‑20 (TBS/T). HSC‑T6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; RNA isolation and reverse transcription‑quantitative poly‑ both Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, merase chain reaction (RT‑qPCR). Total cellular RNA was USA), 100 U/ml penicillin, and 100 µg/ml streptomycin. extracted with TRIzol reagent (Invitrogen; Thermo Fisher Culture medium was changed every 24 h, and the ROCK1 Scientific, Inc.), and cDNA synthesis was performed using inhibitor, Y‑27632 (100 µM) (Merck KGaA, Darmstadt, random hexamers with 1.0 mg RNA. RT‑qPCR was performed Germany ), was added. using SYBR‑Green I (Toyobo Life Science, Osaka, Japan). The reaction mixture volume was 20 µl. Primer sequences siRNA transfection. One day prior to transfection, HSC‑T6 were as follows: cells were seeded in 6‑well plates at a density of 2x105 cells per well or in 96‑well plates at a density of 1x104 cells β‑actin (131) forward, 5 ' ‑ C G T AAA GAC CTC TAT GCC AAC per well. Cells were transfected with 100 nM siHIF1‑α A‑3' and reverse, 5 ' ‑ G G A GGA GCA ATG ATC TTG ATC T ‑ 3 ' ; or mock siRNA (as a control) (Shanghai GenePharma HIF‑1α (36) forward, 5 ' ‑ G T C GGA CAG CCT CAC CAA ACA Co., Ltd., Shanghai, China) using Lipofectamine GAG C‑3' and reverse, 5 ' ‑ G T T AAC TTG ATC CAA AGC TCT 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to GAG‑3'; Col1A1 (95) forward, 5 ' ‑ G T A CAT CAG CCC AAA the manufacturer's instructions. Briefly, Lipofectamine‑siRNA CCC CAA G‑3' and reverse, 5 ' ‑ C G G AAC CTT CGC TTC CAT was incubated for 20 min before being added to cells. The ACT C ‑ 3 ' ; Col3A1 (88) forward, 5'‑GAC TGC CCC AAC CCA transfection medium was replaced with fresh medium after 6 h GAG ATC‑3' and reverse, 5 ' ‑ T A C CAT CAG GAA TGA CAG and cells were collected after another 48 h for the follow‑up GAG CAG‑3'; α‑SMA (77) forward, 5'‑GTG CTG TCC CTC tests. TAT GCC TCT GG‑3' and reverse, 5 ' ‑ G G C ACG TTG TGA GTC ACA CCA T C ‑ 3 ' ; ROCK1 (123) forward, 5'‑CGG TAT CTC TAC Enzyme linked immunosorbent assay (ELISA). We measured ATG GTG ATG GAG‑3' and reverse, 5 ' ‑ A T C CAA TGC AAG secreted collagen 3A1 (Col3AI) using a commercially available AAC TAC TTC T G C ‑ 3 ' ELISA kit (Uscn Life Sciences, Inc., Wuhan, China). After cells were transfected with siRNA or treated with the ROCK1 All PCR reactions were normalized to β‑actin. Relative inhibitor, supernatants were collected at 48 h. Samples were quantification of gene expression is shown using the 2‑∆∆Cq added into wells according to the manufacturers' instructions. method. The optical density (OD) values were read at 450 nm with an ELISA reader. The Col3A1 concentration was measured using MTT assay. Cells were cultured at a density of 1.0x104 cells a standard curve diagram. per well in 96‑well plates (each group with 6 duplicates) for 24 h. Then, cells were either transfected with siRNA or Immunofluorescence detection. HSC‑T6 cells were cultured treated with the ROCK1 inhibitor, Y‑27632, and incubated for in hypoxic conditions at 37℃ as described above, and then 48 h. Thereafter, 50 µl of MTT (5 mg/ml) (Nanjing KeyGen collected at 0, 12 and 48 h. The cells were fixed in 4% parafor- Biotech Co., Ltd., Nanjing, China) was added to each well and maldehyde for 15 min, permeabilized with 0.1% Triton X‑100 cells were incubated at 37˚C in an incubator for another 4 h. for 15 min and blocked with 4% bovine serum albumin (BSA) Finally, the medium was discarded and replaced with 150 µl for 1 h.