RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.

(Lexington, KY). Mouse RASAL1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Halo-tag antibody was purchased from Promega.
The Flag (DYKDDDDK)-tag antibody was purchased from Wako.

Immunoprecipitation
For immunoprecipitation, cell lysates were precipitated by incubation with anti-FLAG M2 agarose (Sigma) for 8 h, or by incubation with anti-MKL1 or anti-SRF antibodies, followed by addition of protein A/G agarose (Santa Cruz) overnight. Cell extracts were prepared as described previously (3).

Western blotting
Western blotting was conducted as described previously (3).

Lentivirus production and transduction
cDNA-expressing lentiviruses were produced by transfection of a cDNA-expressing pCDH vector and pPACKH1 packaging plasmid mix (System Biosciences) into 293T cells. After 48 h, virus-containing supernatants were collected and viral particles were concentrated using PEG-it Virus Precipitation Solution according to the manufacturer's protocol (System Biosciences). To generate stable RASAL1-and AGTR1-overexpressing cells, LX2 cells were transduced with RASAL1-or AGTR1-expressing constructs containing lentiviral particles, followed by selection with 2 g/mL puromycin or 600 g/mL neomycin.

Reporter assays
Dual reporter assays using pGL4-TK, a Renilla luciferase construct as a control, were performed basically as described previously (4). The series of -SMA promoter-driven reporters was kindly provided by Dr. Manabe (5). SRF-RE luc, containing a firefly luciferase gene driven by a TATA box plus five repeats of the binding sites for SRF (GTCCATATTAGGAC), was obtained from Stratagene (La Jolla, CA). An AGTR1 6 promoter-driven reporter construct was purchased from GeneCopoeia (Rockville, MD), and was used according to the manufacturer's protocol.

Chemical compound screening
The

Immunocytochemistry
Cells were seeded in the chamber slides (Nalge Nunc, Naperville, IL). The next day, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X100.
Fixed cells were incubated with primary antibodies for 60 min at room temperature, followed by incubation with Alexa Flour 488-or 555-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) for 60 min at room temperature. Slides were mounted using VectaShield with DAPI (Vector Labs, Burlingame, CA). To examine the effects of 7 TGF-, cells were incubated with serum-free DMEM for 24 h and treated with 10 ng/mL TGF- for 60 min before fixation.

Immnohistochemistry
Immnohistochemistry was performed as described previously (3). The area of fibrosis was determined using the ImageJ software.

Ras, RhoA, and Gi activities
Ras and RhoA activities were measured using the Ras and RhoA Activation Pull-down Biochem Kit from Cytoskeleton (Denver, CO) according to the manufacturer's protocols. Briefly, after washing the cells with ice cold PBS, they were dissolved with cell lysis buffer and harvested using a cell scraper. Cell lysates were transferred to sample tubes and centrifuged (10,000 rpm, 4°C, 2 min). GST-tagged Ras binding domain (RBD) of Raf protein beads for Ras activity and GST-tagged Rhotekin protein beads for RhoA activity were added to the equivalent protein amounts of lysate and incubated at 4°C on a rotator for 1 h. The beads were pelleted by centrifugation (3,000g, 1 min, 4°C), and were washed with 500 L of wash buffer and pelleted by centrifugation (3,000g, 3 min, 4°C). After the supernatant was removed carefully and 2× sample buffer was added, the beads samples were boiled for 2 min. Activities of Gαi were measured using the Gαi activation kit from Abcam based on immunoprecipitation using an anti-active Gαi monoclonal antibody according to the manufacturer's protocol.
The amounts of GTP-bound Ras and RhoA, and active Gi proteins, were determined by Western blotting using the specific antibodies provided.

Quantitative real-time PCR (qRT-PCR)
The Fibrosis RT 2 Profiler PCR Array was obtained from QIAGEN (Hilden, Germany) and used for qRT-PCR analysis. qRT-PCR was performed as described previously (3).
Briefly, total RNA was isolated from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized from RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed using the TaqMan gene Halo-tagged AGTR1-expressing plasmid. Cell lysates without transfection were used as a control. Flag-tagged RASAL1 was immunoprecipitated using anti-Flag agarose.
Agarose-conjugated anti-mouse IgG was also used as a negative control.
Co-precipitated proteins were blotted using antibodies against the indicated proteins. Of the total cell lysates, 5% were loaded as an input control. Representative results from three independent experiments are shown. b, Activities of PKC in control, RASAL1-expressing, and RASAL1 + AGTR1-coexpressing LX2 cell lysates were measured by ELISA using phosphor-specific antibodies against PKC substrate peptides.
Data represent the means ± SD of three independent experiments. c, RASAL1 had no effect on the phosphorylation of JAK2. The JAK2 phosphorylation status in control fibrotic areas in control and RASAL1-deficient mice determined using the ImageJ software. Data represent the means ± SD of four mice in one group. *, p < 0.05 (t-test).
d, The degree of liver inflammation was similar in control and RASAL1-deficient mice.
Biochemical parameters reflecting liver inflammation were measured in sera at 8 weeks activated HSCs a, b, Resting HSCs express RASAL1 but activated HSCs do not in human liver tissues. RASAL1 protein was immunostained in green. Resting HSCs were marked by staining Vimentin (a resting marker) in red in normal liver tissues (a). Activated HSCs were by SMA (an activating marker) in red in fibrotic liver tissues (b). Please note: hepatocytes also express RASAL1 but its expression levels are unchanged in normal (a) and fibrotic liver (b). Regions in the white rectangles in upper images are enlarged below. c, The percentage of RASAL1 expressing HSCs in resting (Vimentin+) in normal liver and activating (SMA+) state in liver cirrhosis tissues. Data represent the means ± SD of 12 samples in one group. *, p < 0.05 (t-test).