INTERNATIONAL JOURNAL OF MOleCular meDICine 44: 491-502, 2019 Rap1 regulates hepatic stellate cell migration through the modulation of RhoA activity in response to TGF‑β1 MI-YOUNG MOON1, HEE-JUN KIM2, MO-JONG KIM2, SUNHO UHM1, JI‑WON PARK1, KI-TAE SUK3, JAE‑BONG PARK4, DONG-JUN KIM3 and SUNG-EUN KIM1 1Department of Internal Medicine, Hallym University Sacred Heart Hospital, College of Medicine, Hallym University, Anyang, Gyeonggi 14068; 2Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi 14066; 3Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chuncheon, Gangwon 24253; 4Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Gangwon 24252, Republic of Korea Received November 1, 2018; Accepted May 28, 2019 DOI: 10.3892/ijmm.2019.4215 Abstract. Although the migration of hepatic stellate cells activation of RhoA in TGF‑β1-stimulated HSC‑T6 cells. These (HSCs) is important for hepatic fibrosis, the regulation of this findings suggest that TGF‑β1 regulates Rap1, resulting in the migration is poorly understood. Notably, transforming growth suppression of RhoA, activation of and formation of F‑actin factor (TGF)-β1 induces monocyte migration to sites of injury during the migration of HSCs. or inflammation during the early phase, but inhibits cell migra- tion during the late phase. In the present study, the role of Introduction transforming protein RhoA signaling in TGF-β1-induced HSC migration was investigated. TGF‑β1 was found to increase Hepatic fibrosis is characterized by the excessive deposition the protein and mRNA levels of smooth muscle actin and of extracellular matrix (ECM) mediated by activated hepatic collagen type I in HSC‑T6 cells. The level of RhoA‑GTP in stellate cells (HSCs) (1). Activated HSCs are considered to TGF-β1‑stimulated cells was significantly higher than that in serve a major role via proliferation and the production of control cells. Furthermore, the phosphorylation of cofilin and ECM, and they may migrate to fibrotic regions by trans- formation of filamentous actin (F‑actin) were more marked in forming into myofibroblasts (2). Although the majority of TGF-β1-stimulated cells than in control cells. Additionally, studies investigating hepatic fibrosis have focused on the TGF-β1 induced the activation of nuclear factor‑κB, and the proliferation and production of ECM of activated HSCs, expression of extracellular matrix proteins and several cyto- studies have demonstrated that the migration of HSCs serves kines in HSC‑T6 cells. The active form of Rap1 (Rap1 V12) an important role in hepatic fibrosis, including ECM and suppressed RhoA‑GTP levels, whereas the dominant‑negative growth factor production (3,4). Cell migration is essential in a form of Rap1 (Rap1 N17) augmented RhoA‑GTP levels. variety of biological events. Cell migration is a dynamic and Therefore, the data confirmed that Rap1 regulated the cyclic process that is regulated by small GTPase proteins (5). Cdc42 regulates the direction of movement, and Rac induces membrane protrusion at the leading edge of the cell through actin polymerization and the stimulation of integrin adhesion Correspondence to: Dr Sung-Eun Kim, Department of Internal complexes (6). Rho regulates actin-myosin contraction and Medicine, Hallym University Sacred Heart Hospital, College of retraction in the cell body and lagging edge (7,8). Among Medicine, Hallym University, 22 Gwanpyeong‑ro 17 Beon‑gil, the cytoskeletal components, actin filaments serve a major Dongan, Anyang, Gyeonggi 14068, Republic of Korea role in the formation of stress fibers during cell adhesion E‑mail: [email protected] and migration. Rho GTPase is known to regulate the actin cytoskeleton, cell polarity, gene expression, microtubule Abbreviations: HSC, hepatic stellate cell; TGF, transforming dynamics and vesicular trafficking (9). growth factor; SMA, smooth muscle actin; Col1a1, collagen type I; Transforming growth factor (TGF)‑β1 is known to be GST‑Rhotekin‑RBD, glutathione S‑transferase‑Rhotekin‑Rho‑binding domain; HIS‑RalGDS‑RBD, His‑Ral guanine nucleotide dissociation a multifunctional cytokine that regulates cell proliferation, stimulator‑Rap binding domain differentiation, migration, survival, carcinogenesis, wound healing, immune reactions and fibrosis (10). In particular, Key words: cell migration, hepatic stellate cell, transforming TGF-β1 is the most important profibrotic cytokine in hepatic growth factor‑β1, Rap1, transforming protein RhoA, nuclear fibrosis. Of note, TGF‑β1 activates Rho subfamily GTPases, factor-κB including transforming protein RhoA, Ras‑related C3 botu- linum toxin substrate 1 (Rac1) and Cdc42, in various cell lines, including the HSC‑T6 cell line (11‑14). Furthermore, a previous study demonstrated that the TGF-β1‑induced activation of 492 MOON et al: Rap1 REGULATES HEPATIC STELLATE CELL MIGRATION RhoA in activated HSCs mediates the migration of HSCs via Expression, purification, and transduction of Tat‑C3. The the Smad/JNK/p38 signaling pathway (15). HIV‑1 Tat transduction domain (amino acid residues 49‑57, In addition to the Smad-dependent TGF-β1 signaling KKKRRQRRR) were fused to the N‑terminus of the C3 pathway, TGF‑β1 can induce activation of the nuclear exoenzyme containing a His tag (Tat‑C3) for the purpose factor-κB (NF-κB) signaling pathway, leading to the migra- of efficiently introducing C3 exoenzymes into cells. Tat‑C3 tion of cells (11). NF-κB is a well‑known transcription factor purification was performed following a previously described that regulates the expression of cytokines, chemokines, method (13,23). To analyze the transduction of Tat‑C3, cell adhesion molecules and inducible proteins to control the cells were plated at a density of 2x105 cells per well in inflammation, apoptosis and malignancy (16). Kim et al 6‑well plates and were incubated with 1 µg/ml Tat‑C3 at 37˚C demonstrated that inhibitor of NF‑κB (IκB) kinase (IKK)‑γ with 5% CO2. After 1 h, the Tat‑C3 transduced cells were stimulates the activation of RhoA, which leads to the direct analyzed by western blotting using anti‑RhoA antibody. The phosphorylation of IKKβ and subsequent activation of NF‑κB, Tat‑C3‑transduced cells showed a mobility shift band, which and induces chemokine expression and cell migration in was modified‑RhoA (Arg 41) by Tat‑C3. response to TGF-β1 (17). As a member of the Ras superfamily of small GTPases, Rap1 is regulated by guanine nucleotide Western blot analysis. The cells were collected, washed once exchange factors (GEFs) and GTPase‑activating proteins with ice‑cold phosphate‑buffered saline (PBS) and lysed with (GAPs). Rap1 is associated with the regulation of both inte- RIPA buffer [50 mM Tris‑HCl (pH 7.5), 150 mM NaCl, 1% grin- and cadherin-mediated cell adhesion, and the recycling, Triton X‑100, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM avidity and affinity of integrins via the inside‑out activation Na3VO4, 2 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin, process (18). Rap1 signaling can positively and negatively 1 µg/ml leupeptin and 1 µg/ml pepstatin]. The cell lysates were modulate the activity of Rho family proteins, including centrifuged at 13,000 x g for 20 min at 4˚C, and protein concen- Cdc42, Rac1 and RhoA. Moon et al demonstrated that Rap1 trations in the supernatants were analyzed using a Bradford inhibits cell migration by regulating the activity of RhoA in assay (Bio‑Rad Laboratory, Inc., Hercules, CA, USA). Equal response to TGF-β1 (19). Rap1 is an important modulator of quantities of proteins (40 µg) were separated using SDS‑PAGE the NF-κB signaling pathway (20,21). Although the influence on 10% gels for Col1a1 and fibronectin or 12% gels for all of Rho GTPase signaling on HSC migration during hepatic other proteins, transferred onto PVDF membranes, blocked fibrosis has been reported (15), the role of NF‑κB signaling in with 5% skimmed milk or 5% BSA and probed with the response to TGF-β1 via RhoA GTPase activation has not been appropriate antibodies (Table I). The immunoreactive bands investigated in activated HSCs. Therefore, the present study were visualized on digital images captured with FUSION FX7 investigated the mechanism by which Rap1 regulates the SPECTRA (Vilber Lourmat, Eberhardzell, Germany) using activity of RhoA by NF‑κB signaling during TGF-β1-induced EzWestLumi plus Western blot detection reagent (ATTO HSC migration. Corporation, Tokyo, Japan). The band intensities were quanti- fied using ImageJ software (version 1.50i; National Institutes Materials and methods of Health, Bethesda, MD, USA). Statistical analyses were performed using GraphPad Prism 4 (GraphPad Software, Inc., Materials. Bovine serum albumin (BSA; cat. no. A2058), San Diego, CA, USA). Y27632 (cat. no. Y0503), BAY11‑7085 (cat. no. 196309‑76‑9) and the recombinant TGF‑β1 protein (cat. no. T7039) were Filamentous actin (F‑actin) staining. The HSC‑T6 cells were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, treated with 10 ng/ml TGF-β1 in DMEM for 1 h at 37˚C Germany). Details of antibodies are shown in Table I. under 5% CO2. The cells were washed with PBS, fixed with 4% methanol‑free formaldehyde and permeabilized with 0.2% Cell culture and transfection. The HSC‑T6 cell line (Dr Triton X‑100 in PBS. F‑actin (green) was then stained with Scott Friedman, Division of Liver Diseases, Icahn School of Alexa Fluor 488‑phalloidin (1 µm) for 30 min at room temper- Medicine at Mount Sinai, New York, NY, USA), an immor- ature. DAPI (blue) was used to counterstain the nuclei. The talized rat HSC line, has been described previously (22). The immunolabeled cells were examined using an LSM 700 laser HSC‑T6 cells were grown in Dulbecco's modified Eagle's confocal microscope (Carl Zeiss AG, Oberkochen, Germany). medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% heat‑inactivated Glutathione S‑transferase (GST)‑Rhotekin‑Rho‑binding fetal bovine serum (FBS; HyClone; GE Healthcare Life domain (RBD) pull‑down assays for activated RhoA.