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ROCK-2 signaling pathway
Regulatory Peptides 169 (2011) 13–20
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Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway Hongli Ji a,b, Ying Meng c, Xiaolan Zhang d, Wei Luo a, Pingsheng Wu e, Bing Xiao a, Zhenshu Zhang a,⁎, Xu Li f,⁎⁎ a
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Oncology, 153rd Hospital of People's Liberation Army, Zhengzhou 450042, China Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China d Department of Senile Disease, Taihe Hospital, Hubei Medicine University, Shiyan 442000, China e Department of Cardiovascular Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China f Department of Emergency, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China b c
a r t i c l e
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Article history: Received 24 December 2010 Received in revised form 21 March 2011 Accepted 16 April 2011 Available online 3 May 2011 Keywords: Hepatic stellate cell Aldosterone RhoA/ROCK-2 signaling pathway Contraction Cirrhosis
a b s t r a c t The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT1R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver disease [1]. Following liver injury, HSCs activate and transform into myofibroblastic cells and then undergo a variety of functional changes including matrix remodeling, chemotaxis, and contraction [2]. The role of HSCs in matrix remodeling and chemotaxis has been intensively studied, nevertheless, little attention has been paid to HSC contractibility in the pathogenesis of cirrhosis and portal hypertension. HSC contractility, due to cytoskeletal reorganization within the cells, affects cells' shape and motility, contributing to intraphepatic resistance and portal hypertensin which are responsible for much of the morbidity in cirrhosis[3]. Thus a better understanding of the molecular mechanism that underlies HSC activation and contractibility is an important prerequisite for the development of new therapeutic goals for cirrhosis and portal hypertension. ⁎ Corresponding author. Tel.: + 86 020 61641546; fax: + 86 020 87280770. ⁎⁎ Corresponding author. Tel./fax: + 86 020 87283517. E-mail addresses: [email protected] (Z. Zhang), [email protected] (X. Li). 0167-0115/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2011.04.010
Aldosterone is one of the main peptides in the renin–angiotensin– aldosterone-system (RAAS) [4,5]. As a mineralocorticoid hormone, aldosterone is secreted by the cells of the zona glomerulosa upon stimulation with angiotensin II (Ang II), adrenocorticotropic hormone (ACTH), or potassium levels [6,7]. Originally described the function of aldosterone is to increase the reabsorption of sodium and water and the secretion of potassium in the kidney, thereby increasing system blood pressure and electrolytic balance [8]. Besides this hormonal effect, aldosterone can be synthesized in extra-adrenal tissues, e.g. the heart, kidney, liver and blood vessels [9]. Other studies revealed that aldosterone is now considered a key player in cellular processes underlying liver fibrosis [10,11]. Recently, large clinical trials have shown that anti-aldosteronic drug like spironolactone, a mineralocorticoid receptor (MR) antagonist, reduces fibrogenic processes in the liver, resulting in decreased tissue remodeling with long-term benefits for patients at risk for cirrhosis and portal hypertension [12]. The mechanism responsible for the remodeling of hepatic architecture partly depends on HSC contraction has been reported [13]. However, the effect of aldosterone in HSC contraction and its exact molecular mechanism are presently unclear.
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It is well-known that Ang II and aldosterone are both important peptides in the RAAS [14–16]. According to recent research, endogenous Ang II binds to angiotensin II type 1 receptor (AT1R), a surface G proteincoupled receptor, which can further activate the cytosolic small GTPase Rho, named ROCK-2 [17], leading to HSC contraction (Fig. 1). However, whether aldosterone affects HSC contraction, or the contraction may also be due to the activation of the RhoA/ROCK-2 signaling pathway is unclear. Therefore, the major objective of this proposed investigation was to examine these matters.
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (500–700 g; Animal Experiment Center, Southern Medical University, China) fed a commercial pelleted diet and given water ad libitum, were used for primary HSC isolation. All experiments and animal handling were performed according to the Association for the Accreditation and Assessment of Laboratory Animal Care.
2.3. Gel contraction assay Contractility of the activated HSCs was evaluated using collagen gel lattices in a plastic 6-well culture plate as previously described, with some modifications [19]. Firstly, HSCs were cultured for 12 h under serum-free conditions and the cells were treated by various reagents as described in Section 2.2. In addition, collagen gels were prepared by mixing Type I rat-tail collagen (3.69 mg/mL; BD Bioscience, Bedford, MA), DMEM (without serum) and a HSC cell suspension so that the final mixture resulted in a physiological ionic strength of DMEM, 0.75 mg/ml collagen and 400,000 cells/mL. A 1550 μL aliquot of the collagen gel solution was then added into each well of a 6-well culture plate and the plate was incubated for 1 h at 37 °C to allow gelation. The collagen gel was detached from the lattice through gentle circumferential dislodgement using a 200 μL micropipette tip. Each gel was photographed every hour for 6 h and the surface area of the gel was measured using digital Adobe Photoshop software 3.0. The contraction of the gel was expressed as a percentage according to the following formula: [(gel surface area of gel surface area of test substance) / (gel surface area of gel surface area of aldosterone)] × 100%. 2.4. Confocal immunofluorescence
2.2. Cell isolation and culture HSCs were isolated via in situ pronase/collagenase (Sigma-Aldrich, St. Louis, MO) perfusion followed by density gradient centrifugation as previously described[18]. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA) with antibiotics and 10% fetal bovine serum (FBS; Invitrogen) in a 37 °C humidified incubator with a mixture of 95% air and 5% CO2. To evaluate whether aldosterone affects HSCs in a time- and dosedependent manner, aldosterone was added into the cells for 5, 15, 30, 60, or 120 min, and at concentrations ranging from 0.01 to 100 nM under serum-free conditions. In all other experiments, 1 nM aldosterone was added to the cell cultures and incubated for 15 min. Y27632 (a ROCK-2 inhibitor), irbesartan (an AT1R inhibitor), and spironolactone (a MR inhibitor) were used at a concentration of 10 nM,1 h prior to aldosterone (1 nM) addition. All of these reagents were obtained from Sigma.
For phalloidin staining, HSCs were cultured on slides at a density of 5 × 104 cells/cm2 and incubated 24 h to permit cell attachment. Under serum-free conditions, the cells were treated by various reagents as described in Section 2.2. After that, the cells were fixed with 4% paraformaldehyde for 10 min. After washing with phosphate buffered saline (PBS), the cells were treated with 0.1% Triton X-100 for 5 min to permeabilize. To visualize actin fibers in the cells, the cells were stained with 2 μg/mL of TRITC-labeled phalloidin (Sigma) for 20 min at room temperature. After mounting, cell samples were observed under an Olympus-FL 500 confocal laser-scanning microscope (Tokyo, Japan). 2.5. Western blotting Total cellular protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech, Nantong, China) containing protease inhibitors (Roche, Switzerland). Protein samples (40 μg) were heated at 100 °C for 10 min before loading and being separated on 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After that, the membrane was blocked with 5% skimmed milk powder in TTBS buffer (20 mM Tris, 500 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature. Membranes were then incubated for 16 h at 4 °C with various primary antibodies in a 5% skimmed milk buffer at a dilution specified by the manufacturers. The following primary antibodies were used: rabbit anti-phospho-MLC, rabbit anti-phospho-moesin, rabbit anti-MLC and rabbit anti-moesin (all from Cell Signaling Technology, Danvers, MA, USA). On the next day, the membranes were washed with TTBS buffer and incubated in the appropriate secondary antibody solution at a 1:5000 dilution (Zhongshan Biotech, Beijing, China). Membranes were further incubated with an enhanced chemiluminescence system (Pierce, Rockford, IL) to visualize the positive protein bands. The density of the individual bands was then quantified using a densitometric scanner with Quantity One software (Bio-Rad). 2.6. RhoA pull-down assay
Fig. 1. Contractile pathway in HSCs. Agonist activation of receptors coupled to Gproteins can activate the RhoA/ROCK-2 signaling pathway, resulting in increased phosphorylation of myosin light chain (P-MLC) and thus induction of HSC contraction.
RhoA activation was assessed by a pull-down assay with a RhoA activation assay kit (Cytoskeleton, Denver, CO) according to the manufacturer's protocol [20]. Briefly, cells were lysed with a lysis buffer
H. Ji et al. / Regulatory Peptides 169 (2011) 13–20
from the kit and total protein concentration was measured using a protein assay kit from Bio-Rad. Each sample containing 500 μg protein was incubated with agarose-conjugated rhotekin-RBD at 4 °C overnight. The mixtures were briefly centrifuged and the pellets resuspended in a 5x Laemmli-reducing buffer (Sigma) and boiled for 5 min. After briefly centrifuging, the supernatants were loaded and run on a 12% SDS-PAGE and transferred onto a PVDF membrane. Proteins were then analyzed by Western blotting using an anti-RhoA antibody from the kit at a 1:500 dilution.
2.7. Statistical analyses Data were summarized as mean ± standard error of the mean (S.E.M.) based on experiments repeated in triplicate. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS) 13.0
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software (Chicago, IL). A probability (P)-value b 0.05 was considered statistically significant. 3. Results 3.1. Aldosterone induction of collagen gel contraction Utilizing a hydrated collagen gel assay, we examined whether aldosterone could induce collagen gel contraction in HSCs. Fig. 2 displays a representative experiment with collagen lattice contraction. The Fig. 2A control shows a gel exposed to buffer only, resulting in a minimal decrease of the collagen surface area. Compared with the control, aldosterone at 1 nM caused an obvious decrease in surface gel area. Addition of the inhibitors spironolactone, Y-27632, or irbesartan to the HSCs for 1 h prior to a 15 minute exposure to aldosterone, resulted in significant loss of gel contraction compared with aldosterone alone (Fig. 2A). The quantitation of percentage gel contraction is shown in Fig. 2B.
Fig. 2. Aldosterone induction of collagen gel contraction in HSCs. The contraction of collagen gel in HSCs exposed to a combination of 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) and 1 nM aldosterone (ALD) compared with the cells exposed to 1 nM aldosterone (ALD) alone, as described in Section 2. (A) Photograph of hydrated collagen lattices. (B) Mean percentage of the collagen gel contraction. *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD.
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Fig. 3. Aldosterone induction of actin stress fiber formation. (A) Staining HSCs with TRITC-phalloidin showed actin fibers (red) and DAPI (blue), demonstrating flat morphology and actin stress fibers. (B) Fifteen minutes after exposure to 1 nM aldosterone (ALD), the cells have increased numbers of actin stress fibers, and developed actin fiber positive projections. After that, the cells were pre-incubated for 1 h with (C) spironolactoue (Sp), (D) Y-27632 (Y), or (E) irbesartan (Irb) at a concentration of 10 nM and then co-treated with aldosterone for 15 min. Aldosterone-induced increase of action stress fiber formation was partially reversed by these inhibitors.
3.2. Aldosterone induction of actin stress fiber formation HSCs in culture develop a cuboidal shape, with a regular arrangement of actin stress fibers. Within 15 min after adding aldosterone, staining of the actin stress fibers using phalloidin showed
a significant increase in terms of quantity, and reorganization in morphology, in response to aldosterone compared with the control (Fig. 3A-B). Aldosterone-induced increase of the action stress fiber formation was partially reversed when performed in the presence of spironolactone, Y27632, or irbesartan (Fig. 3A-D).
Fig. 4. Aldosterone induction of P-MLC expression and actin fiber formation in a time- and dose- dependent manner in HSCs. (A) HSCs were treated with 1 nM aldosterone (ALD) at different time points. The cells were then lysed, and P-MLC and MLC expression were assessed by Western blot analysis and quantified as described in Section 2. (B) HSCs were treated with indicated concentrations of aldosterone for 15 min, and P-MLC and MLC expression were assessed by Western blotting. *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD. (C and D) Actin fibers stained with TRITC-labeled phalloidin. The treatment of cells was similar to (A) and (B) and were then subjected to stain actin fibers and nuclei.
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Fig. 4 (continued).
3.3. Aldosterone activation of the actin-regulatory protein, P-MLC To assess the effect of aldosterone on phosphorylated myosin light chain (P-MLC) expression, we exposed HSCs to different dose ranges and time points as described in Section 2, then measured P-MLC expression using Western blots. As shown in Fig. 4A and B, aldosterone induced P-MLC expression in HSCs in a time- and dose- dependent manner. Fig. 4A shows that aldosterone can induces an increase in P-MLC as early as 5 min and this increase reaches to its peak in 15 min. Fig. 4B shows the
dose- dependent effect of aldosterone on P-MLC expression from 0.01 nM to 100 nM compared with the control and the effect is remarkable at a concentration of 1 nM. As previously reported, P-MLC locates downstream of ROCK-2, which regulates cell contraction by a change in actin fiber formation [21]. Thus, to confirm the role of P-MLC in aldosteroneinduced actin cytoskeleton remodeling, we observed the change of actin stress fiber formation and quantity staining with TRITC-phalloidin. As shown in Fig. 4C and D, aldosterone induced the change in actin fiber formation and recombination in a time- and dose- dependent manner.
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Fig. 5. Aldosterone activation of the RhoA/ROCK-2 signaling pathway. HSCs were incubated with serum-free culture medium for 12 h. The starved cells were treated with 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) for 1 h before 1 nM aldosterone (ALD) was added for 15 min. After that, the cells were subjected to RhoA GTP pull-down assay and Western blot analysis of RhoA GTP and total RhoA protein expression (A). The treated cells by various reagents was similar to (A), total cellular protein was extracted and subjected to Western blot analysis of P-moesin and moesin protein expression (B). *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD.
3.4. Aldosterone activation of the RhoA/ROCK-2 signaling pathway Next, we performed western blots to determine any change of protein expression of the RhoA/ROCK-2 pathway with aldosterone stimulation. RhoA is a small, monomeric guanosine triphosphatebinding protein in the Rho family, with GDP-bounding inactive and
GTP-bounding active forms which can transform each other under the regulation of Rho guanine–nucleotide exchange factors (Rho GEFs). As shown in Fig. 5A, aldosterone markedly upregulated the active RhoA (RhoA GTP) protein expression in HSCs after a treatment of 15 min, as compared to the control. The effect was suppressed by both the MR inhibitor spironolactone and the ROCK-2 inhibitor Y27632 (Fig. 5A). Interestingly, the AT1R inhibitor irbesartan has an inhibitory effect on the activity of aldosterone in HSCs, suggesting that a crosseffect exists between aldosterone and Ang II. In addition, ROCK-2 activity is assessed by phosphorylation of the endogenous ROCK-2 substrate, moesin, at Thr-558 [22,23]. Using the Western blotting, our data showed that aldosterone induction of phospho-moesin (Pmoesin) expression was also suppressed by either spironolactone, Y27632 or irbesartan (Fig. 5B). Finally, aldosterone was able to induce an increase in the protein expression level of P-MLC, a downstream effector of ROCK-2, whereas the effect could be blocked by spironolactone, Y-27632, or irbesartan (Fig. 6). The blockage ability of irbesartan on aldosterone is similar to the results mentioned above. 4. Discussion
Fig. 6. Aldosterone induction of P-MLC expression via the RhoA/ROCK-2 signaling pathway. Serum-starved HSCs were incubated with 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) for 1 h prior to 1 nM aldosterone addition, and then co-incubated for 15 min. Total cellular protein was extracted and subjected to Western blot analysis of P-MLC and MLC protein expression. *P b 0.05 compared with the untreated cells; #Pb 0.05 compared with the cells treated with ALD.
Aldosterone origionly described as an important regulator of blood pressure and electrolytic balance. Nowdays, aldosterone is considered a key player in cellular processes underlying portal hypertension and liver fibrosis. A previous in vivo study demonstrated that expression of CYP11B2, a key gene in aldosterone synthesis, was upregulated in HSCs during CCl4-induced hepatic fibrogenesis [24]. Another in vitro study showed that aldosterone at both physiological (10− 9–10− 10 M) and pharmacological levels (10− 6 M) enhanced the synthesis of types I and IV procollagen in quiescent HSCs [25]. In the current study, we determined a role for aldosterone in HSC contraction and the potential mechanism. Our data showed for the first time that aldosterone at pharmacological levels could induce HSC contraction, and the RhoA/ ROCK-2 signaling pathway appeared to be essential in the regulation of this process.
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Collagen gel contraction is considered to be an in vitro equivalent of the tissue contraction that occurs during wound healing [26]. This procedure is associated with the dynamic morphological change in cells seeded on the gel [27,28]. It has been reported that transforming growth factor-β (TGF-β) and lysophosphatidic acid (LPS) enhance collagen gel contraction in HSCs [29,30]. To find out whether aldosterone has the same effect in HSCs as TGF-β and LPS, we further examined the effect of aldosterone on the ability of HSCs to contract collagen gels. Our results showed that aldosterone could stimulate contraction of native collagen gels. During collagen gel contraction, the cells progress from a round to a spread phenotype with the formation and rearrangement of actin filaments along the long axis of the cells [29]. The resultant increase in traction forces generated from these cellular extensions leads to a significant increase in cell contractility [31,32]. In the current study, we observed that aldosterone induced an increase in HSC actin stress fiber formation that contributed to the strength of cell contractility. The RhoA/ROCK-2 signaling pathway is involved in vasoconstrictor-induced contraction of vascular smooth muscle. After activation of G-protein-coupled receptors by vasoconstrictors, RhoA activates ROCK-2, which then phosphorylates and thereby activates MLC, leading to increase P-MLC and cell contraction [21]. In evaluating whether aldosterone can activate this signal transduction pathway, we investigated expressions of RhoA and ROCK-2 in HSCs after treatment with or without aldosterone. There was a strong upregulation of active RhoA (RhoA GTP) protein expression induced by aldosterone. It is well known that the phosphorylation state of moesin can measure the functional activity of ROCK-2 [22,23]. Our data showed that aldosterone upregulation of active RhoA in HSCs resulted in an increase in P-moesin, reflecting increased activity of ROCK-2 in the cells. The specific ROCK-2 inhibitor, Y27632, could inhibit these protein expressions induced by aldosterone, signifying that the RhoA/ ROCK-2 signaling pathway has an important role in this process. Numerous studies have demonstrated the pivotal role of P-MLC in cell contraction based on the observations that HSCs become contractile coincident with the expression of P-MLC [21]. P-MLCinduced actin depolymerization and reassembly toward the cell membrane edge [33] is responsible for the formation of cortical actin complexes contributing to the contraction of cells. A recent preliminary report showed that HSCs isolated from Sprague-Dawley rats can be activated to contraction when stimulated by Ang II [34]. As an adjunct to this study, we showed that aldosterone, another peptide in the RAAS, induced P-MLC expression in a time- and dose-dependent manner associated with cell contraction. Consistent with P-MLC expression level, HSC contractility mediated by actin stress fiber formation also acted in a time- and dose-dependent fashion. These data indicated that aldosterone could not only induce an increase in PMLC expression but also link myosin and actin to enable HSC contraction through the recombination of actin fibers in HSCs. Furthermore, to evaluate whether P-MLC is involved in aldosteroneinduced contraction via the RhoA/ROCK-2 signaling pathway, we made use of specific inhibitor in the signaling pathway to note differences in the protein expression. We observed that Y27632, the ROCK-2 inhibitor, was able to abrogate aldosterone-induced P-MLC expression. This further supports the idea that P-MLC plays an important role in mediating HSC contraction through the RhoA/ROCK2 signaling pathway. Ang II and aldosterone are both important peptides in the RAAS and can interact and regulate each other [35]. The combination of Ang II with AT1R stimulates both systemic and hepatic aldosterone production and an increase of aldosterone in turn amplifies Ang II effects by increasing the expression of AT1R and angiotensin converting enzyme (ACE) [36]. Montezano et al. demonstrated that the interaction between aldosterone and Ang II in vascular cells may lead to an amplification of cellular responses that contribute to vascular remodeling in cardiovascular diseases [37]. To explore whether the
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effect exists between aldosterone and Ang II in HSCs, we used the AT1R inhibitor irbesartan and the MR inhibitor spironolactone to examine changes in actin fiber formation and protein expressions induced by aldosterone. Interestingly, our current data showed that these inhibitors could not only decrease actin fiber formation, but also effectively inhibit the expression of RhoA GTP, P-moesin and P-MLC, indicating the potential effect of Ang II and aldosterone in HSCs. However, the precise molecular mechanism of how the AT1R inhibitor irbesartan exerted a suppressive effect on aldosterone in HSCs needs to be further studied. In addition, several previous studies have offered evidence that Ang II can evoke the RhoA/ROCK-2 signaling pathway in mammalian cells [38,39]. Considering our present data and these reports, we hypothesized that the integration of aldosterone with MR to induce HSC contraction may partly result from potentiated AngII/ AT1R-mediated actions via the RhoA/ROCK-2 signaling pathway. In summary, our data elucidate a novel molecular mechanism by which aldosterone triggers the RhoA/ROCK-2 signaling pathway to regulate HSC contraction. Targeting some of these signaling events using receptor antagonists may provide therapeutic strategy for patients suffering from cirrhosis and portal hypertension. Acknowledgements This work was supported by grants from the National Natural Scientific Foundation of China (#30871155, #30770974). References [1] Cheng JH, She H, Han YP, Wang J, Xiong S, Asahina K, Tsukamoto H. Wnt antagonism inhibits hepatic stellate cell activation and liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2008;294:G39–49. [2] Massimo P. Liver fibrosis. Springer Semin Immunopathol 2000;21:475–90. [3] Reynaert H, Thompson MG, Thomas T, Geerts A. Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension. Gut 2002;50:571–81. [4] Nagata D, Takahashi M, Sawai K, Tagami T, Usui T, Shimatsu A, Hirata Y, Naruse M. Molecular mechanism of the inhibitory effect of aldosterone on endothelial NO synthase activity. Hypertension 2006;48:165–71. [5] McGrath MF, de Bold AJ. Determinants of natriuretic peptide gene expression. Peptides 2005;26:933–43. [6] Shapiro BA, Olala L, Arun SN, Parker PM, George MV, Bollag WB. Angiotensin IIactivated protein kinase D mediates acute aldosterone secretion. Mol Cell Endocrinol 2010;317:99–105. [7] Taira M, Toba H, Murakami M, Iga I, Serizawa R, Murata S, Kobara M, Nakata T. Spironolactone exhibits direct renoprotective effects and inhibits renal renin– angiotensin–aldosteronesystem in diabetic rats. Eur J Pharmacol 2008;589: 264–71. [8] Ruilope LM. Aldosterone, hypertension and cardiovascular disease: an endless story. Hypertension 2008;52:207–8. [9] Delcayre C, Swynghedauw B. Molecular mechanisms of myocardial remodeling. The role of aldosterone. J Mol Cell Cardiol 2002;34:1577–841. [10] Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008;214: 199–210. [11] Struthers AD, MacDonald TM. Review of aldosterone- and angiotensin II-induced target organ damage and prevention. Cardiovasc Res 2004;61:663–70. [12] Caligiuri A, De Franco RM, Romanelli RG, Gentilini A, Meucci M, Failli P, Mazzetti L, Rombouts K, Geerts A, Vanasia M, Gentilini P, Marra F, Pinzani M. Anti fibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells. Gastroenterology 2003;124:504–20. [13] Kmieć Z. Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol 2001;161:III–XIII 1–151. [14] Li X, Meng Y, Wu P, Zhang Z, Yang X. Angiotensin II and aldosterone stimulating NF-κB and AP-1 activation in hepatic fibrosis of rat. Regul Pept 2007;138:15–25. [15] Sakai H, Nishimura A, Watanabe Y, Nishizawa Y, Hashimoto Y, Chiba Y, Misawa M. Involvement of Src family kinase activation in angiotensin II-induced hyperresponsiveness of rat bronchial smooth muscle. Peptides 2010;31:2216–21. [16] Li L, Zhou Y, Wang C, Zhao YL, Zhang ZG, Fan D, et al. Src tyrosine kinase regulates angiotensin II-induced protein kinase Czeta activation and proliferation in vascular smooth muscle cells. Peptides 2010;31:1159–64. [17] Rattan S, Puri RN, Fan YP. Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II. Exp Biol Med 2003;228: 972–81. [18] Liu C, Gaca MD, Swenson ES, Vellucci VF, Reiss M, Wells RG. Smads2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. J Biol Chem 2003;278:11721–8. [19] Yanase M, Ikeda H, Ogata I, Matsui A, Noiri E, Tomiya T, Arai M, Inoue Y, Tejima K, Nagashima K, Nishikawa T, Shibata M, Ikebe M, Rojkind M, Fujiwara K. Functional diversity between Rho-kinase- and MLCK-mediated cytoskeletal actions in a
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[30] Yanase M, Ikeda H, Matsui A, Maekawa H, Noiri E, Tomiya T, Arai M, Yano T, Shibata M, Ikebe M, Fujiwara K, Rojkind M, Ogata I. Lysophosphatidic acid enhances collagen gel contraction by hepatic stellate cells: association with Rhokinase. Biochem Biophys Res Commun 2000;277:72–8. [31] Arora PD, Narani N, McCulloch CA. The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am J Pathol 1999;154:871–82. [32] Hocking DC, Sottile J, Langenbach KJ. Stimulation of integrin-mediated cell contractility by fibronectin polymerization. J Biol Chem 2000;275:10673–82. [33] Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V, Schneeberger EE, Turner JR. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci 2006;119:2095–106. [34] Brown NJ, Nakamura S, Ma L, Nakamura I, Donnert E, Freeman M, Vaughan DE, Fogo AB. Aldosterone modulates plasminogen activator inhibitor-1 and glomerulosclerosis in vivo. Kidney Int 2000;58:1219–27. [35] Sohail MA, Hashmi AZ, Hakim W, Watanabe A, Zipprich A, Groszmann RJ, Dranoff JA, Torok NJ, Mehal WZ. Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition. Hepatology 2009;49:185–94. [36] Touyz RM. Intracellular mechanisms involved in vascular remodelling of resistance arteries in hypertension: role of angiotensin II. Exp Physiol 2005;90:449–55. [37] Montezano AC, Callera GE, Yogi A, He Y, Tostes RC, He G, Schiffrin EL, Touyz RM. Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways. Arterioscler Thromb Vasc Biol 2008;28:1511–8. [38] Savoia C, Tabet F, Yao G, Schiffrin EL, Touyz RM. Negative regulation of RhoA /Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats. J Hypertens 2005;23:1037–45. [39] Huang Z, Taylor L, Liu B, Yu J, Polgar P. Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2006;290:L1291–9.
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway Hongli Ji a,b, Ying Meng c, Xiaolan Zhang d, Wei Luo a, Pingsheng Wu e, Bing Xiao a, Zhenshu Zhang a,⁎, Xu Li f,⁎⁎ a
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Oncology, 153rd Hospital of People's Liberation Army, Zhengzhou 450042, China Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China d Department of Senile Disease, Taihe Hospital, Hubei Medicine University, Shiyan 442000, China e Department of Cardiovascular Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China f Department of Emergency, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China b c
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 21 March 2011 Accepted 16 April 2011 Available online 3 May 2011 Keywords: Hepatic stellate cell Aldosterone RhoA/ROCK-2 signaling pathway Contraction Cirrhosis
a b s t r a c t The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT1R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver disease [1]. Following liver injury, HSCs activate and transform into myofibroblastic cells and then undergo a variety of functional changes including matrix remodeling, chemotaxis, and contraction [2]. The role of HSCs in matrix remodeling and chemotaxis has been intensively studied, nevertheless, little attention has been paid to HSC contractibility in the pathogenesis of cirrhosis and portal hypertension. HSC contractility, due to cytoskeletal reorganization within the cells, affects cells' shape and motility, contributing to intraphepatic resistance and portal hypertensin which are responsible for much of the morbidity in cirrhosis[3]. Thus a better understanding of the molecular mechanism that underlies HSC activation and contractibility is an important prerequisite for the development of new therapeutic goals for cirrhosis and portal hypertension. ⁎ Corresponding author. Tel.: + 86 020 61641546; fax: + 86 020 87280770. ⁎⁎ Corresponding author. Tel./fax: + 86 020 87283517. E-mail addresses: [email protected] (Z. Zhang), [email protected] (X. Li). 0167-0115/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2011.04.010
Aldosterone is one of the main peptides in the renin–angiotensin– aldosterone-system (RAAS) [4,5]. As a mineralocorticoid hormone, aldosterone is secreted by the cells of the zona glomerulosa upon stimulation with angiotensin II (Ang II), adrenocorticotropic hormone (ACTH), or potassium levels [6,7]. Originally described the function of aldosterone is to increase the reabsorption of sodium and water and the secretion of potassium in the kidney, thereby increasing system blood pressure and electrolytic balance [8]. Besides this hormonal effect, aldosterone can be synthesized in extra-adrenal tissues, e.g. the heart, kidney, liver and blood vessels [9]. Other studies revealed that aldosterone is now considered a key player in cellular processes underlying liver fibrosis [10,11]. Recently, large clinical trials have shown that anti-aldosteronic drug like spironolactone, a mineralocorticoid receptor (MR) antagonist, reduces fibrogenic processes in the liver, resulting in decreased tissue remodeling with long-term benefits for patients at risk for cirrhosis and portal hypertension [12]. The mechanism responsible for the remodeling of hepatic architecture partly depends on HSC contraction has been reported [13]. However, the effect of aldosterone in HSC contraction and its exact molecular mechanism are presently unclear.
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It is well-known that Ang II and aldosterone are both important peptides in the RAAS [14–16]. According to recent research, endogenous Ang II binds to angiotensin II type 1 receptor (AT1R), a surface G proteincoupled receptor, which can further activate the cytosolic small GTPase Rho, named ROCK-2 [17], leading to HSC contraction (Fig. 1). However, whether aldosterone affects HSC contraction, or the contraction may also be due to the activation of the RhoA/ROCK-2 signaling pathway is unclear. Therefore, the major objective of this proposed investigation was to examine these matters.
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (500–700 g; Animal Experiment Center, Southern Medical University, China) fed a commercial pelleted diet and given water ad libitum, were used for primary HSC isolation. All experiments and animal handling were performed according to the Association for the Accreditation and Assessment of Laboratory Animal Care.
2.3. Gel contraction assay Contractility of the activated HSCs was evaluated using collagen gel lattices in a plastic 6-well culture plate as previously described, with some modifications [19]. Firstly, HSCs were cultured for 12 h under serum-free conditions and the cells were treated by various reagents as described in Section 2.2. In addition, collagen gels were prepared by mixing Type I rat-tail collagen (3.69 mg/mL; BD Bioscience, Bedford, MA), DMEM (without serum) and a HSC cell suspension so that the final mixture resulted in a physiological ionic strength of DMEM, 0.75 mg/ml collagen and 400,000 cells/mL. A 1550 μL aliquot of the collagen gel solution was then added into each well of a 6-well culture plate and the plate was incubated for 1 h at 37 °C to allow gelation. The collagen gel was detached from the lattice through gentle circumferential dislodgement using a 200 μL micropipette tip. Each gel was photographed every hour for 6 h and the surface area of the gel was measured using digital Adobe Photoshop software 3.0. The contraction of the gel was expressed as a percentage according to the following formula: [(gel surface area of gel surface area of test substance) / (gel surface area of gel surface area of aldosterone)] × 100%. 2.4. Confocal immunofluorescence
2.2. Cell isolation and culture HSCs were isolated via in situ pronase/collagenase (Sigma-Aldrich, St. Louis, MO) perfusion followed by density gradient centrifugation as previously described[18]. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA) with antibiotics and 10% fetal bovine serum (FBS; Invitrogen) in a 37 °C humidified incubator with a mixture of 95% air and 5% CO2. To evaluate whether aldosterone affects HSCs in a time- and dosedependent manner, aldosterone was added into the cells for 5, 15, 30, 60, or 120 min, and at concentrations ranging from 0.01 to 100 nM under serum-free conditions. In all other experiments, 1 nM aldosterone was added to the cell cultures and incubated for 15 min. Y27632 (a ROCK-2 inhibitor), irbesartan (an AT1R inhibitor), and spironolactone (a MR inhibitor) were used at a concentration of 10 nM,1 h prior to aldosterone (1 nM) addition. All of these reagents were obtained from Sigma.
For phalloidin staining, HSCs were cultured on slides at a density of 5 × 104 cells/cm2 and incubated 24 h to permit cell attachment. Under serum-free conditions, the cells were treated by various reagents as described in Section 2.2. After that, the cells were fixed with 4% paraformaldehyde for 10 min. After washing with phosphate buffered saline (PBS), the cells were treated with 0.1% Triton X-100 for 5 min to permeabilize. To visualize actin fibers in the cells, the cells were stained with 2 μg/mL of TRITC-labeled phalloidin (Sigma) for 20 min at room temperature. After mounting, cell samples were observed under an Olympus-FL 500 confocal laser-scanning microscope (Tokyo, Japan). 2.5. Western blotting Total cellular protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech, Nantong, China) containing protease inhibitors (Roche, Switzerland). Protein samples (40 μg) were heated at 100 °C for 10 min before loading and being separated on 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After that, the membrane was blocked with 5% skimmed milk powder in TTBS buffer (20 mM Tris, 500 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature. Membranes were then incubated for 16 h at 4 °C with various primary antibodies in a 5% skimmed milk buffer at a dilution specified by the manufacturers. The following primary antibodies were used: rabbit anti-phospho-MLC, rabbit anti-phospho-moesin, rabbit anti-MLC and rabbit anti-moesin (all from Cell Signaling Technology, Danvers, MA, USA). On the next day, the membranes were washed with TTBS buffer and incubated in the appropriate secondary antibody solution at a 1:5000 dilution (Zhongshan Biotech, Beijing, China). Membranes were further incubated with an enhanced chemiluminescence system (Pierce, Rockford, IL) to visualize the positive protein bands. The density of the individual bands was then quantified using a densitometric scanner with Quantity One software (Bio-Rad). 2.6. RhoA pull-down assay
Fig. 1. Contractile pathway in HSCs. Agonist activation of receptors coupled to Gproteins can activate the RhoA/ROCK-2 signaling pathway, resulting in increased phosphorylation of myosin light chain (P-MLC) and thus induction of HSC contraction.
RhoA activation was assessed by a pull-down assay with a RhoA activation assay kit (Cytoskeleton, Denver, CO) according to the manufacturer's protocol [20]. Briefly, cells were lysed with a lysis buffer
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from the kit and total protein concentration was measured using a protein assay kit from Bio-Rad. Each sample containing 500 μg protein was incubated with agarose-conjugated rhotekin-RBD at 4 °C overnight. The mixtures were briefly centrifuged and the pellets resuspended in a 5x Laemmli-reducing buffer (Sigma) and boiled for 5 min. After briefly centrifuging, the supernatants were loaded and run on a 12% SDS-PAGE and transferred onto a PVDF membrane. Proteins were then analyzed by Western blotting using an anti-RhoA antibody from the kit at a 1:500 dilution.
2.7. Statistical analyses Data were summarized as mean ± standard error of the mean (S.E.M.) based on experiments repeated in triplicate. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS) 13.0
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software (Chicago, IL). A probability (P)-value b 0.05 was considered statistically significant. 3. Results 3.1. Aldosterone induction of collagen gel contraction Utilizing a hydrated collagen gel assay, we examined whether aldosterone could induce collagen gel contraction in HSCs. Fig. 2 displays a representative experiment with collagen lattice contraction. The Fig. 2A control shows a gel exposed to buffer only, resulting in a minimal decrease of the collagen surface area. Compared with the control, aldosterone at 1 nM caused an obvious decrease in surface gel area. Addition of the inhibitors spironolactone, Y-27632, or irbesartan to the HSCs for 1 h prior to a 15 minute exposure to aldosterone, resulted in significant loss of gel contraction compared with aldosterone alone (Fig. 2A). The quantitation of percentage gel contraction is shown in Fig. 2B.
Fig. 2. Aldosterone induction of collagen gel contraction in HSCs. The contraction of collagen gel in HSCs exposed to a combination of 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) and 1 nM aldosterone (ALD) compared with the cells exposed to 1 nM aldosterone (ALD) alone, as described in Section 2. (A) Photograph of hydrated collagen lattices. (B) Mean percentage of the collagen gel contraction. *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD.
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Fig. 3. Aldosterone induction of actin stress fiber formation. (A) Staining HSCs with TRITC-phalloidin showed actin fibers (red) and DAPI (blue), demonstrating flat morphology and actin stress fibers. (B) Fifteen minutes after exposure to 1 nM aldosterone (ALD), the cells have increased numbers of actin stress fibers, and developed actin fiber positive projections. After that, the cells were pre-incubated for 1 h with (C) spironolactoue (Sp), (D) Y-27632 (Y), or (E) irbesartan (Irb) at a concentration of 10 nM and then co-treated with aldosterone for 15 min. Aldosterone-induced increase of action stress fiber formation was partially reversed by these inhibitors.
3.2. Aldosterone induction of actin stress fiber formation HSCs in culture develop a cuboidal shape, with a regular arrangement of actin stress fibers. Within 15 min after adding aldosterone, staining of the actin stress fibers using phalloidin showed
a significant increase in terms of quantity, and reorganization in morphology, in response to aldosterone compared with the control (Fig. 3A-B). Aldosterone-induced increase of the action stress fiber formation was partially reversed when performed in the presence of spironolactone, Y27632, or irbesartan (Fig. 3A-D).
Fig. 4. Aldosterone induction of P-MLC expression and actin fiber formation in a time- and dose- dependent manner in HSCs. (A) HSCs were treated with 1 nM aldosterone (ALD) at different time points. The cells were then lysed, and P-MLC and MLC expression were assessed by Western blot analysis and quantified as described in Section 2. (B) HSCs were treated with indicated concentrations of aldosterone for 15 min, and P-MLC and MLC expression were assessed by Western blotting. *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD. (C and D) Actin fibers stained with TRITC-labeled phalloidin. The treatment of cells was similar to (A) and (B) and were then subjected to stain actin fibers and nuclei.
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Fig. 4 (continued).
3.3. Aldosterone activation of the actin-regulatory protein, P-MLC To assess the effect of aldosterone on phosphorylated myosin light chain (P-MLC) expression, we exposed HSCs to different dose ranges and time points as described in Section 2, then measured P-MLC expression using Western blots. As shown in Fig. 4A and B, aldosterone induced P-MLC expression in HSCs in a time- and dose- dependent manner. Fig. 4A shows that aldosterone can induces an increase in P-MLC as early as 5 min and this increase reaches to its peak in 15 min. Fig. 4B shows the
dose- dependent effect of aldosterone on P-MLC expression from 0.01 nM to 100 nM compared with the control and the effect is remarkable at a concentration of 1 nM. As previously reported, P-MLC locates downstream of ROCK-2, which regulates cell contraction by a change in actin fiber formation [21]. Thus, to confirm the role of P-MLC in aldosteroneinduced actin cytoskeleton remodeling, we observed the change of actin stress fiber formation and quantity staining with TRITC-phalloidin. As shown in Fig. 4C and D, aldosterone induced the change in actin fiber formation and recombination in a time- and dose- dependent manner.
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Fig. 5. Aldosterone activation of the RhoA/ROCK-2 signaling pathway. HSCs were incubated with serum-free culture medium for 12 h. The starved cells were treated with 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) for 1 h before 1 nM aldosterone (ALD) was added for 15 min. After that, the cells were subjected to RhoA GTP pull-down assay and Western blot analysis of RhoA GTP and total RhoA protein expression (A). The treated cells by various reagents was similar to (A), total cellular protein was extracted and subjected to Western blot analysis of P-moesin and moesin protein expression (B). *P b 0.05 compared with the untreated cells; #P b 0.05 compared with the cells treated with ALD.
3.4. Aldosterone activation of the RhoA/ROCK-2 signaling pathway Next, we performed western blots to determine any change of protein expression of the RhoA/ROCK-2 pathway with aldosterone stimulation. RhoA is a small, monomeric guanosine triphosphatebinding protein in the Rho family, with GDP-bounding inactive and
GTP-bounding active forms which can transform each other under the regulation of Rho guanine–nucleotide exchange factors (Rho GEFs). As shown in Fig. 5A, aldosterone markedly upregulated the active RhoA (RhoA GTP) protein expression in HSCs after a treatment of 15 min, as compared to the control. The effect was suppressed by both the MR inhibitor spironolactone and the ROCK-2 inhibitor Y27632 (Fig. 5A). Interestingly, the AT1R inhibitor irbesartan has an inhibitory effect on the activity of aldosterone in HSCs, suggesting that a crosseffect exists between aldosterone and Ang II. In addition, ROCK-2 activity is assessed by phosphorylation of the endogenous ROCK-2 substrate, moesin, at Thr-558 [22,23]. Using the Western blotting, our data showed that aldosterone induction of phospho-moesin (Pmoesin) expression was also suppressed by either spironolactone, Y27632 or irbesartan (Fig. 5B). Finally, aldosterone was able to induce an increase in the protein expression level of P-MLC, a downstream effector of ROCK-2, whereas the effect could be blocked by spironolactone, Y-27632, or irbesartan (Fig. 6). The blockage ability of irbesartan on aldosterone is similar to the results mentioned above. 4. Discussion
Fig. 6. Aldosterone induction of P-MLC expression via the RhoA/ROCK-2 signaling pathway. Serum-starved HSCs were incubated with 10 nM spironolactoue (Sp), Y-27632 (Y), or irbesartan (Irb) for 1 h prior to 1 nM aldosterone addition, and then co-incubated for 15 min. Total cellular protein was extracted and subjected to Western blot analysis of P-MLC and MLC protein expression. *P b 0.05 compared with the untreated cells; #Pb 0.05 compared with the cells treated with ALD.
Aldosterone origionly described as an important regulator of blood pressure and electrolytic balance. Nowdays, aldosterone is considered a key player in cellular processes underlying portal hypertension and liver fibrosis. A previous in vivo study demonstrated that expression of CYP11B2, a key gene in aldosterone synthesis, was upregulated in HSCs during CCl4-induced hepatic fibrogenesis [24]. Another in vitro study showed that aldosterone at both physiological (10− 9–10− 10 M) and pharmacological levels (10− 6 M) enhanced the synthesis of types I and IV procollagen in quiescent HSCs [25]. In the current study, we determined a role for aldosterone in HSC contraction and the potential mechanism. Our data showed for the first time that aldosterone at pharmacological levels could induce HSC contraction, and the RhoA/ ROCK-2 signaling pathway appeared to be essential in the regulation of this process.
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Collagen gel contraction is considered to be an in vitro equivalent of the tissue contraction that occurs during wound healing [26]. This procedure is associated with the dynamic morphological change in cells seeded on the gel [27,28]. It has been reported that transforming growth factor-β (TGF-β) and lysophosphatidic acid (LPS) enhance collagen gel contraction in HSCs [29,30]. To find out whether aldosterone has the same effect in HSCs as TGF-β and LPS, we further examined the effect of aldosterone on the ability of HSCs to contract collagen gels. Our results showed that aldosterone could stimulate contraction of native collagen gels. During collagen gel contraction, the cells progress from a round to a spread phenotype with the formation and rearrangement of actin filaments along the long axis of the cells [29]. The resultant increase in traction forces generated from these cellular extensions leads to a significant increase in cell contractility [31,32]. In the current study, we observed that aldosterone induced an increase in HSC actin stress fiber formation that contributed to the strength of cell contractility. The RhoA/ROCK-2 signaling pathway is involved in vasoconstrictor-induced contraction of vascular smooth muscle. After activation of G-protein-coupled receptors by vasoconstrictors, RhoA activates ROCK-2, which then phosphorylates and thereby activates MLC, leading to increase P-MLC and cell contraction [21]. In evaluating whether aldosterone can activate this signal transduction pathway, we investigated expressions of RhoA and ROCK-2 in HSCs after treatment with or without aldosterone. There was a strong upregulation of active RhoA (RhoA GTP) protein expression induced by aldosterone. It is well known that the phosphorylation state of moesin can measure the functional activity of ROCK-2 [22,23]. Our data showed that aldosterone upregulation of active RhoA in HSCs resulted in an increase in P-moesin, reflecting increased activity of ROCK-2 in the cells. The specific ROCK-2 inhibitor, Y27632, could inhibit these protein expressions induced by aldosterone, signifying that the RhoA/ ROCK-2 signaling pathway has an important role in this process. Numerous studies have demonstrated the pivotal role of P-MLC in cell contraction based on the observations that HSCs become contractile coincident with the expression of P-MLC [21]. P-MLCinduced actin depolymerization and reassembly toward the cell membrane edge [33] is responsible for the formation of cortical actin complexes contributing to the contraction of cells. A recent preliminary report showed that HSCs isolated from Sprague-Dawley rats can be activated to contraction when stimulated by Ang II [34]. As an adjunct to this study, we showed that aldosterone, another peptide in the RAAS, induced P-MLC expression in a time- and dose-dependent manner associated with cell contraction. Consistent with P-MLC expression level, HSC contractility mediated by actin stress fiber formation also acted in a time- and dose-dependent fashion. These data indicated that aldosterone could not only induce an increase in PMLC expression but also link myosin and actin to enable HSC contraction through the recombination of actin fibers in HSCs. Furthermore, to evaluate whether P-MLC is involved in aldosteroneinduced contraction via the RhoA/ROCK-2 signaling pathway, we made use of specific inhibitor in the signaling pathway to note differences in the protein expression. We observed that Y27632, the ROCK-2 inhibitor, was able to abrogate aldosterone-induced P-MLC expression. This further supports the idea that P-MLC plays an important role in mediating HSC contraction through the RhoA/ROCK2 signaling pathway. Ang II and aldosterone are both important peptides in the RAAS and can interact and regulate each other [35]. The combination of Ang II with AT1R stimulates both systemic and hepatic aldosterone production and an increase of aldosterone in turn amplifies Ang II effects by increasing the expression of AT1R and angiotensin converting enzyme (ACE) [36]. Montezano et al. demonstrated that the interaction between aldosterone and Ang II in vascular cells may lead to an amplification of cellular responses that contribute to vascular remodeling in cardiovascular diseases [37]. To explore whether the
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effect exists between aldosterone and Ang II in HSCs, we used the AT1R inhibitor irbesartan and the MR inhibitor spironolactone to examine changes in actin fiber formation and protein expressions induced by aldosterone. Interestingly, our current data showed that these inhibitors could not only decrease actin fiber formation, but also effectively inhibit the expression of RhoA GTP, P-moesin and P-MLC, indicating the potential effect of Ang II and aldosterone in HSCs. However, the precise molecular mechanism of how the AT1R inhibitor irbesartan exerted a suppressive effect on aldosterone in HSCs needs to be further studied. In addition, several previous studies have offered evidence that Ang II can evoke the RhoA/ROCK-2 signaling pathway in mammalian cells [38,39]. Considering our present data and these reports, we hypothesized that the integration of aldosterone with MR to induce HSC contraction may partly result from potentiated AngII/ AT1R-mediated actions via the RhoA/ROCK-2 signaling pathway. In summary, our data elucidate a novel molecular mechanism by which aldosterone triggers the RhoA/ROCK-2 signaling pathway to regulate HSC contraction. Targeting some of these signaling events using receptor antagonists may provide therapeutic strategy for patients suffering from cirrhosis and portal hypertension. Acknowledgements This work was supported by grants from the National Natural Scientific Foundation of China (#30871155, #30770974). References [1] Cheng JH, She H, Han YP, Wang J, Xiong S, Asahina K, Tsukamoto H. Wnt antagonism inhibits hepatic stellate cell activation and liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2008;294:G39–49. [2] Massimo P. Liver fibrosis. Springer Semin Immunopathol 2000;21:475–90. [3] Reynaert H, Thompson MG, Thomas T, Geerts A. Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension. Gut 2002;50:571–81. [4] Nagata D, Takahashi M, Sawai K, Tagami T, Usui T, Shimatsu A, Hirata Y, Naruse M. Molecular mechanism of the inhibitory effect of aldosterone on endothelial NO synthase activity. Hypertension 2006;48:165–71. [5] McGrath MF, de Bold AJ. Determinants of natriuretic peptide gene expression. Peptides 2005;26:933–43. [6] Shapiro BA, Olala L, Arun SN, Parker PM, George MV, Bollag WB. Angiotensin IIactivated protein kinase D mediates acute aldosterone secretion. Mol Cell Endocrinol 2010;317:99–105. [7] Taira M, Toba H, Murakami M, Iga I, Serizawa R, Murata S, Kobara M, Nakata T. Spironolactone exhibits direct renoprotective effects and inhibits renal renin– angiotensin–aldosteronesystem in diabetic rats. Eur J Pharmacol 2008;589: 264–71. [8] Ruilope LM. Aldosterone, hypertension and cardiovascular disease: an endless story. Hypertension 2008;52:207–8. [9] Delcayre C, Swynghedauw B. Molecular mechanisms of myocardial remodeling. The role of aldosterone. J Mol Cell Cardiol 2002;34:1577–841. [10] Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008;214: 199–210. [11] Struthers AD, MacDonald TM. Review of aldosterone- and angiotensin II-induced target organ damage and prevention. Cardiovasc Res 2004;61:663–70. [12] Caligiuri A, De Franco RM, Romanelli RG, Gentilini A, Meucci M, Failli P, Mazzetti L, Rombouts K, Geerts A, Vanasia M, Gentilini P, Marra F, Pinzani M. Anti fibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells. Gastroenterology 2003;124:504–20. [13] Kmieć Z. Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol 2001;161:III–XIII 1–151. [14] Li X, Meng Y, Wu P, Zhang Z, Yang X. Angiotensin II and aldosterone stimulating NF-κB and AP-1 activation in hepatic fibrosis of rat. Regul Pept 2007;138:15–25. [15] Sakai H, Nishimura A, Watanabe Y, Nishizawa Y, Hashimoto Y, Chiba Y, Misawa M. Involvement of Src family kinase activation in angiotensin II-induced hyperresponsiveness of rat bronchial smooth muscle. Peptides 2010;31:2216–21. [16] Li L, Zhou Y, Wang C, Zhao YL, Zhang ZG, Fan D, et al. Src tyrosine kinase regulates angiotensin II-induced protein kinase Czeta activation and proliferation in vascular smooth muscle cells. Peptides 2010;31:1159–64. [17] Rattan S, Puri RN, Fan YP. Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II. Exp Biol Med 2003;228: 972–81. [18] Liu C, Gaca MD, Swenson ES, Vellucci VF, Reiss M, Wells RG. Smads2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. J Biol Chem 2003;278:11721–8. [19] Yanase M, Ikeda H, Ogata I, Matsui A, Noiri E, Tomiya T, Arai M, Inoue Y, Tejima K, Nagashima K, Nishikawa T, Shibata M, Ikebe M, Rojkind M, Fujiwara K. Functional diversity between Rho-kinase- and MLCK-mediated cytoskeletal actions in a
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