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Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3β inactivation
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation Shohei Furukawa a, b, Momoka Yamaguchi a, *, Akira Ooka a, Haruhisa Kikuchi c, Tomohisa Ishikawa a, Shin-ya Saito a a
Department of Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga Ward, Shizuoka City, Shizuoka, 422-8526, Japan Kyoto Pharmaceutical Industries, Ltd., 38 Nishinokyo, Tsukinowa-cho, Nakagyo-ku, Kyoto, 604-8444, Japan c Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2019 Accepted 26 September 2019 Available online xxx
Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3b (GSK3b) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3b (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of b-catenin and reduced the level of non-phospho (active) b-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/b-catenin signaling pathway through dephosphorylation of GSK3b. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Hepatic stellate cell Transdifferentiation Liver fibrosis Differentiation-inducing factor-1 Glycogen synthase kinase 3b
1. Introduction Liver cirrhosis is the end-stage of chronic liver diseases, such as hepatitis B and C, as well as nonalcoholic fatty liver disease. Furthermore, fibrotic liver microenvironment has been implicated in the pathogenesis of hepatocarcinoma. Under normal physiological conditions, hepatic stellate cells (HSCs) located in the space of Disseda gap comprising hepatocytes and sinusoidal endothelial cellsdconstitute approximately 5%e8% of total cells in the liver. HSCs play important roles in the storage of vitamin A in lipid droplets in vivo [1e3]. During liver injury, HSCs are transdifferentiated from quiescent to activated HSCs, the latter of which are similar to myofibroblasts and are characterized by increased a-
Abbreviations: DIF-1, differentiation-inducing factor-1; GSK3b, glycogen synthase kinase 3b; HSC, hepatic stellate cell; PDE-1, phosphodiesterase-1; a-SMA, asmooth muscle actin; TGFb, transforming growth factor-b; FBS, fetal bovine serum. * Corresponding author. E-mail address: [email protected] (M. Yamaguchi).
smooth muscle actin (a-SMA) expression [4]. Activated HSCs produce cytokines and extracellular matrix, promoting liver fibrosis [1]. Therefore, inhibition of HSC activation could be a therapeutic target for liver fibrosis. The cellular slime mold Dictyostelium discoideum is a eukaryote that transitions from a unicellular amoeba into a multicellular slug and bears a fruiting body with spores and stalk cells [5,6]. Differentiation-inducing factor-1 (DIF-1; Fig. 1A)da morphogen produced by Dictyostelium discoideumdis an important regulator of stalk differentiation and chemotaxis during its development [7]. Recently, considerable attention has been paid to antitumor properties of DIF-1 [8]. DIF-1 reduces the movement of proliferating cells toward energy sources, thus exerting antitumor effects [9e11]. Moreover, DIF-1 attenuates proliferation of cancer cells by upregulating the MEK/ERK-dependent pathway [9,12]. DIF-1 exhibits pharmacological activities by inhibiting phosphodiesterase-1 (PDE-1) [13] or activating glycogen synthase kinase 3b (GSK3b) [8]. These activities might lead to a decline in HSC activation [14,15]. Therefore, the present study examined the potential of DIF-1 to inhibit HSC activation.
https://doi.org/10.1016/j.bbrc.2019.09.117 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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comprising of 0.0223% pronase (Merck-Millipore, Tokyo, Japan), 137 mM NaCl, 5.37 mM KCl, 3.18 mM CaCl2, 0.565 mM NaH2PO4, 0.336 mM Na2HPO4, 4.17 mM NaHCO3, 10 mM HEPES, and 0.001% DNase I (Roche, Basel, Switzerland) and solution B comprising 0.011% collagenase (Yakult, Tokyo, Japan), 137 mM NaCl, 5.37 mM KCl, 3.18 mM CaCl2, 0.565 mM NaH2PO4, 0.336 mM Na2HPO4, 4.17 mM NaHCO3, 10 mM HEPES, and 0.001% DNase I. Cells were then subjected to density gradient centrifugation with 13% Histodenz (Sigma Aldrich, St. Louis, MO, USA). Obtained HSCs were cultured in DMEM (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 C in a humidified atmosphere with 5% CO2. On the following day, reagents were added to the medium. The culture medium was changed every 3 days. 2.4. Immunostaining
Fig. 1. Effect of DIF-1 on the activation of hepatic stellate cells (HSCs) isolated from mice. (A) Chemical structure of DIF-1. (B) Freshly isolated HSCs were cultured for 7 days in a medium containing 10% fetal bovine serum in the absence (DMSO) or presence of DIF-1 (10, 50 and 100 mM). DIF-1 was applied at the 2nd day of culture. Cells were then immunostained with anti-a-smooth muscle actin (a-SMA) antibody (red) and stained with phalloidin (F-actin; green) and Hoechst 33342 (nuclei; blue). (C) Concentrationeresponse curve for the inhibitory effect of DIF-1 on HSC activation. aSMA-positive staining was used as a marker for activated HSCs. Relative a-SMA-positive area was calculated by dividing the red fluorescence area by cell number and normalized to the control (DMSO). Data are expressed as mean ± S.E.M. (n ¼ 4e12). **P < 0.01 vs. the control (DMSO) by Dunnett’s multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods 2.1. Drugs DIF-1 was synthesized in our laboratory (Fig. 1A). SQ22536, a monoclonal anti-a-smooth muscle actin antibody produced in mouse, a monoclonal anti-b-actin antibody produced in mouse, and goat anti-mouse IgG antibody conjugated peroxidase were purchased from Sigma Aldrich; anti-b-catenin antibody (#138400), Alexa Fluor 488 phalloidin (#A12379) and Alexa Fluor 546 goat anti-mouse IgG antibodies (#A11003) were obtained from Invitrogen; anti-phospho-GSK3b (Ser9) antibody (#9323), antiGSK3b antibody (#12456), anti-non-phospho (active) b-catenin (Ser33/37/Thr41) antibody (#8814) and goat anti-rabbit IgG antibody conjugated peroxidase (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA); and ODQ, TWS119, and other chemicals were obtained from Wako Pure Chemical. 2.2. Animals ddY mice (retired, 40e60 g) were purchased from Nihon SLC (Hamamatsu, Japan). All mice were housed in a 12-h light, 12-h dark cycle with food and water ad libitum. The animal use protocols of this study were submitted to and approved by an Institutional Animal Care and Use Committee of the University of Shizuoka (approval number 136055 and 186358) and according to the Guidelines for Animal Experiments established by the Japanese Pharmacological Society. 2.3. Cell isolation and culture HSCs were isolated from ddY mice via digestion with solution A
HSCs were cultured on 96-well plates for the indicated period and fixed with 2% paraformaldehyde in PBS (137 mM NaCl, 8.10 mM Na2HPO4$12H2O, 2.68 mM KCl, aa8nd 147 mM KH2PO4) for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 60 min and blocked with 3% BSA fraction V (Roche, Basel, Switzerland) for 30 min. Next, HCSs were incubated with the primary antibody against a-smooth muscle actin (a-SMA; 1:1000, Sigma Aldrich) or b-catenin (1:1000, Cell Signaling) in 1% BSA/PBS overnight at 4 C. Cells were subsequently incubated with goat anti-mouse IgG antibody-conjugated Alexa Fluor 546 (1:750, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 488-conjugated phalloidin (1:750, Invitrogen), and Hoechst 33342 (1:2000, Dojindo, Kumamoto, Japan) in 1% BSA/PBS for 60 min at room temperature. Fluorescence microscopy images were obtained. The staining area per cell was analyzed using the Image J software (NIH, Bethesda, MD, USA). 2.5. Western blotting HSCs were treated with DIF-1 for 24 h and homogenized with lysis buffer composed of 0.25 M sucrose, 2 mM EDTA, 50 mg/mL PMSF, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 50 mM NaF, and 0.5% SDS. Cell lysates were boiled at 95 C for 5 min. The amount of protein was measured using the BCA™ protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 5e10 ng of protein was separated by 7.5% SDSepolyacrylamide gel electrophoresis, and separated proteins were transferred onto a polyvinylidene difluoride membrane. After blocking with 3% BSA/TBS-Tween buffer (10 mM Tris, 137 mM NaCl, and 0.1% Tween 20) for 60 min at room temperature, the membrane was incubated with anti-phospho-GSK3b (Ser9) rabbit antibodies, anti-GSK3b rabbit antibodies, anti-non-phospho (active) b-catenin (Ser33/37/Thr41) rabbit antibodies, or anti-b-actin mouse antibodies in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan) overnight at 4 C. After the free antibodies were washed away, the membrane was incubated with goat anti-rabbit or goat anti-mouse IgG antibodies-conjugated peroxidase in Can Get Signal immunoreaction enhancer solution 2 (Toyobo) for 1 h (total-GSK3b, nonphospho b-catenin, and b-actin) or 2 h (phospho-GSK3b) at room temperature. Signals were detected using ImmunoStar LD (Wako Pure Chemical, Osaka, Japan) and analyzed using a C-DiGit Blot Scanner (Li-COR Biosciences, Japan). After the signals of phosphoGSK3b were detected, the membrane treated with WB stripping solution (Nacalai tesque, Kyoto, Japan) for 10 min and incubation in anti-GSK3b antibodies. Non-phospho (active) b-catenin (Ser33/37/ Thr41) rabbit antibody recognizes the stabilized form of b-catenin when residues Ser33, Ser37 and Thr41 are not phosphorylated. This antibody has been demonstrated to specifically recognize non-
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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phospho (active) b-catenin in colon carcinoma [16,17].
3.3. GSK3b dephosphorylation is involved in the DIF-1 effect
2.6. Statistical analysis
Next, the possible involvement of GSK3b in the inhibitory effect of DIF-1 on HSC activation was investigated. TWS119 (1 mM), a GSK3b inhibitor [18], nearly abolished the inhibitory effect of DIF-1 (50 mM) on HSC activation (Fig. 2A and B). These results suggest that GSK3b is involved in the DIF-1 effect. GSK3b is inactivated via phosphorylation at Ser9 [19]. Western blot analysis showed that the phosphorylation level of GSK3b at Ser9 was high after 24 h of culture, which was significantly decreased by DIF-1 (50 mM; Fig. 3A and B). These results suggest that DIF-1 inhibits GSK3b inactivation through reducing its phosphorylation at Ser9, which is increased during the transdifferentiation from quiescent to activated HSCs.
Results are expressed as mean ± S.E.M. Student’s t-test or Dunnett’s multiple comparison test was used to analyze differences in means. P < 0.05 was considered statistically significant. 3. Results 3.1. DIF-1 prevents HSC activation The effect of DIF-1 on the activation of HSCs isolated from mice was assessed by a-SMA immunostaining. Isolated mouse HSCs were transformed into activated HSCs expressing a-SMA by culturing in a medium supplemented with 10% FBS on hard plastic plates for 7 days. DIF-1 applied at the second day of culture decreased a-SMA-positive staining area in a concentrationdependent manner (Fig. 1B). These results suggest that DIF-1 inhibits HSC activation in FBS-containing medium. 3.2. The effect of DIF-1 on HSC activation is independent of PDE1 inhibition DIF-1 has been shown to have several pharmacological effects in mammalian cells, including PDE1 inhibition and GSK3b activation [13]. Thus, we first investigated the possible involvement of PDE1 inhibition in the inhibitory effects of DIF-1 on HSC activation. The inhibition of HSC activation by DIF-1 (50 mM) was not affected by either SQ22536 (100 mM), an inhibitor of adenylate cyclase, or ODQ (3 mM), an inhibitor of guanylate cyclase (Fig. 2A and B). These results suggest that increased intracellular cAMP or cGMP levels due to PDE1 inhibition contributes little to the inhibitory effect of DIF-1 on HSC activation.
Fig. 2. Possible involvement of phosphodiesterase inhibition or glycogen synthase kinase 3b (GSK3b) in the inhibitory effect of DIF-1 on the activation of mouse HSCs. (A) Immunostaining for a-SMA (red) in HSCs cultured for 7 days in the absence (DMSO) or presence of DIF-1 (50 mM) with or without SQ22536 (100 mM), an adenylate cyclase inhibitor, ODQ (3 mM), a guanylate cyclase inhibitor, or TWS119 (1 mM), a GSK3b inhibitor. The cells were also stained with phalloidin (F-actin; green) and Hoechst 33342 (nuclei; blue). DIF-1 was applied at the 2nd day of culture. (B) Relative a-SMApositive area was calculated by dividing the red fluorescence area by cell number and normalized to the control without DIF-1. Data are expressed as mean ± S.E.M. (n ¼ 4e8). **P < 0.01 by Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. DIF-1 inhibits b-catenin signaling in HSCs GSK3b has been shown to phosphorylate b-catenin [20]. Phosphorylated b-catenin is ubiquitinated and degraded by proteasomes in the cytoplasm. Conversely, non-phospho b-catenin is an active form and translocated into the nucleus to perform functions. The possible involvement of b-catenin in the DIF-1 effect was thus investigated. Immunostaining with anti-b-catenin antibody demonstrated the translocation of b-catenin into the nucleus in HSCs cultured for 4 days (Control in Fig. 3C). The nuclear translocation of b-catenin was apparently reduced by the treatment with DIF-1 (50 mM) for 72 h (Fig. 3C). Moreover, western blot analysis clearly demonstrated that the treatment with DIF-1 (50 mM) for 24 h significantly decreased the level of non-phospho (active) bcatenin (Fig. 3D and E). These results suggest that DIF-1 inhibits bcatenin signaling through inhibiting GSK3b inactivation in HSCs. 4. Discussion Liver fibrosis is a wound-healing response caused by chronic inflammation of the liver, which occurs in advanced stages of chronic liver diseases. In inflamed liver, HSCs are transdifferentiated from quiescent to activated forms, the latter of which look like myofibroblasts. Activated HSCs produce extracellular matrix proteins and inflammatory cytokines, thereby promoting liver fibrosis [1]. The present study clearly demonstrated that DIF-1 is a potent inhibitor of HSC activation, which is likely mediated by GSK3b dephosphorylation and subsequent disruption of b-catenin signaling. The involvement of GSK3b activation in the inhibitory effect of DIF-1 on HSC activation is supported by the present results that the DIF-1 effect was attenuated by the GSK3b inhibitor TWS119 and that DIF-1 decreased the phosphorylation level of GSK3b at Ser9. GSK3b is inactivated by phosphorylation at Ser9 [19], which inhibits the association of substrates with the binding domain of GSK3b [21]. The antitumor effect of DIF-1 has also been reported to be accompanied by reduction in Ser9-phosphorylated GSK3b levels in tumor tissues [8]. The present results also showed that DIF-1 inhibited nuclear translocation of b-catenin and reduced the level of non-phospho b-catenin in mouse HSCs. b-catenin phosphorylated by GSK3b is ubiquitinated and degraded by proteasomes [22], whereas the accumulation of non-phospho b-catenin in the cytoplasm prevents the degradation of b-catenin, leading to the translocation of b-catenin into the nucleus [23]. Taken together, DIF-1 is likely to inhibit the Wnt/b-catenin signaling pathway through inhibiting GSK3b inactivation through reducing the phosphorylation at Ser9, thereby preventing HSC activation. Several recent studies have suggested that the Wnt/b-catenin signaling pathway is one of the signaling cascades that induce HSC activation [24,25]. In the HSC cell line HSC-T6, a decrease in b-catenin expression with
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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Fig. 3. Effect of DIF-1 on GSK3b/b-catenin signaling in mouse HSCs. (A) Western blot analysis for total (t-GSK3b) and Ser9-phosphorylated GSK3b (p-GSK3b) in HSCs treated with or without DIF-1 (50 mM) for 24 h. DIF-1 was applied at the 2nd day of culture. (B) Phosphorylation of GSK3b was calculated by dividing the densitometric value of p-GSK3b by that of t-GSK3b and expressed as a percentage of the control without DIF-1. Data are expressed as mean ± S.E.M. (n ¼ 4). **P < 0.01 by Student’s t-test. (C) Immunostaining for b-catenin (red) in HSCs treated with or without DIF-1 (50 mM) for 72 h. Nuclei were stained with Hoechst 33342 (blue). Right panels show merged images. Figure C-2 shows higher magnification views of the boxed areas in Figure C-1. Right panels show merged images. Arrows indicates the localization of b-catenin in the nuclei. (D) Western blot analysis for non-phospho (active) b-catenin in HSCs treated with or without DIF-1 (50 mM) for 24 h. (E) Relative expression of non-phospho (active) b-catenin was normalized to b-actin and expressed as a percentage of the control without DIF-1. DIF-1 was applied at the 2nd day of culture. Data are expressed as mean ± S.E.M. (n ¼ 4). **P < 0.01 by Student’s t-test.
siRNA suppressed HSC activation and collagen synthesis, whereas enhanced collagen degradation [25]. Moreover, DKK-1dan inhibitor of the Wnt/b-catenin signaling pathwaydsuppressed HSC activation in rats [24]. Elevated phosphorylation of GSK3b at Ser9 was observed 24 h after primary culture of mouse HSCs. GSK3b phosphorylation at Ser9 has been reported to be induced by Akt [21]. Under the current culture condition including FBS on hard plastic plates, Akt could be activated through various signals, such as transforming growth factor-b (TGFb) [26], insulin-like growth factors-1 [27], integrin [28], and focal adhesion kinase [29]. Several studies have shown that the PI3-kinase/Akt signaling pathway contributes to the activation of rat HSCs [26,29e31]. Such culture condition might induce the activation of HSCs via the PI3-kinase/Akt signaling pathway. DIF-1 inhibits calmodulin-dependent PDE1 [13]dan enzyme that degrades cAMP and cGMP [32]. Several studies have suggested that cAMP mediates the inhibition of HSC activation by decreasing TGFb expression through the EpaceRap1 signaling pathway [33,34]. In contrast, DIF-1 has also been shown to activate GbpB, a PDE in Dictyostelium, and to decrease intracellular cGMP levels, thereby inhibiting Dictyostelium chemotaxis [35]. It is less likely, however, that PDE activation is involved in the inhibition of HSC activation by DIF-1 since cGMP analogs can suppress a-SMA expression, a marker for activated HSCs, in rats [36]. In addition, the
present results indicated less possibility of the involvement of PDE inhibition in the effect of DIF-1 on HSC activation since neither SQ22536 nor ODQ influenced this effect. Notably, however, the production of cyclic nucleotides per se might be low under the present experimental conditions since we have previously observed that caffeine, a nonselective PDE inhibitor, did not increase intercellular cAMP levels in mouse quiescent HSCs under similar experimental conditions [37]. Thus, the present results do not necessarily eliminate the possibility that PDE inhibition leads to the inhibition of HSC activation. At least under the present experimental conditions, the inhibitory effect of DIF-1 on PDE is unlikely to contribute to its inhibitory effect on HSC activation. In summary, DIF-1 suppressed HSC activation in a concentration-dependent manner, which was likely mediated through inhibiting GSK3b inactivation, which in turn inhibits the Wnt/b-catenin signaling pathway. Orally administered DIF-1 is absorbed via the digestive tract to elevate its blood concentration in mice [8]. We thus propose that DIF-1 would be a possible candidate as a therapeutic agent for liver fibrosis. Funding sources This study was supported by grants for the research projects from University of Shizuoka and by JSPS KAKENHI Grant Number
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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JP17H07000 and JP19K20186. Declaration of competing interest The authors declare no competing interests.
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Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation Shohei Furukawa a, b, Momoka Yamaguchi a, *, Akira Ooka a, Haruhisa Kikuchi c, Tomohisa Ishikawa a, Shin-ya Saito a a
Department of Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga Ward, Shizuoka City, Shizuoka, 422-8526, Japan Kyoto Pharmaceutical Industries, Ltd., 38 Nishinokyo, Tsukinowa-cho, Nakagyo-ku, Kyoto, 604-8444, Japan c Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2019 Accepted 26 September 2019 Available online xxx
Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3b (GSK3b) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3b (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of b-catenin and reduced the level of non-phospho (active) b-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/b-catenin signaling pathway through dephosphorylation of GSK3b. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Hepatic stellate cell Transdifferentiation Liver fibrosis Differentiation-inducing factor-1 Glycogen synthase kinase 3b
1. Introduction Liver cirrhosis is the end-stage of chronic liver diseases, such as hepatitis B and C, as well as nonalcoholic fatty liver disease. Furthermore, fibrotic liver microenvironment has been implicated in the pathogenesis of hepatocarcinoma. Under normal physiological conditions, hepatic stellate cells (HSCs) located in the space of Disseda gap comprising hepatocytes and sinusoidal endothelial cellsdconstitute approximately 5%e8% of total cells in the liver. HSCs play important roles in the storage of vitamin A in lipid droplets in vivo [1e3]. During liver injury, HSCs are transdifferentiated from quiescent to activated HSCs, the latter of which are similar to myofibroblasts and are characterized by increased a-
Abbreviations: DIF-1, differentiation-inducing factor-1; GSK3b, glycogen synthase kinase 3b; HSC, hepatic stellate cell; PDE-1, phosphodiesterase-1; a-SMA, asmooth muscle actin; TGFb, transforming growth factor-b; FBS, fetal bovine serum. * Corresponding author. E-mail address: [email protected] (M. Yamaguchi).
smooth muscle actin (a-SMA) expression [4]. Activated HSCs produce cytokines and extracellular matrix, promoting liver fibrosis [1]. Therefore, inhibition of HSC activation could be a therapeutic target for liver fibrosis. The cellular slime mold Dictyostelium discoideum is a eukaryote that transitions from a unicellular amoeba into a multicellular slug and bears a fruiting body with spores and stalk cells [5,6]. Differentiation-inducing factor-1 (DIF-1; Fig. 1A)da morphogen produced by Dictyostelium discoideumdis an important regulator of stalk differentiation and chemotaxis during its development [7]. Recently, considerable attention has been paid to antitumor properties of DIF-1 [8]. DIF-1 reduces the movement of proliferating cells toward energy sources, thus exerting antitumor effects [9e11]. Moreover, DIF-1 attenuates proliferation of cancer cells by upregulating the MEK/ERK-dependent pathway [9,12]. DIF-1 exhibits pharmacological activities by inhibiting phosphodiesterase-1 (PDE-1) [13] or activating glycogen synthase kinase 3b (GSK3b) [8]. These activities might lead to a decline in HSC activation [14,15]. Therefore, the present study examined the potential of DIF-1 to inhibit HSC activation.
https://doi.org/10.1016/j.bbrc.2019.09.117 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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comprising of 0.0223% pronase (Merck-Millipore, Tokyo, Japan), 137 mM NaCl, 5.37 mM KCl, 3.18 mM CaCl2, 0.565 mM NaH2PO4, 0.336 mM Na2HPO4, 4.17 mM NaHCO3, 10 mM HEPES, and 0.001% DNase I (Roche, Basel, Switzerland) and solution B comprising 0.011% collagenase (Yakult, Tokyo, Japan), 137 mM NaCl, 5.37 mM KCl, 3.18 mM CaCl2, 0.565 mM NaH2PO4, 0.336 mM Na2HPO4, 4.17 mM NaHCO3, 10 mM HEPES, and 0.001% DNase I. Cells were then subjected to density gradient centrifugation with 13% Histodenz (Sigma Aldrich, St. Louis, MO, USA). Obtained HSCs were cultured in DMEM (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 C in a humidified atmosphere with 5% CO2. On the following day, reagents were added to the medium. The culture medium was changed every 3 days. 2.4. Immunostaining
Fig. 1. Effect of DIF-1 on the activation of hepatic stellate cells (HSCs) isolated from mice. (A) Chemical structure of DIF-1. (B) Freshly isolated HSCs were cultured for 7 days in a medium containing 10% fetal bovine serum in the absence (DMSO) or presence of DIF-1 (10, 50 and 100 mM). DIF-1 was applied at the 2nd day of culture. Cells were then immunostained with anti-a-smooth muscle actin (a-SMA) antibody (red) and stained with phalloidin (F-actin; green) and Hoechst 33342 (nuclei; blue). (C) Concentrationeresponse curve for the inhibitory effect of DIF-1 on HSC activation. aSMA-positive staining was used as a marker for activated HSCs. Relative a-SMA-positive area was calculated by dividing the red fluorescence area by cell number and normalized to the control (DMSO). Data are expressed as mean ± S.E.M. (n ¼ 4e12). **P < 0.01 vs. the control (DMSO) by Dunnett’s multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods 2.1. Drugs DIF-1 was synthesized in our laboratory (Fig. 1A). SQ22536, a monoclonal anti-a-smooth muscle actin antibody produced in mouse, a monoclonal anti-b-actin antibody produced in mouse, and goat anti-mouse IgG antibody conjugated peroxidase were purchased from Sigma Aldrich; anti-b-catenin antibody (#138400), Alexa Fluor 488 phalloidin (#A12379) and Alexa Fluor 546 goat anti-mouse IgG antibodies (#A11003) were obtained from Invitrogen; anti-phospho-GSK3b (Ser9) antibody (#9323), antiGSK3b antibody (#12456), anti-non-phospho (active) b-catenin (Ser33/37/Thr41) antibody (#8814) and goat anti-rabbit IgG antibody conjugated peroxidase (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA); and ODQ, TWS119, and other chemicals were obtained from Wako Pure Chemical. 2.2. Animals ddY mice (retired, 40e60 g) were purchased from Nihon SLC (Hamamatsu, Japan). All mice were housed in a 12-h light, 12-h dark cycle with food and water ad libitum. The animal use protocols of this study were submitted to and approved by an Institutional Animal Care and Use Committee of the University of Shizuoka (approval number 136055 and 186358) and according to the Guidelines for Animal Experiments established by the Japanese Pharmacological Society. 2.3. Cell isolation and culture HSCs were isolated from ddY mice via digestion with solution A
HSCs were cultured on 96-well plates for the indicated period and fixed with 2% paraformaldehyde in PBS (137 mM NaCl, 8.10 mM Na2HPO4$12H2O, 2.68 mM KCl, aa8nd 147 mM KH2PO4) for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 60 min and blocked with 3% BSA fraction V (Roche, Basel, Switzerland) for 30 min. Next, HCSs were incubated with the primary antibody against a-smooth muscle actin (a-SMA; 1:1000, Sigma Aldrich) or b-catenin (1:1000, Cell Signaling) in 1% BSA/PBS overnight at 4 C. Cells were subsequently incubated with goat anti-mouse IgG antibody-conjugated Alexa Fluor 546 (1:750, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 488-conjugated phalloidin (1:750, Invitrogen), and Hoechst 33342 (1:2000, Dojindo, Kumamoto, Japan) in 1% BSA/PBS for 60 min at room temperature. Fluorescence microscopy images were obtained. The staining area per cell was analyzed using the Image J software (NIH, Bethesda, MD, USA). 2.5. Western blotting HSCs were treated with DIF-1 for 24 h and homogenized with lysis buffer composed of 0.25 M sucrose, 2 mM EDTA, 50 mg/mL PMSF, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 50 mM NaF, and 0.5% SDS. Cell lysates were boiled at 95 C for 5 min. The amount of protein was measured using the BCA™ protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 5e10 ng of protein was separated by 7.5% SDSepolyacrylamide gel electrophoresis, and separated proteins were transferred onto a polyvinylidene difluoride membrane. After blocking with 3% BSA/TBS-Tween buffer (10 mM Tris, 137 mM NaCl, and 0.1% Tween 20) for 60 min at room temperature, the membrane was incubated with anti-phospho-GSK3b (Ser9) rabbit antibodies, anti-GSK3b rabbit antibodies, anti-non-phospho (active) b-catenin (Ser33/37/Thr41) rabbit antibodies, or anti-b-actin mouse antibodies in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan) overnight at 4 C. After the free antibodies were washed away, the membrane was incubated with goat anti-rabbit or goat anti-mouse IgG antibodies-conjugated peroxidase in Can Get Signal immunoreaction enhancer solution 2 (Toyobo) for 1 h (total-GSK3b, nonphospho b-catenin, and b-actin) or 2 h (phospho-GSK3b) at room temperature. Signals were detected using ImmunoStar LD (Wako Pure Chemical, Osaka, Japan) and analyzed using a C-DiGit Blot Scanner (Li-COR Biosciences, Japan). After the signals of phosphoGSK3b were detected, the membrane treated with WB stripping solution (Nacalai tesque, Kyoto, Japan) for 10 min and incubation in anti-GSK3b antibodies. Non-phospho (active) b-catenin (Ser33/37/ Thr41) rabbit antibody recognizes the stabilized form of b-catenin when residues Ser33, Ser37 and Thr41 are not phosphorylated. This antibody has been demonstrated to specifically recognize non-
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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phospho (active) b-catenin in colon carcinoma [16,17].
3.3. GSK3b dephosphorylation is involved in the DIF-1 effect
2.6. Statistical analysis
Next, the possible involvement of GSK3b in the inhibitory effect of DIF-1 on HSC activation was investigated. TWS119 (1 mM), a GSK3b inhibitor [18], nearly abolished the inhibitory effect of DIF-1 (50 mM) on HSC activation (Fig. 2A and B). These results suggest that GSK3b is involved in the DIF-1 effect. GSK3b is inactivated via phosphorylation at Ser9 [19]. Western blot analysis showed that the phosphorylation level of GSK3b at Ser9 was high after 24 h of culture, which was significantly decreased by DIF-1 (50 mM; Fig. 3A and B). These results suggest that DIF-1 inhibits GSK3b inactivation through reducing its phosphorylation at Ser9, which is increased during the transdifferentiation from quiescent to activated HSCs.
Results are expressed as mean ± S.E.M. Student’s t-test or Dunnett’s multiple comparison test was used to analyze differences in means. P < 0.05 was considered statistically significant. 3. Results 3.1. DIF-1 prevents HSC activation The effect of DIF-1 on the activation of HSCs isolated from mice was assessed by a-SMA immunostaining. Isolated mouse HSCs were transformed into activated HSCs expressing a-SMA by culturing in a medium supplemented with 10% FBS on hard plastic plates for 7 days. DIF-1 applied at the second day of culture decreased a-SMA-positive staining area in a concentrationdependent manner (Fig. 1B). These results suggest that DIF-1 inhibits HSC activation in FBS-containing medium. 3.2. The effect of DIF-1 on HSC activation is independent of PDE1 inhibition DIF-1 has been shown to have several pharmacological effects in mammalian cells, including PDE1 inhibition and GSK3b activation [13]. Thus, we first investigated the possible involvement of PDE1 inhibition in the inhibitory effects of DIF-1 on HSC activation. The inhibition of HSC activation by DIF-1 (50 mM) was not affected by either SQ22536 (100 mM), an inhibitor of adenylate cyclase, or ODQ (3 mM), an inhibitor of guanylate cyclase (Fig. 2A and B). These results suggest that increased intracellular cAMP or cGMP levels due to PDE1 inhibition contributes little to the inhibitory effect of DIF-1 on HSC activation.
Fig. 2. Possible involvement of phosphodiesterase inhibition or glycogen synthase kinase 3b (GSK3b) in the inhibitory effect of DIF-1 on the activation of mouse HSCs. (A) Immunostaining for a-SMA (red) in HSCs cultured for 7 days in the absence (DMSO) or presence of DIF-1 (50 mM) with or without SQ22536 (100 mM), an adenylate cyclase inhibitor, ODQ (3 mM), a guanylate cyclase inhibitor, or TWS119 (1 mM), a GSK3b inhibitor. The cells were also stained with phalloidin (F-actin; green) and Hoechst 33342 (nuclei; blue). DIF-1 was applied at the 2nd day of culture. (B) Relative a-SMApositive area was calculated by dividing the red fluorescence area by cell number and normalized to the control without DIF-1. Data are expressed as mean ± S.E.M. (n ¼ 4e8). **P < 0.01 by Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. DIF-1 inhibits b-catenin signaling in HSCs GSK3b has been shown to phosphorylate b-catenin [20]. Phosphorylated b-catenin is ubiquitinated and degraded by proteasomes in the cytoplasm. Conversely, non-phospho b-catenin is an active form and translocated into the nucleus to perform functions. The possible involvement of b-catenin in the DIF-1 effect was thus investigated. Immunostaining with anti-b-catenin antibody demonstrated the translocation of b-catenin into the nucleus in HSCs cultured for 4 days (Control in Fig. 3C). The nuclear translocation of b-catenin was apparently reduced by the treatment with DIF-1 (50 mM) for 72 h (Fig. 3C). Moreover, western blot analysis clearly demonstrated that the treatment with DIF-1 (50 mM) for 24 h significantly decreased the level of non-phospho (active) bcatenin (Fig. 3D and E). These results suggest that DIF-1 inhibits bcatenin signaling through inhibiting GSK3b inactivation in HSCs. 4. Discussion Liver fibrosis is a wound-healing response caused by chronic inflammation of the liver, which occurs in advanced stages of chronic liver diseases. In inflamed liver, HSCs are transdifferentiated from quiescent to activated forms, the latter of which look like myofibroblasts. Activated HSCs produce extracellular matrix proteins and inflammatory cytokines, thereby promoting liver fibrosis [1]. The present study clearly demonstrated that DIF-1 is a potent inhibitor of HSC activation, which is likely mediated by GSK3b dephosphorylation and subsequent disruption of b-catenin signaling. The involvement of GSK3b activation in the inhibitory effect of DIF-1 on HSC activation is supported by the present results that the DIF-1 effect was attenuated by the GSK3b inhibitor TWS119 and that DIF-1 decreased the phosphorylation level of GSK3b at Ser9. GSK3b is inactivated by phosphorylation at Ser9 [19], which inhibits the association of substrates with the binding domain of GSK3b [21]. The antitumor effect of DIF-1 has also been reported to be accompanied by reduction in Ser9-phosphorylated GSK3b levels in tumor tissues [8]. The present results also showed that DIF-1 inhibited nuclear translocation of b-catenin and reduced the level of non-phospho b-catenin in mouse HSCs. b-catenin phosphorylated by GSK3b is ubiquitinated and degraded by proteasomes [22], whereas the accumulation of non-phospho b-catenin in the cytoplasm prevents the degradation of b-catenin, leading to the translocation of b-catenin into the nucleus [23]. Taken together, DIF-1 is likely to inhibit the Wnt/b-catenin signaling pathway through inhibiting GSK3b inactivation through reducing the phosphorylation at Ser9, thereby preventing HSC activation. Several recent studies have suggested that the Wnt/b-catenin signaling pathway is one of the signaling cascades that induce HSC activation [24,25]. In the HSC cell line HSC-T6, a decrease in b-catenin expression with
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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Fig. 3. Effect of DIF-1 on GSK3b/b-catenin signaling in mouse HSCs. (A) Western blot analysis for total (t-GSK3b) and Ser9-phosphorylated GSK3b (p-GSK3b) in HSCs treated with or without DIF-1 (50 mM) for 24 h. DIF-1 was applied at the 2nd day of culture. (B) Phosphorylation of GSK3b was calculated by dividing the densitometric value of p-GSK3b by that of t-GSK3b and expressed as a percentage of the control without DIF-1. Data are expressed as mean ± S.E.M. (n ¼ 4). **P < 0.01 by Student’s t-test. (C) Immunostaining for b-catenin (red) in HSCs treated with or without DIF-1 (50 mM) for 72 h. Nuclei were stained with Hoechst 33342 (blue). Right panels show merged images. Figure C-2 shows higher magnification views of the boxed areas in Figure C-1. Right panels show merged images. Arrows indicates the localization of b-catenin in the nuclei. (D) Western blot analysis for non-phospho (active) b-catenin in HSCs treated with or without DIF-1 (50 mM) for 24 h. (E) Relative expression of non-phospho (active) b-catenin was normalized to b-actin and expressed as a percentage of the control without DIF-1. DIF-1 was applied at the 2nd day of culture. Data are expressed as mean ± S.E.M. (n ¼ 4). **P < 0.01 by Student’s t-test.
siRNA suppressed HSC activation and collagen synthesis, whereas enhanced collagen degradation [25]. Moreover, DKK-1dan inhibitor of the Wnt/b-catenin signaling pathwaydsuppressed HSC activation in rats [24]. Elevated phosphorylation of GSK3b at Ser9 was observed 24 h after primary culture of mouse HSCs. GSK3b phosphorylation at Ser9 has been reported to be induced by Akt [21]. Under the current culture condition including FBS on hard plastic plates, Akt could be activated through various signals, such as transforming growth factor-b (TGFb) [26], insulin-like growth factors-1 [27], integrin [28], and focal adhesion kinase [29]. Several studies have shown that the PI3-kinase/Akt signaling pathway contributes to the activation of rat HSCs [26,29e31]. Such culture condition might induce the activation of HSCs via the PI3-kinase/Akt signaling pathway. DIF-1 inhibits calmodulin-dependent PDE1 [13]dan enzyme that degrades cAMP and cGMP [32]. Several studies have suggested that cAMP mediates the inhibition of HSC activation by decreasing TGFb expression through the EpaceRap1 signaling pathway [33,34]. In contrast, DIF-1 has also been shown to activate GbpB, a PDE in Dictyostelium, and to decrease intracellular cGMP levels, thereby inhibiting Dictyostelium chemotaxis [35]. It is less likely, however, that PDE activation is involved in the inhibition of HSC activation by DIF-1 since cGMP analogs can suppress a-SMA expression, a marker for activated HSCs, in rats [36]. In addition, the
present results indicated less possibility of the involvement of PDE inhibition in the effect of DIF-1 on HSC activation since neither SQ22536 nor ODQ influenced this effect. Notably, however, the production of cyclic nucleotides per se might be low under the present experimental conditions since we have previously observed that caffeine, a nonselective PDE inhibitor, did not increase intercellular cAMP levels in mouse quiescent HSCs under similar experimental conditions [37]. Thus, the present results do not necessarily eliminate the possibility that PDE inhibition leads to the inhibition of HSC activation. At least under the present experimental conditions, the inhibitory effect of DIF-1 on PDE is unlikely to contribute to its inhibitory effect on HSC activation. In summary, DIF-1 suppressed HSC activation in a concentration-dependent manner, which was likely mediated through inhibiting GSK3b inactivation, which in turn inhibits the Wnt/b-catenin signaling pathway. Orally administered DIF-1 is absorbed via the digestive tract to elevate its blood concentration in mice [8]. We thus propose that DIF-1 would be a possible candidate as a therapeutic agent for liver fibrosis. Funding sources This study was supported by grants for the research projects from University of Shizuoka and by JSPS KAKENHI Grant Number
Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117
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JP17H07000 and JP19K20186. Declaration of competing interest The authors declare no competing interests.
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Please cite this article as: S. Furukawa et al., Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3b inactivation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.117