Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells

Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and plateletderived growth factor in rat hepatic stellate cells KIM R. BRIDLE, LIN LI, ROSEMARY O’NEILL, ROBERT S. BRITTON, and BRUCE R. BACON SAINT LOUIS, MISSOURI

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3kinase (PI3-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, PI3-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that PI3-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-3␤. Coordinate activation of ERK, PI3-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis. (J Lab Clin Med 2006;147:234 –241) Abbreviations: CDK ⫽ cyclin-dependent kinase; ERK ⫽ extracellular signal-regulated kinase; GSK-3␤ ⫽ glycogen synthase kinase-3␤; HSC ⫽ hepatic stellate cells; IGF-1 ⫽ insulin-like growth factor-1; MAP ⫽ mitogen-activated protein; MEK ⫽ MAP/ERK kinase; p70-S6-K ⫽ p70-S6 kinase; PDGF ⫽ platelet-derived growth factor; PI3-K ⫽ phosphatidylinositol 3-kinase; PKB ⫽ protein kinase B

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epatic stellate cell (HSC) proliferation is an important event in the development of hepatic fibrosis. Proliferation of HSC is mediated by growth factors such as insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor (PDGF).1–7 Insulin-like growth factors are known to play a role in growth and differentiation of a wide variety of cell types.8 The liver is a major source of IGF-1,8 and HSC and myofibroblasts express components of the IGF axis.9 IGF-1 has recently been shown to play a major

role in liver fibrogenesis and regeneration.10 It has previously been demonstrated that extracellular signalregulated kinase (ERK) and phosphatidylinositol 3kinase (PI3-K) signaling pathways are involved in the proliferative effect of PDGF on HSC.11–13 Previous reports have shown IGF-1 can also induce ERK, PI3-K and p70-S6 kinase (p70-S6-K),7,14,15 although the crosstalk between these pathways in HSC has not been fully examined. The ERK pathway has been extensively studied, and

From the Division of Gastroenterology and Hepatology, Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri. Supported by a grant from the U.S. Public Health Service to B.R.B. (R01 DK-41816). K.R.B. was the recipient of a Postdoctoral Research Fellowship from the American Liver Foundation (Robert E. Rossel Memorial Award). Submitted for publication July 26, 2005; revision submitted December 5, 2005; accepted for publication December 29, 2005.

Reprint requests: Bruce R. Bacon, M.D., Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, 3635 Vista Avenue, St. Louis, MO 63110-0250. e-mail: [email protected].

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plays a role in cell proliferation, differentiation and transformation.16 –18 In fibroblasts, it has been demonstrated that growth factor-induced activation of ERK is an absolute requirement for the proliferative response19 and it may be responsible for increases in cyclin D levels.20,21 Occupation and dimerization of a tyrosine kinase receptor (such as the PDGF receptor) results in autophosphorylation of tyrosines in the intracellular domain of the receptor. Motifs containing these phosphorylated tyrosines can recruit adaptor proteins such as Grb2, which has a binding site for Sos, a guanine nucleotide exchange factor.22,23 The association of Sos with the small GTP-binding protein Ras converts Ras to its GTP-bound active form, which in turn activates Raf kinase. Raf kinase can then activate MAP/ERK kinase (MEK) by phosphorylation, and MEK phosphorylates ERK on specific threonine and tyrosine residues. Upon activation, ERK translocates to the nucleus where it phosphorylates transcription factors such as Elk-1/TCF, increasing their transcriptional efficacy.16,17 The PI3-K pathway has also been implicated in mitogenic signaling in a variety of cell types.24 –31 Important downstream components of this signaling pathway include protein kinase B (PKB, also known as Akt) and p70-S6-K. PI3-K is composed of a 110 kDa catalytic subunit and an 85 kDa adaptor subunit: the latter binds to phosphorylated tyrosine residues, thus serving to recruit the catalytic subunit to tyrosine kinase signaling pathways.24,25 When activated, PI3-K converts its preferred substrate phosphatidylinositol (4,5)-bisphosphate to phosphatidylinositol (3,4,5)-triphosphate (PI-3,4, 5-P3).26,27 PI-3,4,5-P3 and its dephosphorylation product PI-3,4-P2 can recruit PKB and its upstream kinase PDK1 to the membrane, resulting in PDK1 activating PKB.24,25,32–35 In addition to the role of growth factors as mitogens, it is now recognized that some growth factors play important roles as cell survival factors. In particular, IGF-1 has been shown to have survival properties in various cell types.36 –38 The PI3-K/PKB pathway, in addition to being involved in cell proliferation, plays a role as an anti-apoptotic pathway. Phosphorylation catalyzed by PKB results in the inactivation of glycogen synthase kinase-3␤ (GSK-3␤) which inhibits the proapoptotic effect of GSK-3␤. Activation of p70-S6-K is a highly conserved cellular response to mitogenic stimuli.39 – 42 This serine/threonine kinase was originally described as the enzyme that phosphorylates the S6 protein of the 40S ribosomal subunit, which may regulate the initiation of translation of specific mRNA species.39,42– 44 Subsequent observations suggest that p70-S6-K may regulate a wider array of cellular processes, including gene transcription (through CREM transcription factor) and cell

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proliferation by influencing the levels of cyclins D and E.39,41– 43,45 A number of positive and negative regulatory cell cycle components play an important role in progression through the cell cycle and therefore cell proliferation.46,47 Progression through the cell cycle is dependent on the formation of active cyclin/cyclin dependent kinase (CDK) complexes. Different cyclin/CDK complexes regulate different stages of the cell cycle.48 Cyclin D1/CDK4 regulates G1 progression through the restriction point, while cyclin E/CDK2 is active during G1/S transition.49,50 Thus, the aims of the current study were to examine the roles of the ERK, PI3-K and p70-S6-K signaling pathways in IGF-1- and PDGF-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. We also investigated whether IGF-1 and PDGF would increase the levels of cyclins D1 and E, and whether they would induce phosphorylation of GSK-3␤. METHODS Hepatic stellate cell isolation and culture. HSC were isolated from male Sprague-Dawley rat livers by sequential pronase and collagenase digestion.51,52 All procedures using animals were approved by the Animal Care Committee of Saint Louis University. After density-gradient centrifugation, HSC were cultured on uncoated tissue culture plastic in DMEM with 10% fetal calf serum and were used at passage 2-6. When cells had reached 70-80% confluence, they were serumstarved for 48 hr prior to the experiments. Some HSC were treated with inhibitors of p70-S6-K, MEK (upstream ERK activating kinase) and PI3-K (or compounds that inhibit phosphorylation of the kinases); rapamycin (5 nM), PD-98059 (50 ␮M), wortmannin (500 nM), respectively, starting 30 min before stimulation with IGF-1 (100 ng/ml) or PDGF (10 ng/ml). The concentrations of IGF-1 (100 ng/ml) and PDGF (10 ng/ml) used were the lowest concentrations found to produce maximum stimulation of HSC proliferation (data not shown). To evaluate the potential toxicity of the inhibitors and to ensure equal protein loading, we performed Western blotting for pan-PKB (1:1000, Cell Signaling Technology) that demonstrated that IGF-1, PDGF or the inhibitors did not change the levels of PKB (data not shown). We also examined any effects on total cellular protein following inhibitor treatment and this was also unchanged (data not shown). 3 Hepatic stellate cell proliferation. H-thymidine incorporation to measure DNA synthesis was used as an index of HSC proliferation in the presence of PDGF and IGF-1.53 Following a 48 hr incubation in serumfree medium, HSC were incubated with methyl-3H-thy-

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midine for 18 hr after treatment with IGF-1 or PDGF (with or without inhibitor treatment). Phospho-kinase western blotting. HSC lysates were harvested 30 min after treatment with growth factors. After SDS-PAGE, Western blotting was performed using overnight incubation with a monoclonal phospho p44/42 ERK (Thr202/Tyr204) antibody (1:2000), polyclonal phospho PKB/Akt (Ser473) antibody (1:1000), polyclonal phospho p70-S6-K (Thr389) antibody (1: 1000), or phospho-GSK-3␤ (Ser9) antibody (1:1000): these antibodies were purchased from Cell Signaling Technology. Immunoreactive bands were detected using enhanced chemiluminescence (ECL, Amersham), and quantitated by scanning laser densitometry. For analysis of phospho-ERK levels both the p44 and p42 bands were quantitated. Cyclin western blotting. HSC were cultured as described above and lysates were harvested 18 h after treatment with growth factors. Western blotting was performed after SDS-PAGE using polyclonal antibodies against cyclin D1 and cyclin E (1:3000; Santa Cruz). The immunoreactive bands were detected using enhanced chemiluminescence, and quantitated by scanning laser densitometry. Statistical analysis. Statistical significance was measured between groups using a two-tailed Student’s ttest, and a p value of ⬍0.05 was considered to be significant. Values are presented as mean ⫾ SE.

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Fig 1. Effect of PDGF and IGF-1 on HSC proliferation, and the influence of selective inhibitors. Serum-starved HSC were treated with PD-98059 (PD) (50 ␮M), wortmannin (WT) (500 nM), rapamycin (RAP) (5 nM) or vehicle prior to treatment with PDGF (10 ng/ml) or IGF-1 (100 ng/ml). Proliferation was measured by 3Hthymidine incorporation. Results are expressed as fold-increase from control and are shown as mean ⫾ SE of three experiments performed in triplicate.

RESULTS Hepatic stellate cell proliferation. IGF-1 and PDGF were mitogenic for HSC, although the response to PDGF (6.6 ⫾ 1.9-fold) was substantially greater than for IGF-1 (1.4 ⫾ 0.1-fold). Inhibitors of the ERK (PD-98059), PI3-K (wortmannin) or p70-S6-K (rapamycin) pathways suppressed IGF-1- and PDGFinduced proliferation of HSC (Fig 1). PD-98059 reduced IGF-1- and PDGF-induced proliferation by 50% and 68%, respectively, and wortmannin decreased IGF-1- and PDGF-induced proliferation by 36% and 68%, respectively. Rapamycin had a strong inhibitory effect on PDGF- and IGF-1-induced proliferation, reducing proliferation almost to control levels (Fig 1). Phospho-ERK levels. Both IGF-1 and PDGF increased phospho-ERK levels (Fig. 2), but the PDGF-induced increase (4.3 ⫾ 0.9-fold) was larger than that of IGF-1 (1.2 ⫾ 0.1-fold). As anticipated, treatment of HSC with PD-98059 effectively inhibited both IGF-1- and PDGFinduced phospho-ERK levels (100% and 77%, respectively). Rapamycin treatment had no significant effect on phospho-ERK levels. However, inhibition of PI3-K using wortmannin partially suppressed PDGF-induced phospho-ERK levels (36%), while having no effect on IGF-1-induced phospho-ERK levels.

Fig 2. Effect of PDGF and IGF-1 on the levels of phospho-ERK in HSC. Serum-starved HSC were treated with PD-98059 (50 ␮M), wortmannin (500 nM) or rapamycin (5 nM) alone or prior to treatment with PDGF (10 ng/ml) or IGF-1 (100 ng/ml). Results are expressed as fold-increase over control and are the mean ⫾ SE of at least three separate HSC preparations. Representative Western blots are shown for each group. *p⬍0.05; #p⬍0.05 compared to PDGF or IGF-1 alone, respectively.

p70-S6-K activity. The levels of phospho-p70-S6-K were used as an index of p70-S6-K activity. As shown in Fig 3, both IGF-1 and PDGF caused a marked increase in p70-S6-K activity, 14.4 ⫾ 2.7-fold and 16.9 ⫾ 3.2-fold, respectively. As expected, rapamycin completely inhibited this IGF-1- and PDGF-induced increase in p70-S6-K activity. The PI3-K inhibitor wortmannin also markedly reduced both IGF-1- and PDGFinduced p70-S6-K activity (100% and 79%, respectively). Inhibition of the ERK pathway using

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Fig 3. Effect of IGF-1 and PDGF on the levels of phospho-p70-S6-K in HSC. Serum-starved HSC were treated with PD-98059 (50 ␮M), wortmannin (500 nM) or rapamycin (5 nM) alone or prior to treatment with IGF-1 (100 ng/ml) or PDGF (10 ng/ml). Results are expressed as fold-increase over control and are the mean ⫾ SE of at least three separate HSC preparations. Representative Western blots are shown for each group. *p⬍0.05; #p⬍0.05 compared to PDGF or IGF-1 alone, respectively.

Fig 4. Effect of IGF-1 and PDGF on the levels of phospho-PKB (Akt). Serum-starved HSC were treated with PD-98059 (50 ␮M), wortmannin (500 nM) or rapamycin (5 nM) alone or prior to treatment with IGF-1 (100 ng/ml) or PDGF (10 ng/ml). Results are expressed as fold-increase over control and are the mean ⫾ SE of at least three separate HSC preparations. Representative Western blots are shown for each group. *p⬍0.05; #p⬍0.05 compared to PDGF or IGF-1 alone, respectively.

PD-98059 did not significantly affect IGF-1- or PDGFinduced p70-S6-K activity. PI3-K activity. The levels of phospho-PKB were used as an index of p70-S6-K activity. PI3-K activity was also increased substantially by treatment with both IGF-1 (6.5 ⫾ 2.0-fold) and PDGF (11.9 ⫾ 4.0-fold) (Fig 4). Treatment of HSC with wortmannin effectively inhibited both IGF-1- and PDGF-induced PI3-K activity, 100% and 81%, respectively. Rapamycin did not

Fig 5. Effect of PDGF and IGF-1 on (A) cyclin D1 and (B) cyclin E levels in HSC. Serum-starved HSC were treated with PD-98059 (50 ␮M), wortmannin (500 nM) or rapamycin (5 nM) prior to treatment with PDGF (10 ng/ml) or IGF-1 (100 ng/ml). Cyclin D1 and E levels were determined by Western blotting. Results are expressed as fold-increase over control and are the mean ⫾ SE of at least three separate HSC preparations. Representative Western blots are shown for each group. *p⬍0.05; #p⬍0.05 compared to PDGF or IGF-1 alone, respectively.

affect PI3-K activity, while PD-98059 significantly decreased IGF-1- and PDGF-induced PI3-K activity (37% and 45%, respectively). Cyclin D1 levels. PDGF treatment of HSC resulted in a 4.2 ⫾ 1.4-fold increase in cyclin D1 levels while IGF-1-treatment increased cyclin D1 by 1.4 ⫾ 0.1-fold (Fig 5, A). The ERK pathway inhibitor, PD-98059, decreased PDGF- and IGF-1-induced cyclin D1 levels by 60% and 64%, respectively. Treatment of HSC with wortmannin also reduced PDGF- and IGF-1-induced cyclin D1 levels by 45% and 50%, respectively. PDGFand IGF-1-induced cyclin D1 levels were decreased by rapamycin by 54% and 31%, respectively.

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Fig 6. Effect of IGF-1 and PDGF on the levels of phospho-GSK-3␤ in HSC. Serum-starved HSC were treated with wortmannin (500 nM) or vehicle prior to treatment with IGF-1 (100 ng/ml) or PDGF (10 ng/ml). Phospho-GSK-3␤ levels were determined by Western blotting and the results are expressed as fold-increase over control (mean ⫾ SE of at least three separate HSC preparations). Representative Western blots are shown for each group. *p⬍0.05 vs. control, **p⬍0.05 vs. PDGF alone, #p⬍0.05 vs. control, ##p⬍0.05 vs. IGF-1 alone.

Cyclin E levels. PDGF increased cyclin E levels by 2.1 ⫾ 0.3-fold, but the effect of the inhibitors was less pronounced than the response for cyclin D1 (Fig 5, B). PDGF-induced cyclin E levels were significantly decreased by rapamycin (48%), but not by PD-98059 or wortmannin. IGF-1 had only a small stimulatory effect on cyclin E levels (1.2 ⫾ 0.2-fold). Phospho-GSK-3␤ levels. IGF-1 and PDGF increased phosphorylation of GSK-3␤ by 1.6-fold and 2.2-fold, respectively (Fig 6). Following inhibition of the PI3-K/ PKB pathway with wortmannin, there was a significant decrease in the phosphorylation of GSK-3␤ induced by IGF-1 and PDGF (55% and 44%, respectively).

DISCUSSION

Both IGF-1 and PDGF have been implicated in the pathogenesis of hepatic fibrosis.2,4 –7 The results of this study demonstrate the mitogenic effects of both PDGF and IGF-1 on activated HSC, with PDGF having a more potent effect. For the first time, this study has directly compared the effects of both IGF-1 and PDGF on three intracellular signaling pathways, namely the ERK, PI3-K and p70-S6-K pathways. In concert with the proliferative response, we have shown that activities of ERK, PKB and p70-S6-K are upregulated by IGF-1 and PDGF. Selective inhibition of the ERK, PI3-K and p70-S6-K pathways decreased HSC proliferation; thus, IGF-1- and PDGF-induced mitogenesis of HSC is dependent on coordinate activation of these three pathways. As expression of cyclins is an important factor in progression of the cell cycle, we examined cyclin D1 and E levels in HSC. PDGF was more efficacious than IGF-1 in increasing the expression of

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both cyclins. In addition to the mitogenic effects of IGF-1 and PDGF, we investigated phospho-GSK-3␤ expression following growth factor stimulation. Both IGF-1 and PDGF increased the phosphorylation of GSK-3␤ which suggests that they may also act as survival factors, in addition to their role as proliferative agents. The actions of PDGF on intracellular signaling in HSC have been the focus of research interest, because PDGF is the most potent mitogen for activated HSC,1,54,55 and the expression of PDGF and its receptor are increased during acute and chronic liver injury.56 Interestingly, quiescent HSC have negligible numbers of PDGF receptors and thus are unresponsive to its mitogenic effects. However, it has been demonstrated that induction of the expression of the PDGF receptor (especially the ␤ isoform) is part of the activation process.57,58 In culture-activated HSC, PDGF has been shown to increase mitogenesis, stimulate chemotaxis, and influence the expression of a number of genes such as egr, fos and jun.5,12,13,59,60 Thus, PDGF is an example of a growth factor that triggers intracellular signaling and proliferation of already activated HSC, but is less likely to be involved in the initiation of HSC activation, since PDGF receptors are not expressed in quiescent HSC. In contrast, IGF-1 is an example of a growth factor that may be involved in the initiation of HSC activation, since quiescent HSC express IGF-1 receptors.6,61 Gressner and colleagues have proposed that IGF-1 released by hepatocytes during liver damage may play a role in triggering HSC activation.4,61– 63 Activated HSC have IGF receptors, albeit at lower numbers than primary HSC61 and respond to IGF-1 by proliferating.64,65 Our current results agree with previous reports that PDGF1,11–13,55,59,60 and IGF-17,14,15 stimulate both ERK activity and proliferation in HSC. Marra and co-workers have shown that activation of the ERK signaling pathway is essential for PDGF-induced proliferation of HSC while being only partially responsible for chemotaxis induced by PDGF.13 Marra et al11–13 and Gentilini et al15 have demonstrated that PDGF and IGF-1 stimulate PI3-K activity in HSC, and that inhibition of PI3-K suppresses PDGF’s and IGF-1’s mitogenic effect. However, the downstream components of IGF-1-stimulated PI3-K signaling, including cyclin levels, have not been investigated in HSC. Individually the ERK and PI3-K pathways strongly influence HSC proliferative responses; however, significant crosstalk between pathways can also occur. Marra and colleagues11 have shown that PI3-K is partially responsible for PDGF-induced ERK activation, as inhibiting the PI3-K pathway resulted in a 40-50% reduction in ERK activity. The current study also dem-

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onstrated a 36% reduction in PDGF-induced ERK activity following inhibition of PI3-K with wortmannin. In contrast, inhibition of PI3-K did not decrease IGF-1-induced ERK activity in our study. This latter observation is in agreement with the findings of Svegliati-Baroni and co-workers7 who likewise failed to find any effect of PI3-K inhibition on IGF-1-induced ERK activity in HSC and those of Kuemmerle and Bushman66 who examined these pathways in intestinal smooth muscle cells. Consideration of such crosstalk between HSC signaling pathways is important when evaluating potential inhibitors for use as antifibrotic agents. p70-S6-K is a downstream component of the PI3-K pathway which is strongly inhibited by rapamycin. In agreement with the current study, Gäbele and coworkers67 have recently demonstrated that rapamycin inhibits PDGF-induced p70-S6-K phosphorylation but does not influence PKB phosphorylation. We have shown that rapamycin has a strong antiproliferative effect on HSC at a low concentration (5 nM), and that both IGF-1- and PDGF-induced p70-S6-K activity is dependent on PI3-K. A previous report has also shown that inhibiting the PI3-K pathway with wortmannin significantly decreases IGF-1-induced p70-S6-K activity in HSC.7 In addition, rapamycin effectively inhibits CCl4induced fibrosis in vivo.60 Taken together these findings suggest that the p70-S6-K pathway may be a potential target for novel antifibrotic agents. Cyclin expression and cyclin/CDK complex formation are critical steps in the cell cycle. Thus, in the current study, we examined levels of cyclin D1 and E after stimulation with PDGF and IGF-1, and we studied the potential contributions of ERK, PI3-K and p70S6-K to this effect. Cyclin D1 and E levels were significantly increased by PDGF, while the response to IGF-1 was not as strong. Inhibition of the ERK, PI3-K and p70-S6-K pathways significantly decreased the growth factor-induced response. These finding are consistent with those of Kawada and coworkers who demonstrated increased cyclin levels during cultureinduced activation of HSC, and suppression of this effect after inhibition of PI3-K.68,69 This study has demonstrated that IGF-1 is not as potent as PDGF in stimulating mitogenesis in activated HSC. However, both IGF-1 and PDGF may play a significant role in cell survival in addition to cell proliferation. Recent studies have highlighted the role of IGF-1 as a cell survival factor in other cell types36 –38 and in HSC.70 PI3-K and PKB are important mediators of cell survival. In the current study we examined GSK-3␤, a downstream substrate of PKB which is known to play a role in the PI3-K/PKB cell survival pathway. GSK-3␤ is inhibited by phosphorylation

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caused by growth factor stimulation. We demonstrated for the first time that IGF-1 and PDGF increase the levels of phospho-GSK-3␤ in HSC, suggesting inhibition of GSK-3␤ activity and therefore an anti-apoptotic response. Further studies are required to fully elucidate the role of GSK-3␤ in the cell survival pathways of HSC. In summary, this study has demonstrated that IGF-1and PDGF-induced proliferation of activated HSC is dependent on the ERK, PI3-K and p70-S6-K pathways. PDGF is a stronger mitogen than IGF-1, and is more efficacious in stimulating ERK and increasing cyclin levels. Activation of p70-S6-K is dependent on PI3-K, and there is crosstalk between the PI3-K pathway and the ERK pathway after PDGF stimulation. In addition, the PI3-K pathway plays a role in growth factorinduced phosphorylation of GSK-3␤, which may promote HSC survival. Therefore, coordinate activation of ERK, PI3-K and p70-S6-K by growth factors such as IGF-1 and PDGF may be important for perpetuating the activated state of HSC during fibrogenesis.

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