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Thymosin-β4 (Tβ4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells
The American Journal of Pathology, Vol. 178, No. 5, May 2011 Copyright © 2011 Published by Elsevier Inc. on behalf of American Society for Investigative Pathology. DOI: 10.1016/j.ajpath.2011.01.025
Gastrointestinal, Hepatobiliary, and Pancreatic Pathology
Thymosin-4 (T4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells
Karina Reyes-Gordillo,* Ruchi Shah,* Anastas Popratiloff,† Sidney Fu,‡ Anna Hindle,§ Frederick Brody,§ and Marcos Rojkind* From the Department of Biochemistry and Molecular Biology,* the Center for Microscopy and Image Analysis,† the Division of Genomic Medicine, Department of Medicine,‡ and the Department of General Surgery,§ The George Washington University Medical Center, Washington, DC
Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)- receptor and the proliferative response to platelet-derived growth factor- (PDGF) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-4 (T4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that T4 inhibited cell proliferation and migration and prevented the expression of PDGF- receptor (PDGF-r), ␣-smooth muscle actin and ␣1(I) collagen mRNAs. T4 also inhibited the reappearance of PDGF-r after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-r phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that T4 prevented this effect. T4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco’s modified Eagle’s medium or Dulbecco’s minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that T4 by
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sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, T4 has the potential to be an antifibrogenic agent. (Am J Pathol 2011, 178:2100 –2108; DOI: 10.1016/j.ajpath.2011.01.025)
Great progress has been made in understanding the pathophysiology of liver fibrosis and several forms of therapy have evolved in the attempts to prevent and/or revert the disease. Most therapies are derived from our current knowledge of the molecular mechanisms involved in the activation of hepatic stellate cells (HSC) and in the increased production of type I collagen.1–3 Activation of HSC results in the expression of multiple genes including the up-regulation of the platelet-derived growth factor- receptor (PDGF-r) and ␣-smooth muscle actin (␣-SMA).1,2 In this regard, several studies have shown that interference with the expression of the PDGF-r results in inhibition of HSC activation and amelioration of liver fibrosis in animal models.4 – 6 Others have focused the therapy on the inhibition of oxidative stress,7,8 the main process involved in the activation of HSC and collagen production, or on the stimulation of hepatocyte regeneration after the administration of hepatocyte growth factor (HGF).9 –12 More recently, liver fibrosis in rats has been inhibited, directly targeting a collagen
This work was supported by the National Institute on Alcohol Abuse and Alcoholism grant RO1 10541 (M.R.). Synthetic T4 used for this work was gifted by RegeneRx. Author contributions: M.R. led the project and wrote the manuscript; K.R.G. and R.S. performed experiments and revised the manuscript; A.H. and F.B. provided human hepatic stellate cells. S.F. provided advice and interpretation of quantitative PCR analysis; A.P performed the confocal microscopy experiments and provided advice in interpretation of findings. Accepted for publication January 18, 2011. Supplemental material for this article can be found at http://ajp. amjpathol.org and at doi: 10.1016/j.ajpath.2011.01.025. Address reprint requests to Marcos Rojkind, M.D., Ph.D., Professor of Biochemistry and Molecular Biology and Pathology, The George Washington University Medical Center, 2300 Eye Street, NW, Ross Hall 225, Washington, DC 20037. E-mail: [email protected].
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chaperon protein by delivering a small interfering RNA against glycoprotein 46.13 Although HGF has been successfully used in the treatment of various models of liver fibrosis in rats and mice, the molecular mechanisms whereby it exerts its actions remains to be determined. Some results suggest that HGF inhibits the expression of transforming growth factor- (TGF)- and collagen, whereas other findings suggest that it enhances liver regeneration by promoting the recruitment of bone marrow cells or by preventing epithelial mesenchymal transition.11 Studies have demonstrated that HGF induces the expression of thymosin-4 (T4) in endothelial cells.14 T4 belongs to a class of small molecular weight proteins that are expressed by all cell types (except erythrocytes).15,16 T4 has been shown to be anti-inflammatory in the eye17,18 and to prevent fibrosis of the heart after a myocardial infarction.19,20 Because HSC express T4,21 we tested the antifibrogenic properties of T4. Preliminary studies revealed that T4 prevented the expression of PDGF-r in cultured HSC. Based on this information, we tested whether T4 could prevent the activation of HSC, and we investigated the molecular mechanisms involved. In this communication, we show that T4 inhibits the expression of PDGF-r in human HSC and prevents the activation of freshly plated rat HSC. We further show that this effect is mediated by preventing the phosphorylation of AKT by a phosphoinositide 3-kinase (PI3K)-independent mechanism.12
Materials and Methods Cell Culture All of the experiments were performed with human HSC that were isolated as described by collagenase and protease digestion and fractionation on an Optiprep (Sigma, Saint Louis, MO) gradient22 from human liver biopsies of patients with morbid obesity that were subject to bypass surgery by an approved protocol (IRB 070701). Cultured HSC were used at passage 4 to 6 and grown in Dulbecco’s modified Eagle’s medium (Gibco; Invitrogen, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) amino acids (Cellgro, Herndon, VA), and 1% (v/v) penicillin streptomycin (Gibco; Invitrogen, Grand Island, NY). Human HSC were maintained in fetal bovine serum-containing medium until 16 hours before start of the experiments, at which time they were washed with phosphate buffered saline (PBS), and the medium was replaced with a serum-free medium that contained 0.1% fetal bovine serum. Cells were treated with 50 ng/mL of PDGF-BB, 10 ng/mL of T4, or both, and maintained at 37°C and in a 5% CO2 incubator.
Western Blotting Human HSC were lysed in buffer containing 1 mol/L Tris (pH 8), 5 mol/L NaCl, 0.5 mol/L EDTA, 0.5 mol/L NaF, 100 mmol/L sodium pyrophosphate, 100 mmol/L Na3VO4, 200 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were determined by bicinchoninic acid as-
say, according to the manufacturer’s instructions (Pierce Chemical Rockford, IL). The lysates were centrifuged at 14,000 ⫻ g for 10 minutes at 4°C, and aliquots containing 8 g of proteins were used for Western blot analysis, as previously described.23 A 1:1000 dilution of polyclonal rabbit anti-human antibodies against Akt, phosphor-Akt (Thr308), phosphor-Akt (Ser473), phosphatase and tensin homolog (PTEN), phospho-PTEN, PDK1, phosphoPDK1, proline-rich Akt substrate (PRAS)-40, phosphoPRAS-40, mammalian target of rapamycin (mTOR), PI3K(p85) and monoclonal mouse anti-human PDGF-r (Cell Signaling Technology, Inc., Danvers, MA). Antigen antibody complexes were detected by chemiluminescence enhanced chemiluminescence detection system (NEN Life Sciences Products, Boston, MA).
RNA Extraction and Quantitative RT-PCR These experiments were performed as previously described.24 The primer sequences used for quantitative PCR amplification of PDGF- receptor mRNA forward 5=-GGCTACATGGACATGAGCAA-3= and reverse 5=-TCGGCAGGTCCTCTCAG-3=; for ␣-SMA were: forward 5=-CTGAGCGTGGCTATTCCTTC-3= and reverse 5=-GCAGTGGCCATCTCATTTTC-3=; for ␣1(I) collagen were: forward 5=GAGAGCATGACCGATGGATT-3= and reverse 5=-ATGTAGGCCACGCTGTTCTT-3=; and for glyceraldehyde-3-phosphate dehydrogenase were forward 5=-GGCCTCCAAGGAGTAAGACC-3= and reverse 5=-CTGTGAGGAGGGGAGATTTCA3=. All reagents were purchased from Applied Biosystems (Foster City, CA). Relative gene expression was calculated as 2⫺⌬Ct (⌬Ct ⫽ Ct of glyceraldehyde-3-phosphate dehydrogenase).
Growth Proliferation Assay Cell proliferation was assessed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were plated in 96-well tissue culture plates at a concentration of 3000 cells/well. After 24 hours of quiescence, the cells were cultured for 24 hours and 48 hours with media containing 0.1% fetal bovine serum. At the end of the treatment, 20 L MTT solution (5 mg/mL in PBS) was added to each well and incubated for an additional 2 hours at 37°C. The colored formazan product was then dissolved in 150 L of MTT solvent (4 mmol/L HCl and 0.1% Nonidet P-40 in isopropanol). The mitochondrial activity was evaluated by measuring the optical density at 570.
Cell Migration Assay The migration of cells was measured in a scratch-wound assay in which the cells migrate from a confluent area to an area that has been mechanically denuded of cells. Initially, the human HSC were grown to a confluent monolayer and were then serum deprived for 24 hours. After the medium was discarded, a scratch wound was inflicted in a straight line across the cells with a pipette tip. The plates were then rinsed with PBS and incubated with Dulbecco’s modified Eagle’s medium supplemented with 50 ng/mL PDGF-BB, 10 ng/mL T4, or both. Wound closure was monitored and
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photographed after 24 hours under a light microscope (Nikon Inc., Melville, NY) and was analyzed using Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA). The distances between the edges of the HSC migrating from both sides were measured.
Immunofluorescent Staining Human HSC were grown on coverslips and treated with 50 ng/mL PDGF-BB, 10 ng/mL T4, or both for 30 minutes. They were fixed with 4% paraformaldehyde and permeabilized by incubating with absolute ethanol. Nonspecific binding sites were blocked for 30 minutes with 3% bovine serum albumin (Sigma, Saint Louis, MO) diluted in PBS. The cells were incubated for 2 hours at 37°C with primary antibody Akt (Cell Signaling Technology Inc.) at 1:500. A fluorescent labeled AlexaFluor 488 (Molecular Probes, Eugene, OR) goat anti-rabbit secondary antibody was used to stain the cells. Actin filaments were stained using AlexaFluor 568 Phalloidin and DAPI (Molecular Probes) was used to stain the nucleus and were subsequently scanned with a confocal microscope (Zeiss 510, Carl Zeiss, Thornwood, NY).
a pan AKT antibody (Cell Signaling Technology Inc.) and the presence of actin (1:4000) was determined in the immunoprecipitate by Western analysis. A reverse experiment was also performed where actin was immunoprecipitated with a -actin antibody and the presence of AKT (1:1000) was determined in the immunoprecipitate by Western analysis. In addition, PDGF-r was immunoprecipitated and the presence of phosphor-tyrosine (1:3000) was detected (Cell Signaling Technology Inc.) using Western analysis.
Flow Cytometry Apoptosis was analyzed by flow cytometry using an Annexin V-FITC/PI kit (BioVision, Inc., Mountain View, CA). The 1 ⫻ 105 cells were plated and treated with 50 ng/mL PDGFBB, 10 ng/mL T4, or both, for 30 minutes. Cells were resuspended in PBS, the staining was performed according to the manufacturer’s instructions, and flow cytometry was conducted on a FACS Calibur (BD Biosciences, San Jose, CA). Cells that were annexin V (⫺) and PI (⫺) were considered viable cells. Cells that were annexin V (⫹) and PI (⫺) were considered early apoptotic cells. Cells that were annexin V (⫹) and PI (⫹) were considered late apoptotic cells.
Immunoprecipitation
Statistical Analysis
The immunoprecipitation experiments were performed as previously described.25 AKT was immunoprecipitated with
All of the experiments were performed (at least) in triplicate, and data are expressed as mean ⫾ SE. Statistical
Figure 1. Quantitative RT-PCR analysis performed with total RNA obtained from human hepatic stellate cells (HSC) (5 to 6 passage) treated for 24 hours with platelet-derived growth factor (PDGF)-BB (50 ng/ mL), in the presence or absence of thymosin-4 (TB4) (10 ng/mL) (A, B, C). The figure also shows results obtained with freshly isolated rat HSC activated after culturing for 10 days with Dulbecco’s modified Eagle’s medium media (DMEM) containing 10% fetal bovine serum (FBS) in the presence or absence of 10 ng/mL of T4 (D, E, F). As shown in the figure, the expression of PDGF-r, smooth muscle actin (␣-SMA), and ␣1(I) collagen mRNA was induced in human HSC treated with PDGF-BB. Incubation with TB4, inhibited the expression of these mRNAs. Similarly, the expression of PDGF-r and ␣–SMA was induced in freshly isolated rat HSC cultured with DMEM, the expression of the ␣1(I) collagen gene was not inhibited by TB4. Western analysis of proteins extracted from rat HSC show that the culture-induced expression of PDGF-r was inhibited by TB4 (G). Values are means of triplicate experiments ⫾ SE after correcting for the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (PCR) or actin (Western). * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
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Figure 2. Time-course analysis of the expression of platelet-derived growth factor (PDGF)- receptor (PDGF-r) in total protein extracted from human hepatic stellate cells (HSC) (5 to 6 passage) treated with PDGF-BB and cultured in the presence or absence of thymosin-4 (TB4) for (A) 30 minutes, (B) 1 hour, (C) 3 hours, (D) 24 hours, (E) 48 hours and (F) 72 hours. Expression of the PDGF-r was determined by Western analysis. As shown in the figure, PDGF-BB down-regulated the expression of the PDGF-r and by 3 hours, no receptor was detected (C). The PDGF-r reappeared after 72 hours in culture (F). The figure also shows that T4 prevented the reappearance of the PDGF-r (F). Values are means of triplicate experiments ⫾ SE and were corrected for difference in loading after reprobing with an antibody to -actin. * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
differences between experimental groups were analyzed by Student’s t-test and P ⬍ 0.05 was considered to be significantly different (Microsoft Excel 2003, Microsoft Corporation, Redmond, WA).
Results T4 Inhibits HSC Activation as Determined by the Expression of ␣-SMA and PDGF- Receptor To investigate the antifibrogenic properties of T4, we first determined whether the chemokine had any effect on the expression of markers of HSC activation and type I collagen gene expression. To this end, we incubated human HSC (4 to 5 passage) with 50 ng/mL of PDGF-BB in the presence or absence of 10 ng/mL of T4. As controls, we used untreated HSC. As illustrated in Figure 1A, PDGF-BB induced the expression of PDGF-r, ␣-SMA, and ␣1(I) collagen mRNAs by 24 hours. T4 alone did not have a significant effect on the expression of these mRNAs; however, it significantly inhibited the PDGF-BB-dependent up-regulation of PDGF-r by 71.6% ⫾ 5.4 (P ⱕ 0.036) (Figure 1A), ␣-SMA by 22.2% ⫾ 1.2 (P ⱕ 0.037) (Figure 1B), and ␣1(I) collagen by 49% ⫾ 7.7 (P ⱕ 0.003) (Figure 1C) mRNAs. To confirm that T4 inhibits activation of HSC, freshly isolated rat HSC were placed in culture for 10 days using Dulbecco’s modified Eagle’s medium containing 10% serum with or without T4. As illustrated in Figure 1, D, E, and F, culture-activated HSC expressed high levels of PDGF-r, ␣-SMA,
and ␣1(I) collagen mRNA. Although T4 significantly inhibited the expression of PDGF-r by 77.9% ⫾ 1.7 (P ⱕ 0.0002) and ␣-SMA by 91.6% ⫾ 1.4 (P ⱕ 0.0005); it had a small, but not significant, effect on the expression of ␣1(I) collagen mRNA (13.9% ⫾ 1.2) (Figure 1F). T4 also inhibited the expression of PDGF-r protein by 67.1% ⫾ 0.13 (P ⱕ 0.008) (Figure 1G).
T4 Prevents the Up-Regulation of PDGF-r Protein that Occurs after the PDGF-BB-Induced Degradation of the Protein Once established that T4 inhibited the expression of markers of activation in freshly cultured rat HSC and that PDGF-BB-induced changes in PDGF-r, ␣-SMA, and ␣1(I) collagen mRNAs in cultured human HSC, we investigated changes in the expression of the PDGF-r protein in human HSC. We have already shown that in mouse HSC, the administration of PDGF-BB results in the lysosomal degradation of the receptor. Interestingly, there is no internal pool of the receptor and it takes as much as 24 to 48 hours to reappear.26 A time-dependent study performed with the human HSC treated with PDGF-BB showed a similar pattern to that previously observed in the mouse, albeit the timing of disappearance was different. As observed in Figure 2, PDGF-BB down-regulated the expression of the receptor by 50% ⫾ 9.8 (P ⱕ 0.02) by 1 hour (Figure 2B), and by 3 hours the protein was not detectable by Western analysis (Figure 2C). As also shown in the figure, T4 by itself has a minor and insignificant initial effect on the expression of the re-
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ceptor. However, the protein decreased with time, and by 3 hours it was not detectable. In HSC treated with PDGF-BB and T4, the receptor decreased with time and followed a similar pattern, as the cells were treated only with PDGF-BB. However, while in the PDGFtreated HSC, the receptor reappeared after 72 hours in cells receiving T4, the receptor was barely detectable after 72 hours, and was 20.1% ⫾ 7.0 (P ⱕ 0.011) of levels found in cells treated with only PDGF-BB (Figure 2F). These findings suggested that T4 inhibits the reappearance of the receptor protein once it is degraded. The data also showed that HSC does not have a pool of receptors, as the protein disappears completely after exposure to PDGF-BB. Because PDGF-r is the main receptor involved in HSC proliferation and migration, and its pharmacological inhibition results in amelioration of liver fibrosis,4 – 6 we investigated whether T4 had any effect on HSC proliferation and migration.
T4 Inhibits PDGF-BB-Dependent Proliferation and Migration of Cultured HSC HSC were plated in 96 multi-well culture dishes and the actual number of cells determined by the MTT assay. As illustrated in Figure 3A, HSC proliferated with a duplication time of approximately 24 hours after treatment with 50 ng/mL of PDGF-BB. As expected, neither controluntreated cells nor the HSC treated with T4 proliferated. As also shown in the figure, T4 significantly inhibited HSC proliferation, and by 48 hours, the inhibition was 67% ⫾ 1.2 (P ⱕ 0.0009) as compared to PDGF-BB treated cells. Thus, T4 inhibits the expression of the PDGF-r and inhibits PDGF-BB-induced proliferation. As shown in Figure 3B, T4 did not induce phosphorylation of the PDGF-r and did not prevent autophosphorylation of the PDGF-r. In addition, as shown as follows, T4 did not interfere with PDGF-BB-dependent activation of PI3K. To rule out the possibility that HSC were proliferating when treated with both PDGF-BB and T4, (but with levels that remained the same due to the induction of apoptosis or cell death), we explored using flow cytometry to determine whether T4 was inducing apoptosis and/or cell death. Our findings revealed that T4 was not inducing necrosis or early or late apoptosis of the HSC (see Supplemental Figure S1 at http://ajp.amjpathol.org). To investigate possible changes in HSC migration, we used the wound healing system induced by removing cells from a confluent culture with the tip of a micropipette and analyzing the closing of the wound after 24 hours. As can be seen in Figure 3C, in cells treated with PDGF-BB, the wound was filling up with cells and by 24 hours only 34.5% ⫾ 5.1 (P ⱕ 0.0008) of the wound remained open. However, in HSC treated with T4 (108.3% ⫾ 2.4) or PDGF-BB plus T4 (120.7 ⫾ 5.1; P ⱕ 0.001), the wound was even wider than that observed in the untreated controls. Quantification pertaining to the size of the wound is illustrated in Figure 3D.
Figure 3. A: Hepatic stellate cell proliferation of human hepatic stellate cells (HSC) treated with 50 ng/mL of platelet-derived growth factor (PDGF)-BB and cultured in the presence or absence of thymosin-4 (TB4). B: Western blot (WB) analysis performed with a phosphotyrosine-specific antibody of a total protein extract immunoprecipitated (IP) with an antibody to PDGF-r. The result of the PDGF-r immunoprecipitation experiment is representative of three independent experiments carried out under the same experimental conditions. TB4 did not interfere with PDGF-r autophosphorylation. Cell migration was determined after wounding a confluent culture (C) and measuring the denuded area at the time points 0 hour and 24 hours as indicated in the figure (D). As shown in the figure TB4 prevented PDGF-BB-induced HSC proliferation and migration. Values are means of triplicate experiments ⫾ SE.
T4 Inhibits PDGF-BB-Dependent Phosphorylation of AKT It has already been established that activation of the PI3K/ AKT pathway is required for HSC proliferation.27 We have further shown that the expression of the PDGF- receptor is dependent on the activation of AKT.26 Because T4 inhibits HSC proliferation and migration and prevents the reappearance of the receptor after PDGF-BB administration, we considered it important to measure the phosphorylation of AKT in cells treated with T4, PDGF alone, or the combination of both. As illustrated in Figure 4A, total AKT, as determined by Western analysis using a pan AKT antibody, did not change significantly in any of the groups. However, although PDGF-BB induced the phosphorylation of AKT at residues
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T4 Inhibits the Phosphorylation of PRAS-40 AKT is responsible for the phosphorylation and inactivation of PRAS-40. Because the phosphorylation of AKT was blocked by T4, we determined whether this effect resulted in the lack of phosphorylation of PRAS-40. As illustrated in Figure 5E, the phosphorylation of PRAS-40 was enhanced by PDGF-BB, but was blunted by T4. Thus, whereas signaling from upstream of AKT phosphorylation is unaffected by T4, downstream signaling is blocked.
PDGF-BB Induces AKT Binding to Actin and T4 Blunts this Effect
Figure 4. Western analysis of total and phosphorylated AKT performed with total proteins extracted from human hepatic stellate cells (HSC) treated with platelet-derived growth factor (PDGF)-BB in the presence or absence of thymosin-4 (TB4). As shown in panel A, the total concentration of AKT was similar in all of the samples. However, although PDGF-BB induced phosphorylation of AKT at residues S473 (B) and T308 (C), TB4 blunted these effects. Values are means of triplicate experiments ⫾ SE and were corrected for possible differences in loading after re-probing with an antibody to -actin.
S473 (Figure 4B) and T308 (Figure 4C), T4 blunted AKT phosphorylation at both sites.
T4 Did Not Prevent Phosphorylation of Kinases Upstream of AKT Because AKT is downstream of PI3K signaling pathway, we analyzed the degree of phosphorylation of selected components of this pathway. As illustrated in Figure 5A, T4 had no effect on the PDGF-BB-dependent increased phosphorylation of the regulatory subunit of PI3K (p85). Thus, T4 is not acting by preventing the activation of the PI3K pathway by PDGF-BB. We further analyzed the expression and phosphorylation of PTEN, the phosphatase that prevents PI3K-mediated effects by hydrolyzing phosphatidylinositol (3,4,5)-trisphosphate to phosphatidylinositol 4,5-bisphosphate. As shown in Figure 5D, levels of total and phosphorylated PTEN were the same in all of the experimental conditions investigated. Similarly, mTOR and the phosphorylation of mTOR (Figure 5B), the kinase that phosphorylates AKT at S473, and the phosphorylation of PDK1, the kinase that phosphorylates AKT at T308, were not modified by T4 (Figure 5C).
Altogether, our findings suggest that the inhibition of AKT phosphorylation is not due to the inactivation of several kinases of the PI3K pathway. Because AKT is phosphorylated at the plasma membrane28,29 and T4 could prevent its translocation by sequestering actin, we investigated whether PDGF-BB induced the binding of AKT to actin and whether T4 could prevent this action. Using confocal immunofluorescent microscopy with cells stained for actin (red) and AKT (green), we determined whether the two proteins co-localized in the cell. As shown in Figure 6A, AKT is distributed around the nucleus in control untreated cells. In HSC treated with PDGF-BB, AKT is bound to actin (arrows) (Figure 6B). Interestingly, T4 prevented the co-localization of AKT and actin (Figure 6, C and D). To confirm the confocal microscopy findings, we immunoprecipitated AKT with a pan AKT antibody (rabbit) and determined the presence of actin in the immunoprecipitate by Western analysis using a -actin antibody. As shown in Figure 6E, actin co-precipitated with AKT in control and PDGF-BB treated HSC. However, T4, the main actin sequestering protein prevented binding of AKT to actin, and therefore the actin was not detected in the Western blot. We also performed the reverse experiment immunoprecipitating actin and testing for the presence of AKT in the immunoprecipitate. As shown in Figure 6F, AKT was not present in the actin immunoprecipitate, and therefore they did not form a complex in HSC treated with T4. In summary, our findings suggest that T4 inhibits the expression of markers of HSC activation induced by PDGF-BB in cultured human HSC and prevents culture activation of rat HSC. In addition, T4 prevents HSC proliferation and migration and blocks the expression of the PDGF-r. These effects are mediated by preventing binding of Akt to actin and the subsequent inhibition of AKT phosphorylation.
Discussion Myofibroblasts play a key role in the production and contraction of the scar tissue of the cirrhotic liver.1,2 Although they may be derived from bone marrow cells or epithelial-mesenchymal transition,30,31 current evidence suggests that the main source of liver myofibroblasts is the activation/transdifferentiation of HSC.1,2 On activation, HSC switch phenotype and express and/or upregu-
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Figure 5. Western analysis of the expression of p85-PI3K regulatory subunit (A), total, and phosphorylated mammalian target of rapamycin (mTOR) (B), pyruvate dehydrogenase kinase isozyme 1 (PDK1) (C), phosphatase and tensin homolog (PTEN) (D), and proline-rich Akt substrate (PRAS)-40 (E). Analysis was performed with total protein extracted from human HSC treated with 50 ng/mL of platelet-derived growth factor (PDGF) in the presence or absence of thymosin-4 (TB4). The figure shows that none of these upstream kinases of the PI3K pathway were inhibited by TB4. Values are means of triplicate experiments ⫾ SE and were corrected for possible differences in loading after reprobing with an antibody to -actin. * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
late a large variety of genes. Among these, the expression of PDGF-r has been accepted as a bona fide marker of the activation process.1,2 The activated HSC/ myofibroblasts proliferate and migrate in response to PDGF-BB,32 a process regulated by the PI3K/AKT pathway.33 AKT (serine/threonine protein kinase) is a key kinase that regulates multiple cellular processes, and therefore it is an ideal target for preventing the activation/ transdifferentiation of HSC and scar formation in the injured liver. AKT is phosphorylated by PDK1 and mTOR, whereas PDK1 phosphorylates T308, and mTOR phoshorylates S473. As shown in this communication, the administration of PDGF-BB to culture activated HSC/myofibroblats induced the phosphorylation of AKT at T308 and S473. This
phosphorylation was prevented by the addition of T4 to the cultured cells. Interestingly, the upstream kinases that activate AKT, including the regulatory subunit of PI3K (p85PI3K), phosphor-PTEN, phosphor-PDK1, and pMTOR were not affected by T4. These and other findings, namely that T4 did not induce PDGF-r autophosphorylation and did not prevent PDGF-r-dependent autophosphorylation suggest that T4 is not interfering directly with initial events of PDGF-BB signaling, but prevents events occurring after AKT phosphorylation. Therefore, our findings suggest an alternative mode of action of T4. Phosphorylation of AKT is a complex process that involves the membrane localization of PI3K with the formation of phosphatidylinositol (3,4,5)-trisphosphate.33,34 If
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Figure 6. Human hepatic stellate cells (HSC) were grown, fixed, and permeabilized (see Materials and Methods). Cells were stained with a rabbit anti-AKT and were detected with green-fluorescent AlexaFluor 488 goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Actin was labeled with AlexaFluor 568 Phalloidin (Molecular Probes), and the nucleus were stained with blue-fluorescent DAPI and were subsequently scanned with a confocal microscope. The figure shows that in plateletderived growth factor (PDGF)-BB-treated human HSC, AKT is recruited to the membrane as indicated by the arrows (B). However, in cells treated with thymosin-4 (TB4) alone or in combination with PDGF-BB, AKT remains in the cytoplasm (C and D). Panel A corresponds to untreated controls. As shown in Panels E and F, immunoprecipitation of either actin (E) or AKT (F) followed by Western analysis for the detection of AKT (E) and actin (F), respectively, demonstrated that AKT and actin were present in a complex in HSC treated with 50 ng/mL of PDGF-BB. However, in HSC treated with PDGF-BB and TB4, no complexes were detected. The results are representative of three independent experiments performed under the same experimental conditions. IP, immunoprecipitation; WB, Western Blot.
phosphatidylinositol (3,4,5)-trisphosphate is not dephosphorylated by PTEN, it recruits AKT to the plasma membrane via its plekstrin homology domain.34 This is followed by the phosphorylation of AKT by PDK1 and mTOR. Thymosins are a group of small molecular weight proteins that have been used both in experimental models, as well as in the treatment of human disease.15,16,18 T4, a member of the group, has interesting pharmacological actions and is the main sequestering protein of cellular actin.18 Thus, T4 could regulate many cellular activities by modifying the ration of F/G actin.18 Recent data have shown that T4 is anti-inflammatory in the eye17, and it prevents scarring of the myocardium following an infarction.35 Serum levels of T4 also have prognostic value. In patients with fatal hepatitis B infection, levels of T4 are significantly decreased as compared to patients that survive.36 Thus T4 has multiple pharmacological activities, however, its main mechanism of action remains to be fully established. T4 binds to actin, and therefore it may prevent binding of AKT to actin. As shown in this communication, confocal microscopy revealed that PDGF-BB induced the colocalization of AKT to actin. This was further confirmed by the co-precipitation of actin and AKT when antibodies to either AKT or actin were used. These findings suggest the possibility that actin filaments are required for the recruitment of AKT to the plasma membrane. Therefore, sequestration of actin by T4 prevented AKT binding and this resulted in a lack of phosphorylation. Alternatively, a lack of AKT phosphorylation may prevent its binding to actin.37 As a consequence of this inhibition, HSC/myofibroblasts did not migrate or proliferate when PDGF-BB was added to the culture. We have previously shown that PDGF-r is completely eliminated on interaction with PDGF-BB and that 24 to 48 hours are required for the reappearance of the receptor.
This event is also regulated by the PI3K/AKT signaling pathway.26 As expected from these findings, T4 prevented or significantly delayed the reappearance of the receptor. Because the expression of this receptor is a hallmark of HSC activation/transdifferentiation, we measured whether T4 could prevent the activation of freshly isolated HSC. As also shown in this communication, T4 prevented the expression of ␣-SMA and PDGF-r, two markers of HSC activation. Although T4 did not significantly inhibit the expression of collagen during HSC activation in culture, it did inhibit its expression by 13.93% ⫾ 6.25 in PDGF-BB treated HSC. This inhibition is insignificant, and further work to understand why T4 inhibits other markers of HSC activation and has little effect on collagen is needed. HGF induces hepatocyte proliferation and prevents hepatic fibrosis.9 However, T4 at the concentrations used in this communication did not induce the expression of HGF (see Supplemental Figure S2 at http://ajp.amjpathol.org). Therefore T4 is not acting via the induction of HGF, and additional experiments to investigate its effects on hepatocyte function and proliferation are needed. Altogether our findings show that T4 is an effective agent in preventing the phosphorylation of AKT and thus, the activation of HSC. Therefore, T4 has great potential as an antifibrogenic agent. However, additional experiments in vivo are required to determine whether the antifibrogenic activities of T4 observed with cultured HSC can be reproduced in an animal model of liver fibrosis.
Acknowledgments We thank Dr. Allan L. Goldstein for his encouragement and advice and Dr. Hynda K. Kleinman for her advice.
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References 1. Safadi R, Friedman SL: Hepatic fibrosis - role of hepatic stellate cell activation. Med Gen Med 2002, 4:27 2. Rojkind M, Reyes Gordillo K. Hepatic Stellate Cells: The liver biology and pathobiology. Edited by Arias IM, Wolkoff AW, Boyer JL, Cohen DE, Shafritz DA, Fausto N, Alter HJ. Willey Blackwell, Oxford, UK 2009, 407– 422 3. Ghiassi-Nejad Z, Friedman SL: Advances in antifibrotic therapy. Expert Rev Gastroenterol Hepatol 2008, 2:803– 816 4. Borkham-Kamphorst E, Stoll D, Gressner AM, Weiskirchen R: Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochem Biophys Res Commun 2004, 321:413– 423 5. Borkham-Kamphorst E, Stoll D, Gressner AM, Weiskirchen R: Inhibitory effect of soluble PDGF-beta receptor in culture-activated hepatic stellate cells. Biochem Biophys Res Commun 2004, 317:451– 462 6. Borkham-Kamphorst E, Herrmann J, Stoll D, Treptau J, Gressner AM, Weiskirchen R: Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis. Lab Invest 2004, 84:766 –777 7. Vercelino R, Crespo I, de Souza GF, Cuevas MJ, de Oliveira MG, Marroni NP, González-Gallego J, Tuñón MJ: S-nitroso-N-acetylcysteine attenuates liver fibrosis in cirrhotic rats. J Mol Med 2010, 88: 401– 411 8. Tsai MK, Lin YL, Huang YT: Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats. Toxicol Appl Pharmacol 2010, 242:155–164 9. Xia JL, Dai C, Michalopoulos GK, Liu Y: Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation. Am J Pathol 2006, 168:1500 –1512 10. Kanemura H, Iimuro Y, Takeuchi M, Ueki T, Hirano T, Horiguchi K, Asano Y, Fujimoto J: Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats. Hepatol Res 2008, 38:930 –939 11. Asano Y, Iimuro Y, Son G, Hirano T, Fujimoto J: Hepatocyte growth factor promotes remodeling of murine liver fibrosis, accelerating recruitment of bone marrow-derived cells into the liver. Hepatol Res 2007, 37:1080 –1094 12. Inagaki Y, Higashi K, Kushida M, Hong YY, Nakao S, Higashiyama R, Moro T, Itoh J, Mikami T, Kimura T, Shiota G, Kuwabara I, Okazaki I: Hepatocyte growth factor suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7. Gastroenterology 2008, 134:1180 –1190 13. Sato Y, Murase K, Kato J, Kobune M, Sato T, Kawano Y, Takimoto R, Takada K, Miyanishi K, Matsunaga T, Takayama T, Niitsu Y: Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotechnol 2008, 26:431– 442 14. Oh IS, So SS, Jahng KY, Kim HG: Hepatocyte growth factor upregulates thymosin beta4 in human umbilical vein endothelial cells. Biochem Biophys Res Commun 2002, 296:401– 405 15. Goldstein AL, Hannappel E, Kleinman HK: Thymosin beta 4: actinsequestering protein moonlights to repair injured tissues. Trends Mol Med 2005, 11:421– 429 16. Sosne G, Qiu P, Goldstein AL, Wheater M: Biological activities of thymosin beta 4 defined by active sites in short peptide sequences. FASEB J 2010, 24:2144 –2151 17. Sosne G, Qiu P, Kurpakus-Wheater M: Thymosin beta 4: a novel corneal wound healing and anti-inflammatory agent. Clin Ophthalmol 2007, 1:201–207 18. Dunn SP, Heidemann DG, Chow CY, Crockford D, Turjman N, Angel J, Allan CB, Sosne G: Treatment of chronic nonhealing neurotrophic corneal epithelial defects with thymosin beta 4. Arch Ophthalmol 2010, 128:636 – 638 19. Bock-Marquette I, Saxena A, White MD, Dimaio JM, Srivastava D: Thymosin beta4 activates integrin-linked kinase and promotes car-
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Gastrointestinal, Hepatobiliary, and Pancreatic Pathology
Thymosin-4 (T4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells
Karina Reyes-Gordillo,* Ruchi Shah,* Anastas Popratiloff,† Sidney Fu,‡ Anna Hindle,§ Frederick Brody,§ and Marcos Rojkind* From the Department of Biochemistry and Molecular Biology,* the Center for Microscopy and Image Analysis,† the Division of Genomic Medicine, Department of Medicine,‡ and the Department of General Surgery,§ The George Washington University Medical Center, Washington, DC
Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)- receptor and the proliferative response to platelet-derived growth factor- (PDGF) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-4 (T4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that T4 inhibited cell proliferation and migration and prevented the expression of PDGF- receptor (PDGF-r), ␣-smooth muscle actin and ␣1(I) collagen mRNAs. T4 also inhibited the reappearance of PDGF-r after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-r phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that T4 prevented this effect. T4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco’s modified Eagle’s medium or Dulbecco’s minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that T4 by
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sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, T4 has the potential to be an antifibrogenic agent. (Am J Pathol 2011, 178:2100 –2108; DOI: 10.1016/j.ajpath.2011.01.025)
Great progress has been made in understanding the pathophysiology of liver fibrosis and several forms of therapy have evolved in the attempts to prevent and/or revert the disease. Most therapies are derived from our current knowledge of the molecular mechanisms involved in the activation of hepatic stellate cells (HSC) and in the increased production of type I collagen.1–3 Activation of HSC results in the expression of multiple genes including the up-regulation of the platelet-derived growth factor- receptor (PDGF-r) and ␣-smooth muscle actin (␣-SMA).1,2 In this regard, several studies have shown that interference with the expression of the PDGF-r results in inhibition of HSC activation and amelioration of liver fibrosis in animal models.4 – 6 Others have focused the therapy on the inhibition of oxidative stress,7,8 the main process involved in the activation of HSC and collagen production, or on the stimulation of hepatocyte regeneration after the administration of hepatocyte growth factor (HGF).9 –12 More recently, liver fibrosis in rats has been inhibited, directly targeting a collagen
This work was supported by the National Institute on Alcohol Abuse and Alcoholism grant RO1 10541 (M.R.). Synthetic T4 used for this work was gifted by RegeneRx. Author contributions: M.R. led the project and wrote the manuscript; K.R.G. and R.S. performed experiments and revised the manuscript; A.H. and F.B. provided human hepatic stellate cells. S.F. provided advice and interpretation of quantitative PCR analysis; A.P performed the confocal microscopy experiments and provided advice in interpretation of findings. Accepted for publication January 18, 2011. Supplemental material for this article can be found at http://ajp. amjpathol.org and at doi: 10.1016/j.ajpath.2011.01.025. Address reprint requests to Marcos Rojkind, M.D., Ph.D., Professor of Biochemistry and Molecular Biology and Pathology, The George Washington University Medical Center, 2300 Eye Street, NW, Ross Hall 225, Washington, DC 20037. E-mail: [email protected].
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chaperon protein by delivering a small interfering RNA against glycoprotein 46.13 Although HGF has been successfully used in the treatment of various models of liver fibrosis in rats and mice, the molecular mechanisms whereby it exerts its actions remains to be determined. Some results suggest that HGF inhibits the expression of transforming growth factor- (TGF)- and collagen, whereas other findings suggest that it enhances liver regeneration by promoting the recruitment of bone marrow cells or by preventing epithelial mesenchymal transition.11 Studies have demonstrated that HGF induces the expression of thymosin-4 (T4) in endothelial cells.14 T4 belongs to a class of small molecular weight proteins that are expressed by all cell types (except erythrocytes).15,16 T4 has been shown to be anti-inflammatory in the eye17,18 and to prevent fibrosis of the heart after a myocardial infarction.19,20 Because HSC express T4,21 we tested the antifibrogenic properties of T4. Preliminary studies revealed that T4 prevented the expression of PDGF-r in cultured HSC. Based on this information, we tested whether T4 could prevent the activation of HSC, and we investigated the molecular mechanisms involved. In this communication, we show that T4 inhibits the expression of PDGF-r in human HSC and prevents the activation of freshly plated rat HSC. We further show that this effect is mediated by preventing the phosphorylation of AKT by a phosphoinositide 3-kinase (PI3K)-independent mechanism.12
Materials and Methods Cell Culture All of the experiments were performed with human HSC that were isolated as described by collagenase and protease digestion and fractionation on an Optiprep (Sigma, Saint Louis, MO) gradient22 from human liver biopsies of patients with morbid obesity that were subject to bypass surgery by an approved protocol (IRB 070701). Cultured HSC were used at passage 4 to 6 and grown in Dulbecco’s modified Eagle’s medium (Gibco; Invitrogen, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) amino acids (Cellgro, Herndon, VA), and 1% (v/v) penicillin streptomycin (Gibco; Invitrogen, Grand Island, NY). Human HSC were maintained in fetal bovine serum-containing medium until 16 hours before start of the experiments, at which time they were washed with phosphate buffered saline (PBS), and the medium was replaced with a serum-free medium that contained 0.1% fetal bovine serum. Cells were treated with 50 ng/mL of PDGF-BB, 10 ng/mL of T4, or both, and maintained at 37°C and in a 5% CO2 incubator.
Western Blotting Human HSC were lysed in buffer containing 1 mol/L Tris (pH 8), 5 mol/L NaCl, 0.5 mol/L EDTA, 0.5 mol/L NaF, 100 mmol/L sodium pyrophosphate, 100 mmol/L Na3VO4, 200 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were determined by bicinchoninic acid as-
say, according to the manufacturer’s instructions (Pierce Chemical Rockford, IL). The lysates were centrifuged at 14,000 ⫻ g for 10 minutes at 4°C, and aliquots containing 8 g of proteins were used for Western blot analysis, as previously described.23 A 1:1000 dilution of polyclonal rabbit anti-human antibodies against Akt, phosphor-Akt (Thr308), phosphor-Akt (Ser473), phosphatase and tensin homolog (PTEN), phospho-PTEN, PDK1, phosphoPDK1, proline-rich Akt substrate (PRAS)-40, phosphoPRAS-40, mammalian target of rapamycin (mTOR), PI3K(p85) and monoclonal mouse anti-human PDGF-r (Cell Signaling Technology, Inc., Danvers, MA). Antigen antibody complexes were detected by chemiluminescence enhanced chemiluminescence detection system (NEN Life Sciences Products, Boston, MA).
RNA Extraction and Quantitative RT-PCR These experiments were performed as previously described.24 The primer sequences used for quantitative PCR amplification of PDGF- receptor mRNA forward 5=-GGCTACATGGACATGAGCAA-3= and reverse 5=-TCGGCAGGTCCTCTCAG-3=; for ␣-SMA were: forward 5=-CTGAGCGTGGCTATTCCTTC-3= and reverse 5=-GCAGTGGCCATCTCATTTTC-3=; for ␣1(I) collagen were: forward 5=GAGAGCATGACCGATGGATT-3= and reverse 5=-ATGTAGGCCACGCTGTTCTT-3=; and for glyceraldehyde-3-phosphate dehydrogenase were forward 5=-GGCCTCCAAGGAGTAAGACC-3= and reverse 5=-CTGTGAGGAGGGGAGATTTCA3=. All reagents were purchased from Applied Biosystems (Foster City, CA). Relative gene expression was calculated as 2⫺⌬Ct (⌬Ct ⫽ Ct of glyceraldehyde-3-phosphate dehydrogenase).
Growth Proliferation Assay Cell proliferation was assessed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were plated in 96-well tissue culture plates at a concentration of 3000 cells/well. After 24 hours of quiescence, the cells were cultured for 24 hours and 48 hours with media containing 0.1% fetal bovine serum. At the end of the treatment, 20 L MTT solution (5 mg/mL in PBS) was added to each well and incubated for an additional 2 hours at 37°C. The colored formazan product was then dissolved in 150 L of MTT solvent (4 mmol/L HCl and 0.1% Nonidet P-40 in isopropanol). The mitochondrial activity was evaluated by measuring the optical density at 570.
Cell Migration Assay The migration of cells was measured in a scratch-wound assay in which the cells migrate from a confluent area to an area that has been mechanically denuded of cells. Initially, the human HSC were grown to a confluent monolayer and were then serum deprived for 24 hours. After the medium was discarded, a scratch wound was inflicted in a straight line across the cells with a pipette tip. The plates were then rinsed with PBS and incubated with Dulbecco’s modified Eagle’s medium supplemented with 50 ng/mL PDGF-BB, 10 ng/mL T4, or both. Wound closure was monitored and
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photographed after 24 hours under a light microscope (Nikon Inc., Melville, NY) and was analyzed using Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA). The distances between the edges of the HSC migrating from both sides were measured.
Immunofluorescent Staining Human HSC were grown on coverslips and treated with 50 ng/mL PDGF-BB, 10 ng/mL T4, or both for 30 minutes. They were fixed with 4% paraformaldehyde and permeabilized by incubating with absolute ethanol. Nonspecific binding sites were blocked for 30 minutes with 3% bovine serum albumin (Sigma, Saint Louis, MO) diluted in PBS. The cells were incubated for 2 hours at 37°C with primary antibody Akt (Cell Signaling Technology Inc.) at 1:500. A fluorescent labeled AlexaFluor 488 (Molecular Probes, Eugene, OR) goat anti-rabbit secondary antibody was used to stain the cells. Actin filaments were stained using AlexaFluor 568 Phalloidin and DAPI (Molecular Probes) was used to stain the nucleus and were subsequently scanned with a confocal microscope (Zeiss 510, Carl Zeiss, Thornwood, NY).
a pan AKT antibody (Cell Signaling Technology Inc.) and the presence of actin (1:4000) was determined in the immunoprecipitate by Western analysis. A reverse experiment was also performed where actin was immunoprecipitated with a -actin antibody and the presence of AKT (1:1000) was determined in the immunoprecipitate by Western analysis. In addition, PDGF-r was immunoprecipitated and the presence of phosphor-tyrosine (1:3000) was detected (Cell Signaling Technology Inc.) using Western analysis.
Flow Cytometry Apoptosis was analyzed by flow cytometry using an Annexin V-FITC/PI kit (BioVision, Inc., Mountain View, CA). The 1 ⫻ 105 cells were plated and treated with 50 ng/mL PDGFBB, 10 ng/mL T4, or both, for 30 minutes. Cells were resuspended in PBS, the staining was performed according to the manufacturer’s instructions, and flow cytometry was conducted on a FACS Calibur (BD Biosciences, San Jose, CA). Cells that were annexin V (⫺) and PI (⫺) were considered viable cells. Cells that were annexin V (⫹) and PI (⫺) were considered early apoptotic cells. Cells that were annexin V (⫹) and PI (⫹) were considered late apoptotic cells.
Immunoprecipitation
Statistical Analysis
The immunoprecipitation experiments were performed as previously described.25 AKT was immunoprecipitated with
All of the experiments were performed (at least) in triplicate, and data are expressed as mean ⫾ SE. Statistical
Figure 1. Quantitative RT-PCR analysis performed with total RNA obtained from human hepatic stellate cells (HSC) (5 to 6 passage) treated for 24 hours with platelet-derived growth factor (PDGF)-BB (50 ng/ mL), in the presence or absence of thymosin-4 (TB4) (10 ng/mL) (A, B, C). The figure also shows results obtained with freshly isolated rat HSC activated after culturing for 10 days with Dulbecco’s modified Eagle’s medium media (DMEM) containing 10% fetal bovine serum (FBS) in the presence or absence of 10 ng/mL of T4 (D, E, F). As shown in the figure, the expression of PDGF-r, smooth muscle actin (␣-SMA), and ␣1(I) collagen mRNA was induced in human HSC treated with PDGF-BB. Incubation with TB4, inhibited the expression of these mRNAs. Similarly, the expression of PDGF-r and ␣–SMA was induced in freshly isolated rat HSC cultured with DMEM, the expression of the ␣1(I) collagen gene was not inhibited by TB4. Western analysis of proteins extracted from rat HSC show that the culture-induced expression of PDGF-r was inhibited by TB4 (G). Values are means of triplicate experiments ⫾ SE after correcting for the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (PCR) or actin (Western). * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
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Figure 2. Time-course analysis of the expression of platelet-derived growth factor (PDGF)- receptor (PDGF-r) in total protein extracted from human hepatic stellate cells (HSC) (5 to 6 passage) treated with PDGF-BB and cultured in the presence or absence of thymosin-4 (TB4) for (A) 30 minutes, (B) 1 hour, (C) 3 hours, (D) 24 hours, (E) 48 hours and (F) 72 hours. Expression of the PDGF-r was determined by Western analysis. As shown in the figure, PDGF-BB down-regulated the expression of the PDGF-r and by 3 hours, no receptor was detected (C). The PDGF-r reappeared after 72 hours in culture (F). The figure also shows that T4 prevented the reappearance of the PDGF-r (F). Values are means of triplicate experiments ⫾ SE and were corrected for difference in loading after reprobing with an antibody to -actin. * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
differences between experimental groups were analyzed by Student’s t-test and P ⬍ 0.05 was considered to be significantly different (Microsoft Excel 2003, Microsoft Corporation, Redmond, WA).
Results T4 Inhibits HSC Activation as Determined by the Expression of ␣-SMA and PDGF- Receptor To investigate the antifibrogenic properties of T4, we first determined whether the chemokine had any effect on the expression of markers of HSC activation and type I collagen gene expression. To this end, we incubated human HSC (4 to 5 passage) with 50 ng/mL of PDGF-BB in the presence or absence of 10 ng/mL of T4. As controls, we used untreated HSC. As illustrated in Figure 1A, PDGF-BB induced the expression of PDGF-r, ␣-SMA, and ␣1(I) collagen mRNAs by 24 hours. T4 alone did not have a significant effect on the expression of these mRNAs; however, it significantly inhibited the PDGF-BB-dependent up-regulation of PDGF-r by 71.6% ⫾ 5.4 (P ⱕ 0.036) (Figure 1A), ␣-SMA by 22.2% ⫾ 1.2 (P ⱕ 0.037) (Figure 1B), and ␣1(I) collagen by 49% ⫾ 7.7 (P ⱕ 0.003) (Figure 1C) mRNAs. To confirm that T4 inhibits activation of HSC, freshly isolated rat HSC were placed in culture for 10 days using Dulbecco’s modified Eagle’s medium containing 10% serum with or without T4. As illustrated in Figure 1, D, E, and F, culture-activated HSC expressed high levels of PDGF-r, ␣-SMA,
and ␣1(I) collagen mRNA. Although T4 significantly inhibited the expression of PDGF-r by 77.9% ⫾ 1.7 (P ⱕ 0.0002) and ␣-SMA by 91.6% ⫾ 1.4 (P ⱕ 0.0005); it had a small, but not significant, effect on the expression of ␣1(I) collagen mRNA (13.9% ⫾ 1.2) (Figure 1F). T4 also inhibited the expression of PDGF-r protein by 67.1% ⫾ 0.13 (P ⱕ 0.008) (Figure 1G).
T4 Prevents the Up-Regulation of PDGF-r Protein that Occurs after the PDGF-BB-Induced Degradation of the Protein Once established that T4 inhibited the expression of markers of activation in freshly cultured rat HSC and that PDGF-BB-induced changes in PDGF-r, ␣-SMA, and ␣1(I) collagen mRNAs in cultured human HSC, we investigated changes in the expression of the PDGF-r protein in human HSC. We have already shown that in mouse HSC, the administration of PDGF-BB results in the lysosomal degradation of the receptor. Interestingly, there is no internal pool of the receptor and it takes as much as 24 to 48 hours to reappear.26 A time-dependent study performed with the human HSC treated with PDGF-BB showed a similar pattern to that previously observed in the mouse, albeit the timing of disappearance was different. As observed in Figure 2, PDGF-BB down-regulated the expression of the receptor by 50% ⫾ 9.8 (P ⱕ 0.02) by 1 hour (Figure 2B), and by 3 hours the protein was not detectable by Western analysis (Figure 2C). As also shown in the figure, T4 by itself has a minor and insignificant initial effect on the expression of the re-
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ceptor. However, the protein decreased with time, and by 3 hours it was not detectable. In HSC treated with PDGF-BB and T4, the receptor decreased with time and followed a similar pattern, as the cells were treated only with PDGF-BB. However, while in the PDGFtreated HSC, the receptor reappeared after 72 hours in cells receiving T4, the receptor was barely detectable after 72 hours, and was 20.1% ⫾ 7.0 (P ⱕ 0.011) of levels found in cells treated with only PDGF-BB (Figure 2F). These findings suggested that T4 inhibits the reappearance of the receptor protein once it is degraded. The data also showed that HSC does not have a pool of receptors, as the protein disappears completely after exposure to PDGF-BB. Because PDGF-r is the main receptor involved in HSC proliferation and migration, and its pharmacological inhibition results in amelioration of liver fibrosis,4 – 6 we investigated whether T4 had any effect on HSC proliferation and migration.
T4 Inhibits PDGF-BB-Dependent Proliferation and Migration of Cultured HSC HSC were plated in 96 multi-well culture dishes and the actual number of cells determined by the MTT assay. As illustrated in Figure 3A, HSC proliferated with a duplication time of approximately 24 hours after treatment with 50 ng/mL of PDGF-BB. As expected, neither controluntreated cells nor the HSC treated with T4 proliferated. As also shown in the figure, T4 significantly inhibited HSC proliferation, and by 48 hours, the inhibition was 67% ⫾ 1.2 (P ⱕ 0.0009) as compared to PDGF-BB treated cells. Thus, T4 inhibits the expression of the PDGF-r and inhibits PDGF-BB-induced proliferation. As shown in Figure 3B, T4 did not induce phosphorylation of the PDGF-r and did not prevent autophosphorylation of the PDGF-r. In addition, as shown as follows, T4 did not interfere with PDGF-BB-dependent activation of PI3K. To rule out the possibility that HSC were proliferating when treated with both PDGF-BB and T4, (but with levels that remained the same due to the induction of apoptosis or cell death), we explored using flow cytometry to determine whether T4 was inducing apoptosis and/or cell death. Our findings revealed that T4 was not inducing necrosis or early or late apoptosis of the HSC (see Supplemental Figure S1 at http://ajp.amjpathol.org). To investigate possible changes in HSC migration, we used the wound healing system induced by removing cells from a confluent culture with the tip of a micropipette and analyzing the closing of the wound after 24 hours. As can be seen in Figure 3C, in cells treated with PDGF-BB, the wound was filling up with cells and by 24 hours only 34.5% ⫾ 5.1 (P ⱕ 0.0008) of the wound remained open. However, in HSC treated with T4 (108.3% ⫾ 2.4) or PDGF-BB plus T4 (120.7 ⫾ 5.1; P ⱕ 0.001), the wound was even wider than that observed in the untreated controls. Quantification pertaining to the size of the wound is illustrated in Figure 3D.
Figure 3. A: Hepatic stellate cell proliferation of human hepatic stellate cells (HSC) treated with 50 ng/mL of platelet-derived growth factor (PDGF)-BB and cultured in the presence or absence of thymosin-4 (TB4). B: Western blot (WB) analysis performed with a phosphotyrosine-specific antibody of a total protein extract immunoprecipitated (IP) with an antibody to PDGF-r. The result of the PDGF-r immunoprecipitation experiment is representative of three independent experiments carried out under the same experimental conditions. TB4 did not interfere with PDGF-r autophosphorylation. Cell migration was determined after wounding a confluent culture (C) and measuring the denuded area at the time points 0 hour and 24 hours as indicated in the figure (D). As shown in the figure TB4 prevented PDGF-BB-induced HSC proliferation and migration. Values are means of triplicate experiments ⫾ SE.
T4 Inhibits PDGF-BB-Dependent Phosphorylation of AKT It has already been established that activation of the PI3K/ AKT pathway is required for HSC proliferation.27 We have further shown that the expression of the PDGF- receptor is dependent on the activation of AKT.26 Because T4 inhibits HSC proliferation and migration and prevents the reappearance of the receptor after PDGF-BB administration, we considered it important to measure the phosphorylation of AKT in cells treated with T4, PDGF alone, or the combination of both. As illustrated in Figure 4A, total AKT, as determined by Western analysis using a pan AKT antibody, did not change significantly in any of the groups. However, although PDGF-BB induced the phosphorylation of AKT at residues
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T4 Inhibits the Phosphorylation of PRAS-40 AKT is responsible for the phosphorylation and inactivation of PRAS-40. Because the phosphorylation of AKT was blocked by T4, we determined whether this effect resulted in the lack of phosphorylation of PRAS-40. As illustrated in Figure 5E, the phosphorylation of PRAS-40 was enhanced by PDGF-BB, but was blunted by T4. Thus, whereas signaling from upstream of AKT phosphorylation is unaffected by T4, downstream signaling is blocked.
PDGF-BB Induces AKT Binding to Actin and T4 Blunts this Effect
Figure 4. Western analysis of total and phosphorylated AKT performed with total proteins extracted from human hepatic stellate cells (HSC) treated with platelet-derived growth factor (PDGF)-BB in the presence or absence of thymosin-4 (TB4). As shown in panel A, the total concentration of AKT was similar in all of the samples. However, although PDGF-BB induced phosphorylation of AKT at residues S473 (B) and T308 (C), TB4 blunted these effects. Values are means of triplicate experiments ⫾ SE and were corrected for possible differences in loading after re-probing with an antibody to -actin.
S473 (Figure 4B) and T308 (Figure 4C), T4 blunted AKT phosphorylation at both sites.
T4 Did Not Prevent Phosphorylation of Kinases Upstream of AKT Because AKT is downstream of PI3K signaling pathway, we analyzed the degree of phosphorylation of selected components of this pathway. As illustrated in Figure 5A, T4 had no effect on the PDGF-BB-dependent increased phosphorylation of the regulatory subunit of PI3K (p85). Thus, T4 is not acting by preventing the activation of the PI3K pathway by PDGF-BB. We further analyzed the expression and phosphorylation of PTEN, the phosphatase that prevents PI3K-mediated effects by hydrolyzing phosphatidylinositol (3,4,5)-trisphosphate to phosphatidylinositol 4,5-bisphosphate. As shown in Figure 5D, levels of total and phosphorylated PTEN were the same in all of the experimental conditions investigated. Similarly, mTOR and the phosphorylation of mTOR (Figure 5B), the kinase that phosphorylates AKT at S473, and the phosphorylation of PDK1, the kinase that phosphorylates AKT at T308, were not modified by T4 (Figure 5C).
Altogether, our findings suggest that the inhibition of AKT phosphorylation is not due to the inactivation of several kinases of the PI3K pathway. Because AKT is phosphorylated at the plasma membrane28,29 and T4 could prevent its translocation by sequestering actin, we investigated whether PDGF-BB induced the binding of AKT to actin and whether T4 could prevent this action. Using confocal immunofluorescent microscopy with cells stained for actin (red) and AKT (green), we determined whether the two proteins co-localized in the cell. As shown in Figure 6A, AKT is distributed around the nucleus in control untreated cells. In HSC treated with PDGF-BB, AKT is bound to actin (arrows) (Figure 6B). Interestingly, T4 prevented the co-localization of AKT and actin (Figure 6, C and D). To confirm the confocal microscopy findings, we immunoprecipitated AKT with a pan AKT antibody (rabbit) and determined the presence of actin in the immunoprecipitate by Western analysis using a -actin antibody. As shown in Figure 6E, actin co-precipitated with AKT in control and PDGF-BB treated HSC. However, T4, the main actin sequestering protein prevented binding of AKT to actin, and therefore the actin was not detected in the Western blot. We also performed the reverse experiment immunoprecipitating actin and testing for the presence of AKT in the immunoprecipitate. As shown in Figure 6F, AKT was not present in the actin immunoprecipitate, and therefore they did not form a complex in HSC treated with T4. In summary, our findings suggest that T4 inhibits the expression of markers of HSC activation induced by PDGF-BB in cultured human HSC and prevents culture activation of rat HSC. In addition, T4 prevents HSC proliferation and migration and blocks the expression of the PDGF-r. These effects are mediated by preventing binding of Akt to actin and the subsequent inhibition of AKT phosphorylation.
Discussion Myofibroblasts play a key role in the production and contraction of the scar tissue of the cirrhotic liver.1,2 Although they may be derived from bone marrow cells or epithelial-mesenchymal transition,30,31 current evidence suggests that the main source of liver myofibroblasts is the activation/transdifferentiation of HSC.1,2 On activation, HSC switch phenotype and express and/or upregu-
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Figure 5. Western analysis of the expression of p85-PI3K regulatory subunit (A), total, and phosphorylated mammalian target of rapamycin (mTOR) (B), pyruvate dehydrogenase kinase isozyme 1 (PDK1) (C), phosphatase and tensin homolog (PTEN) (D), and proline-rich Akt substrate (PRAS)-40 (E). Analysis was performed with total protein extracted from human HSC treated with 50 ng/mL of platelet-derived growth factor (PDGF) in the presence or absence of thymosin-4 (TB4). The figure shows that none of these upstream kinases of the PI3K pathway were inhibited by TB4. Values are means of triplicate experiments ⫾ SE and were corrected for possible differences in loading after reprobing with an antibody to -actin. * means significantly different from control untreated group. # means significantly different from PDGF-BB treated group.
late a large variety of genes. Among these, the expression of PDGF-r has been accepted as a bona fide marker of the activation process.1,2 The activated HSC/ myofibroblasts proliferate and migrate in response to PDGF-BB,32 a process regulated by the PI3K/AKT pathway.33 AKT (serine/threonine protein kinase) is a key kinase that regulates multiple cellular processes, and therefore it is an ideal target for preventing the activation/ transdifferentiation of HSC and scar formation in the injured liver. AKT is phosphorylated by PDK1 and mTOR, whereas PDK1 phosphorylates T308, and mTOR phoshorylates S473. As shown in this communication, the administration of PDGF-BB to culture activated HSC/myofibroblats induced the phosphorylation of AKT at T308 and S473. This
phosphorylation was prevented by the addition of T4 to the cultured cells. Interestingly, the upstream kinases that activate AKT, including the regulatory subunit of PI3K (p85PI3K), phosphor-PTEN, phosphor-PDK1, and pMTOR were not affected by T4. These and other findings, namely that T4 did not induce PDGF-r autophosphorylation and did not prevent PDGF-r-dependent autophosphorylation suggest that T4 is not interfering directly with initial events of PDGF-BB signaling, but prevents events occurring after AKT phosphorylation. Therefore, our findings suggest an alternative mode of action of T4. Phosphorylation of AKT is a complex process that involves the membrane localization of PI3K with the formation of phosphatidylinositol (3,4,5)-trisphosphate.33,34 If
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Figure 6. Human hepatic stellate cells (HSC) were grown, fixed, and permeabilized (see Materials and Methods). Cells were stained with a rabbit anti-AKT and were detected with green-fluorescent AlexaFluor 488 goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Actin was labeled with AlexaFluor 568 Phalloidin (Molecular Probes), and the nucleus were stained with blue-fluorescent DAPI and were subsequently scanned with a confocal microscope. The figure shows that in plateletderived growth factor (PDGF)-BB-treated human HSC, AKT is recruited to the membrane as indicated by the arrows (B). However, in cells treated with thymosin-4 (TB4) alone or in combination with PDGF-BB, AKT remains in the cytoplasm (C and D). Panel A corresponds to untreated controls. As shown in Panels E and F, immunoprecipitation of either actin (E) or AKT (F) followed by Western analysis for the detection of AKT (E) and actin (F), respectively, demonstrated that AKT and actin were present in a complex in HSC treated with 50 ng/mL of PDGF-BB. However, in HSC treated with PDGF-BB and TB4, no complexes were detected. The results are representative of three independent experiments performed under the same experimental conditions. IP, immunoprecipitation; WB, Western Blot.
phosphatidylinositol (3,4,5)-trisphosphate is not dephosphorylated by PTEN, it recruits AKT to the plasma membrane via its plekstrin homology domain.34 This is followed by the phosphorylation of AKT by PDK1 and mTOR. Thymosins are a group of small molecular weight proteins that have been used both in experimental models, as well as in the treatment of human disease.15,16,18 T4, a member of the group, has interesting pharmacological actions and is the main sequestering protein of cellular actin.18 Thus, T4 could regulate many cellular activities by modifying the ration of F/G actin.18 Recent data have shown that T4 is anti-inflammatory in the eye17, and it prevents scarring of the myocardium following an infarction.35 Serum levels of T4 also have prognostic value. In patients with fatal hepatitis B infection, levels of T4 are significantly decreased as compared to patients that survive.36 Thus T4 has multiple pharmacological activities, however, its main mechanism of action remains to be fully established. T4 binds to actin, and therefore it may prevent binding of AKT to actin. As shown in this communication, confocal microscopy revealed that PDGF-BB induced the colocalization of AKT to actin. This was further confirmed by the co-precipitation of actin and AKT when antibodies to either AKT or actin were used. These findings suggest the possibility that actin filaments are required for the recruitment of AKT to the plasma membrane. Therefore, sequestration of actin by T4 prevented AKT binding and this resulted in a lack of phosphorylation. Alternatively, a lack of AKT phosphorylation may prevent its binding to actin.37 As a consequence of this inhibition, HSC/myofibroblasts did not migrate or proliferate when PDGF-BB was added to the culture. We have previously shown that PDGF-r is completely eliminated on interaction with PDGF-BB and that 24 to 48 hours are required for the reappearance of the receptor.
This event is also regulated by the PI3K/AKT signaling pathway.26 As expected from these findings, T4 prevented or significantly delayed the reappearance of the receptor. Because the expression of this receptor is a hallmark of HSC activation/transdifferentiation, we measured whether T4 could prevent the activation of freshly isolated HSC. As also shown in this communication, T4 prevented the expression of ␣-SMA and PDGF-r, two markers of HSC activation. Although T4 did not significantly inhibit the expression of collagen during HSC activation in culture, it did inhibit its expression by 13.93% ⫾ 6.25 in PDGF-BB treated HSC. This inhibition is insignificant, and further work to understand why T4 inhibits other markers of HSC activation and has little effect on collagen is needed. HGF induces hepatocyte proliferation and prevents hepatic fibrosis.9 However, T4 at the concentrations used in this communication did not induce the expression of HGF (see Supplemental Figure S2 at http://ajp.amjpathol.org). Therefore T4 is not acting via the induction of HGF, and additional experiments to investigate its effects on hepatocyte function and proliferation are needed. Altogether our findings show that T4 is an effective agent in preventing the phosphorylation of AKT and thus, the activation of HSC. Therefore, T4 has great potential as an antifibrogenic agent. However, additional experiments in vivo are required to determine whether the antifibrogenic activities of T4 observed with cultured HSC can be reproduced in an animal model of liver fibrosis.
Acknowledgments We thank Dr. Allan L. Goldstein for his encouragement and advice and Dr. Hynda K. Kleinman for her advice.
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