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NEUROREGULATION AND MOTILITY
receptor upregulation post-PDGF-BB treatment in mouse HSC
1Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York; 2Experimental Pathology Section, Department of Clinical Investigation, Walter Reed Army Medical Center; and 3Departments of Biochemistry and Molecular Biology and Pathology, The George Washington University, Washington, District of Columbia; and 4Departamento de Ciencias de la Salud, División de Ciencias Básicas y de la Salud, Universidad Autónoma Metropolitana, Iztapalapa, México
Submitted 9 February 2005 ; accepted in final form 19 July 2006
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ABSTRACT |
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receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF- receptor by cathepsin B, thus suggesting that the absence of PDGF- receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF- receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).
hepatic stellate cells; phosphatidylinositol 3-kinase; platelet-derived growth factor- receptor; platelet-derived growth factor-BB
-smooth muscle actin and PDGF- receptor, proliferation and migration in response to PDGF-BB, and production of type I collagen. These changes in HSC have been recognized as key events in the fibrogenic process (7, 14, 30, 35) and result in the formation of the scar tissue observed in fibrotic and cirrhotic livers.
One of the key events in the activation of HSC is the expression of PDGF- receptor, which is responsible, in part, for their increased proliferative and migratory response to this growth factor (8, 9, 28, 29). PDGF-BB induces HSC migration and proliferation by a complex pathway involving phosphatidylinositol 3-kinase (PI3K) and the activation of PKB (Akt) (29). This kinase is an important survival factor that prevents HSC apoptosis and enhances their proliferation. Recent studies have shown that focal adhesion kinase, a 125-kDa tyrosine protein kinase involved in integrin signaling, also plays a key role in HSC migration and is upstream of PI3K (31).
Data from several laboratories, including ours, have suggested that oxidative stress plays a key role in inducing the activation of HSC and in their increased capacity to produce type I collagen (6, 10, 13, 24, 25). It has been demonstrated that fibrogenic effects induced by TGF-1, acetaldehyde, and ethanol are mediated by H2O2 formation (6, 10, 13, 24, 25). Moreover, PDGF-BB growth response in fibroblast is H2O2 mediated (1, 21, 32), and antioxidants such as N-acetyl-L-cysteine induce the extracellular degradation of PDGF- receptor in HSC and, thus, desensitize the cells to respond to this growth factor (26, 37). This effect appears to be mediated by the release of cathepsin B. In view of the key role of the PDGF- receptor in HSC activation, we considered it important to further investigate its fate in HSC treated with recombinant PDGF-BB. In this communication, we show that PDGF-BB administration induces a rapid time- and dose-dependent degradation of its cognate receptor in mouse HSC by a mechanism dependent, in part, on its intrinsic autophosphorylation and lysosomal degradation but independent of proteasomal degradation and/or extracellular proteolysis. We also show that there is no internal pool of receptor protein. Therefore, reappearance of PDGF- receptor protein after PDGF-BB treatment is de novo protein synthesis dependent and requires the activation of PI3K and Akt pathways.
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MATERIALS AND METHODS |
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Proteasome inhibitor I, lactacystin, PD98059, SB203580, LY294002, wortmannin, rapamycin, and tyrphostin 1296 were purchased from Calbiochem-Novabiochiem (San Diego, CA). Chloroquine, cycloheximide, and actinomycin D were purchased from Sigma Chemical (St. Louis, MO). PDGF-BB was purchased from Boehringer Mannheim-Roche (Indianapolis, IN). Polyclonal antibody against PDGF- receptor was purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal (Ser473) phospho-Akt, (Thr308) phospho-Akt, (Ser241) phospho-PDK1, total PI3K p85, phospho-tyr, total Akt, (Thr389) phospho-p70S6k, and total p70S6k were purchased from Cell Signaling-New England BioLabs (Beverly, MA).
Cell Culture
All the experiments were performed with mouse HSC (MHSC) that were isolated from adult B6D2F2 mice and used in their activated phenotype (passages 5–7) as previously described (16). In brief, cells were cultured in minimum essential medium (Cellgro, Herndon, VA) supplemented with 10% FBS (HyClone, Logan, UT), nonessential amino acids, kanamycin, and penicillin-streptomycin (Life Technologies, Grand Island, NY). MHSC were maintained in FBS-containing medium until 16 h before start of the experiments, at which time the medium was replaced for a serum-free medium that contained 0.2% bovine serum albumin (fraction V, Sigma Chemical, St. Louis, MO). All incubations were performed in this medium, and the cells were maintained at 37°C in a 5% CO2 incubator. In some cultures, actinomycin D (10 µg/ml) or cycloheximide (0.3 mmol/l) was added 30 min before PDGF-BB administration.
For all the experiments described below and unless otherwise indicated, PDGF-BB (50 ng/ml) was added to the culture media and the cells were harvested at the various time points indicated in each experiment. When protein kinase inhibitors were used, they were added 30 min before PDGF-BB administration. The inhibitors and concentrations used were the following: SB203580, 10 µM (p38-MAPK); PD98059, 50 µM (ERK1/2); LY294002, 50 µM, and wortmannin, 200 nM (PI3K); rapamycin, 50 nM (p70S6k kinase); tyrphostin-1296, 10 µM (PDGF- receptor autophosphorylation inhibitor); and chloroquine, 100 µM (lysosomal inhibitor); lactacystin, 10 µM, and proteasome inhibitor I, 1 µM (proteasomal inhibitors), were added 1 h before growth factor administration.
Western Blotting
MHSC were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, and 1 µg/ml each chymostatin, leupeptin, antipain, and pepstatin. Protein concentrations were determined by bicinchoninic acid assay according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). The lysates were centrifuged at 16,000 g for 5 min at 4°C, and aliquots containing 15 µg protein were used for Western blot analysis as previously described (10). A 1:5,000 dilution of the primary antibody to actin (Santa Cruz Biotechnology, Santa Cruz, CA, C-2 antibody) was used. All other primary antibodies were used at a 1:1,000 dilution. Antigen antibody complexes were detected by chemiluminescence (ECL Renaissance System; NEN Life Science Products, Boston, MA). Signal intensities were corrected for possible differences in protein loading after probing with the anti-actin antibody and/or after Coomassie blue staining and densitometric analysis of the bands.
Protein Kinase Assays
The PI3K pathway was analyzed measuring the phosphorylation state of PDK1, Akt, and p70S6K by using the antibodies described in Materials above and following the manufacturers’ instructions. PI3K activity was measured as previously described (4, 19, 36). In brief, cells were washed once with ice-cold 0.9% NaCl and immediately homogenized in lysis medium containing 50 mM HEPES (pH 7.4), 10 mM Na4P2O7, 100 mM NaF, 10 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM benzamidine, and 2 mM PMSF. Cell lysates were centrifuged (10,000 g for 30 min), and the supernatants were incubated overnight with an antibody against p85 regulatory subunit (Upstate Biotechnologies, Waltham, MA) (1/100 dilution). Immunocomplexes formed were captured with protein A-agarose (Sigma Chemical), as indicated by the manufacturer. All the procedures were carried out at 4°C. PI3K activity was assayed in the immunocomplexes by 32P incorporation into phosphatidylinositol as previously described (4, 19, 36).
RNA Extraction and Quantitative RT-PCR
Amplification of PDGF- receptor, -smooth muscle actin, and S14 riboprotein mRNAs.
Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 µg RNA was reverse transcribed at 37°C for 1 h by using SuperScript reverse transcriptase from Invitrogen (34). The primer sequences used for quantitative PCR amplification of PDGF- receptor mRNA were 5'-CAG CAA GAG TGG CAG AGA AG-3' and 5'-GGC AGT TGA GGT GGT AAT CC-3' and those for S18 were 5'-GCC GCT AGA GGT GAA ATT CTT-3' and 5'-CAT TCT TGG CAA ATG CTT TGC-3'. Amplification reactions were performed using a Light Cycler and the LightCycler FastStart DNA MasterPlus SYBR Green I kit from Roche Molecular Biochemicals.
Subcellular localization of PDGF- receptor after treatment of HSC with PDGF-BB.
Activated HSC were cultured in four-well Permanox slides (Nalge Nunc International, Rochester, NY) until they were semiconfluent. The evening before the experiment, culture medium was removed, the cells were washed twice with PBS, and the medium was replaced with a serum-free medium containing 0.2% albumin but without phenol red. The next morning, the cells were treated with 50 nM lysotracker green DND-26 (Molecular Probes, Eugene, OR) and 30 min later recombinant PDGF-BB, 50 ng/ml, was added. Cells were fixed with formaldehyde at 0, 15, and 30 min after addition of PDGF-BB. After permeabilization of the cells with cold ethanol, cultured cells were incubated for 2 h with a polyclonal antibody against PDGF- receptor (Upstate Biotechnologies) and washed several times with TBS followed by 2-h incubation with a Texas-red-labeled goat anti-rabbit antibody (4010-07) (Southern Biotechnology Associates, Birmingham, AL). Cells were washed three times with Tris-buffered saline, covered with glass slides using ProLong-Gold antifade reagent (Invitrogen), and analyzed by confocal microscopy.
Confocal Microscopy
A Bio-Rad MRC 1024 confocal laser-scanning microscope (Hercules, CA) equipped with a krypton-argon laser and an Olympus IX-70 inverted microscope (Melville, NY) were used for image localization of FITC (488-nm laser line excitation; 522/35 emission filter) and Texas red (568-nm excitation; 605/32 emission filter). Optical sections (Z = 0.5 µm) of confocal epifluorescence images were sequentially acquired using a x60 (numerical aperture = 1.40) with Bio-Rad LaserSharp v3.2 software. Confocal Assistant 4.02 and Image J (National Institutes of Health) software were subsequently used to merge images and perform orthogonal studies, respectively. Merged images were processed in Photoshop 7.0 with minimal manipulations of contrast.
Statistical Analysis
All the experiments were performed at least in triplicate, and data are expressed as means ± SE. Statistical differences between experimental groups were analyzed by Student's t-test, and P < 0.05 was considered to be significantly different (Microsoft Excel 2000).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSDISCLAIMERREFERENCES |
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Expression of PDGF- receptor in HSC plays a key role in their activation and transformation into myofibroblasts. Thus, to investigate molecular mechanisms involved in regulating the fate of the PDGF- receptor in PDGF-BB-treated HSC, we incubated cells with 50 ng/ml of recombinant PDGF-BB (see MATERIALS AND METHODS) and measured total receptor expression and phosphorylation. As illustrated in Fig. 1A, within 5 min after the addition of human recombinant PDGF-BB, levels of PDGF- receptor decreased to 61 ± 6% (P < 0.05) of pretreatment values. At the same time, it was maximally phosphorylated. Total receptor concentration continued to decrease with time and was 41 ± 7% (P < 0.001) of control by 10 min and 8 ± 2% (P < 0.001) by 60 min. Similarly, the amount of phosphorylated receptor also decreased and by 30 min it was barely detectable (see Fig. 1A). Levels of PDGF- receptor remained very low for at least 6 h, after which time they started to increase and returned to close to normal values by 24–48 h (Fig. 1, B and C). The recovery time was dose dependent, and receptor levels in activated HSC treated with 10 ng/ml PDGF-BB were 0.65 ± 0.17 (P < 0.05) by 24 h and reached 0.92 ± 0.15 (P = not significant) of control values by 48 h. However, receptor levels in activated HSC treated with 50 ng/ml of PDGF-BB were 0.32 ± 0.21 by 24 h (P < 0.05) and only reached 0.68 ± 0.33 (P = not significant) of control values by 48 h.
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Receptor mRNA
We first measured the time course of expression of PDGF- receptor mRNA in control untreated cells maintained in a serum-free, albumin-containing culture medium as described in MATERIALS AND METHODS. As shown in Fig. 2A, quantitative PCR amplification (Lightcycler, Roche) in control, untreated cells revealed a steady increase in mRNA levels that was 2.5-fold above time 0 levels by 12 h (P < 0.05). When activated HSC cultured in serum-free medium were treated with 10 µg/ml of actinomycin D, receptor mRNA levels decreased to 80% of control values by 1 h (P < 0.05), and these values remained unchanged for up to 12 h (see Fig. 2B). Treatment with PDGF-BB alone decreased PDGF- receptor mRNA to 50 and 30% of controls by 1 and 3 h, respectively (P < 0.05), and values remained below 50% of controls for up to 24 h (Fig. 2C). Results obtained with the combination of PDGF-BB plus actinomycin D did not differ from those obtained with actinomycin alone (Fig. 2D).
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Receptor Autophosphorylation Delays Its Degradation
Experiments described above suggest that, upon interaction with the growth factor, PDGF- receptor protein and mRNA are degraded. To determine whether a specific phosphorylation event played a role in receptor degradation, we measured first the effect of PDGF-BB on the expression and/or activity of several protein kinases and then determined whether specific inhibitors of the PDGF-BB-activated kinases prevented receptor degradation. As illustrated in Fig. 3, A and B, PDGF-BB induced a time-dependent change in p38 MAPK and p42/44 MAPK activities as determined by their capacity to phosphorylate ATF-2 and ELK-1, respectively. Protein kinase activities increased within 5 min of the addition of the growth factor, reached their maximal levels between 5 (ELK-1: 9.2 ± 2.3-fold, P < 0.01) and 10 min (ATF-2: 8.9 ± 2-fold, P < 0.05), and started to decrease by 30 and 15 min, respectively. After 10 min of PDGF-BB administration, PI3K, PKB, and p70s6k activities were also induced, as demonstrated by the increased levels of phosphorylated Ser473 Akt (values ranged between 5.8 ± 1.8-fold in one set of experiments and 7.6 ± 2.3-fold in a second group of experiments, P < 0.05; Fig. 3, C and D) and p70s6k (10.7 ± 0.5-fold, P < 0.01; Fig. 3E). Direct measurements of PI3K activity performed after immunoprecipitation of the enzyme with an antibody against the p85 regulatory subunit and measuring 32P-radiolabeled phosphatidylinositol triphosphate revealed a significant increase in kinase activity (Fig. 3G). We showed by Western analysis of the samples used for the immunoprecipitation with phosphor-tyrosine, that 308-threonine Akt was also increased in PDGF-BB-treated cultures as well as Ser241 PDK1, the enzyme directly involved in phosphorylation of Thr308 in Akt (Fig. 3F). After determining the changes in kinase activities, we investigated whether wortmannin (Fig. 3, C, F, and G), LY294002 (Fig. 3D), and rapamycin (Fig. 3E) blocked their activities and whether they had any effect on PDGF- receptor degradation. Although each of the inhibitors blocked its cognate kinase activity, none of them had any effect on PDGF- receptor protein degradation (Fig. 4, A–C).
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receptor was required to induce its degradation. To this end, we used 10 µM tyrphostin 1296, a specific inhibitor of PDGF- receptor autophosphorylation, and measured total levels of receptor and its phosphorylation. As illustrated in Fig. 5, receptor phosphorylation was completely prevented by the inhibitor. However, receptor degradation was retarded by tyrphostin, and by 60 min levels were 55.3 ± 8.8% compared with 16.7 ± 4.3% (P < 0.05) observed in control cells treated without the inhibitor. However, because low levels of PDGF- receptor synthesis may take place at these early time points and may contribute to the levels of receptor determined by Western analysis, the actual inhibitory effect of tyrphostin 1296 on receptor degradation maybe underestimated.
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Receptor Degradation Is Not Mediated by Proteasomes But Is Eliminated by Lysosomal Degradation
To study possible sites of PDGF- receptor degradation, we preincubated MHSC with two proteasomal inhibitors (lactacystin and proteasome inhibitor 1) and with chloroquine to study proteasomal and lysosomal degradation, respectively. Although none of the proteasomal inhibitors prevented PDGF- receptor degradation (Fig. 6, A and B), chloroquine prevented its degradation when used at a 100 µM concentration (Fig. 6C). To further establish the role of lysosomes in receptor degradation after incubation with PDGF-BB, we performed confocal microscopy on cells treated with 50 nM lysotracker green DND-26 followed by immunolocalization of the receptor (see Confocal Microscopy under MATERIALS AND METHODS). As shown in Fig. 7, a1–a3, at time 0, PDGF- receptor (red) was localized to areas associated with the plasma membrane and there was little or no localization to lysosomes (green). It formed numerous clusters throughout the cell membrane. Fifteen minutes after the administration of PDGF-BB, the receptor was colocalized with the lysotracker (Fig. 7, b1–b3) and by 30 min most of the receptors appeared to be degraded as determined by the decrease in red immunofluorescence (Fig. 7, c1–c3).
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Receptor Is Dependent on Gene Transcription, De Novo Protein Synthesis, and PI3K Activity
PDGF- receptor reappearance after PDGF-BB administration is time and dose dependent (see Fig. 1B). Therefore, it was important to investigate molecular events involved in recovery of the receptor. To this end, cells were treated with PDGF-BB for 6 h, after which time the medium was removed and the cells were washed and then incubated with either medium alone or medium containing inhibitors of transcription, protein synthesis, or protein kinases (see below). Levels of receptor present at 6 h were used as controls to assure that, indeed, PDGF-BB downregulated its expression. Cells were incubated for additional 42 h, after which time levels of receptor were determined by Western blot analysis (see MATERIALS AND METHODS). Because measurements were performed with total cell extracts and not with isolated membrane fractions, the lack of immunoreactive PDGF- receptor by 6 h suggested that no internal pool of receptor was present. Thus PDGF- receptor reappearance had to be de novo synthesized. To confirm this suggestion, we performed experiments with actinomycin D or cycloheximide to prevent gene transcription and protein production and analyzed the amount of receptor that reappeared after incubation with PDGF-BB. As illustrated in Fig. 8, actinomycin D and cycloheximide completely blocked receptor reappearance (0.03 ± 0.01-fold, P < 0.001, and 0.03 ± 0.005-fold, P < 0.001, respectively), thus suggesting that gene transcription and de novo protein synthesis were required. To investigate signal transduction pathways involved in the reappearance of PDGF- receptor, we performed experiments similar to those described above using the inhibitors of p70S6k, ERK1/2, p38MAPK, and PI3K (see Fig. 9, A and B). Although all the inhibitors blocked kinase activity, only wortmannin and LY294002 prevented reappearance of PDGF- receptor (0.23 ± 0.17-fold and 0.41 ± 0.25-fold, P < 0.05, respectively; Fig. 9B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSDISCLAIMERREFERENCES |
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receptor degradation in HSC, the molecular mechanisms involved in PDGF- receptor fate after exposure to its ligand, as well as the mechanisms involved in its subsequent upregulation, remain to be fully elucidated. Results presented in this communication addressed some of these questions. Our findings demonstrated that HSC do not contain an internal pool of PDGF- receptor protein and that PDGF-BB administration results in a complete degradation of the receptor. Receptor protein degradation is in part dependent on its autophosphorylation because tyrphostin, a specific inhibitor of receptor autophosphorylation, prevented, albeit did not completely inhibit, receptor degradation. Although we may be underestimating receptor values due to additional PDGF- receptor synthesis, our findings could also be interpreted as suggesting that an additional event following ligand receptor interaction and autophosphorylation of the receptor is required to trigger its degradation. However, attempts to establish additional key factors required for degradation failed to produce significant results. Inhibitors of the various kinases that are induced by PDGF-BB, namely inhibitors of p38 MAPK, ERK1/2, p70s6k, PI3K, and PKB kinase, failed to prevent receptor degradation.
Studies to determine the role of proteasomes in degradation of PDGF- receptor protein after PDGF-BB administration failed to show a significant role of proteasomes in receptor elimination. Our findings revealed that PDGF- receptor ligand interaction induced lysosomal degradation of the receptor. Although complete protection of receptor degradation was not obtained, the effect was dose dependent, and 100 mM chloroquine was effective in preventing degradation. Nonetheless, additional mechanisms involved in PDGF- receptor degradation need to be investigated because chloroquine did not inhibit receptor degradation completely. Work is in progress to determine whether matrix metalloproteinase-2 can degrade PDGF- receptor as shown for N-cadherin during gliotoxin-induced apoptosis (23).
The lack of an internal pool of PDGF- receptor protein strongly suggested that its expression and reappearance had to be an active process and thus required transcription and de novo protein synthesis. This was confirmed in experiments in which receptor expression was inhibited with actinomycin D or cycloheximide. These findings suggest a more complex mechanism of receptor regulation and that the newly expressed PDGF- receptor is derived from newly transcribed gene.
Regarding signaling pathways involved in receptor reexpression, our results indicated that the PI3K pathway is required after stimulation with the growth factor. Therefore, the de novo expression of the PDGF- receptor is a PI3K-dependent process irrespectively of whether the receptor is expressed during activation (29, 30) or reappears after its degradation (this communication). Our data showed that N-acetylcysteine whereas two inhibitors of the PI3K pathway, namely, wortmannin and LY294002, blocked the reappearance of receptors, inhibitors of p38 MAPK, ERK1/2, and p70s6k had no effect.
PDGF- receptor is degraded in the presence of antioxidants such as N-acetylcysteine (26, 37), thus suggesting that antioxidant defense mechanisms may favor receptor degradation and thus prevent its expression and accumulation on the plasma membrane. Consequently, quiescent HSC will not proliferate in response to PDGF-BB. On the basis of these findings, one could infer that activation of HSC in culture and/or perhaps in vivo could result from cell distress and a failure of their antioxidant defense mechanisms to inactivate excess reactive oxygen species. This will result in a decrease in PDGF- receptor degradation with increased membrane accumulation and response to PDGF-BB. Once the receptor is expressed, its interaction with PDGF-BB results in a pepstatin-independent, lysosomal-dependent degradation. Altogether, these findings suggest that PDGF- receptor regulation is complex and follows at least two distinct pathways. One, occurring in quiescent HSC, is regulated by the antioxidant defense mechanisms, resulting in the extracellular degradation of the receptor by cathepsin B (26, 37). The second one, occurring primarily in already-activated HSC, is mediated by receptor ligand interaction and results in receptor phosphorylation and lysosomal degradation. Accordingly, on the basis of the fact that some PDGF-BB- (1, 21, 32), TGF-1-, and acetaldehyde-mediated events are H2O2 dependent (10, 13), we can suggest that reactive oxygen species in general and H2O2 formation in particular are key mediators of the fibrogenic cascade. Accordingly, a better understanding of key molecular mechanisms whereby oxidative stress induces PDGF- receptor expression and degradation in HSC could lead to the development of novel antifibrogenic therapies.
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DISCLAIMER |
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ACKNOWLEDGMENTS |
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Present address for C. G. Lechuga: Servicio de Endocrinología Experimental, Hospital Universitario Puerta de Hierro, Madrid, España.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* C. G. Lechuga and Z. H. Hernández-Nazara contributed equally to this work. 
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSDISCLAIMERREFERENCES |
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1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k. Am J Physiol Gastrointest Liver Physiol 287: G974–G987, 2004.This article has been cited by other articles:
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  S. L. Friedman Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver Physiol Rev, January 1, 2008; 88(1): 125 - 172. [Abstract] [Full Text] [PDF]
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