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Adenosine A_2A Receptor Occupancy Stimulates Collagen Expression by Hepatic Stellate Cells via Pathways Involving Protein Kinase A, Src, and Extracellular Signal-Regulated Kinases 1/2 Signaling Cascade or p38 Mitogen-Activated Protein Kinase Signaling Pathway

Division of Clinical Pharmacology, Department of Medicine, New York University School of Medicine, New York, New York

Received June 6, 2007; accepted September 14, 2007

	Abstract

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Prior studies indicate that adenosine and the adenosine A_2A receptor play a role in hepatic fibrosis by a mechanism that has been proposed to involve direct stimulation of hepatic stellate cells (HSCs). The objective of this study was to determine whether primary hepatic stellate cells produce collagen in response to adenosine (via activation of adenosine A_2A receptors) and to further determine the signaling mechanisms involved in adenosine A_2A receptor-mediated promotion of collagen production. Cultured primary HSCs increase their collagen production after stimulation of the adenosine A_2A receptor in a dose-dependent fashion. Likewise, LX-2 cells, a human HSC line, increases expression of procollagen I and procollagen III mRNA and their translational proteins, collagen type I and type III, in response to pharmacological stimulation of adenosine A_2A receptors. Based on the use of pharmacological inhibitors of signal transduction, adenosine A_2A receptor-mediated stimulation of procollagen I mRNA and collagen type I collagen expression were regulated by signal transduction involving protein kinase A, src, and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (erk), but surprisingly, adenosine A_2A receptor-mediated stimulation of procollagen III mRNA and collagen type III protein expression depend on the activation of p38 mitogen-activated protein kinase (MAPK), findings confirmed by small interfering RNA-mediated knockdown of src, erk1, erk2, and p38 MAPK. These results indicate that adenosine A_2A receptors signal for increased collagen production by multiple signaling pathways. These results provide strong evidence in support of the hypothesis that adenosine receptors promote hepatic fibrosis, at least in part, via direct stimulation of collagen expression and that signaling for collagen production proceeds via multiple pathways.

Based on the prior observation that adenosine, acting at adenosine A_2A receptors, plays a role in wound healing (Montesinos et al., 1997, 2002; Victor-Vega et al., 2002) we hypothesized that adenosine and adenosine A_2A receptors might play a role in excess fibrosis in the skin and hepatic fibrosis. Indeed, endogenously released adenosine and adenosine A_2A receptors play a critical role in the development of liver and skin fibrosis after toxic insults based on studies in animal models (Chan et al., 2006b). Thus, both adenosine A_2A receptor antagonists and adenosine A_2A receptor deletion protect mice from developing skin and liver fibrosis (Chan et al., 2006a,b).

Signaling for enhanced collagen production after adenosine A_2A receptor activation has been explored only superficially, and inhibition of protein kinase A and erk1/2 partially inhibits overall collagen production by the LX-2 hepatic stellate cell line and primary dermal fibroblasts (Chern et al., 1995; Seidel et al., 1999; Arslan and Fredholm, 2000; Mori et al., 2004; Chan et al., 2006a,b). Adenosine A_2A receptors may signal through other pathways as well, including src kinase (Lee and Chao, 2001; Schulte and Fredholm, 2003; Rajagopal and Chao, 2006).

The aim of the present study is to determine whether collagen production by HSCs is regulated by adenosine A_2A receptors and to better characterize the signal-transduction mechanisms involved in adenosine A_2A receptor-mediated regulation of matrix production. We present evidence here that occupancy of adenosine A_2A receptors regulates the transcription and translation of collagen type I via activation of PKA, src kinase, and erk1/2 MAPK and collagen type III via p38 MAPK signaling pathway.

	Materials and Methods

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Reagents and Drugs. Adenosine A_2A receptor agonist CGS21680 was purchased from Research Biochemicals (Natick, MA). Adenosine A_2A receptor antagonist ZM241385 was from Tocris (Ellisville, MO). PKAI, a PKA inhibitor that corresponds to the amino acids 5 to 24 of the naturally occurring PKA inhibitor (Kemp et al., 1991), was from Promega (Madison, WI). srcI, MEK/erk MAPK inhibitors, U0126, PD98059, and p38 MAPK inhibitor SB202190 were from Calbiochem (San Diego, CA), and their structures are described in the manufacturer's catalog. CellTiter 96 proliferation assay kit was from Promega. Sirius red F3B and Fast Green FCF were from Sigma (St. Louis, MO). Sircol collagen assay kit was from Accurate Chemical (Westbury, NY). TRIzol and SuperScript III RT kit were from Invitrogen (Carlsbad, CA). Brilliant SYBR Green QPCR Master Mix was from Stratagene (La Jolla, CA).

Cell Culture. Cultured normal human hepatic primary HSCs isolated from single donor human liver were purchased from Scien-Cell Research Laboratories (San Diego, CA). To activate hepatic primary HSCs, cells were cultured in Stellate Cell Medium (SteCM; ScienCell Research Laboratories) supplemented with 2% fetal bovine serum (Invitrogen), 1% of stellate cell growth supplement (ScienCell Research Laboratories), 100 U/ml penicillin (Invitrogen, Carlsbad, CA), and 100 U/ml streptomycin (Invitrogen) on uncoated plastic flasks (BD Biosciences, San Jose, CA) for 14 days (Ikeda et al., 1999; Marra et al., 2000) and were then used for biological studies.

The LX-2 human hepatic stellate cell line was characterized previously by and kindly provided by Dr. S. L. Friedman, (Mount Sinai School of Medicine, New York, NY) (Xu et al., 2005). LX-2 cells were grown and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (all from Invitrogen). Cells were cultured in 75-cm² flasks until 80 to 90% confluence and were then used for biological studies.

Cell Proliferation Assay. The effect of CGS21680 and ZM241385 on proliferation of the LX-2 cells was analyzed by morphological analysis and CellTiter 96 proliferation assay kit according to the manufacturer's instructions. In brief, the cells were seeded into 96-well plates (5 x 103 cells/well). Six wells were used for each concentration of each compound (n = 5 for each concentration). The CellTiter 96 Aqueous One solution [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and phenazine ethosulfate] was added to the wells, and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was reduced into a colored formazan product by the living cells. The quantity of formazan product was measured 3 h later at a wavelength of 490 nm, because the optical density of the dye at 490 nm is directly proportional to the number of living cells (Zolnai et al., 1998). The results were confirmed by three independent experiments.

Sircol Collagen Assay. The collagen content in ice-cold methanol-fixed primary HSCs and LX-2 cells was determined by the differential binding of Sirius red F3B and Fast Green FCF to collagen and noncollagenous proteins, respectively, in the presence of picric acid (Jimenez et al., 1985; López-De León and Rojkind, 1985). Collagen content of primary HSCs and LX-2 cells was determined by a modification of the original method (Freeman et al., 2002; Bennett et al., 2003). The cells were plated in 24-well plates at a density that resulted in 80 to 90% confluence after a 24-h incubation in medium containing 2% (for primary HSCs) or 10% fetal bovine serum (for LX-2 cells). Culture medium was then replaced with serum-free medium supplemented with 1% of stellate cell growth supplement, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin for 24 h, and then cells were treated with various adenosine receptor agonists and/or antagonists for 24 h. After treatment, the medium was harvested, and the cells were washed and fixed in cold methanol for 15 min. Cells were stained at room temperature as follows: 1) 15 min in 0.5 ml of a solution containing 0.01% Fast Green FCF and 0.5% picric acid in distilled water; 2) 15 min in 0.5 ml of a solution containing 0.04% Fast Green FCF, 0.1% Sirius red, and 0.5% picric acid in distilled water; and 3) washing in tap water for 5 min until the elution fluid was completely clear. Cells were allowed to dry before elution of the bound dyes with 1 ml of 50% methanol containing 50 mM NaOH. After a spectrum scan to establish the peak absorbance for each dye, the absorbance of the solution at 630 and 540 nm was determined by spectrophotometer (SmartSpec Plus Spectrophotometer; Bio-Rad Laboratories, Hercules, CA). Calculation of collagen and protein content was performed as described previously (Jimenez et al., 1985), and the data are expressed as the change in the content of soluble collagen production (microgram per milligram of total proteins) over the treatment interval to correct for collagen present in the cultures without the addition of the test compounds.

Total soluble collagen concentration in the cell supernatants was also measured using the Sircol collagen assay kit. In brief, a 200-µl aliquot of supernatant was added to 1 ml of Sircol dye and incubated at room temperature for 30 min and then centrifuged to pack the collagen-dye complex at the bottom of the tube. The pellet obtained was dissolved in alkali reagent (0.5 M NaOH solution) to release the collagen-dye complex whose absorbance was measured at 540 nm. The concentration of collagen production was determined from a collagen standard curve provided with the assay.

Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction. Total RNA was isolated from 3 x 10⁸ LX-2 cells using the TRIzol method. RNA (1.0 µg) was reverse-transcribed in 20 µl of buffer containing 50 µM oligo(dT)20, 25 mM MgCl₂, 0.1 M dithiothreitol, 40 U/µl RNaseOUT, and 200 U/µl SuperScript III RT for 50 min at 50°C. The reaction was stopped by incubating the samples at 85°C for 5 min, and 40 µl of nuclease-free water was added. Real-time PCR was performed by using the Brilliant SYBR Green QPCR Master Mix. The PCR template source was either 30 ng of first-strand cDNA or purified DNA standard. Primer sequences used to amplify the desired cDNA are detailed in Table 1. Amplification was performed with a spectrofluorometric thermal cycler (Stratagene). After an initial denaturation step at 95°C for 10 min, amplification was performed using 40 cycles of denaturation (95°C for 30 s), annealing (56°C for 1 min), and extension (72°C for 1 min). For each run, a standard curve was generated from purified DNA ranging from 10⁶ to 10¹² copies. To standardize mRNA levels, we amplified GAPDH, a housekeeping gene, as an internal control. Gene expression was normalized by calculating the ratio between the number of cDNA copies of collagen type I, type III, and that of GAPDH. After amplification, a final melting curve was recorded by cooling the PCR mixture to 65°C for 30 s and then slowly heating it to 95°C at 30 s. Fluorescence was measured continuously during the slow temperature increase to monitor the dissociation of double-stranded DNA. Specificity of the expected Light Cycler products was demonstrated by melting curve analysis. Amplification products formed in the Light Cycler were checked by electrophoresis on 1.5% ethidium bromide-stained agarose gel. The estimated size of the amplified fragments matched the calculated size.

Western Blot Analysis. Cells were rinsed with ice-cold phosphate-buffered saline, and total cell protein extracts were prepared using a cell lysis buffer (Cell Signaling Technology, Danvers, MA) containing 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, 1% Protease Inhibitor Cocktail (Sigma). Protein concentrations were measured by a BCA assay (Pierce, Rockford, IL). Ten micrograms of protein extracts were boiled in Laemmli sample buffer and was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane (0.2 µm; Bio-Rad) using a Bio-Rad gel-blotting apparatus. Membranes were stained with Ponceau red to confirm bands existed on the membranes, followed by blocking of the membranes in 5% nonfat milk in TBST (10 mM Tris HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h. Blots were probed with monoclonal antibodies directed against mouse collagen type I (Chemicon, Temecula, CA), type III (Abcam, Cambridge, MA), and phosphorylated and total src [P-Y(416), activated src; Upstate, Charlottesville, VA] diluted 1:500 with 0.05% TBST or polyclonal antibodies directed against rabbit total erk1 (Chemicon), total erk2 (Upstate), phosphorylated erk1/2, and phosphorylated and total p38 MAPK (Biosource, Camarillo, CA) diluted 1:1000, and incubated overnight at 4°C. After 3 x 10-min wash, blots were incubated for 1 h at room temperature with alkaline phosphatase-conjugated anti-mouse IgG (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) or alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody (Sigma) diluted 1:1000 with TBST for 1 h at room temperature. After each incubation step, membranes were washed in TBST for 20 min. Proteins of interest were visualized by enhanced cyan fluorescent substrate (GE Healthcare, Braunschweig, Germany) and scanned on fluorescent scanning instrument (Storm 860 Molecular Imager; GE Healthcare) for appropriate times.

RNA Interference. siRNA sequences against src, erk1, erk2, and p38 were designed using criteria described previously by Elbashir et al. (2002), and the sequences are shown in Table 2. Parallel scrambled siRNAs as the negative control were also designed differing from the siRNAs by three nucleotides. These sequences did not match human genes as checked via the National Center for Biotechnology Information standard nucleotide-nucleotide BLAST program. Sequences were purchased from Sigma. Transfection of siRNAs into LX-2 cells was performed 24 h after plating. In brief, LX-2 cells were seeded at a density of 1 x 10⁷ cells/100-mm Petri dishes the day before transfection to achieve 50 to 60% confluence. Transfections were carried out with 50 nmol siRNA duplex using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 6-h incubation, the transfection medium was removed, and cells were changed to serum-free medium. After 48 h, transfected cells were treated with stimuli or medium for 24 h followed by lysis, harvest of proteins, and Western blot analysis. For real-time PCR, stimulation by CGS21680 for 12 h was carried out 60 h after transfection, and then cells were collected for extraction of total RNA.

Statistical Analysis. All experiments were performed at least three times, and results were analyzed by analysis of variance with repeated measures and post hoc analysis using Bonferroni correction.

	Results

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Proliferation of LX-2 Cells Was Not Regulated by Adenosine A_2A Receptor Agonists and Antagonists. To exclude the effect of adenosine agonists and antagonists on cell viability, the morphology of LX-2 cells and the proliferation of LX-2 cells was examined after 24-h treatment by microscope and CellTiter 96 proliferation assay, respectively. After treatment with CGS21680 (1 nM to 1 µM) and ZM241385 (1 nM to 1 µM), the morphology of the LX-2 cells seemed similar to that of the cells without the treatment (data not shown). Likewise, proliferation of LX-2 cells was unaffected by either the adenosine A_2A receptor agonist or antagonist at these concentrations (data not shown).

Adenosine A_2A Receptor Occupancy Increased Total Soluble Collagen Production by LX-2 Cells. In our initial studies, we assessed the effect of varying doses of CGS21680 on collagen production by LX-2 cells and compared this with a known stimulus for collagen production, TGF. The adenosine A_2A receptor agonist CGS21680 significantly increased both soluble and cell-associated collagen production by as much as 2-fold in a dose-dependent fashion (EC₅₀ = 79 nM for supernatant total soluble collagen production; EC₅₀ = 82 nM for cell-associated total soluble collagen production). The magnitude of the CGS21680-induced increase was similar to that induced by TGF (Fig. 1). The A_2A receptor antagonist ZM241385 completely abrogated the effect of CGS21680 on collagen production (Fig. 2). These results are consistent with the hypothesis that adenosine A_2A receptors, when occupied, promote collagen production.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Determination of increased total soluble collagen production both in supernatant and LX-2 cells. LX-2 cells were treated with the indicated concentration of adenosine A2A receptor agonist, CGS21680, as described for 24 h before measurement of collagen. CGS21680 increased collagen production measured both in the cells and supernatant (P < 0.01, one-way analysis of variance). The cells were also treated with 40 ng/ml TGF as a positive control. A, CGS21680-induced collagen production in supernatant. B, CGS21680-induced collagen production in LX-2 cells. *, P < 0.01 versus cells alone; n = 3.

View larger version (11K): [in this window] [in a new window]   Fig. 2. ZM241385 inhibited CGS21680-induced total soluble collagen production. LX-2 cells were treated with TGF (40 ng/ml, positive control), 1 µM CGS21680, 1 µM ZM241385, or 1 µM CGS21680 plus 1 µM ZM241385 for 24 h before harvesting and measurement of collagen in supernatant and cells, as described. The data are presented as mean ± S.D. of triplicate cultures in four separate experiments. *, P < 0.01 versus cells alone; **, P < 0.01 versus CGS21680-treated cells.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Correspondence of collagen production between primary HSCs and LX-2 cells. Primary HSCs and LX-2 cells were treated with TGF (40 ng/ml, positive control), 1 µM CGS21680, 1 µM ZM241385, or 1 µM CGS21680 plus 1 µM ZM241385 for 24 h before harvesting and measurement of collagen production in supernatant and cells as described. The CGS21680-induced increase in collagen production measured in primary cells did not differ from that observed in LX-2 cells. The data are presented as mean ± S.D. of triplicate cultures carried out in duplicate in six separate experiments. A, CGS21680-induced collagen production in supernatant. B, CGS21680-induced collagen production in LX-2 cells.

Effect of CGS21680 and ZM241385 on Procollagen I and Procollagen III mRNA Expression.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of CGS21680 and ZM241385 on the expression of procollagen I and procollagen III mRNA in LX-2 cells. LX-2 cells were cultured in serum-free DMEM containing stellate cell growth supplement and treated with TGF, (40 ng/ml), 1 µM CGS21680, 1 µM ZM241385, or 1 µM CGS21680 plus 1 µM ZM241385 for the indicated periods of time before the RNA was harvested. After reverse transcription, the number of copies of mRNA for the two transcripts was determined by real-time PCR. Data were normalized to GAPDH mRNA and are expressed as the mean ± S.D. (n = 4). A, procollagen I mRNA. B, procollagen III mRNA. *, P < 0.01, **, P < 0.05 versus cells alone. , P < 0.01; , P < 0.05 versus CGS21680-treated cells.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Western blot analyses of LX-2 cell lysates for collagen types I and III. LX-2 cells were treated with 40 ng/ml TGF,1 µM CGS21680, 1 µM ZM241385, or 1 µM CGS21680 plus 1 µM ZM241385 for 24 h before the proteins were harvested. Lysates were subject to SDS-PAGE and immunoblotting. Data are expressed as the mean ± S.D. (n = 3). A, collagen type I; B, collagen type III. *, P < 0.01 versus cells alone. **, P < 0.01 versus CGS21680-treated cells.

Involvement of Protein Kinases in CGS21680-Stimulated Soluble Collagen Production. To better understand how adenosine A_2A receptor activation regulates collagen production, we examined the signaling mechanisms downstream of receptor activation that might affect collagen production using a series of kinase inhibitors. We were surprised to find that the inhibitors of PKA (PKAI), src kinase (srcl), MEK/erk (U0126 and PD98059), and p38 MAPK (SB202190) inhibited the adenosine A_2A receptor-mediated increase in supernatant and cell-associated total soluble collagen production (Fig. 6A). Because a number of signaling mechanisms were implicated in these studies, we further dissected the effect of protein kinase inhibitors on procollagen I and procollagen III mRNA expression by real-time reverse-transcriptase polymerase chain reaction. Treatment of the cells with PKAI, srcI, U0126, and PD98059 significantly attenuated procollagen I mRNA up-regulation by CGS21680 but SB202190 did not. In contrast, only SB202190 completely abolished CGS21680-induced procollagen III mRNA expression (Fig. 6B). In a similar fashion, inhibition of PKA, src kinase, and MEK/erk MAPK but not p38 MAPK reversed the effect of adenosine A_2A receptor occupancy on collagen type I but not collagen type III protein expression (Fig. 6C-1), and inhibition of p38 MAPK reversed the effect of receptor occupancy on collagen type III but not collagen type I (Fig. 6C-2). These findings suggest that adenosine A_2A receptors regulate both collagen type I and type III production but do so by different signaling mechanisms.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Effects of PKI, srcI, U0126, PD98059, and SB202190 on CGS21680-stimulated collagen expression in LX-2 cells. LX-2 cells were treated with 1 µM CGS21680 alone or in combination with 1 µM PKI, 1 µM srcI, 1 µM U0126, 1 µM PD98059, or SB202190, respectively, for 24 h. Collagen production, procollagen I, and procollagen III mRNA and collagen types I and III were measured as described. Data are expressed as the mean ± S.D. A, collagen production. B, procollagen, C-1, collagen type I. C-2, collagen type III. *, P < 0.01, **, P < 0.05 versus cells alone. , P < 0.01; , P < 0.05 versus CGS21680-treated cells.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Effects of PKI, srcI, U0126, PD98059, and SB202190 on the phosphorylation of src, erk1/2, and P38 MAPK in LX-2 cells treated with CGS21680. LX-2 cells were treated with 1 µM CGS21680 alone or in combination with 1 µM PKI, 1 µM srcI, 1 µM U0126, and 1 µM PD98059 or SB202190 for 10 min. Phosphorylated (Tyr416) and total src, erk1/2, and p38 MAPK were detected by immunoblot of lysates as described. Data are expressed as the mean ± S.D. (n = 3). A, src kinase. B, erk1/2 MAPK. C, p38 MAPK. *, P < 0.01 versus cells alone. , P < 0.01 versus CGS21680-treated cells.

siRNA-Mediated Knockdown of src, erk1, erk2, and p38 Decreased CGS21680-Stimulated Collagen Types I and III. To confirm that the signaling events indicated by the pharmacological inhibitors were indeed responsible for adenosine A_2A receptor-mediated stimulation of collagens I and III, we diminished expression of src, erk1, erk2, and p38 MAPK by use of siRNA. Appropriately sequenced siRNA diminished expression of the corresponding kinases (Fig. 8) and reversed the CGS21680-mediated increase in phosphorylated src (Fig. 8A), erk1 (Fig. 8B), erk2 (Fig. 8C), and p38 MAPK (Fig. 8D). More importantly, knockdown of src and erk1 significantly diminished procollagen I mRNA and reversed the CGS21680-mediated increase in procollagen I (Fig. 9, A and B). Knockdown of erk2 also diminished procollagen I mRNA and reversed CGS21680-mediated increase in procollagen I mRNA, but the differences observed did not achieve statistical significance (Fig. 9C). Knockdown of both erk1 and erk2 by siRNA led to greater reversal of the effect of CGS21680 on the expression of procollagen than knockdown of either kinase alone (Fig. 9D). Knockdown of p38 MAPK diminished procollagen III mRNA and reversed CGS21680-mediated procollagen III mRNA (Fig. 9E). These results parallel and confirm the effects of the pharmacological inhibitors on adenosine A_2A receptor signaling events involved in promoting collagen production.

View larger version (63K): [in this window] [in a new window]   Fig. 8. siRNA-mediated knockdown of src, erk1, erk2, and p38 decreased total and phosphorylated src, erk1, erk2, and p38 expression in nonstimulated and CGS21680-stimulated LX-2 cells. LX-2 cells were transiently transfected with src, erk1, erk2, and p38 siRNAs (50 nM) or scrambled siRNAs for 48 h and then treated with or without CGS21680 (1 µM) for 24 h. siRNA significantly reduced the level of corresponding total and phosphorylated src, erk1, erk2, and p38 proteins. Results are expressed as comparisons between siRNA-transfected cells and wild type or cells transfected with corresponded scrambled siRNAs (n = 3 separate experiments). A, src siRNA-transfected. B, erk1 siRNA-transfected. C, erk2, siRNA-transfected. D, p38 siRNA-transfected. **, P < 0.05; *, P < 0.01.

View larger version (32K): [in this window] [in a new window]   Fig. 9. siRNA-mediated knockdown of src, erk1, erk2, and p38 down-regulated the mRNA levels of procollagen I or procollagen III in LX-2 cells. LX-2 cells were transiently transfected with src, erk1, erk2, erk1/2, and p38 siRNAs (50 nM) or scrambled siRNAs (50 nM) individually. At 60 h after transfection, cells were treated with or without CGS21680 (1 µM) for 12 h. Total RNA was extracted, reverse-transcribed, and real-time PCR was performed to determine procollagen I and procollagen III mRNA levels. src (A), erk1 (B), erk2 (C), and erk1/2 (D) siRNAs significantly reduced the expression of both basal and CGS21680-stimulated procollagen I mRNA. In contrast, p38 siRNA significantly reduced the expression of procollagen III mRNA (E). Results are expressed as comparisons between siRNA-transfected cells and wild type or corresponded scrambled siRNAs and represent the means (± S.D.) of three separate determinations. **, P < 0.05; *, P < 0.01.

	Discussion

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We report here evidence that adenosine A_2A receptor occupancy directly stimulates collagen production by both primary human hepatic stellate cells and the human hepatic stellate cell line LX-2. More strikingly, we have observed that adenosine A_2A receptors stimulate both collagen type I and type III expression but by distinct signaling pathways: via PKA–src–erk1/2 for collagen type I and via p38 MAPK for collagen type III.

One important limitation in studying hepatic stellate cell function and the regulation of this function by receptor ligands or other agents has been the difficulty in obtaining these cells in a consistent state of activation. To obviate this problem, Xu et al. (2005) established the human hepatic cell line LX-2 and extensively characterized them, demonstrating that they retain the key features of stellate cells, including cytokine signaling, neuronal gene expression, retinoid metabolism, and fibrogenesis, making them highly suitable for culture-based studies of human hepatic fibrosis as a valuable tool (Xu et al., 2005). Because the effects of adenosine A_2A receptor occupation on collagen production were identical in primary hepatic cells and cultured LX-2 cells, we concluded that LX-2 cells were an excellent model for studying signaling at adenosine A_2A receptors, leading to collagen production by hepatic stellate cells.

Topical application of adenosine A_2A receptor agonists have been shown previously to promote wound healing in part by stimulating the production of matrix in the wound. More recent results indicate that adenosine A_2A receptors stimulate collagen type I and type III production by primary human dermal fibroblasts as well and that this stimulation plays a role in dermal fibrosis in response to bleomycin treatment (Chan et al., 2006a). These findings are consistent with those reported here and previously (Chan et al., 2006b).

Classic signal transduction at adenosine A_2A receptors involves coupling of the receptor to G_s signaling proteins to activate adenylate cyclase, thereby elevating the level of cAMP and activating PKA in response to ligand binding (Klinger et al., 2002a). It has also been reported that activation of adenosine A_2A receptor increases the activity of erk1/2 MAPK (Chern et al., 1995; Seidel et al., 1999; Arslan and Fredholm, 2000; Schulte and Fredholm, 2003). In probing signal transduction at adenosine A_2A receptors, we used a series of kinase inhibitors shown previously to disrupt signal transduction via inhibition of protein phosphorylation of appropriate substrates. The results indicate that in LX-2 cells, adenosine A_2A receptor-stimulated increases of production of collagen type I proceeds via cAMP-dependent PKA-mediated activation of src kinase and erk1/2 MAPK. Further evidence for involvement of this pathway in signal transduction at adenosine A_2A receptors is provided by the demonstration that adenosine A_2A receptor occupation leads to phosphorylation (activation) of erk1/2. Previous studies demonstrate that the well-characterized cAMP/PKA and MAPK pathways exhibit cross-talk (Stork and Schmitt, 2002), although in general, emphasis has been placed on the ability of PKA-dependent phosphorylation to disrupt the interaction between p21^ras and c-RAF, which results in cAMP-mediated suppression of the MAPK pathway (Burgering et al., 1993). Nonetheless, stimulation of G_S-coupled receptors may also result in both increased cellular cAMP and with stimulation of MAPK (Daaka et al., 1997; Keiper et al., 2004), as we have observed in LX-2 cells.

Prior experiments carried out in Chinese hamster ovary cells indicate that adenosine A_2A receptors activate erk1/2 in a cAMP-dependent fashion that relies on src or an src-like kinase downstream of PKA for MAPK activation (Seidel et al., 1999; Klinger et al., 2002b). This sequence of signaling events is also most consistent with our results because treatment of the cells with forskolin, which directly stimulates cAMP production, leads to phosphorylation of src and erk (Supplemental Figs. S1 and S2) and treatment of LX-2 cells with the src inhibitor prevented phosphorylation of erk1/2 (Fig. 7B), but inhibitors of erk activation did not affect src activation. Thus, the sequence of signaling most consistent with our results is cAMP–PKA–src–erk1/2.

We were surprised to find that adenosine A_2A receptor activation mediates an increase in collagen type III production expression via activation of p38 MAPK but independent of PKA, src, MEK, and erk MAPK. There are few reports that link adenosine A_2A receptors to p38 MAPK and fewer clues as to which mediator is involved between receptors and p38 MAPK activation. A recent report (Rahman et al., 2004) demonstrated that cAMP inhibits p38 MAPK activation in human umbilical vein endothelial cells. In contrast, PKA was found to activate p38 MAPK in macrophages (Chio et al., 2004). Furthermore, the signaling pathways up- and downstream of p38 MAPK pathway are diverse, which may explain why p38 MAPK pathway can be activated by various stimuli (Han et al., 1994; Moriguchi et al., 1996; Pietersma et al., 1997; Wang et al., 1997; Craxton et al., 1998).

We also demonstrated that RNAi-mediated knockdown of src, erk1, erk2, and erk1/2 was sufficient to inhibit baseline and CGS21680-stimulated procollagen I mRNA expression. In addition, RNAi-mediated p38 knockdown significantly down-regulated baseline and CGS21680-stimulated procollagen III mRNA expression. The results of the studies with siRNA-mediated knockdown of relevant signaling molecules were clearly consistent with the results with pharmacological inhibitors of the same kinases, providing a second level of confidence in the results.

Based on studies in knockout mice as well as pharmacological evidence, we have reported previously that endogenously released adenosine and adenosine A_2A receptors play a major role in the development of hepatic fibrosis in experimental murine models. These observations may shed particular light on alcohol-induced hepatic fibrosis/cirrhosis. Prior studies demonstrate that ethanol treatment increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter (Nagy et al., 1990). Short-term treatment with ethanol increases extracellular adenosine concentrations in vitro (Nagy et al., 1989), which causes the release of adenosine from rat cerebellar synaptosomes (Clark and Dar, 1989). Ethanol-induced accumulation of extracellular adenosine activates adenosine A_2A receptor to stimulate cAMP production (Nagy et al., 1989). Likewise, treatment of mice with hepatotoxins leads to increased adenosine release ex vivo (Chan et al., 2006b). Because ethanol promotes purine release in humans as well (Puig and Fox, 1984), the previous studies suggest that ethanol-induced increases in extracellular adenosine levels promote hepatic fibrosis by stimulating enhanced production of collagen by otherwise activated hepatic stellate cells. Moreover, these observations suggest a mechanism for the observation that coffee-drinking protects against death from hepatic cirrhosis in that caffeine, contained in coffee in pharmacologically relevant concentrations, is a well known adenosine receptor antagonist (Klatsky et al., 1993; Corrao et al., 1994, 2001; Sharp et al., 1999; Gallus et al., 2002; Ruhl and Everhart, 2005).

We conclude that in hepatic stellate cells, signaling for collagen production via adenosine A_2A receptors is complex. Although generally believed to be nearly exclusively signaling via G_s/cAMP/PKA and their related downstream signaling events, these results indicate that stimulation of adenosine A_2A receptors activates p38 MAPK as well. Moreover, both of these signals contribute to stimulating the expression of collagen by hepatic stellate cells. Although it is likely that other events also contribute, adenosine, released as a result of hepatic injury or exposure to agents like ethanol, contributes to the development of hepatic cirrhosis via stimulating hepatic stellate cell production of collagen.

	Footnotes

 
This work was supported by grants from the National Institutes of Health (AA13336, AR41911, and GM56268), King Pharmaceuticals, the General Clinical Research Center (M01-RR00096), and the Kaplan Cancer Center of New York University School of Medicine.

B.N.C. holds intellectual property (patents on the use of adenosine A_2A receptor agonists to promote wound healing and use of A_2A receptor antagonists to inhibit fibrosis; patent on testing for single nucleotide polymorphisms in the adenosine A₁ receptor in patients with fibromyalgia; patent on the use of adenosine A₁ receptor antagonists to treat osteoporosis and other diseases of bone) and has served as a consultant to the following companies: King Pharmaceutical (licensee of patents above), CanFite Biopharmaceuticals, Bristol-Myers Squibb, Cellzome, Tap Pharmaceuticals, Prometheus Laboratories, Regeneron (Westat, DSMB), Sepracor, Amgen, and Endocyte. In addition, B.N.C. has received honoraria for speaking from Tap Pharmaceuticals and Amgen. He holds stock in CanFite Biopharmaceuticals and receives research grants from King Pharmaceuticals.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.107.038760.

ABBREVIATIONS: HSC, hepatic stellate cell; siRNA, small interfering RNA; PKA, protein kinase A; PKAI: protein kinase A peptide inhibitor; srcI, src kinase inhibitor II; CGS21680, 2-(p-(2-carbonylethyl) phenylethylamino)-5-N-ethylcarboxamido adenosine; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol; MEK, mitogen-activated protein kinase kinase; erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBST, Tris-buffered saline/Tween 20; TGF, transforming growth factor ; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; PD98059, 2'-amino-3'-methoxyflavone; SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Bruce N. Cronstein, Department of Medicine, New York University School of Medicine, 550 First Avenue, NBV16N1, New York, NY 10016. E-mail: cronsb01{at}med.nyu.edu

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