Abstract
Prior studies indicate that adenosine and the adenosine A2A 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 A2A receptors) and to further determine the signaling mechanisms involved in adenosine A2A receptor-mediated promotion of collagen production. Cultured primary HSCs increase their collagen production after stimulation of the adenosine A2A receptor in a dose-dependent fashion. Likewise, LX-2 cells, a human HSC line, increases expression of procollagen αI and procollagen αIII mRNA and their translational proteins, collagen type I and type III, in response to pharmacological stimulation of adenosine A2A receptors. Based on the use of pharmacological inhibitors of signal transduction, adenosine A2A receptor-mediated stimulation of procollagen αI 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 A2A receptor-mediated stimulation of procollagen αIII 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 A2A 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.
Footnotes
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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.
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B.N.C. holds intellectual property (patents on the use of adenosine A2A receptor agonists to promote wound healing and use of A2A 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.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.038760.
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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; TGFβ, transforming growth factor β; 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.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received June 6, 2007.
- Accepted September 14, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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