Halofuginone Induces Matrix Metalloproteinases in Rat Hepatic Stellate Cells via Activation of p38 and NFκB

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen α1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB(NFκB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFκB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFκB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies. The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen α1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB(NFκB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFκB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFκB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies. Liver cirrhosis as a consequence of many forms of chronic liver diseases is associated with a high morbidity and mortality. Treatment for advanced liver fibrosis and cirrhosis is unsatisfactory or inefficient. Thus, there is an urgent need for antifibrotic treatments that can prevent, halt, or even reverse advanced fibrosis. In recent years, significant progress has been made in our understanding of hepatic fibrosis as a dynamic process, characterized by an imbalance between collagen production and degradation, finally leading to distortion of normal hepatic architecture and loss of function (1McCrudden R. Iredale J.P. Histol. Histopathol. 2000; 15: 1159-1168PubMed Google Scholar, 2Kossakowska A.E. Edwards D.R. Lee S.S. Urbanski L.S. Stabbler A.L. Zhang C.L. Phillips B.W. Zhang Y. Urbanski S.J. Am. J. Pathol. 1998; 153: 1895-1902Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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In hepatic fibrosis the excessive extracellular matrix is produced by activated mesenchymal cells which resemble myofibroblasts and derive from quiescent hepatic stellate cells (HSCs) 4The abbreviations used are: HSC, hepatic stellate cell; HAL, halofuginone; IκB-α, inhibitory κB-α protein; MF, myofibroblast-like cell; MMP, matrix metalloproteinase; NFκB, nuclear factor κB; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; FCS, fetal calf serum; BrdUrd, bromodeoxyuridine; PDGF, platelet-derived growth factor; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APMA, 4-aminophenyl mercuric acetate; TRITC, tetramethylrhodamine isothiocyanate; CREB, cAMP-response element-binding protein; TAA, thioacetamide; HYP, hydroxyproline; TIMP-1, tissue inhibitor of metalloproteinase-1; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase.4The abbreviations used are: HSC, hepatic stellate cell; HAL, halofuginone; IκB-α, inhibitory κB-α protein; MF, myofibroblast-like cell; MMP, matrix metalloproteinase; NFκB, nuclear factor κB; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; FCS, fetal calf serum; BrdUrd, bromodeoxyuridine; PDGF, platelet-derived growth factor; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APMA, 4-aminophenyl mercuric acetate; TRITC, tetramethylrhodamine isothiocyanate; CREB, cAMP-response element-binding protein; TAA, thioacetamide; HYP, hydroxyproline; TIMP-1, tissue inhibitor of metalloproteinase-1; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase. and periportal or perivenular fibroblasts (7Reeves H.L. Friedman S.L. Front. Biosci. 2002; 7: d808-d826Crossref PubMed Scopus (424) Google Scholar, 8Knittel T. Kobold D. Piscaglia F. Saile B. Neubauer K. Mehde M. Timpl R. Ramadori G. Histochem. Cell Biol. 1999; 112: 387-401Crossref PubMed Scopus (162) Google Scholar, 9Cassiman D. Libbrecht L. Desmet V. Denef C. Roskams T. J. Hepatol. 2002; 36: 200-209Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). These myofibroblasts are the major target for antifibrotic therapies. Halofuginone (HAL), a semisynthetic quinazolinone alkaloid originally derived from the plant Dichroa febrifuga, has been used to prevent coccidiosis in poultry, when its antifibrotic properties were discovered accidentally, since skin tearing and loss of skin integrity were observed in broilers that received HAL in the diet (10Granot I. Bartov I. Plavnik I. Wax E. Hurwitz S. Pines M. Poult. Sci. 1991; 70: 1559-1563Crossref PubMed Scopus (66) Google Scholar). During the last decade HAL was investigated as an antifibrotic agent in various in vivo fibrosis models (reviewed in Ref. 11Pines M. Nagler A. Gen. Pharmacol. 1998; 30: 445-450Crossref PubMed Scopus (141) Google Scholar) and suggested to be a specific inhibitor of procollagen type I expression for various collagen-expressing cells (12Granot I. Halevy O. Hurwitz S. Pines M. Biochim. Biophys. Acta. 1993; 1156: 107-112Crossref PubMed Scopus (117) Google Scholar, 13Halevy O. Nagler A. Levi-Schaffer F. Genina O. Pines M. Biochem. Pharmacol. 1996; 52: 1057-1063Crossref PubMed Scopus (85) Google Scholar, 14McGaha T.L. Kodera T. Spiera H. Stan A.C. Pines M. Bona C.A. Arthritis Rheum. 2002; 46: 2748-2761Crossref PubMed Scopus (35) Google Scholar, 15Nagler A. Miao H.Q.A.H. Pines M. Genina O. Vlodavsky I. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 194-202Crossref PubMed Scopus (69) Google Scholar). More recently, HAL was shown to partly reverse established thioacetamide-induced rat liver fibrosis (16Bruck R. Genina O. Aeed H. Alexiev R. Nagler A. Avni Y. Pines M. Hepatology. 2001; 33: 379-386Crossref PubMed Scopus (138) Google Scholar). However, inhibition of procollagen I synthesis alone can hardly explain the ability of HAL to reverse pre-established fibrosis in rats. So far, there exist no experimental studies that explain the mode of action of HAL in hepatic fibrosis and, especially, toward the hepatic fibrogenic effector cells, i.e. activated HSCs and myofibroblasts. Here we present an extensive analysis of the in vitro antifibrotic potential of HAL, study the signal transduction pathways triggered by the drug in HSCs/myofibroblasts, and provide in vivo evidence of its pro-fibrolytic mode of action in a rat model of fibrosis reversal. We show that HAL is a prominent activator of fibrolytic matrix metalloproteinase (MMP)-3 and -13 in HSCs, a hitherto unique property for an antifibrotic agent, which is mediated by activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB(NFκB). HSC Isolation and Culture—CFSC-2G (17Greenwel P. Rubin J. Schwartz M. Hertzberg E.L. Rojkind M. Lab. Invest. 1993; 69: 210-216PubMed Google Scholar), HSC line obtained from cirrhotic rat liver (kind gift from Dr. M. Rojkind, Washington, D. C.) and culture-activated primary rat HSCs were seeded onto 12-, 24-, or 96-well plastic plates at a density 40,000 cells per ml at 95% air/5% CO2 in a humidified atmosphere. Cells were maintained in Dulbecco's modified Eagle's medium (10% FCS, 1% penicillin/streptomycin) and used for experiments at low (0.5% FCS) serum conditions after 12–24 h unless specified otherwise. HAL (kind gift of Dr. M. Pines, Rehovot, Israel) was added directly to the were from not otherwise. HSCs were from to a T. K. P. K. V. D. M. G. A. Hepatology. 1999; PubMed Scopus Google Scholar). the liver was with and type collagenase in Dulbecco's modified Eagle's medium for by with and at for and by a a and of at for Cell was by and was of HSC was by their stellate and at with cells, as by the ability to was after and after the Cells were used or after the not otherwise. cells were obtained by from primary HSC to Cell synthesis was an and culture-activated primary HSCs were seeded at a density of in 96-well plates in growth medium for h of in FCS, cells were by for h in the of concentrations of was added the last and its was an experiments were with in and at of cells were seeded at 40,000 in plates and maintained in growth medium for h by of in were with a Cell migration was by the of a made in a Cells were in plates and in medium for a was in the by a plastic to and medium was to the between the was with a the as a platelet-derived growth or and HAL at concentrations were and migration was the 12–24 per were in for and are as a of was as Y. E. P. M. D. J. Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. V. B. J. Y. D. S. Hepatology. PubMed Scopus Google Scholar, A. D. Y. R. V. M. D. Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar). was from the or liver and of was reverse reverse with of and of mRNA transcript levels were a the The was in a for and were the as in are as of to of and at and and at its with and at the with were at and and D. V. B. J. Y. D. S. Hepatology. PubMed Scopus Google Scholar). mRNA transcript levels were in as after to and used in in a protein production was by as B. N. D. P. Rojkind M. Am. J. Pathol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) in or in of cells with or HAL for from of rat as and from MMP-3 from and were by and of was by of after protein for were activated with 4-aminophenyl mercuric at for h to interstitial collagenase of type I collagen as of was with for h at and were to a 96-well by to the human was used as a of NFκB were seeded in in medium for and with HAL at for various of in at and for cells were in nuclear for and for were at for at The nuclear was in of MAPK activation for and at The was in nuclear by MAPK activation an from protein was by from in obtained after of cells were in modified Eagle's medium for h and with HAL for with and in for NFκB was by with the for by of was and were a inhibit NFκB nuclear C. C. Y. L. Hepatology. 1998; PubMed Scopus Google Scholar), the inhibitor was MAPK cells were with for with or with HAL for Cell were 1% and inhibitor for and at inhibit activation of specific inhibitors were (p38 and from and and were by and Cell of by was a onto and with to protein and were with in at with primary and for h with their were the were from The for was by the of was induced in by of in a for induction and were not in the and which received of as of cirrhotic were of and HAL and with HAL, was after the last and the HAL received halofuginone in by for the the received of for were and fibrosis were and of was from of liver as Y. E. P. M. D. J. Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar, B. H. M. D. Gastroenterology. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mRNA expression was by from from liver of as for tissue was in liver from of to the of of of were are as The of was the Halofuginone Cell and dose dependently inhibited proliferation as by with a inhibition at in and primary rat HSCs and of synthesis was with in The of HAL migration of HSCs, as in an in vitro was in the of HAL, with significant inhibition at migration was inhibited Cell as by and viability, as by were not by the of HAL added for h was reversible after of the drug Halofuginone of in cells HAL up-regulated MMP-3 and MMP-13 mRNA expression between 10- and and MMP-13 mRNA was at h after and to levels after h whereas MMP-3 mRNA at h In culture-activated HSCs MMP-13 mRNA was in and cells not whereas HAL induced MMP-3 mRNA at h to its in obtained by from primary HSCs were to be for MMP-13 HAL up-regulated MMP-3 and -13 mRNAs and respectively, after h of and of and an increase in MMP-3 and -13 protein respectively, with HAL and In cells and culture-activated HSCs, HAL procollagen α1(I) and MMP-2 2- and 3-fold, respectively, and h were obtained for myofibroblasts These by in fibroblasts and a rat HSC line for procollagen α1(I) (12Granot I. Halevy O. Hurwitz S. Pines M. Biochim. Biophys. Acta. 1993; 1156: 107-112Crossref PubMed Scopus (117) Google Scholar, R. Genina O. Aeed H. Alexiev R. Nagler A. Avni Y. Pines M. Hepatology. 2001; 33: 379-386Crossref PubMed Scopus (138) Google Scholar), and in cells for MMP-2 M. R. Nagler A. E. Pines M. N. A. Vlodavsky I. 1999; Google Scholar). Procollagen expression was suppressed as procollagen α1(I) whereas growth factor tissue growth and and transcript levels remained in cells and primary HSCs not Halofuginone of was in of obtained by from primary HAL of collagen type I by hepatic myofibroblasts in a and with a 2- to 3-fold increase by HAL after h and was the of our Halofuginone via of p38 MAPK and the mechanisms that to MMP-3 and MMP-13 induction by HAL, we activation of and NFκB. of cells with HAL alone by did not of as compared with However, of the cells with of for after with HAL for h induced a increase of p38 MAPK a of extracellular signal-regulated kinase as compared with cells, with alone of or was by HAL In HAL a increase of nuclear compared with as by cells to HAL nuclear of at h and at nuclear was by with the inhibitor was by of at MAPK and NFκB are involved in the induction of MMP-13 in of of the inhibitor and the inhibitor in to of MMP-13 induction by and Cells were with HAL for h after of inhibitors in Dulbecco's modified Eagle's and specific inhibition of by with inhibitors at as used in Cells were with for and as and p38 MAPK as a inhibition of activation of p38 MAPK and its target by in cells in the of and The of was after of with or fetal calf serum MMP-13 mRNA expression in cells with HAL at for h in of after a with concentrations of the specific p38 MAPK and and mRNA are of experiments in to the pathways are involved in the observed of fibrolytic expression by HAL, cells were with specific inhibitors of their of activation by HAL, inhibition of or by their specific inhibitors and did not affect MMP-13 mRNA which was used as for whereas inhibitors of p38 MAPK and of NFκB nuclear inhibitor induction of MMP-13 mRNA expression inhibition of and NFκB was and specific and Thus, inhibited activation of p38 and of its the experimental conditions as used for the MMP-13 MMP-13 expression was inhibited dose dependently by and that pathways are of collagenase induction by HAL in Liver via of induction by HAL in vitro is in cirrhosis was induced in with for by HAL for for advanced liver with a and increase in and hepatic HYP, and the of cirrhosis of there was a toward a hepatic to liver as was by an increase in liver However, hepatic remained no which was by tissue in liver no in collagen Treatment with HAL for after in a significant of and liver was by of which was a in a HAL in and hepatic collagen in with and its HAL for as compared to the of fibrosis HYP, as compared to the liver HYP, as compared to the as compared to the of fibrosis as compared to the in a the of procollagen TIMP-1, and MMP-13 mRNA were up-regulated and of procollagen α1(I) mRNA by whereas and MMP-13 were by In the as compared with the procollagen α1(I) expression remained and whereas MMP-3 and -13 mRNA were up-regulated and 2-fold, respectively, and was and study the effects of halofuginone (HAL) activated hepatic stellate cells (HSCs). HAL suppressed HSC proliferation and which was by of extracellular In HAL induced a of MMP-3 and -13 which in a 2- to 3-fold interstitial activity. MMP-3 and interstitial are to a in and potential of liver fibrosis T. M. S. K. T. M. A. K. I. J. Hepatol. 2000; 33: Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; Google Scholar, T. Mehde M. A. Saile B. Ramadori G. Histochem. Cell Biol. 2000; PubMed Scopus Google Scholar). the of interstitial with the expression of an of MMP-13 as hepatic interstitial was by of I and and MMP-2 MMP-2 is to to of collagen M.J. Liver 2001; PubMed Scopus Google Scholar), which as for and S.L. J. J. Biol. Full Text PDF PubMed Google Scholar, R. K. Iredale J.P. Biol. 2003; PubMed Scopus Google Scholar). MMP-2 is up-regulated by the profibrogenic growth in to interstitial and MMP-3 and which are by In MMP-2 is in HSC migration and proliferation M. Iredale J.P. M.J. Hepatology. 1999; 30: PubMed Scopus Google Scholar, E. K. L. Friedman S.L. J. 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These are in with our that a and of HSC and expression by we were to inhibit MMP-13 induction by specific inhibitors of p38 and NFκB, not of or PI3K, that the mode of action of HAL in HSCs is we did not experiments the of activation of p38 and NFκB, that p38 may be involved in HSC proliferation and migration inhibition induced by HAL, whereas NFκB is of p38 to So far, is the or extracellular or of is to our HAL is the agent with a fibrolytic in vivo and in its mode of action is from that of potential which via their are as to their antifibrotic been in fibrosis models that fibrosis to chronic which be in models with no or as rat cirrhosis D. Y. E. M. P. G. B. and in the Scholar) or in a model of fibrosis as was used in the present HAL was in vitro at low between and that with in vivo M. D. S. Nagler A. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) the be to at a low Thus, I may in the In conclusion, our show that HAL a of HSCs and myofibroblasts at submicromolar leading to significant in cirrhotic rat In we p38 MAPK and NFκB as signal activated by HAL that in that HAL is a promising agent for the of liver the antifibrotic of which be in combination with other that target profibrogenic We V. of for G. of for in of the and to and Dr. C. for in MAPK