Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt
Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85α subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)surf modulates the inflammatory response. Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85α subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)surf modulates the inflammatory response. Biliverdin reductase (BVR) 2The abbreviations used are: BVRbiliverdin reductaseDMSOdimethyl sulfoxideLPSlipopolysaccharidePI3Kphosphatidylinositol 3-kinaseILinterleukinHEKhuman embryonic kidneyPBSphosphate-buffered salinesiRNAsmall interfering RNARNAiinterfering RNAAdadenovirusTIRFtotal internal reflective fluorescenceBSAbovine serum albuminFITCfluorescein isothiocyanateBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMES4-morpholineethanesulfonic acidELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sorterBRbilirubinWTwild type. mediates the rapid conversion of biliverdin to bilirubin (1McDonagh A.F. Nat. Struct. Biol. 2001; 8: 198-200Crossref PubMed Scopus (107) Google Scholar, 2Doré S. Takahashi M. Ferris C.D. Zakhary R. Hester L.D. Guastella D. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 2445-2450Crossref PubMed Scopus (623) Google Scholar). BVR also functions as a dual tyrosine and serine/threonine kinase (3Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7109-7114Crossref PubMed Scopus (122) Google Scholar) and as a transcription factor that binds promoters within Ap-1 sites (5Ahmad Z. Salim M. Maines M.D. J. Biol. Chem. 2002; 277: 9226-9232Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Biliverdin and bilirubin both possess potent cytoprotective properties in a variety of animal models (6Sarady-Andrews J.K. Liu F. Gallo D. Nakao A. Overhaus M. Ollinger R. Choi A.M. Otterbein L.E. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1131-1137Crossref PubMed Scopus (171) Google Scholar, 7Baranano D.E. Rao M. Ferris C.D. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16093-16098Crossref PubMed Scopus (888) Google Scholar) including those for ischemia/reperfusion injury following small bowel or liver transplantation (8Nakao A. Otterbein L.E. Overhaus M. Sarady J.K. Tsung A. Kimizuka K. Nalesnik M.A. Kaizu T. Uchiyama T. Liu F. Murase N. Bauer A.J. Bach F.H. Gastroenterology. 2004; 127: 595-606Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9Fondevila C. Katori M. Lassman C. Carmody I. Busuttil R.W. Bach F.H. Kupiec-Weglinski J.W. Transplant. Proc. 2003; 35: 1798-1799Crossref PubMed Scopus (26) Google Scholar, 10Fondevila C. Shen X.D. Tsuchiyashi S. Yamashita K. Csizmadia E. Lassman C. Busuttil R.W. Kupiec-Weglinski J.W. Bach F.H. Hepatology. 2004; 40: 1333-1341Crossref PubMed Scopus (145) Google Scholar), vascular injury (11Ollinger R. Bilban M. Erat A. Froio A. McDaid J. Tyagi S. Csizmadia E. Graça-Souza A.V. Liloia A. Soares M.P. Otterbein L.E. Usheva A. Yamashita K. Bach F.H. Circulation. 2005; 112: 1030-1039Crossref PubMed Scopus (212) Google Scholar), and endotoxic shock (6Sarady-Andrews J.K. Liu F. Gallo D. Nakao A. Overhaus M. Ollinger R. Choi A.M. Otterbein L.E. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1131-1137Crossref PubMed Scopus (171) Google Scholar, 12Wang W.W. Smith D.L. Zucker S.D. Hepatology. 2004; 40: 424-433Crossref PubMed Scopus (186) Google Scholar). The mechanisms underlying these effects are still poorly understood and to date have not been linked to BVR activity per se, but attributed rather to the antioxidant power of biliverdin and bilirubin. biliverdin reductase dimethyl sulfoxide lipopolysaccharide phosphatidylinositol 3-kinase interleukin human embryonic kidney phosphate-buffered saline small interfering RNA interfering RNA adenovirus total internal reflective fluorescence bovine serum albumin fluorescein isothiocyanate 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 4-morpholineethanesulfonic acid enzyme-linked immunosorbent assay fluorescence-activated cell sorter bilirubin wild type. The concept that BVR may be an ideal docking protein for SH-2 domains (4Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7109-7114Crossref PubMed Scopus (122) Google Scholar) combined with the known anti-inflammatory effects of the bile pigments and rapid conversion of BV to BR in vivo, led us to hypothesize that BVR is expressed on the membrane and through its kinase activity regulates Akt signaling via recruitment of phosphatidylinositol 3-kinase (PI3K). We present novel findings that the ability of biliverdin to prevent LPS-induced morbidity and mortality most likely requires BVR surface expression for specific activation of PI3K-Akt and IL-10. We suggest that this is the major pathway by which biliverdin inhibits the inflammatory response and that these molecular events are natural protective mechanisms involved in the innate immune response to bacterial endotoxin that enables homeostasis. The mouse macrophage cell line, RAW 264.7 (RAW), and HEK cells were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 50 μg/ml gentamicin (Invitrogen). For treatment, cells were seeded 24 h before experiment. Biliverdin (Frontier Scientific) was freshly prepared in DMSO (Sigma) and kept in the dark before and during treatment. Final concentration of DMSO in medium was <0.01%. LPS (Escherichia coli serotype 0127:B08, Sigma) was dissolved in PBS and used for treatment at concentrations ranging from 1 to 1000 ng/ml. GGTI-287, a selective inhibitor of geranylgeranyl transferase I (Calbiochem), geranylgeranyl pyrophosphate (Sigma), and dl-Β-hydroxymyristic acid (Sigma) were dissolved in DMSO and used at concentrations of 1, 3, and 100 μm, respectively. LY294002 (Sigma; 10 μm) was used as a selective inhibitor of PI3K. Bone marrow-derived macrophages were isolated and cultured as previously described (13Chin B.Y. Jiang G. Wegiel B. Wang H.J. Macdonald T. Zhang X.C. Gallo D. Cszimadia E. Bach F.H. Lee P.J. Otterbein L.E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5109-5114Crossref PubMed Scopus (179) Google Scholar). Adenovirus containing Cre recombinase was used at 50 multiplicity of infection on the third day of culture. Macrophages were treated and harvested on the fifth day of culture. C57BL6/J mice were purchased from The Jackson Laboratories. PI3K p85Β−/−/p85α loxp mice were kindly provided by Prof. Lewis Cantley (BIDMC, Harvard Medical School). All animals were held under pathogen-free conditions and the experiments were approved by the BIDMC Animal Care and Use Committee. Lung, liver, and spleen as well as blood samples were harvested for immunohistochemical and immunostaining analyses from control and LPS-injected mice (5 mg/kg, intraperitoneal) for 6 h. Biliverdin/bilirubin was freshly dissolved in 0.2 n NaOH, adjusted to a final pH of 7.4 with HCl, and kept in the dark. Mice were administered biliverdin or bilirubin (35 mg/kg, intraperitoneal) 16 h and again 2 h prior to LPS/d-galactosamine (250 μg/kg, intraperitoneal/750 mg/kg, intraperitoneal; E. coli serotype 0127:08, Sigma). Serum bilirubin levels were evaluated spectrophotometrically (Sigma Kit), according to the manufacturer’s protocol in a separate group of non-LPS-treated mice. For adenovirus experiments, mice were administered either Ad-BVR-siRNA or Ad-Y5 (2 × 109 plaque-forming units intraperitoneally) 5 days prior to LPS/d-Gal. Liver, lung, and spleen tissue samples were embedded in freezing medium and stored at −80 °C. Five-μm sections were fixed in cold acetone and embedded in paraffin followed by immunohistochemistry using fluorescently tagged or horseradish peroxidase-tagged secondary antibody as previously described (10Fondevila C. Shen X.D. Tsuchiyashi S. Yamashita K. Csizmadia E. Lassman C. Busuttil R.W. Kupiec-Weglinski J.W. Bach F.H. Hepatology. 2004; 40: 1333-1341Crossref PubMed Scopus (145) Google Scholar). RAW cells were seeded on glass (Fisher) and treated with 10–100 ng/ml LPS for various time points as described. For detection of surface antigens, cells were rinsed in PBS and blocked with 0.5% BSA (bovine serum albumin, RIA grade, Sigma) in PBS for 1 h followed by overnight incubation with primary antibodies at 4 °C. Staining of intracellular antigens was performed by membrane permeabilizers with Triton X-100 or methanol, followed by rinsing with PBS and blocking with 0.5% BSA in PBS. Fluorescent-labeled secondary antibodies were applied for 1 h at room temperature. Finally, cells were washed, fixed with 0.25% paraformaldehyde, and covered with 4′,6-diamidino-2-phenylindole (+/−) mounting medium. Fluorescence was with a at or For of surface antigens, cells were × for 5 and in PBS. were blocked in 0.5% BSA in PBS for followed by incubation with primary cells were with blocking and in fluorescently antibodies and in the dark at room for followed by with PBS. were by were performed on cells and as for with cells were seeded on the glass and treatment, with PBS. were fixed with for followed by with Triton X-100 for were with 5 of PBS and blocked for with cells were washed, with for and with and with a The following antibodies were used for analyses where reductase mouse mouse and mouse p85α For fluorescence fluorescein and were and membrane cell were isolated following the manufacturer’s protocol of the cells were rinsed in in and on for 1 h. total membrane cell or liver was at × for 10 The was and at × for plasma membrane the was in a followed by 5 incubation on The was with the by at × for 5 The was and in 5 of for 5 on The of the plasma membrane protein was by for 10 × were to analyses by and for biliverdin reductase activity assay as previously described Maines M.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was applied for of in and followed by to and The following were used with the and 1 and 4 were used to a of and in 2 and a of the BVR with in the with 1 and and were by and The and were as previously described and A. D. M. T. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). was as a the and sites of and the was using the following C. was the and sites of using the following and Akt and control were a from RAW cells were using or according to the HEK cells were with according to the manufacturer’s by was and and control were purchased from was used for with 5 of in medium according to the manufacturer’s experiments with 2 days of incubation was BVR protein was 100 h as previously described B. A. Z. J. PubMed Scopus Google Scholar). tissue samples were in tissue (250 5 Triton 10 pH containing the inhibitor were by a in 50 pH 1 pH 1 and the inhibitor were for at × at 4 and were concentration was using the of protein was on in (Invitrogen). For of protein in pH 50 2 pH 10 and the inhibitor were with antibodies and of and for h at 4 °C. The were with and 5 in The were to followed by to membrane The were blocked with with primary antibodies followed by horseradish secondary antibodies at a of and using or followed by to the IL-10 was in medium using according to the manufacturer’s are as the S.D. was performed by of We including and to that BVR is expressed on the external plasma cell of BVR activity rapid conversion of biliverdin to bilirubin at which was lost in the of and us to hypothesize that BVR may be a membrane protein with an present on the external of the with BVR and the antibodies the of surface BVR with the well surface in RAW 264.7 macrophages BVR and cells were by with The expression of BVR by LPS on the cell surface was via which specific detection of expressed We the effects of LPS on BVR expression in macrophages to potentially the anti-inflammatory of biliverdin that and we have (6Sarady-Andrews J.K. Liu F. Gallo D. Nakao A. Overhaus M. Ollinger R. Choi A.M. Otterbein L.E. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1131-1137Crossref PubMed Scopus (171) Google Scholar). was a of BVR on the cell surface as by of RAW cells LPS in a and increase in BVR surface expression as as treatment with activation of BVR and LPS on the of total BVR not it We the increase in membrane BVR LPS treatment by the of membrane in macrophages was specific for membrane expression as in BVR expression in total was our in were in vivo, we performed immunohistochemical for BVR in the of mice treated with Staining of BVR in macrophages 6 h LPS with surface expression by with antibodies to BVR and the macrophage Finally, to BVR membrane and we performed experiments with membrane of biliverdin to cells in rapid of bilirubin with an increase in at at 1 h from to activity in the an increase from to in membrane Collectively, the in 1, 2 BVR on the external of the plasma A of BVR using the B. Sci. PubMed Scopus Google Scholar) a acid The of BVR specific sites for geranylgeranyl transferase which in to sites may be for recruitment and of BVR to the membrane 3, A and of to to the plasma membrane T. Biol. 2005; PubMed Scopus Google Scholar). Inhibition of or LPS-induced cell surface BVR expression of in of LPS-induced BVR surface expression that BVR is from the to the cell membrane by protein and The tyrosine phosphorylation is also present in the BVR as previously described (4Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7109-7114Crossref PubMed Scopus (122) Google Scholar) the in not internal and external BVR the in 1, 2 the concept that BVR is to the external plasma our that surface BVR with a downstream kinase to the membrane via its tyrosine phosphorylation we a BVR and the p85α subunit of PI3K in RAW The p85α subunit of PI3K with BVR in RAW cells the total BVR or p85α expression in the biliverdin treatment, BVR is on tyrosine and is of p85α to BVR of the tyrosine that is for the kinase activity or in a of BVR with p85α in response to BV The within BVR to be the for the p85α subunit of PI3K to of Akt. of BVR or either on BVR or expression that Akt is downstream of we the effects of biliverdin on Akt BV a and phosphorylation of Akt in RAW cells A and and in marrow-derived macrophages the of BV-induced activation of Akt is dependent on a we used and The of BV on Akt phosphorylation is blocked by a selective inhibitor of PI3K We that the of biliverdin is by the p85α subunit of PI3K but not the subunit by marrow-derived macrophages from and loxp p85α PI3K mice. The of p85α in an of BV to BV was to Akt in macrophages in wild cells was phosphorylation of Akt expression of of a BVR blocked Akt phosphorylation not and the activation of Akt by we BVR on BV-induced downstream We phosphorylation of protein as of Akt which was by of PI3K not of a in a in the ability of BVR to biliverdin to bilirubin in the of p85α to PI3K to BVR and of phosphorylation in cells treated with BV that the activity of BVR is for the of We also an BVR which is but the of the for BV and is expression of this we of Akt BV signaling we have shown that BV IL-10 expression in macrophages and in vivo in we evaluated a Akt signaling and production of anti-inflammatory of BVR 10% led to IL-10 production in RAW macrophages the BVR blocked BV-induced signaling through PI3K-Akt to increase IL-10 of a Akt of IL-10 LPS rapid of and biliverdin as a for BVR as is the of BVR in IL-10 we or BVR of BVR enhanced LPS-induced production of IL-10 of BVR or Akt with or led to of IL-10 production and suggest that BV in part via a pathway to its anti-inflammatory effects in response to LPS through expression of IL-10 and for in to its antioxidant The regulation of IL-10 expression by is not and we that IL-10 regulation is part of the mechanism by which BV modulates the inflammatory response to in the animal models described We evaluated the in vivo conversion of biliverdin to bilirubin and the in a of endotoxic Biliverdin as a to mice was to a concentration of bilirubin of 50 7.4 at 5 time from a of 6 at 5 and to by 6 h not We also the ability of biliverdin to prevent acute following a of LPS or mg/kg, intraperitoneal) to acute liver One group of mice biliverdin (35 mg/kg, intraperitoneal) 16 and 2 h prior to mice or saline in both the with LPS all mice were by 24 of mice We our with treatment and in the group the by biliverdin the of we blocked BVR with an adenovirus on the D.E. Rao M. Ferris C.D. Snyder S.H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16093-16098Crossref PubMed Scopus (888) Google Scholar). of mice were administered 2 × 109 plaque-forming of either Ad-BVR-siRNA or Ad-Y5 control in 5 the time expression is and in and specific expression of the in the liver, we administered in the and of to serum was and the were harvested and for treated with BV or control in as by serum levels of BV, but not bilirubin (BR) at the blocked in those animals in which BVR was blocked with expressed serum levels with BVR was by in liver our in that the by BV requires the activity of We describe a novel that the that converts biliverdin to is a surface protein that initiates a signaling cascade the of biliverdin activation of and downstream production of IL-10. We suggest that the anti-inflammatory effects of previously attributed to the antioxidant properties of biliverdin and are in part via this novel mechanism (6Sarady-Andrews J.K. Liu F. Gallo D. Nakao A. Overhaus M. Ollinger R. Choi A.M. Otterbein L.E. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1131-1137Crossref PubMed Scopus (171) Google Scholar, 12Wang W.W. Smith D.L. Zucker S.D. Hepatology. 2004; 40: 424-433Crossref PubMed Scopus (186) Google Scholar). We that BVR functions in part as a signaling protein that tyrosine kinase and as is involved in regulation of the innate immune response. We this membrane as BVR been with to functions other biliverdin to bilirubin. been shown by that BVR as a kinase (3Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7109-7114Crossref PubMed Scopus (122) Google Scholar) and as a transcription factor involved in the regulation of (5Ahmad Z. Salim M. Maines M.D. J. Biol. Chem. 2002; 277: 9226-9232Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). BVR is present in various of the cell including the cell membrane in cells in with M.D. N. J. 2001; Google Scholar, Wang F. Choi A.M. J. 2004; PubMed Scopus Google Scholar). are to date in the demonstrating the ability of biliverdin to the inflammatory response. The protective of from a of of the R. 2004; PubMed Scopus Google Scholar, L.E. Soares M.P. Yamashita K. Bach F.H. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the as a cytoprotective molecule, the by which it functions unclear. on and and of these have to on the mechanisms of as a (8Nakao A. Otterbein L.E. Overhaus M. Sarady J.K. Tsung A. Kimizuka K. Nalesnik M.A. Kaizu T. Uchiyama T. Liu F. Murase N. Bauer A.J. Bach F.H. Gastroenterology. 2004; 127: 595-606Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, Otterbein Liu F. Choi A.M. Bach F.H. Otterbein L.E. J. 2003; PubMed Scopus Google Scholar, G. S. Soares M. Yamashita K. E. G. Hepatology. 2004; 40: PubMed Scopus Google Scholar, Yamashita K. Csizmadia E. Bach F.H. 2005; PubMed Scopus Google Scholar). The of in BVR is with our findings the signaling cascade by The tyrosine of BVR present on the the of factor for the p85α subunit of PI3K. We suggest that biliverdin BVR phosphorylation of tyrosine which is the within the membrane that enables BVR to the p85α subunit of PI3K and to Akt. The regulation of this is and in part regulation to by the cell during and levels of biliverdin the BVR and in the is to be for biliverdin to bilirubin which in the external of the our using the of BVR this as important for conversion of biliverdin to bilirubin and for of signaling by The for the reduction of biliverdin may either from by membrane J. A. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) or from other as of Google Scholar). The not conversion of biliverdin to however, it the of the of and in the which likely the of with BVR as and by A. D. M. T. Nat. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar). the of biliverdin is as is the with BVR not the signaling to Akt and its downstream as by the suggest that conversion of BV to BR to a that is important in the of BVR may a as that with the BVR in a to be and to the to as a The expression of BVR on the surface is an important finding and a of the in our of this and for the cytoprotective in The of surface BVR a novel and cell signaling mechanism by which biliverdin as a anti-inflammatory effects other via its antioxidant we elucidate a signaling pathway by which biliverdin as a for BVR an anti-inflammatory in to its antioxidant we present in vivo findings a for BVR in the immune response to prevent injury and in response to We suggest that this is a major natural mechanism used by the for and to and homeostasis. We G. A. and Yamashita for with the animal experiments and for to the analyses and We the at the of the BIDMC for with