Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Leo E. Otterbein,David Gallo,Jawed Alam,Eva Csizmadia,Elzbieta Kaczmarek,Ardavan Akhavan,Fritz H. Bach,Beek Y. Chin,Brian S. Zuckerbraun,Jeffrey R. Scott,Catherine J. Baty,Barbara Wegiel

doi:10.1074/jbc.m109.027433

Abstract

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 p85alpha 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.

Highlights

Sine and serine/threonine kinase [3, 4] and as a transcription factor that binds promoters within Ap-1 sites [5]
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
Membrane-associated Biliverdin Reductase Is a Functional Surface Antigen in Macrophages—We employed several techniques, including Total Internal Reflective Fluorescence (TIRF) microscopy, immunoblotting, immunofluorescence, and flow cytometry (FACS) to demonstrate that Biliverdin reductase A (BVR) is expressed on the external plasma membrane

Summary

Cell Culture and Treatment

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 ␮g/ml gentamicin (Invitrogen). 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 Ͻ0.01%. LPS (Escherichia coli serotype 0127:B08, Sigma) was dissolved in PBS and used for treatment at concentrations ranging from 1 to 1000 ng/ml. Biliverdin Induces PI3K-Akt Signaling geranylgeranyl pyrophosphate (Sigma), and DL-␤-hydroxymyristic acid (Sigma) were dissolved in DMSO and used at concentrations of 1, 3, and 100 ␮M, respectively. LY294002 (Sigma; 10 ␮M) was used as a selective inhibitor of PI3K. Bone marrow-derived macrophages were isolated and cultured as previously described [13]. 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

Animal Treatment

Source of Antibodies

Cell Fractionations

Plasmids and Transfections

RESULTS

DISCUSSION