Tihomir Miralem

Small Interference RNA-mediated Gene Silencing of Human Biliverdin Reductase, but Not That of Heme Oxygenase-1, Attenuates Arsenite-mediated Induction of the Oxygenase and Increases Apoptosis in 293A Kidney Cells

BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4–5-fold) after treatment of 293A cells with 5 μm As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 μm As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation.

Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

Activation of the MEK/ERK/Elk-signaling cascade is a mechanism for relaying mitogenic and stress stimuli for gene activation. MEK1 is the proximate kinase for activation of ERK1/2, and nuclear targeting of ERK1/2 is obligatory for Elk1 transcriptional activity. Human biliverdin reductase (hBVR) is a recently described Ser/Thr/Tyr kinase in the MAPK insulin/insulin-like growth factor 1 (IGF1)-signaling cascade. Using 293A cells and in vitro experiments, we detail the formation of a ternary complex of MEK/ERK/hBVR, activation of MEK1 and ERK1/2 kinase activities by hBVR, and phosphorylation of hBVR by ERK1/2. hBVR is nearly as effective as IGF1 in activating ERK; intact hBVR ATP-binding domain is necessary for Elk1 activation, whereas protein–protein interaction is the basis for hBVR activation of MEK1 and ERK. The two MAPK docking consensus sequences present in hBVR, F162GFP and K275KRILHCLGL (C- and D-box, respectively), are ERK interactive sites; interaction at each site is critical for ERK/Elk1 activation. Transfection with mutant hBVR-P165 or peptides corresponding to the C- or D-box blocked activation of ERK by IGF1. Transfection with D-box mutant hBVR prevented the activation of ERK by wild-type protein and dramatically decreased Elk1 transcriptional activity. hBVR is a nuclear transporter of ERK; experiments with hBVR nuclear export signal (NES) and nuclear localization signal (NLS) mutants demonstrated its critical role in the nuclear localization of IGF-stimulated ERK for Elk1 activation. These findings, together with observations that si-hBVR blocked activation of ERK and Elk1 by IGF1 and prevented formation of ternary complex between MEK/ERK/hBVR, define the critical role of hBVR in ERK signaling and nuclear functions of the kinase.

Human Biliverdin Reductase, a Previously Unknown Activator of Protein Kinase C βII

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) βII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084–17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109–7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC βII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC βII, but not the reductase and PKC ζ, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC βII K371R mutant protein (“kinase-dead”) was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC βII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC βII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC βII nor PKC ζ activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR·PKC βII immunocomplex. The potential biological relevance of the hBVR activation of PKC βII was suggested by the finding that in cells transfected with the PKC βII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC βII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC βII underscores its potential function in propagation of signals relayed through PKCs.

Regulation of TNF-α-activated PKC-ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) ‐ζ, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC‐ζ autophosphorylation, stimulation by TNF‐α, as well as cytokine stimulation of NF‐κB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVRs docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC‐ζ. Two hBVR‐based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S→AorC→A derivatives, respectively, blocked PKC‐ζ stimulation by TNF‐α and its membrane translocation. The C‐terminal‐based peptide KY‐CCSRK296 (#3), enhanced PKC‐ζ stimulation by TNF‐α; for this, Lys296 was essential. In metabolically 32P‐labeled HEK293 cells transfected with hBVR or PKC‐ζ, TNF‐α increased hBVR phosphorylation. TNF‐α did not stimulate PKC‐ζ in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide‐binding loop (G17), S149,orS230; this was similar to the response of “kinase‐dead” PKC‐ζK281R. We suggest peptide #1 blocks PKC‐ζ‐docking site interaction, peptide #2 disrupts function of the PKC‐ζ C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC‐ζ activity and cellular response to cyto‐kines.— Lerner‐Marmarosh, N., Miralem, T., Gibbs, P. E. M., Maines, M. D. Regulation of TNF‐α‐activated PKC‐ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase. FASEB J. 21, 3949–3962 (2007)

Cadmium and calcium-dependent c-fos expression in mesangial cells.

Cadmium is a carcinogenic metal known to increase the expression of several protooncogenes in a variety of cells. although the underlying mechanisms are unknown. Renal mesangial cells are smooth muscle cells in which Ca2+ signaling pathways regulate the induction of c-fos through both cAMP-dependent and mitogen-activated protein kinase- (MAPK-) dependent pathways. We report that c-fos is induced in these cells by both protein kinase C- (PKC-) dependent (phorbol ester, platelet-derived growth factor), and independent (serum, ionomycin) mechanisms. In all cases, prevention of an increase in cytosolic [Ca2+] with the chelator BAPTA prevented this induction. CdCl2 (10 microM) caused an accumulation of c-fos mRNA over 30 min that was sustained for at least 8 h. Cycloheximide inhibits turnover of c-fos mRNA and shows a synergistic effect with Cd2+ on transcript levels. Together with a similar half life of the transcript whether accumulated in response Cd2+ or induced by phorbol ester, this suggests induction of c-fos by Cd2+ rather than an effect of Cd2+ on transcript stability. Cadmium increased MAPK activity by 5 min; this was sustained for at least 8 h, consistent with the time course of c-fos mRNA accumulation. The MAPK kinase inhibitor PD98059 caused a marked decrease in the induction of c-fos by Cd2+, but did not eliminate the phenomenon completely. Although Cd2+ has been reported to activate PKC in vitro, no effect was found on PKC activity in Cd2+ -treated cells, indicating the activation of MAPK by Cd2+ is through an unidentified PKC-independent pathway. We conclude that Cd2+ can cause a sustained induction of c-fos in part through sustained activation of MAPK, that contrasts with the transient activation of these species in response to physiological mitogenic stimuli.

Formation of ternary complex of human biliverdin reductase-protein kinase Cδ-ERK2 protein is essential for ERK2-mediated activation of Elk1 protein, nuclear factor-κB, and inducible nitric-oxidase synthase (iNOS).

Background: ERK2 activation by PKCδ relays cell growth signals. hBVR is a bridge/scaffold protein and nuclear transporter of ERK. Results: hBVR forms a ternary complex with PKCδ and ERK2; this requires specific hBVR sequences. Corresponding peptides inhibit PKCδ/ERK2 interaction. PKCδ/ERK-mediated transcriptional activation is hBVR-dependent. Conclusion: hBVR is essential for ERK2 activation by PKCδ and MEK1/2. Significance: hBVR-based peptides are useful in regulating PKCδ/ERK signaling. Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKCδ and MEK1/2. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase and intracellular scaffold/bridge/anchor, is a nuclear transporter of MEK1/2-stimulated ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E., and Maines, M. D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6870–6875). hBVR, PKCδ, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKCδ-ERK2 ternary complex that is essential for ERK2 signal transduction and activation of genes linked to cell proliferation and cancer. MEK1/2 and the protein phosphatase PP2A were also present in the complex. When cells were stimulated with insulin-like growth factor-1 (IGF-1), an increased interaction between hBVR and PKCδ was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKCδ; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKCδ binding; hBVR was required for the activation of PKCδ and its interaction with ERK2. The C-terminal phenylalanine residues of PKCδ (Phe660, Phe663, and Phe665) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKCδ-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(δ)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKCδ/ERK2 interaction. Phorbol ester- and TNF-α-dependent activation of the ERK-regulated transcription factors Elk1 and NF-κB and expression of the iNOS gene were suppressed by hBVR siRNA; those activities were rescued by hBVR. The findings reveal the direct input of hBVR in PKCδ/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.

Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF-α-activated NF-κB

hBVR is a Ser/Thr/Tyr kinase/scaffold protein/transcription factor/intracellular transporter of regulators. hBVR is an upstream activator of the insulin/IGF‐1/MAPK/PI3K signaling pathway, and of NF‐κB. As a reductase, it converts biliverdin to the antioxidant, bilirubin. hBVR gene has 8 exons; exon 1 is not translated. We report the characterization of hBVR promoter and its negative and positive regulation, respectively, by TNF‐α and hypoxia. The 5′ end of exon 1 was defined by primer extension analyses; deletion of an inhibitor sequence 350–425 bp upstream of this exon enhanced the promoter activity. One of two NF‐κB binding sites in the 836‐bp promoter was functional; the P65 subunit of NF‐κB and TNF‐α acted as inhibitors. On the basis of EMSA and ChIP assays, TNF‐α treatment increases binding of NF‐κB to its regulatory element. Overexpression of IκB increased hBVR mRNA. Biliverdin, but not bilirubin, was as effective as TNF‐α in inhibiting hBVR promoter activity. Only one of 4 hypoxia responsive elements (HREs) bound to HIF‐1α and ARNT expressed in HEK293A cells. An abasic site was introduced at the 3′ G of the HRE. This element bound HIF‐1 in the gel shift and in in‐cell luciferase assays. hBVR was detected in the nucleus at 1, 2, and 4 h after hypoxia (1% O2), at which times its kinase and reductase activities were increased. Because hypoxia positively influences hBVR promoter and phosphorylation and TNF‐α activated NF‐κB inhibits the promoter, while biliverdin inhibits both NF‐κB activity and hBVR promoter, we propose a regulatory mechanism for NF‐κB by hypoxia and TNF‐α centered on hBVR/biliverdin.—Gibbs, P. E. M., Miralem, T., Maines, M. D. Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF‐α‐activated NF‐κB. FASEB J. 24, 3239–3254 (2010). www.fasebj.org

Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity THE REDUCTASE REGULATES TUMOR NECROSIS FACTOR-α-NF-κB-DEPENDENT GPBP EXPRESSION

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity THE PEPTIDES ARE INHIBITORS OR SUBSTRATE FOR THE PROTEIN KINASE C

Background: hBVR reduces biliverdin to antioxidant bilirubin. PKCδ promotes tumorigenesis and apoptosis. Results: Complex formation between PKCδ and hBVR results in transactivation. hBVR-based peptides are identified as substrates or inhibitors of the PKC in vitro and in the cell. Biliverdin inhibits PKCδ. Conclusion: A regulatory loop links PKCδ and hBVR in cell signaling. Significance: hBVR-based peptides can be used to regulate PKCδ signaling. PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066–1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser230 and Ser237), flanking the ATP-binding motif (Ser21) and in PHPS sequence (Ser33) as targets of PKCδ. Ser21 and Ser230 were also phosphorylated in hBVR-based peptides. The Ser230-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.

Biliverdin reductase: a target for cancer therapy?

Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1, and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ, and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.