Jian Kang Chen

Cytochrome P450 Epoxygenase Metabolism of Arachidonic Acid Inhibits Apoptosis

ABSTRACT The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H2O2, etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase–Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation.

Epoxyeicosatrienoic Acids and Their Sulfonimide Derivatives Stimulate Tyrosine Phosphorylation and Induce Mitogenesis in Renal Epithelial Cells

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14,15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14,15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14,15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymidine incorporation. Moreover, immunoblotting indicated that 14,15-EET stimulated tyrosine phosphorylation of the specific pp60c- src substrate p120 and c-Src association with EGFR. 14,15-EET increased src kinase activity within 1 min. Our data indicate that EETs are potent mitogens for renal epithelial cells, and the mitogenic effects of the EETs are mediated, at least in part, by the activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade.

EGFR Signaling Promotes TGFβ-Dependent Renal Fibrosis

The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFβ signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFβ expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFβ expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFβ-mediated fibrogenesis. We conclude that TGFβ-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

Transfection of an Active Cytochrome P450 Arachidonic Acid Epoxygenase Indicates That 14,15-Epoxyeicosatrienoic Acid Functions as an Intracellular Second Messenger in Response to Epidermal Growth Factor

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241.82 ng/108 cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.

Role of Mammalian Target of Rapamycin Signaling in Compensatory Renal Hypertrophy

Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNX-induced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.

Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells

In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.

Importance of Functional EGF Receptors in Recovery from Acute Nephrotoxic Injury

Previous studies have demonstrated increased renal expression of EGF receptor (EGFR) and EGFR ligands in response to acute toxic or ischemic renal tubular injury and have indicated that exogenous administration of EGF accelerates recovery from such injury. However, no studies to date have proved definitively an essential role for EGFR-mediated responses in regeneration after tubule injury. To this end, waved-2 (wa-2) mice, which contain a point mutation in EGFR that reduces receptor tyrosine kinase activity by >90%, were studied. These mice have a mild phenotype (wavy coat, curly whiskers, and runted stature) and normally developed kidneys. Acute nephrotoxic injury was induced in wa-2 and wild-type mice with HgCl(2). One day after HgCl(2) injection, functional renal compromise was comparable in wild-type and wa-2 mice. However, the rates of recovery of serum blood urea nitrogen and creatinine levels were markedly slower in wa-2 mice. Histologic evidence of tubular injury also was more severe and persisted longer in wa-2 mice. Furthermore, their kidneys demonstrated reduced levels of DNA synthesis and increased TdT-mediated dUTP nick-end labeling staining. These studies indicate that functional EGFR activity is an essential component of the kidneys ability to recover from acute injury and that EGFR may regulate genes involved in growth, repair, and cell survival in the kidney.

Angiotensin II Induces Epithelial-to-Mesenchymal Transition in Renal Epithelial Cells through Reactive Oxygen Species/Src/Caveolin-Mediated Activation of an Epidermal Growth Factor Receptor–Extracellular Signal-Regulated Kinase Signaling Pathway

ABSTRACT Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFRptKO). Ischemia–reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFRptKO and control mice 24 h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFRptKO and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.

Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.