MAPK signaling up-regulates the activity of hypoxia-inducible factors by its effects on p300

Abstract

Hypoxia-inducible factors (HIF) are a family of heterodimeric transcriptional regulators that play pivotal roles in the regulation of cellular utilization of oxygen and glucose and are essential transcriptional regulators of angiogenesis in solid tumor and ischemic disorders. The transactivation activity of HIF complexes requires the recruitment of p300/CREB-binding protein (CBP) by HIF-1 alpha and HIF-2 alpha that undergo oxygen-dependent degradation. HIF activation in tumors is caused by several factors including mitogen-activated protein kinase (MAPK) signaling. Here we investigated the molecular basis for HIF activation by MAPK. We show that MAPK is required for the transactivation activity of HIF-1 alpha. Furthermore, inhibition of MAPK disrupts the HIF-p300 interaction and suppresses the transactivation activity of p300. Overexpression of MEK1, an upstream MAPK activator, stimulates the transactivation of both p300 and HIF-1 alpha. Interestingly, the C-terminal transactivation domain of HIF-1 alpha is not a direct substrate of MAPK, and HIF-1 alpha phosphorylation is not required for HIF-CAD/p300 interaction. Taken together, our data suggest that MAPK signaling facilitates HIF activation through p300/CBP.

Fig. 1. Role of MAPK signaling in endogenous HIF-1 activity

A, schematic representation of MAPK-signaling pathway and the sites for inhibitor action. B, model of the luciferase-reporter system. C and D, effects of genistein on HIF-1 activity. B1 cells were exposed to various doses of genistein, a tyrosine kinase inhibitor, and cultured in normoxia (C) or in a hypoxic work station with 2% O₂ (D) for 8 h. E and F, effects of PDx on HIF-1 activity. B1 cells were exposed to various doses of PDx, MAPK inhibitor, under normoxic or hypoxic conditions (2% O₂) for 8 h. RLU, relative light units.

Fig. 2. Role of MAPK signaling in the transactivation activity of HIF-1α

A and B, effects of PDx on transactivation activity of HIF-1α. HeLa cells were transfected with a plasmid expressing G4.H1α530–826 (2.5 μg) and the luciferase reporter pFR-luc (2.5 μg). Following transfection, the cells were treated with variable doses of PDx and incubated in normoxic (Nmx) on hypoxic (Hpx) conditions for 8 h before luciferase measurements. C and D, effects of PDx on protein levels of G4.H1α530–826 under normoxic and hypoxic conditions. HeLa cells cultured in 100-mm dishes were transfected with pG4.H1α530–826 (5 μg/dish). 24 h after the transfection, the cells were trypsinized, pooled, and evenly reseeded into fresh dishes and cultured for 12 h. The cells were treated with indicated doses of PDx and cultured under either normoxic on hypoxic (2% O₂) conditions for 8 h before harvest. RLU, relative light units.

Fig. 3. Correlation between MAPK phosphorylation and the transactivation activity of HIF-1α

A–F, effects of MAPK inhibitor on the transactivation activity of G4.H1α constructs. Each G4.H1α construct (3 μg) was co-transfected with the pFR-Luc reporter (2 μg), and 24 h later the cells were trypsinized and evenly divided into 12-well culture plates. Immediately before the reporter assays, the indicated doses of PDx were added, and the cells were exposed to either normoxic (A, C, and E) or hypoxic (2% O₂) conditions (B, D, and F) for 8 h before luciferase assays. G, in vitro MAPK assays. HIF-1α fragments were purified as GST fusion proteins and incubated with activated MAPK and [γ-³²P]ATP. The top panel shows the autoradiography, and the bottom panel shows the protein input. GST and myelin basic protein (MBP) were used as negative and positive control, respectively. RLU, relative light units.

Fig. 4. Effects of MAPK on p300 transactivation activity

A, effect of PDx and genistein on p300 transactivation activity. HeLa cells where co-transfected with a plasmid expressing G4.p300 and with the GAL4-luciferase reporter (pFR-luc). The transfected cells were divided evenly and exposed to Me₂SO (DMSO), PDx (75 μM), or genistein for 6 h in normoxia or hypoxia immediately before the luciferase assays. B, dose-dependent effect of PDx on p300 activity. HeLa cells were transfected as in B, and increasing amounts of PDx were used, as indicated (in μM). C, dose-dependent effects of PDx on the transactivation domains of p300TD. HeLa cells were transfected with plasmid G4.p300TD, expressing the transactivation domain of p300 (aa 1751–2414), and the cells were exposed to increasing concentrations of PDx. D, effects of overexpression of MEK1 on the transactivation activity of p300TD. Plasmid expressing MEK1 (2 μg) was co-transfected with pG4.p300TD (2 μg) and the luciferase-reporter (pFR-Luc; 2 μg). Luciferase activity was expressed as -fold increase over the co-transfected vector control. E, direct phosphorylation of p300TD in vitro by MAPK. GST.p300TD was expressed in and purified from BL21 cells and used in MAPK kinase assays. RLU, relative light units.

Fig. 5. MAPK signaling regulates the interaction between p300 and HIF-1αCAD

A, PDx treatments do not affect p300 levels. HeLa cells were incubated with PDx at 100 μM for 6 h, and the cell lysates were analyzed by immunoblotting using anti-p300 antibodies. B, GST pull-down assays. GST and GST.H1α530–826 were incubated with whole cell lysates (WCL) from HeLa that were untreated or treated with Me₂SO (DMSO) or PDx (100 μM) for 6 h. Precipitates were resolved in an 8% SDS-PAGE. Top panel, Coomassie Blue staining shows GST and GST.H1α530–826 proteins used in the pull-down assays. Bottom, Western blotting with anti-p300 monoclonal antibody demonstrating that GST.H1α fails to pull down p300 from PDx-treated cell lysates. C, effects of PDx treatment on the interaction between HIF-1αCAD and p300 in vivo. HeLa cells were transfected with G4.H1α786–826 and treated with or without PDx and cultured under either normoxic or hypoxic conditions for 6 h before harvest. Whole cell lysates (WCL; top three panels) were assayed for the levels of HIF-1α, phosphorylated ERK (ERKp), and G4.H1α786–826 by Western blot (WB). Immunoprecipitations (I.P.) were performed with anti-p300 and anti-Gal4 monoclonal antibody, and the immunoprecipitates were detected with an anti-Gal4 or an anti-p300 monoclonal antibody reciprocally (bottom two panels).

Fig. 6. Effect of MAPK signaling in hypoxia sensing

A, effect of hypoxia and hypoxia mimics on MAPK activity. HeLa cells were maintained in normoxia or exposed to hypoxia (2% O₂), Dfx (130 μM), or DOG (1 mM) for 6 h. 50 μg of whole cell lysates were applied to an SDS-PAGE. The total and activated ERK1/2 (pERK1/2) were measured by immunoblotting. B, effect of overexpression of MEK1 on HIF-1α activity and its responsiveness to hypoxia. 2 μg of pCMV.MEK1 or vector alone were co-transfected with 2 μg of pG4.H1α744–826 and 2 μg of pRR-Luc into HeLa cells in 100-mm culture dishes. 24 h after transfection, cells were trypsinized and evenly split into 12-well culture plates. 12 h after seeding, cells were switched to the indicated culture condition or exposed to the indicated chemicals for 6 h before analyses. Luciferase activity was normalized to co-transfected 0.5 μg of pCMV.β-gal. RLU, relative light units.