Hypoxia-inducible factor 1alpha protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O(2) availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. HIF-1 activity is controlled by the O(2)-regulated expression of the HIF-1alpha subunit. Under nonhypoxic conditions, HIF-1alpha protein is subject to ubiquitination and proteasomal degradation. Here we report that missense mutations and/or deletions involving several different regions of HIF-1alpha result in constitutive expression and transcriptional activity in nonhypoxic cells. We demonstrate that hypoxia results in decreased ubiquitination of HIF-1alpha and that missense mutations increase HIF-1alpha expression under nonhypoxic conditions by blocking ubiquitination.

Figure 1

Effect of C-terminal deletions on HIF-1α protein expression. 293 cells were transfected with expression vectors encoding HIF-1α constructs and 36 h later were exposed to 20% O₂ or 1% O₂ (hypoxia) for 4 h before nuclear extract preparation. Fifteen-microgram aliquots were fractionated by SDS/PAGE and subjected to immunoblot assay by using an anti-HIF-1α Ab. The constructs analyzed encoded the following residues of HIF-1α: (A) 1–826, 1–754, 1–681, 1–608; and (B) 1–729, 1–726, 1–703. Migration of full-length (FL) and deleted (Δ) forms of HIF-1α are indicated (Right).

Figure 2

Summary of the effect of internal deletions on the O₂-dependent regulation of HIF-1α expression. Expression of deletion constructs that were wild type (WT) or contained S551G/T552A missense mutations is summarized (Right). Regulation refers to the presence of increased protein expression under hypoxic as compared with nonhypoxic conditions. Experimental results are shown in Figs. 3 and 4.

Figure 3

Effect of missense mutations on expression of HIF-1α(1–391/512–826). (A) Immunoblot assay of nuclear extracts from 293 cells transfected with expression vectors encoding HIF-1α(1–391/512–826) (lanes 1–2), HIF-1α(1–391/512–826/C520 M) (lanes 3–4), or HIF-1α(1–391/521–826) (lanes 5–6). (B) Analysis of nuclear extracts from cells transfected with empty vector (EV; lanes 1–2) or HIF-1α constructs consisting of residues 1–826 (lanes 3–4) or 1–391/512–826 that was either wild type (lanes 5–6) or contained S515A/S517A (lanes 7–8) or Y519F (lanes 9–10) missense mutations. (C) Analysis of nuclear extracts from cells transfected with HIF-1α constructs consisting of residues 1–391/521–826 (lanes 1–2), 1–391/517–826 (lanes 3–4), or 1–391/512–826 that was either wild type (lanes 5–6) or contained P513G/P516G (lanes 7–8) or E512G/E518G (lanes 9–10) missense mutations. (D) Summary of the data. The amino acid (AA) sequence of HIF-1α residues 512–521 and the effect of missense mutations and deletions (−) on O₂-regulated expression are shown.

Figure 4

Effect of internal deletions and missense mutations on HIF-1α expression. (A) Expression vectors encoding HIF-1α deletion constructs, consisting of residues 1–391 fused to 429–826, 494–826, or 508–826, either wild type (WT) or containing the S551G/T552A missense mutations (MUT), were transfected into 293 cells and exposed to 20% or 1% O₂ for 4 h. Nuclear extracts were prepared, and 15-μg aliquots were subjected to immunoblot assay, which detected both endogenous full-length (FL) and recombinant deleted (Δ) HIF-1α. The expression of the deleted HIF-1α proteins was quantified by densitometry. (B) Cells were cotransfected with pSVβgal and vectors encoding HIF-1α(1–391/512–826) (lanes 1–2), His₆-HIF-1α(1–391/512–826) (lanes 3–4), His₆-HIF-1α(1–391/512–826/S551G/T552A) (lanes 5–6), or His₆-HIF-1α(1–391/512–826/S551D/T552D) (lanes 7–8). Transfected cells were exposed to 20% or 1% O₂ for 4 h, and His-tagged proteins were purified from lysates by metal-affinity resin binding. Aliquots of purified protein (normalized to β-gal expression) were subjected to immunoblot assay by using an anti-HIF-1α polyclonal Ab, and protein expression was quantified by densitometry.

Figure 5

Effect of deletion or missense mutations on HIF-1α-mediated reporter gene transcription. Hep3B cells were transfected with 5 μg of pSVβgal, 10 μg of p2.1 (containing a hypoxia response element upstream of an SV40 promoter-luciferase reporter gene), and 125 ng of expression vectors encoding either no protein (EV), full-length HIF-1α (FL), full-length HIF-1α with S551G/T552A missense mutations (MIS), or HIF-1α(1–391/521–826) (DEL). Transfected cells were incubated under 20% O₂ for 24 h. The luciferase:β-gal activity ratio was determined and normalized to the value obtained from cells transfected with empty vector (EV) to obtain the relative luciferase activity. Results shown represent the mean and standard error for three plates of transfected cells.

Figure 6

Expression of GAL4/HIF-1α constructs. COS cells were transfected with pSVβgal and expression vectors encoding the GAL4 DNA-binding domain (DBD) and either no additional residues (lanes 1–2) or HIF-1α amino acid sequences 429–608 that were either wild type (lanes 3–4) or contained the S551G/T552A mutations (lanes 5–6). Transfected cells were exposed to 20% or 1% O₂ for 4 h. Lysates were prepared and 60-μg aliquots were fractionated by SDS/PAGE and subjected to immunoblot assays by using an Ab against endogenous HIF-1α (Top) or the GAL4 DBD (Middle). The isolated GAL4 DBD (Middle, lanes 1–2) migrated faster than the GAL4/HIF-1α fusion proteins and thus is not within the portion of the immunoblot displayed. Expression of GAL4/HIF-1α fusion proteins was quantified by densitometry (Bottom).

Figure 7

Analysis of HIF-1α ubiquitination. (A) 293 cells were cotransfected with expression vectors encoding HIF-1α and either no protein (lanes 1–2) or His₆-ubiquitin (lanes 3–4). Cells were exposed to 20% or 1% O₂ for 4 h, and lysates were prepared. Two hundred-microgram aliquots of total protein were incubated with nickel resin and washed with buffer containing 75 mM imidazole. Aliquots of unpurified (Top) and nickel resin-purified (Bottom) lysates were subjected to SDS/PAGE and immunoblot assay by using an anti-HIF-1α polyclonal Ab. (B) Levels of total and ubiquitinated HIF-1α in lanes 3–4 were quantified by densitometry. The ratio of ubiquitinated/total HIF-1α was determined and normalized to the result obtained for the lysates analyzed in lane 4 to yield the relative HIF-1α ubiquitination. (C) Cells were cotransfected with: pSVβgal; expression vector encoding HIF-1α(1–391/512–826) that was either wild type (WT; lanes 1–3) or contained the S551G/T552A missense mutations (M; lane 4); and vector encoding either no protein (lanes 1–2) or His₆-ubiquitin (lanes 3–4). Cells were exposed to 20% or 1% O₂ for 4 h, and lysates were prepared. Aliquots of total protein (normalized to β-gal) were incubated with nickel resin and washed with RIPA buffer (0.15 mM NaCl/0.05 mM Tris⋅HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS) containing 75 mM imidazole. Aliquots of unpurified (Top) or resin-purified (Bottom) lysates were subjected to immunoblot assay by using an anti-HIF-1α polyclonal Ab. (D) Levels of total and ubiquitinated wild-type and mutant HIF-1α (1–391/512–826) in lanes 3–4 were quantified by densitometry. The ratio of ubiquitinated/total HIF-1α was determined and normalized to the result obtained for the lysate analyzed in lane 4 to yield the relative HIF-1α ubiquitination.