HAF is the mediator of a novel mechanism of HIF-1α degradation in pVHL-deficient cells. (A) Effect of HAF overexpression on HIF-1 transactivation in RCC4 cells. Cells were transfected with HAF or control DNA together with HRE-firefly luciferase reporter or pGL3 basic vector (HRE and Con, respectively) with pTK-Renilla as a normalization control. Dual-luciferase activity was measured, and the ratio of firefly/Renilla luciferase activity was determined in relative light units (RLU). The data shown are from a representative experiment performed in triplicate (± standard error). (B and C) Western blot showing the effect of F-HAF transfection on HIF-α levels in UMRC6 and RCC4 cells (B) and RCC4/VHL cells (C) ± 5 μM MG132 in normoxia (where applicable) and hypoxia. The blot probed with HAF antibody shows the overexpressed F-HAF band as a slightly shifted band above the endogenous HAF. (D) Effect of HAF overexpression on HIF-1 transactivation in RCC4/VHL cells. The experiment was performed as in panel A. (E) Effect of HAF (siHAF) or the nontargeting control (siCon) siRNA transfection on levels of HIF-α in UMRC6 and RCC4 cells in normoxia. Cells were transfected with 50 nM siRNA for 72 h and then subjected to Western blotting. Untransfected cells were treated with MG132 16 h prior to harvest to show HIF-1α levels detected under proteasomal inhibition. Similar results were obtained for cells in hypoxia. (F) Effect of HAF knockdown in panel E on HIF-1 transactivation in UMRC6 and RCC4 cells. Cells were cotransfected with siRNA and DNA as in panel A. In all studies, significance was determined using Student's t test comparing data points to the vector control. *, P < 0.05; **, P < 0.01.

HAF promotes the ubiquitination of HIF-1α and decreases HIF-1α half-life. (A) Effect of HAF overexpression on HIF-1α ubiquitination. HAF clones and vector control cells were transiently transfected with HA-ubiquitin (HA-Ub) and then incubated in hypoxia with MG132. (Ai) Western blot of whole-cell lysates used for IP. (Aii) IP of HIF-1α from the lysates in panel Ai probed with anti-HA showing levels of ubiquitinated HIF-1α. The arrow indicates the expected migration for unmodified HIF-1α. (B) Sequential IP to demonstrate specific ubiquitination of F-HAF. HT29 cells were transiently transfected with HA-Ub and F-HAF or vector, incubated in hypoxia with MG132, and then subjected to sequential IP as outlined in the schema. IB, immunoblot; NS, nonspecific. (C) Pulse-chase experiment showing the effects of HAF knockdown on HIF-1α half-life in RCC4 cells in normoxia. RCC4 cells were transiently transfected with HAF or control siRNA for 72 h, pulsed with [³⁵S]methionine/cysteine, and chased for 0 to 4 h. Immunoprecipitated HIF-1α was detected by autoradiography and quantified by densitometry, and the HIF-1α half-life (T_1/2) was calculated. (D) Effects of HAF overexpression on HIF-1α half-life. The vector control or HAF-overexpressing stable HT29 clone S2-8 was incubated in hypoxia for 6 h and then treated with 25 μg/ml cycloheximide (Chx), after which the decay of HIF-1α was monitored at the indicated times by Western blotting. HIF-1α intensity was quantified by densitometry and normalized to actin.

Characterization of HAF E3-ubiquitin ligase activity. (A) Western blot of in vitro assay showing E2-dependent autoubiquitination activity of HAF. The assay was performed using GST-HAF on glutathione-agarose beads, E1, ubiquitin (Ub), ATP, and a panel of recombinant E2s. After incubation, beads were washed and polyubiquitinated HAF was detected by SDS-PAGE using anti-Ub. (B) The in vitro autoubiquitination assay was performed as in panel A with UbcH5a and GST-HAF or GST-HAF^396-800. (C) The top portion shows that the HAF C terminus (140-amino-acid region depicted) contains only three cysteine residues, which are shown in boldface capital letters. Below is shown the in vitro autoubiquitination assay of GST-HAF^396-800 (wild type [WT]) and GST-HAF^396-800 C-A mutants with UbcH5a. (D) Mapping of the HAF E3 ligase domain. In vitro ubiquitination assays as in panel A were performed using GST-HAF C-terminal truncations, as indicated using UbcH5a. (E) In vitro ubiquitination assays as in panel D with the addition of GST-HIF-1α-V5 to GST-HAF C-terminal truncations. The presence of higher-molecular-weight polyubiquitinated GST-HIF-1α-V5 was detected using GST antibody. (F) Confirmation of HAF-mediated E2-dependent ubiquitination of HIF-1α. In vitro ubiquitination assays were performed as outlined in the schema, and then GST-HIF-1α-V5 was immunoprecipitated and analyzed by SDS-PAGE using anti-Ub.

The oxygen-independent HAF pathway complements the oxygen-dependent pVHL pathway to determine total levels of HIF-1α and is independent of the RACK1-elongin C pathway. (A) HAF siRNA increases HIF-1α levels in normoxia and hypoxia. Cells were transfected with a nontargeting siRNA control (Con) or two different HAF siRNA constructs (HAF1 and HAF2) for 48 h and then incubated in normoxia or hypoxia for a further 16 h and analyzed by Western blotting. (B) Comparison of the effects of HAF and pVHL knockdown on HIF-1α levels. PANC-1 or PC-3 cells were transfected with HAF (HAF1), pVHL, or Con siRNA and then treated as in panel A. Quantitations of HIF-1α intensities normalized to actin are depicted above the respective bands. The results are representative of at least two independent experiments. (C) Endogenous HAF regulates EGF-induced HIF-1α. Cells were transfected with siRNA as in panel A, serum starved for 24 h, and then induced with EGF for 7 h. (D and E) Effect of knockdown of elongin C (D) or RACK1 (E) on the HAF-mediated decrease in HIF-1α. PANC-1 cells were transfected with control, elongin C, or RACK1 siRNA as indicated and with F-HAF or the vector control. Seventy-two hours later, cells were placed into hypoxia for 16 h and analyzed by Western blotting. HIF-1α intensities normalized to actin are shown above the respective bands.

Effect of HAF knockdown or overexpression on HIF-1α levels and activity in HT29 cells. (A) Effect of HAF or control siRNA (siHAF or siCon, respectively) transfection on HIF-1α levels in normoxia and hypoxia in HT29 cells. (B) Effect of HAF knockdown on HIF-1 transactivation. Cells were cotransfected with siRNA and HRE-firefly luciferase vector (or pGL3 basic vector only) and pTK-Renilla vector as the control. Dual-luciferase activity was measured using the ratio of firefly/Renilla luciferase in relative light units (RLU). The data shown are from a representative experiment performed in triplicate (± standard error). (C) Effect of HAF knockdown on hypoxia-induced VEGF transcription. HT29 cells were transiently transfected with HAF siRNA and then incubated under normoxic or hypoxic conditions for 16 h. VEGF mRNA levels in hypoxia were determined by TaqMan quantitative RT-PCR and normalized to VEGF mRNA levels in normoxia. Data are the means of four independent experiments performed in triplicate, and bars represent ± standard error. **, P = 0.004. (D) Relative increase in HAF mRNA in the HAF-overexpressing stable clones compared to vector control cells determined by quantitative RT-PCR. The data shown are for cells in normoxia, with similar results obtained for cells in hypoxia. (E) Western blots showing the levels of HAF and HIF-1α in the HAF-overexpressing stable clones. (F) Hypoxia-induced VEGF secretion in parental, empty vector, and HAF clones. VEGF concentrations measured by ELISA were determined by subtracting VEGF concentrations in cells grown in normoxia from the corresponding VEGF concentration of cells grown in hypoxia. *, P < 0.1. (G) Western blot showing the effect of proteasome inhibition (5 μM MG132 prior to hypoxic incubation) on the HAF-associated decrease of HIF-1α levels in HAF clones S1-1 and S2-9. (H) Western blots of cell fractionation showing HAF localization in HT29 parental, vector, and HAF clones. “HAF (Long)” indicates increased exposure showing cytoplasmic HAF.

Characterization of the HAF-HIF-1α interaction. (A) Coimmunoprecipitation of HAF with HIF-1α. HT29 cells were cotransfected with F-HAF or empty vector and HIF-1α and incubated in hypoxia with MG132. Lysates were immunoprecipitated with either anti-FLAG or anti-HIF-1α antibodies and then probed for HIF-1α or FLAG, respectively. (B) Co-IP of endogenous HAF with HIF-1α from a panel of human tumor xenografts in vivo. Flash-frozen xenografts were lysed and immunoprecipitated with HIF-1α or control IgG1 antibodies as indicated and probed for HAF. Membranes were stripped and reprobed for HIF-1α. (C) Pull down of in vitro-transcribed/translated HIF-1α (Ci) and P402A P564A N803A HIF-1α (HIF-1α tm) (Cii) by GST-HAF but not GST alone. Recombinant GST-HAF or GST bound to glutathione-agarose beads was incubated with HIF-1α or HIF-1α tm, and then beads were pelleted and bound HIF-1α was detected with anti-HIF-1α antibody. (D) Mapping of the HIF-1α minimal binding domain of HAF. Pull-down assays were performed as in panel C using the indicated GST-HAF truncations and in vitro-transcribed/translated V5-HIF^296-603. Pull down of HIF-1α was detected by immunoblotting (IB) with anti-V5 antibody. Input of GST and GST-HAF truncations are indicated by Ponceau S staining. (E) Mapping of the HAF minimal binding domain of HIF-1α. Pull-down assays were performed as in panel D using GST-HAF^654-800 and HIF-1α-V5 truncations generated based on the known HIF-1α functional domains indicated. The results of the pull-down assays are summarized schematically. Representative blots confirming interaction between HAF^654-800 and HIF-1α^296-603 and HIF-1α^400-603 and HIF-1α^296-400 are depicted.

Effects of HAF overexpression on tumor growth. (A) In vivo growth curves of the HAF or vector control HT29 xenografts in nude mice. Values are the means of four mice per group (± standard error). (B) Photographs of representative tumors demonstrating the differences in vascularization between the HAF and vector clones. (C) Xenograft microvessel density determined via staining with anti-CD105 antibody (± standard error). (D) Western blot showing HIF-1α expression in HT29 xenografts. Samples depicted are from two representative tumor lysates derived from each HAF or vector clone. (E) Densitometric analyses of the Western blot shown in panel D normalized to actin (± standard deviation). (F) Transcription of HIF-1α and key HIF-1α-regulated genes VEGF, GLUT-1, and CA-IX (coding for VEGF, glucose transporter 1, and carbonic anhydrase IX, respectively) in HAF xenografts determined by quantitative RT-PCR normalized to values from pooled vector xenografts RNA (± standard error). *, P < 0.05; **, P < 0.01. (G) Co-IP of HAF with endogenous HIF-1α from xenograft lysates depicted in panel D. (H) In vitro growth curves of HAF clones in normoxia and hypoxia (n = 3 [± standard error]).

HAF and pVHL participate in two overlapping but distinct HIF-1α regulatory pathways. HAF regulates the basal levels of HIF-1α in an oxygen-independent pathway and degrades HIF-1α in normoxia and hypoxia, whereas pVHL maintains low HIF-1α levels only in normoxia in an oxygen-dependent pathway. Together the two parallel but complementary pathways determine the total levels of HIF-1α.