FHL2 interacts with HIF-1α. A, FLAG-HIF-1α co-immunoprecipitates with FHL2. 293T cells were co-transfected with expression vectors encoding FLAG-HIF-1α and either EV or vector encoding Myc epitope-tagged FHL2. At 24 h post-transfection, the cells were lysed and the lysates were immunoprecipitated with anti-Myc antibody. IP products and cell lysates were subjected to WB with anti-Myc or anti-FLAG antibody. B, endogenous FHL2 co-immunoprecipitates with endogenous HIF-1α. Hep3B cells were exposed to 1% O₂ for 6 h, lysed, and subjected to immunoprecipitation with anti-IgG or anti-FHL2 antibody. IP products were subjected to WB with anti-FHL2 or anti-HIF-1α antibody. C, the location of the basic helix-loop-helix domain (bHLH), Per-Arnt-Sim homology domain (PAS), O₂-dependent degradation domain (ODDD), inhibitory domain (ID), and C-TAD of HIF-1α are shown. Sites of prolyl (P) and asparaginyl (N) hydroxylation are indicated. D and E, FHL2 interacts specifically and directly with HIF-1α(429–608). Purified GST and GST fusion proteins containing the indicated amino acid residues of HIF-1α were incubated with in vitro transcribed, in vitro translated, and ³⁵S-labeled FHL2, captured with glutathione-Sepharose beads and analyzed by SDS-PAGE and autoradiography (top panels) or by WB with GST antibody (bottom panel).

FHL2 inhibits HIF-1 transcriptional activity in an O₂-independent manner. A, schematic of HIF reporter assay. B, FHL2 inhibits hypoxia-induced HIF activity. 293 cells were co-transfected with control Renilla luciferase reporter pSV-RL; HIF-1-dependent firefly luciferase reporter p2.1; and either EV or FHL2 expression vector. At 24 h post-transfection, the cells were exposed to either nonhypoxic (20% O₂) or hypoxic (1% O₂) conditions as indicated for another 24 h and then lysed, and the ratio of firefly:Renilla luciferase activity was determined. The results are normalized to cells transfected with EV under nonhypoxic conditions. C, effect of FHL2 on HIF-1α deletion mutants. 293 cells were co-transfected with pSV-RL; p2.1; vector encoding the indicated HIF-1α deletion mutant; and either EV or FHL2 vector. 48 h post-transfection, the cells were lysed, and the ratio of firefly:Renilla activity was measured. D, FHL2 does not affect HIF-1α protein levels. 293T cells were co-transfected with 1 μg of FLAG-HIF-1α expression vector and either 0 (−), 1 (+), or 2 (++) μg of Myc-FHL2 expression vector. 48 h post-transfection, the cells were lysed and probed with anti-FLAG, anti-V5, and anti-β-actin antibodies. E, the effect of FHL2 is independent of HIF-1α hydroxylation. 293 cells were co-transfected with pSV-RL; p2.1; vector encoding the wild-type FLAG-HIF-1α (WT), P402A/P564A double-mutant (DM) HIF-1α, or P402A/P564A/N803A triple mutant (TM) HIF-1α; and either EV or FHL2 vector. 24 h post-transfection, the cells were lysed to determine firefly:Renilla activity. F, binding of FHL2 deletion mutants to HIF-1α. Purified GST or GST-HIF-1α(429–608) fusion protein was incubated with in vitro transcribed, in vitro translated, and ³⁵S-labeled full-length FHL2 or FHL2 deletion construct as indicated. GST proteins were captured with glutathione-Sepharose beads and analyzed by SDS-PAGE and autoradiography or by WB with anti-GST antibody. G, full-length (WT) FHL2 is required to inhibit HIF-1 activity. 293 cells were co-transfected with pSV-RL; p2.1; and EV or vector encoding the indicated FHL2 deletion construct. 24 h post-transfection, the cells were exposed to 1% O₂ for an additional 24 h and then lysed, and the ratio of firefly:Renilla activity was determined. Total plasmid transfected was kept constant in all experiments. The results are shown as the means ± S.D. *, p < 0.01 compared with EV.

All three FHL proteins inhibit HIF-1 transcriptional activity. A, FHL2 is the only FHL family member that binds HIF-1α. 293T cells were co-transfected with FLAG-HIF-1α and either EV or vector encoding Myc-FHL1, Myc-FHL2, or Myc-FHL3. Lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody and probed for FLAG-HIF-1α and Myc-FHL by WB. B, FHL1–3 inhibit HIF-1 activity induced by hypoxia. 293 cells were co-transfected with p2.1, pSV-RL, and EV or vector encoding FHL1, FHL2, or FHL3. At 24 h post-transfection, cells were exposed to either 20% O₂ or 1% O₂ as indicated for another 24 h, then lysed and the ratio of firefly:Renilla activity was determined. (C) FHL1–3 inhibit HIF-1 activity induced by HIF-1α overexpression under nonhypoxic conditions. 293 cells were co-transfected with p2.1, pSV-RL, FLAG-HIF-1α vector or EV, and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were lysed, and the ratio of firefly:Renilla activity was determined. D, FHL1–3 inhibit HIF-1 in a hydroxylation-independent manner. 293 cells were co-transfected with pSV-RL; p2.1; vector encoding wild-type FLAG-HIF-1α (WT) or P402A/P564A/N803A triple mutant (TM); and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were lysed to determine firefly:Renilla activity. E, schematic of HIF-1α TAD assay. F, FHL1–3 inhibit HIF-1α TAD function. 293 cells were co-transfected with pSV-RL; pG5-E1b-Luc; either Gal4-HIF-1α(531–826) or Gal4-EV; and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were exposed to hypoxia for an additional 24 h. The cells were harvested, and the ratio of firefly:Renilla luciferase was determined. The amount of total plasmid transfected was kept constant in all experiments. The results are shown as the means ± S.D. G, HeLa cells were transfected with either empty shRNA vector (shEV) or shRNA targeting FHL1, FHL2, or FHL3. 48 h post-transfection RNA was isolated, and quantitative RT-PCR of the target mRNA was performed. H–J, HeLa cells were transfected with either shEV or shRNA targeting FHL1, FHL2, or FHL3. 24 h post-transfection the cells were exposed to either 20% O₂ or 1% O₂ for an additional 24 h. RNA was isolated, and quantitative RT-PCR was performed against GLUT1 (H), PDK1 (I), and VEGF (J). The results are shown as the means ± S.E. *, p < 0.01 compared with EV or shEV.

FHL1 disrupts binding of HIF-1α to p300/CBP co-activators. A, FHL1 interacts with p300 and CBP. 293T cells transfected with V5-FHL1 were lysed and immunoprecipitated with a control IgG or V5 antibody. Immunoprecipitates were probed with p300, CBP, and Myc antibodies. B, mammalian two-hybrid assay of HIF-1α TAD-p300 interaction. C, FHL1 inhibits interaction between the HIF-1α C-TAD and p300 CH1 domain. 293 cells were co-transfected with Gal4-HIF-1α(786–826) and VP16-CH1 expression vectors; pG5-E1b-Luc and pSV-RL reporter plasmids; and EV, FHL1, or FHL2 vector. 24 h post-transfection, the cells were exposed to an additional 24 h of hypoxic or nonhypoxic conditions. The cells were harvested, and the ratio of firefly:Renilla luciferase was determined. The results are shown as the means ± S.D. *, p < 0.01 compared with EV. D, FHL1 disrupts HIF-1α-p300 interaction. 293T cells were co-transfected with vector encoding FLAG-tagged double mutant (P402A/P564A) HIF-1α (DM) and either EV, FHL1, or FHL2 vector as indicated. 24 h post-transfection, the cells were left untreated or treated with 100 μm DFX for 6 h, then lysed, and subjected to IP with anti-FLAG antibodies. Immunoprecipitates and cell lysates were subjected to WB with antibodies against p300, FLAG, or Myc. E, FHL1 disrupts HIF-1α-CBP interaction. 293T cells were co-transfected with FLAG-HIF-1α(DM) vector and either EV or vector encoding FHL1 or FHL2 as indicated. 24 h post-transfection, the cells were left untreated or treated with 100 μm DFX for 6 h, then lysed, and subjected to IP with anti-FLAG antibodies. Immunoprecipitates and cell lysates were subjected to WB with antibodies against CBP, FLAG, V5, or Myc.

FHL proteins inhibit HIF-1 transcriptional activity in hepatocellular carcinoma cells. A, FHL1–3 inhibit HIF-1 activity in Hep3B cells subjected to hypoxia. Hep3B cells were co-transfected with pSV-RL; p2.1; FLAG-HIF-1α; and either EV or vector encoding FHL1, FHL2, or FHL3. 24 h post-transfection, cells were exposed to an additional 24 h under nonhypoxic or hypoxic conditions. Cells were then lysed and the ratio of firefly:Renilla activity was measured. B, FHL1–3 inhibit HIF-1α TAD function. Hep3B cells were co-transfected with pSV-RL; pG5-E1b-Luc; either Gal4-HIF-1α(531–826) or Gal4-EV; and either EV or FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were lysed to determine firefly:Renilla activity. C–E, FHL1–3 inhibit HIF activity at target gene promoters. Hep3B cells were co-transfected with the indicated luciferase promoter constructs and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection the cells were exposed to an additional 24 h under nonhypoxic or hypoxic conditions. The cells were then lysed, and luciferase activity was measured. F, FHL1–3 knockdown increases HIF-1 activity under hypoxic conditions. Hep3B cells were co-transfected with pSV-RL; p2.1; and either empty vector (shEV) or vector encoding shRNA against FHL1, FHL2, or FHL3. 24 h post-transfection the cells were exposed to an additional 24 h at 20% or 1% O₂. The cells were lysed, and the ratio of firefly:Renilla activity was determined. G, FHL1–3 knockdown increases HIF-1 activity under nonhypoxic conditions. Hep3B cells were co-transfected with pSV-RL; p2.1; FLAG-HIF-1α vector or EV; and either EV or vector encoding shRNA against FHL1, FHL2, or FHL3. 48 h post-transfection the cells were lysed, and the ratio of firefly:Renilla activity was determined. H, FHL1–3 knockdown up-regulates HIF-1α TAD function. Hep3B cells were co-transfected with pSV-RL; pG5-E1b-Luc; either Gal4-HIF-1α(531–826) or Gal4-EV; and either EV or vector encoding shRNA against FHL1, FHL2, or FHL3. 24 h post-transfection the cells were exposed to 24 h of hypoxia and then lysed to determine firefly:Renilla activity. The amount of total plasmid transfected was kept constant in all experiments. The results are shown as the means ± S.D. *, p < 0.01; #, p < 0.05 compared with EV or shEV.

FHL1 and FHL3, but not FHL2, inhibit HIF-2 transcriptional activity. A, FHL2 inhibits HIF-1, but not HIF-2 activity. 293 cells were co-transfected with p2.1, pSV-RL, HIF-1α or HIF-2α expression vector, and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were lysed, and the ratio of firefly:Renilla activity was determined. B-D, FHL1–3 inhibit HIF-1 activity at target gene promoters. Hep3B cells were co-transfected with the indicated luciferase reporter, HIF-1α expression vector, and either EV, FHL1, FHL2, or FHL3 expression vector. 24 h post-transfection cells were lysed, and luciferase activity measured. E–G, FHL1 and FHL3, but not FHL2, inhibit HIF-2 activity at target gene promoters. Hep3B cells were co-transfected with the indicated luciferase reporter, HIF-2α expression vector, and either EV, FHL1, FHL2, or FHL3 expression vector. 24 h post-transfection the cells were lysed, and luciferase activity was measured. The results are shown as the means ± S.D. *, p < 0.01; #, p < 0.05 compared with EV; ns, not significant. H and I, FHL1–3 have no effect on HIF-1α or HIF-2α protein levels. 293T cells were co-transfected with HIF-1α expression vector (H) or HIF-2α expression vector (I) and either EV or V5-tagged FHL1, FHL2, or FHL3 expression vector. 24 h post-transfection the cells were lysed and probed with anti-HIF-1α or anti-HIF-2α, anti-V5, or β-actin antibodies.

Increased FHL expression in hypoxic cells. A, HIF-1α inducers increase expression of FHL1–3 mRNA. Hep3B cells were exposed to 48 h of nonhypoxic (N) or hypoxic (H) conditions or were treated with DFX (100 μm) or cobalt chloride (100 μm). Afterward, RNA was isolated, and quantitative real time RT-PCR of FHL1, FHL2, and FHL3 mRNA was performed. B, HIF-1α inducers increase FHL1 protein levels. Hep3B cells were exposed to 48 h of nonhypoxic (N) or hypoxic conditions (H) or were treated with DFX (100 μm) or DMOG (1 mm). The cells were then lysed, and the lysates were probed with anti-FHL1, anti-HIF-1α, or anti-β-actin antibodies. C, digoxin blocks induction of VEGF by HIF-1. Hep3B cells were left untreated or treated with digoxin for 24 h and treated with DMOG or exposed to hypoxia as indicated. RNA was isolated, and RT-PCR of VEGF mRNA was performed. Fold induction over the untreated and normoxic control is shown. D and E, digoxin blocks induction of FHL family members by HIF-1. Hep3B cells were left untreated or treated with digoxin and then treated with DMOG or vehicle for 24 h (D) or exposed to nonhypoxic or hypoxic conditions for 24 h (E). RNA was isolated, and quantitative RT-PCR of FHL1, FHL2, and FHL3 was performed. Fold induction over the vehicle condition is shown in each case. F and G, Hep3B cells were stably transfected with empty shRNA vector or vector encoding shRNA against HIF-1α or both HIF-1α and HIF-2α. The cells were treated with DMOG or vehicle for 24 h, after which RNA was isolated and quantitative RT-PCR of VEGF (F) and FHL1, FHL2, and FHL3 (G) was performed. The results are shown as the means ± S.E. *, p < 0.01; #, p < 0.05 compared with untreated and nonhypoxic conditions. H, feedback regulation of HIF-1 activity by FHL1–3.