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RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus

RHOBTB3 downregulates HIF1α expression. (A) Protein levels of HIF1α and HIF2α are elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and control (WT) MEFs were maintained in normoxia or exposed to hypoxia (1% O2) for 8 h. Cells were then lysed and analyzed by immunoblotting with antibodies indicated. (B) Re-introduction of RHOBTB3 into RHOBTB3−/− MEFs reduces the HIFα levels. RHOBTB3−/− MEFs stably expressing GFP or RHOBTB3 were maintained in normoxia or exposed to hypoxia for 8 h, and were then lysed and analyzed as described in A. (C) Knockdown of RHOBTB3 increases the protein levels of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting either GFP (control) or RHOBTB3. At 16 h post-infection, cells were exposed to hypoxia for different periods of time as indicated, and were then lysed and analyzed by immunoblotting with antibodies indicated. (D) Ectopic expression of RHOBTB3 in HEK293T cells downregulates HIF1α. HEK293T cells were transfected with pcDNA3.3-MYC-RHOBTB3 or pcDNA3.3-MYC vector as a control. At 16 h post-transfection, cells were exposed to hypoxia for the indicated periods of time and were then lysed, and the protein levels of HIF1α were analyzed. (E) RHOBTB3 does not affect the protein levels of HIF1β/ARNT. HEK293T cells were transfected with RHOBTB3. At 16 h post-transfection, cells were lysed and analyzed by immunoblotting with antibodies indicated. (F) RHOBTB3 has no effect on the mRNA levels of HIF1α or HIF2α under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or hypoxia, were homogenized in Trizol reagent, and total RNAs were purified, and were subjected to real-time PCR analysis for mRNA levels of HIF1α and HIF2α. Values are presented as mean ± SEM; n = 3 for each group; three replicate experiments. N.S., not significant. Statistical analysis was carried out by ANOVA followed by Tukey.
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fig1: RHOBTB3 downregulates HIF1α expression. (A) Protein levels of HIF1α and HIF2α are elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and control (WT) MEFs were maintained in normoxia or exposed to hypoxia (1% O2) for 8 h. Cells were then lysed and analyzed by immunoblotting with antibodies indicated. (B) Re-introduction of RHOBTB3 into RHOBTB3−/− MEFs reduces the HIFα levels. RHOBTB3−/− MEFs stably expressing GFP or RHOBTB3 were maintained in normoxia or exposed to hypoxia for 8 h, and were then lysed and analyzed as described in A. (C) Knockdown of RHOBTB3 increases the protein levels of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting either GFP (control) or RHOBTB3. At 16 h post-infection, cells were exposed to hypoxia for different periods of time as indicated, and were then lysed and analyzed by immunoblotting with antibodies indicated. (D) Ectopic expression of RHOBTB3 in HEK293T cells downregulates HIF1α. HEK293T cells were transfected with pcDNA3.3-MYC-RHOBTB3 or pcDNA3.3-MYC vector as a control. At 16 h post-transfection, cells were exposed to hypoxia for the indicated periods of time and were then lysed, and the protein levels of HIF1α were analyzed. (E) RHOBTB3 does not affect the protein levels of HIF1β/ARNT. HEK293T cells were transfected with RHOBTB3. At 16 h post-transfection, cells were lysed and analyzed by immunoblotting with antibodies indicated. (F) RHOBTB3 has no effect on the mRNA levels of HIF1α or HIF2α under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or hypoxia, were homogenized in Trizol reagent, and total RNAs were purified, and were subjected to real-time PCR analysis for mRNA levels of HIF1α and HIF2α. Values are presented as mean ± SEM; n = 3 for each group; three replicate experiments. N.S., not significant. Statistical analysis was carried out by ANOVA followed by Tukey.

Mentions: In an effort to study the regulation of HIFα, we identified RHOBTB3 as an interacting protein for VHL in a yeast two-hybrid screen (Supplementary information, Figure S1A). To explore the functional linkage, we first generated MEF cells from RHOBTB3−/− mice, and found that RHOBTB3 cells had significantly elevated levels of all three HIFα isoforms (HIF1α-3α) under normoxic and hypoxic conditions and after cobalt chloride (CoCl2) treatment compared with WT MEFs (Figure 1A and Supplementary information, Figure S1B and S1C), suggesting that RHOBTB3 exerts a negative effect on HIFα expression. Transcriptional activities of HIF, measured in an HRE-luciferase reporter activity assay, were also significantly increased in the RHOBTB3- MEFs (Supplementary information, Figure S1D). Consistently, reintroducing RHOBTB3 into RHOBTB3−/− MEFs restored the inhibition on both protein levels and transcriptional activities of HIFα (Figure 1B and Supplementary information, Figure S1D). We also knocked down RHOBTB3 in HEK293T cells, and detected an increase of HIF1α at the protein levels (Figure 1C). Conversely, ectopic expression of RHOBTB3 in HEK293T cells strongly reduced the protein levels and transcriptional activity of HIF1α (Figure 1D and Supplementary information, Figure S1E). Unlike HIF1α, the protein levels of HIF1β/ARNT were not changed by RHOBTB3 overexpression in HEK293T cells (Figure 1E). Compared with RHOBTB3, overexpression of RHOBTB1 or RHOBTB2 had no effect on the protein levels of HIF1α in HEK293T cells (Supplementary information, Figure S1F). Of note, there was no difference in the mRNA levels of HIF1α and HIF2α between WT and RHOBTB3−/− MEFs under both normoxic and hypoxic conditions (Figure 1F), indicating that RHOBTB3 selectively affects the protein levels of HIFα. Overall, these results suggest that RHOBTB3 strongly downregulates the basal protein levels of HIFα and its induction under hypoxia.


RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

RHOBTB3 downregulates HIF1α expression. (A) Protein levels of HIF1α and HIF2α are elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and control (WT) MEFs were maintained in normoxia or exposed to hypoxia (1% O2) for 8 h. Cells were then lysed and analyzed by immunoblotting with antibodies indicated. (B) Re-introduction of RHOBTB3 into RHOBTB3−/− MEFs reduces the HIFα levels. RHOBTB3−/− MEFs stably expressing GFP or RHOBTB3 were maintained in normoxia or exposed to hypoxia for 8 h, and were then lysed and analyzed as described in A. (C) Knockdown of RHOBTB3 increases the protein levels of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting either GFP (control) or RHOBTB3. At 16 h post-infection, cells were exposed to hypoxia for different periods of time as indicated, and were then lysed and analyzed by immunoblotting with antibodies indicated. (D) Ectopic expression of RHOBTB3 in HEK293T cells downregulates HIF1α. HEK293T cells were transfected with pcDNA3.3-MYC-RHOBTB3 or pcDNA3.3-MYC vector as a control. At 16 h post-transfection, cells were exposed to hypoxia for the indicated periods of time and were then lysed, and the protein levels of HIF1α were analyzed. (E) RHOBTB3 does not affect the protein levels of HIF1β/ARNT. HEK293T cells were transfected with RHOBTB3. At 16 h post-transfection, cells were lysed and analyzed by immunoblotting with antibodies indicated. (F) RHOBTB3 has no effect on the mRNA levels of HIF1α or HIF2α under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or hypoxia, were homogenized in Trizol reagent, and total RNAs were purified, and were subjected to real-time PCR analysis for mRNA levels of HIF1α and HIF2α. Values are presented as mean ± SEM; n = 3 for each group; three replicate experiments. N.S., not significant. Statistical analysis was carried out by ANOVA followed by Tukey.
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fig1: RHOBTB3 downregulates HIF1α expression. (A) Protein levels of HIF1α and HIF2α are elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and control (WT) MEFs were maintained in normoxia or exposed to hypoxia (1% O2) for 8 h. Cells were then lysed and analyzed by immunoblotting with antibodies indicated. (B) Re-introduction of RHOBTB3 into RHOBTB3−/− MEFs reduces the HIFα levels. RHOBTB3−/− MEFs stably expressing GFP or RHOBTB3 were maintained in normoxia or exposed to hypoxia for 8 h, and were then lysed and analyzed as described in A. (C) Knockdown of RHOBTB3 increases the protein levels of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting either GFP (control) or RHOBTB3. At 16 h post-infection, cells were exposed to hypoxia for different periods of time as indicated, and were then lysed and analyzed by immunoblotting with antibodies indicated. (D) Ectopic expression of RHOBTB3 in HEK293T cells downregulates HIF1α. HEK293T cells were transfected with pcDNA3.3-MYC-RHOBTB3 or pcDNA3.3-MYC vector as a control. At 16 h post-transfection, cells were exposed to hypoxia for the indicated periods of time and were then lysed, and the protein levels of HIF1α were analyzed. (E) RHOBTB3 does not affect the protein levels of HIF1β/ARNT. HEK293T cells were transfected with RHOBTB3. At 16 h post-transfection, cells were lysed and analyzed by immunoblotting with antibodies indicated. (F) RHOBTB3 has no effect on the mRNA levels of HIF1α or HIF2α under both normoxic and hypoxic conditions. RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or hypoxia, were homogenized in Trizol reagent, and total RNAs were purified, and were subjected to real-time PCR analysis for mRNA levels of HIF1α and HIF2α. Values are presented as mean ± SEM; n = 3 for each group; three replicate experiments. N.S., not significant. Statistical analysis was carried out by ANOVA followed by Tukey.
Mentions: In an effort to study the regulation of HIFα, we identified RHOBTB3 as an interacting protein for VHL in a yeast two-hybrid screen (Supplementary information, Figure S1A). To explore the functional linkage, we first generated MEF cells from RHOBTB3−/− mice, and found that RHOBTB3 cells had significantly elevated levels of all three HIFα isoforms (HIF1α-3α) under normoxic and hypoxic conditions and after cobalt chloride (CoCl2) treatment compared with WT MEFs (Figure 1A and Supplementary information, Figure S1B and S1C), suggesting that RHOBTB3 exerts a negative effect on HIFα expression. Transcriptional activities of HIF, measured in an HRE-luciferase reporter activity assay, were also significantly increased in the RHOBTB3- MEFs (Supplementary information, Figure S1D). Consistently, reintroducing RHOBTB3 into RHOBTB3−/− MEFs restored the inhibition on both protein levels and transcriptional activities of HIFα (Figure 1B and Supplementary information, Figure S1D). We also knocked down RHOBTB3 in HEK293T cells, and detected an increase of HIF1α at the protein levels (Figure 1C). Conversely, ectopic expression of RHOBTB3 in HEK293T cells strongly reduced the protein levels and transcriptional activity of HIF1α (Figure 1D and Supplementary information, Figure S1E). Unlike HIF1α, the protein levels of HIF1β/ARNT were not changed by RHOBTB3 overexpression in HEK293T cells (Figure 1E). Compared with RHOBTB3, overexpression of RHOBTB1 or RHOBTB2 had no effect on the protein levels of HIF1α in HEK293T cells (Supplementary information, Figure S1F). Of note, there was no difference in the mRNA levels of HIF1α and HIF2α between WT and RHOBTB3−/− MEFs under both normoxic and hypoxic conditions (Figure 1F), indicating that RHOBTB3 selectively affects the protein levels of HIFα. Overall, these results suggest that RHOBTB3 strongly downregulates the basal protein levels of HIFα and its induction under hypoxia.

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus