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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 is a negative regulator of the Warburg effect. (A) Knockout of RHOBTB3 elevates the expression of HK2, LDHA GLUT1 and PDK1. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 16 h. Cells were then lysed and the protein extracts were analyzed by immunoblotting with antibodies indicated. (B) RHOBTB3 deficiency leads to increased mRNA levels of GLUT1 and LDHA. Total RNAs from RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or exposed to hypoxia for 16 h, were purified, and analyzed by real-time PCR analysis for the expression levels of GLUT1 and LDHA. Values are presented as mean ± SEM, n = 3 for each group, three replicate experiments. ***P< 0.001 (ANOVA followed by Tukey). (C, D) RHOBTB3 decreases rates of glucose consumption (C) and lactate production (D). RHOBTB3−/− and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h, and glucose consumption rates (C) and lactate production rates (D) were measured. Values are presented as mean ± SEM, n = 3 for each group, ***P< 0.001 (ANOVA followed by Tukey). (E, F) RHOBTB3 and LIMD1 cooperatively decrease glucose consumption (E) and lactate production (F). HEK293T cells were infected with lentiviruses expressing siRNAs targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h and glucose consumption rates (E) and lactate production rates (F) were measured. Values are presented as mean ± SEM, n = 3 for each group, *P< 0.05, **P< 0.01, ***P< 0.001 (ANOVA followed by Tukey).
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fig6: RHOBTB3 is a negative regulator of the Warburg effect. (A) Knockout of RHOBTB3 elevates the expression of HK2, LDHA GLUT1 and PDK1. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 16 h. Cells were then lysed and the protein extracts were analyzed by immunoblotting with antibodies indicated. (B) RHOBTB3 deficiency leads to increased mRNA levels of GLUT1 and LDHA. Total RNAs from RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or exposed to hypoxia for 16 h, were purified, and analyzed by real-time PCR analysis for the expression levels of GLUT1 and LDHA. Values are presented as mean ± SEM, n = 3 for each group, three replicate experiments. ***P< 0.001 (ANOVA followed by Tukey). (C, D) RHOBTB3 decreases rates of glucose consumption (C) and lactate production (D). RHOBTB3−/− and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h, and glucose consumption rates (C) and lactate production rates (D) were measured. Values are presented as mean ± SEM, n = 3 for each group, ***P< 0.001 (ANOVA followed by Tukey). (E, F) RHOBTB3 and LIMD1 cooperatively decrease glucose consumption (E) and lactate production (F). HEK293T cells were infected with lentiviruses expressing siRNAs targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h and glucose consumption rates (E) and lactate production rates (F) were measured. Values are presented as mean ± SEM, n = 3 for each group, *P< 0.05, **P< 0.01, ***P< 0.001 (ANOVA followed by Tukey).

Mentions: We next examined whether RHOBTB3 could suppress the Warburg effect since it could significantly reduce HIFα levels in cells, and levels of HK2, LDHA, GLUT1 and PDK1, known stream targets of HIFs, were elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions (Figure 6A and 6B). The Warburg effect is characterized by high rates of glucose uptake and lactate production regardless of oxygen concentration30. We thus measured the glucose consumption and lactate production rates, and found that they were increased in RHOBTB3−/− MEFs in both normoxia and hypoxia (Figure 6C and 6D). We also explored the possibility that RHOBTB3 and its partner LIMD1 could regulate the Warburg effect cooperatively, and found that knockdown of RHOBTB3 or LIMD1 promoted glucose consumption and lactate production under both normoxic and hypoxic conditions, and their double knockdown in HEK293T cells further enhanced these effects (Figure 6E and 6F). Taken together, our data suggest that RHOBTB3 inhibits the Warburg effect most likely through promoting HIF degradation.


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 is a negative regulator of the Warburg effect. (A) Knockout of RHOBTB3 elevates the expression of HK2, LDHA GLUT1 and PDK1. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 16 h. Cells were then lysed and the protein extracts were analyzed by immunoblotting with antibodies indicated. (B) RHOBTB3 deficiency leads to increased mRNA levels of GLUT1 and LDHA. Total RNAs from RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or exposed to hypoxia for 16 h, were purified, and analyzed by real-time PCR analysis for the expression levels of GLUT1 and LDHA. Values are presented as mean ± SEM, n = 3 for each group, three replicate experiments. ***P< 0.001 (ANOVA followed by Tukey). (C, D) RHOBTB3 decreases rates of glucose consumption (C) and lactate production (D). RHOBTB3−/− and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h, and glucose consumption rates (C) and lactate production rates (D) were measured. Values are presented as mean ± SEM, n = 3 for each group, ***P< 0.001 (ANOVA followed by Tukey). (E, F) RHOBTB3 and LIMD1 cooperatively decrease glucose consumption (E) and lactate production (F). HEK293T cells were infected with lentiviruses expressing siRNAs targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h and glucose consumption rates (E) and lactate production rates (F) were measured. Values are presented as mean ± SEM, n = 3 for each group, *P< 0.05, **P< 0.01, ***P< 0.001 (ANOVA followed by Tukey).
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Related In: Results  -  Collection

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fig6: RHOBTB3 is a negative regulator of the Warburg effect. (A) Knockout of RHOBTB3 elevates the expression of HK2, LDHA GLUT1 and PDK1. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 16 h. Cells were then lysed and the protein extracts were analyzed by immunoblotting with antibodies indicated. (B) RHOBTB3 deficiency leads to increased mRNA levels of GLUT1 and LDHA. Total RNAs from RHOBTB3−/− MEFs and WT MEFs, maintained in normoxia or exposed to hypoxia for 16 h, were purified, and analyzed by real-time PCR analysis for the expression levels of GLUT1 and LDHA. Values are presented as mean ± SEM, n = 3 for each group, three replicate experiments. ***P< 0.001 (ANOVA followed by Tukey). (C, D) RHOBTB3 decreases rates of glucose consumption (C) and lactate production (D). RHOBTB3−/− and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h, and glucose consumption rates (C) and lactate production rates (D) were measured. Values are presented as mean ± SEM, n = 3 for each group, ***P< 0.001 (ANOVA followed by Tukey). (E, F) RHOBTB3 and LIMD1 cooperatively decrease glucose consumption (E) and lactate production (F). HEK293T cells were infected with lentiviruses expressing siRNAs targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h and glucose consumption rates (E) and lactate production rates (F) were measured. Values are presented as mean ± SEM, n = 3 for each group, *P< 0.05, **P< 0.01, ***P< 0.001 (ANOVA followed by Tukey).
Mentions: We next examined whether RHOBTB3 could suppress the Warburg effect since it could significantly reduce HIFα levels in cells, and levels of HK2, LDHA, GLUT1 and PDK1, known stream targets of HIFs, were elevated in RHOBTB3−/− MEFs under both normoxic and hypoxic conditions (Figure 6A and 6B). The Warburg effect is characterized by high rates of glucose uptake and lactate production regardless of oxygen concentration30. We thus measured the glucose consumption and lactate production rates, and found that they were increased in RHOBTB3−/− MEFs in both normoxia and hypoxia (Figure 6C and 6D). We also explored the possibility that RHOBTB3 and its partner LIMD1 could regulate the Warburg effect cooperatively, and found that knockdown of RHOBTB3 or LIMD1 promoted glucose consumption and lactate production under both normoxic and hypoxic conditions, and their double knockdown in HEK293T cells further enhanced these effects (Figure 6E and 6F). Taken together, our data suggest that RHOBTB3 inhibits the Warburg effect most likely through promoting HIF degradation.

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