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HIF-1α and TAZ serve as reciprocal co-activators in human breast cancer cells.

Xiang L, Gilkes DM, Hu H, Luo W, Bullen JW, Liang H, Semenza GL - Oncotarget (2015)

Bottom Line: Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality.Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization.Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality. Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization. Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α. Inhibition of TAZ expression impairs the hypoxic induction of HIF-1 target genes, such as PDK1, LDHA, BNIP3 and P4HA2 in response to hypoxia, whereas inhibition of HIF-1α expression impairs TAZ-mediated transactivation of the CTGF promoter. Taken together, these results complement our previous findings and establish bidirectional crosstalk between HIF-1α and TAZ that increases their transcriptional activities in hypoxic cells.

No MeSH data available.


Related in: MedlinePlus

HIF-1α serves as a co-activator for TAZA. MCF-7 cells were stably transfected with a lentiviral expression vector encoding a non-targeting control shRNA (NTC) or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β; efficiency of knockdown shown in immunoblot at upper right) or TAZ (shT1). The MCF-7 subclones were transiently transfected with pSV-Renilla and either the pGL2-Basic or pGL2-CTGF firefly luciferase reporter, and exposed to 20% or 1% O2 for 24 h. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus NTC at 20% O2;##P < 0.01, ###P < 0.001 versus NTC at 1% O2. B. MCF-7 cells stably transfected with HIF-1β shRNA (sh1β) vector were transiently transfected with pGL2-CTGF, pSV-Renilla, and either pcDNA-EV (empty vector) or pcDNA-HIF-1α-DM (encoding double mutant HIF-1α [P402A/P564A]) in the presence of TAZ shRNA (shT1) or NTC vector. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus lane 1; ###P < 0.001 versus lane 2.
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Figure 2: HIF-1α serves as a co-activator for TAZA. MCF-7 cells were stably transfected with a lentiviral expression vector encoding a non-targeting control shRNA (NTC) or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β; efficiency of knockdown shown in immunoblot at upper right) or TAZ (shT1). The MCF-7 subclones were transiently transfected with pSV-Renilla and either the pGL2-Basic or pGL2-CTGF firefly luciferase reporter, and exposed to 20% or 1% O2 for 24 h. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus NTC at 20% O2;##P < 0.01, ###P < 0.001 versus NTC at 1% O2. B. MCF-7 cells stably transfected with HIF-1β shRNA (sh1β) vector were transiently transfected with pGL2-CTGF, pSV-Renilla, and either pcDNA-EV (empty vector) or pcDNA-HIF-1α-DM (encoding double mutant HIF-1α [P402A/P564A]) in the presence of TAZ shRNA (shT1) or NTC vector. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus lane 1; ###P < 0.001 versus lane 2.

Mentions: We next investigated whether HIF-1α regulates TAZ transcriptional activity. We established MCF-7 breast cancer subclones, which were stably transduced with lentiviral vectors encoding NTC shRNA or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β), or TAZ (shT1), and immunoblot assays confirmed effective knockdown of HIF-1α and TAZ [5] as well as HIF-1β (Figure 2A) protein expression. A 247-bp CTGF proximal promoter sequence, which contains three copies of the TEAD-binding site sequence (5′-GGAATG-3′) and no match to the HIF-1 binding site consensus sequence (5′-RCGTG-3′), was inserted into the promoterless firefly luciferase reporter plasmid pGL2-Basic to generate pGL2-CTGF. MCF-7 subclones were transiently transfected with pSV-Renilla and either pGL2-CTGF or pGL2-Basic. Luciferase activity was increased in response to hypoxia in NTC cells transfected with pGL2-CTGF, whereas knockdown of HIF-1α, HIF-1β, or TAZ significantly decreased luciferase activity under hypoxia and the effect of HIF-1α knockdown was greater than the effect of HIF-1β knockdown (Figure 2A).


HIF-1α and TAZ serve as reciprocal co-activators in human breast cancer cells.

Xiang L, Gilkes DM, Hu H, Luo W, Bullen JW, Liang H, Semenza GL - Oncotarget (2015)

HIF-1α serves as a co-activator for TAZA. MCF-7 cells were stably transfected with a lentiviral expression vector encoding a non-targeting control shRNA (NTC) or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β; efficiency of knockdown shown in immunoblot at upper right) or TAZ (shT1). The MCF-7 subclones were transiently transfected with pSV-Renilla and either the pGL2-Basic or pGL2-CTGF firefly luciferase reporter, and exposed to 20% or 1% O2 for 24 h. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus NTC at 20% O2;##P < 0.01, ###P < 0.001 versus NTC at 1% O2. B. MCF-7 cells stably transfected with HIF-1β shRNA (sh1β) vector were transiently transfected with pGL2-CTGF, pSV-Renilla, and either pcDNA-EV (empty vector) or pcDNA-HIF-1α-DM (encoding double mutant HIF-1α [P402A/P564A]) in the presence of TAZ shRNA (shT1) or NTC vector. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus lane 1; ###P < 0.001 versus lane 2.
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Figure 2: HIF-1α serves as a co-activator for TAZA. MCF-7 cells were stably transfected with a lentiviral expression vector encoding a non-targeting control shRNA (NTC) or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β; efficiency of knockdown shown in immunoblot at upper right) or TAZ (shT1). The MCF-7 subclones were transiently transfected with pSV-Renilla and either the pGL2-Basic or pGL2-CTGF firefly luciferase reporter, and exposed to 20% or 1% O2 for 24 h. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus NTC at 20% O2;##P < 0.01, ###P < 0.001 versus NTC at 1% O2. B. MCF-7 cells stably transfected with HIF-1β shRNA (sh1β) vector were transiently transfected with pGL2-CTGF, pSV-Renilla, and either pcDNA-EV (empty vector) or pcDNA-HIF-1α-DM (encoding double mutant HIF-1α [P402A/P564A]) in the presence of TAZ shRNA (shT1) or NTC vector. The luciferase activity was normalized to lane 1 (mean ± SEM, n = 4). ***P < 0.001 versus lane 1; ###P < 0.001 versus lane 2.
Mentions: We next investigated whether HIF-1α regulates TAZ transcriptional activity. We established MCF-7 breast cancer subclones, which were stably transduced with lentiviral vectors encoding NTC shRNA or shRNA targeting HIF-1α (sh1α), HIF-1β (sh1β), or TAZ (shT1), and immunoblot assays confirmed effective knockdown of HIF-1α and TAZ [5] as well as HIF-1β (Figure 2A) protein expression. A 247-bp CTGF proximal promoter sequence, which contains three copies of the TEAD-binding site sequence (5′-GGAATG-3′) and no match to the HIF-1 binding site consensus sequence (5′-RCGTG-3′), was inserted into the promoterless firefly luciferase reporter plasmid pGL2-Basic to generate pGL2-CTGF. MCF-7 subclones were transiently transfected with pSV-Renilla and either pGL2-CTGF or pGL2-Basic. Luciferase activity was increased in response to hypoxia in NTC cells transfected with pGL2-CTGF, whereas knockdown of HIF-1α, HIF-1β, or TAZ significantly decreased luciferase activity under hypoxia and the effect of HIF-1α knockdown was greater than the effect of HIF-1β knockdown (Figure 2A).

Bottom Line: Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality.Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization.Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality. Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization. Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α. Inhibition of TAZ expression impairs the hypoxic induction of HIF-1 target genes, such as PDK1, LDHA, BNIP3 and P4HA2 in response to hypoxia, whereas inhibition of HIF-1α expression impairs TAZ-mediated transactivation of the CTGF promoter. Taken together, these results complement our previous findings and establish bidirectional crosstalk between HIF-1α and TAZ that increases their transcriptional activities in hypoxic cells.

No MeSH data available.


Related in: MedlinePlus