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Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Chen RQ, Yang QK, Chen YL, Oliveira VA, Dalton WS, Fearns C, Lee JD - J. Biol. Chem. (2009)

Bottom Line: Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis.To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions.Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.

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Depletion of SMG-1 augmented the HIF-1 activity under hypoxic conditions. A, HeLa cells were transfected with Smart-pool siRNAs for individual members of the PIKK family or with control siRNA (Cont) as indicated. After 24 h, cells were transfected with reporter plasmids, incubated in hypoxia chamber, and analyzed for luciferase activity as described in Fig. 1. The normalized luciferase activity of control hypoxic cells was taken as 1. The protein levels of endogenous SMG-1, ATM, ATR, mTOR, and DNA-PK were detected by Western blotting using antibodies specific to each of these proteins (described under “Experimental Procedures”). B, HeLa cells were transfected with four different SMG-1 siRNA duplexes (DP1–4), Smart-pool (SP), or control (Cont) siRNAs. After 24 h, cells were transfected with reporter plasmids, incubated in the hypoxia chamber, and analyzed for luciferase activity as described above. Endogenous SGM-1 protein was detected by Western blotting using an anti-SMG-1 antibody (lower panel). C, HeLa cells were transfected with SMG-1 siRNA. After 24 h, cells were transfected with empty vector or the expression plasmids encoding HA-tagged wild-type SMG-1 (HA-SMG-1-WT) or kinase dead SMG-1 (HA-SMG-1-KD). HRE and pRL-TK luciferase reporter plasmids were also included in the transfection mixture. The luciferase activity in SMG-1-deficient cells transfected with empty vector was taken as 100%. The expression of HA-SMG-1-WT and HA-SMG-1-KD were detected by Western blotting using an anti-HA antibody (lower panel).
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Figure 2: Depletion of SMG-1 augmented the HIF-1 activity under hypoxic conditions. A, HeLa cells were transfected with Smart-pool siRNAs for individual members of the PIKK family or with control siRNA (Cont) as indicated. After 24 h, cells were transfected with reporter plasmids, incubated in hypoxia chamber, and analyzed for luciferase activity as described in Fig. 1. The normalized luciferase activity of control hypoxic cells was taken as 1. The protein levels of endogenous SMG-1, ATM, ATR, mTOR, and DNA-PK were detected by Western blotting using antibodies specific to each of these proteins (described under “Experimental Procedures”). B, HeLa cells were transfected with four different SMG-1 siRNA duplexes (DP1–4), Smart-pool (SP), or control (Cont) siRNAs. After 24 h, cells were transfected with reporter plasmids, incubated in the hypoxia chamber, and analyzed for luciferase activity as described above. Endogenous SGM-1 protein was detected by Western blotting using an anti-SMG-1 antibody (lower panel). C, HeLa cells were transfected with SMG-1 siRNA. After 24 h, cells were transfected with empty vector or the expression plasmids encoding HA-tagged wild-type SMG-1 (HA-SMG-1-WT) or kinase dead SMG-1 (HA-SMG-1-KD). HRE and pRL-TK luciferase reporter plasmids were also included in the transfection mixture. The luciferase activity in SMG-1-deficient cells transfected with empty vector was taken as 100%. The expression of HA-SMG-1-WT and HA-SMG-1-KD were detected by Western blotting using an anti-HA antibody (lower panel).

Mentions: To explore the role of kinases in regulating HIF-1 activity under hypoxia, we depleted kinases in tumor cells using a siRNA kinase library targeting 779 kinases and then analyzed cellular HIF-1 activity under oxygen deprivation using an HRE luciferase reporter assay. In this assay, hypoxia strongly increased HRE luciferase activity and HIF-1α protein stabilization in cells (Fig. 1A). Among the 779 kinases screened by siRNA-mediated silencing, depletion of 18 kinases significantly increased HIF-1 activity and another 10 kinases decreased HIF-1 activity under hypoxic conditions (Fig. 1B). Among these kinases, the depletion of SMG-1 impacted HIF-1 activity most significantly (Fig. 1B). SMG-1 belongs to the PIKK family of protein kinases. We found that siRNA-mediated depletion of SMG-1, but not other members of the PIKK family, ATM, ATR, DNA-PKcs, and mTOR, markedly increased the HIF-1 activity in hypoxic cancer cells (Fig. 2A). To further confirm that the increase in hypoxia-induced HIF-1 activity in SMG-1-depleted cells was directly related to an on-target effect of the siRNA, we repeated this experiment using four different siRNA duplexes to deplete cellular SMG-1 and obtained similar results (Fig. 2B). Next, we forced expression of wild-type (WT) SMG-1 and its kinase-dead (KD) mutant separately in SMG-1-depleted cell and found that forced expression of WT, but not KD SMG-1, could reverse the increased HIF-1 activity observed in hypoxic tumor cells depleted of cellular SMG-1 (Fig. 2C). These results confirmed that SMG-1 deficiency increased HIF-1 activity in hypoxia and suggested that cellular SMG-1 activity is critical in suppressing HIF-1 activation in hypoxic cancer cells.


Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Chen RQ, Yang QK, Chen YL, Oliveira VA, Dalton WS, Fearns C, Lee JD - J. Biol. Chem. (2009)

Depletion of SMG-1 augmented the HIF-1 activity under hypoxic conditions. A, HeLa cells were transfected with Smart-pool siRNAs for individual members of the PIKK family or with control siRNA (Cont) as indicated. After 24 h, cells were transfected with reporter plasmids, incubated in hypoxia chamber, and analyzed for luciferase activity as described in Fig. 1. The normalized luciferase activity of control hypoxic cells was taken as 1. The protein levels of endogenous SMG-1, ATM, ATR, mTOR, and DNA-PK were detected by Western blotting using antibodies specific to each of these proteins (described under “Experimental Procedures”). B, HeLa cells were transfected with four different SMG-1 siRNA duplexes (DP1–4), Smart-pool (SP), or control (Cont) siRNAs. After 24 h, cells were transfected with reporter plasmids, incubated in the hypoxia chamber, and analyzed for luciferase activity as described above. Endogenous SGM-1 protein was detected by Western blotting using an anti-SMG-1 antibody (lower panel). C, HeLa cells were transfected with SMG-1 siRNA. After 24 h, cells were transfected with empty vector or the expression plasmids encoding HA-tagged wild-type SMG-1 (HA-SMG-1-WT) or kinase dead SMG-1 (HA-SMG-1-KD). HRE and pRL-TK luciferase reporter plasmids were also included in the transfection mixture. The luciferase activity in SMG-1-deficient cells transfected with empty vector was taken as 100%. The expression of HA-SMG-1-WT and HA-SMG-1-KD were detected by Western blotting using an anti-HA antibody (lower panel).
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Figure 2: Depletion of SMG-1 augmented the HIF-1 activity under hypoxic conditions. A, HeLa cells were transfected with Smart-pool siRNAs for individual members of the PIKK family or with control siRNA (Cont) as indicated. After 24 h, cells were transfected with reporter plasmids, incubated in hypoxia chamber, and analyzed for luciferase activity as described in Fig. 1. The normalized luciferase activity of control hypoxic cells was taken as 1. The protein levels of endogenous SMG-1, ATM, ATR, mTOR, and DNA-PK were detected by Western blotting using antibodies specific to each of these proteins (described under “Experimental Procedures”). B, HeLa cells were transfected with four different SMG-1 siRNA duplexes (DP1–4), Smart-pool (SP), or control (Cont) siRNAs. After 24 h, cells were transfected with reporter plasmids, incubated in the hypoxia chamber, and analyzed for luciferase activity as described above. Endogenous SGM-1 protein was detected by Western blotting using an anti-SMG-1 antibody (lower panel). C, HeLa cells were transfected with SMG-1 siRNA. After 24 h, cells were transfected with empty vector or the expression plasmids encoding HA-tagged wild-type SMG-1 (HA-SMG-1-WT) or kinase dead SMG-1 (HA-SMG-1-KD). HRE and pRL-TK luciferase reporter plasmids were also included in the transfection mixture. The luciferase activity in SMG-1-deficient cells transfected with empty vector was taken as 100%. The expression of HA-SMG-1-WT and HA-SMG-1-KD were detected by Western blotting using an anti-HA antibody (lower panel).
Mentions: To explore the role of kinases in regulating HIF-1 activity under hypoxia, we depleted kinases in tumor cells using a siRNA kinase library targeting 779 kinases and then analyzed cellular HIF-1 activity under oxygen deprivation using an HRE luciferase reporter assay. In this assay, hypoxia strongly increased HRE luciferase activity and HIF-1α protein stabilization in cells (Fig. 1A). Among the 779 kinases screened by siRNA-mediated silencing, depletion of 18 kinases significantly increased HIF-1 activity and another 10 kinases decreased HIF-1 activity under hypoxic conditions (Fig. 1B). Among these kinases, the depletion of SMG-1 impacted HIF-1 activity most significantly (Fig. 1B). SMG-1 belongs to the PIKK family of protein kinases. We found that siRNA-mediated depletion of SMG-1, but not other members of the PIKK family, ATM, ATR, DNA-PKcs, and mTOR, markedly increased the HIF-1 activity in hypoxic cancer cells (Fig. 2A). To further confirm that the increase in hypoxia-induced HIF-1 activity in SMG-1-depleted cells was directly related to an on-target effect of the siRNA, we repeated this experiment using four different siRNA duplexes to deplete cellular SMG-1 and obtained similar results (Fig. 2B). Next, we forced expression of wild-type (WT) SMG-1 and its kinase-dead (KD) mutant separately in SMG-1-depleted cell and found that forced expression of WT, but not KD SMG-1, could reverse the increased HIF-1 activity observed in hypoxic tumor cells depleted of cellular SMG-1 (Fig. 2C). These results confirmed that SMG-1 deficiency increased HIF-1 activity in hypoxia and suggested that cellular SMG-1 activity is critical in suppressing HIF-1 activation in hypoxic cancer cells.

Bottom Line: Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis.To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions.Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.

Show MeSH
Related in: MedlinePlus