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W2023279237 abstract "Adaptation to hypoxia is a crucial process both physiologically (i.e. in chondrocytes) and pathologically (i.e. in tumor cells). Previous studies have shown that DEC1, a basic helix-loop-helix transcription factor, is induced by hypoxia in glioma cells (Ivanova, A. V., Ivanov, S. V., Danilkovitch-Miagkova, A., and Lerman, M. I. (2001)J. Biol. Chem. 276, 15306–15315). In the present study, we found that hypoxia or CoCl2 enhanced the mRNA expression of DEC2, as well as DEC1, within 24 h in chondrogenic ATDC5, 293T, and HeLa cells. In luciferase assays, the regions between −524 and −401 in the DEC1 promoter, and between −863 and −258 in the DEC2 promoter, were responsible for the hypoxia- or hypoxia-inducible factor-1α (HIF-1α)-induced transcription. In these regions, we identified functional hypoxia response elements (HREs) that bound to HIF-1α and HIF-1β. In addition to an HIF-1 binding site consensus sequence, the DEC1 HRE had cAMP response element-like and CACAG sequences, which were also involved in the transcription activation in response to HIF-1α. Although the DEC2 HRE did not have a cAMP response element-like or CACAG sequence, it showed a higher affinity for HIF-1 than did the DEC1 HRE. Because DEC1 and DEC2 are directly inducible by HIF-1, these transcription factors may be crucial for the adaptation to hypoxia. Adaptation to hypoxia is a crucial process both physiologically (i.e. in chondrocytes) and pathologically (i.e. in tumor cells). Previous studies have shown that DEC1, a basic helix-loop-helix transcription factor, is induced by hypoxia in glioma cells (Ivanova, A. V., Ivanov, S. V., Danilkovitch-Miagkova, A., and Lerman, M. I. (2001)J. Biol. Chem. 276, 15306–15315). In the present study, we found that hypoxia or CoCl2 enhanced the mRNA expression of DEC2, as well as DEC1, within 24 h in chondrogenic ATDC5, 293T, and HeLa cells. In luciferase assays, the regions between −524 and −401 in the DEC1 promoter, and between −863 and −258 in the DEC2 promoter, were responsible for the hypoxia- or hypoxia-inducible factor-1α (HIF-1α)-induced transcription. In these regions, we identified functional hypoxia response elements (HREs) that bound to HIF-1α and HIF-1β. In addition to an HIF-1 binding site consensus sequence, the DEC1 HRE had cAMP response element-like and CACAG sequences, which were also involved in the transcription activation in response to HIF-1α. Although the DEC2 HRE did not have a cAMP response element-like or CACAG sequence, it showed a higher affinity for HIF-1 than did the DEC1 HRE. Because DEC1 and DEC2 are directly inducible by HIF-1, these transcription factors may be crucial for the adaptation to hypoxia. Recently we cloned the cDNA for the novel basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; HIF-1, hypoxia-inducible factor-1; HBS, HIF-1 binding site; HRE, hypoxia response element; CRE, cAMP response element; PPARγ2, peroxisome proliferator-activated receptor-γ2; RT, reverse transcriptase; wt, wild-type; CREB, CRE-binding protein; LDH, lactate dehydrogenase; Epo, erythropoeitin; TK, thymidine kinase 1The abbreviations used are: bHLH, basic helix-loop-helix; HIF-1, hypoxia-inducible factor-1; HBS, HIF-1 binding site; HRE, hypoxia response element; CRE, cAMP response element; PPARγ2, peroxisome proliferator-activated receptor-γ2; RT, reverse transcriptase; wt, wild-type; CREB, CRE-binding protein; LDH, lactate dehydrogenase; Epo, erythropoeitin; TK, thymidine kinase transcription factor DEC1 (BHLHB2), which was expressed at a higher level in human primary chondrocytes than in fibroblastic cells (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar). A mouse ortholog (Stra13) and a rat ortholog (SHARP-2) of DEC1 were identified independently by others in the P19 embryonal carcinoma cells and rat brain, respectively (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar), and the mRNA of DEC1 was found in a variety of embryonic and adult tissues (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar, 2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar). In addition, we cloned a member of the DEC subfamily of bHLH proteins, DEC2 (BHLHB3), by searching the expressed sequence tags data bank (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). DEC2 is similar to SHARP-1 (3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar), which is a truncated molecule of DEC2 produced by a sequencing error or a minor frameshift mutant (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). The bHLH regions of DEC1 and DEC2 exhibit the highest similarities to those ofDrosophila Hairy, Enhancer of split and mammal HES, which are known as transcriptional repressors with the WRPW motif, which interacts with the Groucho family members of corepressors. Although the Stra13/DEC1 and DEC2/SHARP-1 lack the WRPW motif, they also act as transcriptional repressors by a discrete mechanism (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar, 6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). Stra13/DEC1 interacts physically with the components of the basal transcription machineries, such as TATA-binding protein and TFIIB, and can recruit the histone deacetylase 1-Sin3A-NcoR corepressor complex through their carboxyl-terminal repression domain (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar).DEC1/Stra13/SHARP-2 may be involved in the control of the proliferation and/or differentiation of chondrocytes, nerve cells, fibroblasts, and T cells (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar, 2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar, 7Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Google Scholar). The overexpression of DEC1/Stra13 promoted a chondrogenic differentiation of the mesenchymal stem cells, 2M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato, manuscript in preparation.2M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato, manuscript in preparation. and a neural differentiation of the P19 cells (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar). Recently, Stra13-deficient mice were generated, and it was shown that Stra13/DEC1 is a key regulator of lymphocyte activation that is vital for the maintenance of self-tolerance and constraint of autoimmunity. Other phenotypic differences between Stra13−/− mutants and the wild-type littermates have not been reported (7Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Google Scholar). On the other hand, DEC2/SHARP-1 worked as a transcriptional repressor in vitro (6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). The mRNA of DEC2 was expressed ubiquitously, but the expression level of DEC2 was more variable tissue-dependently than that of DEC1 (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). The role of DEC2 in vivo is not known.The transcription factor, hypoxia-inducible factor-1 (HIF-1), functions as a global regulator of O2 homeostasis and the adaptation to O2 deprivation (8Semenza G.L. J. Appl. Physiol. 2000; 88: 1474-1480Google Scholar). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits, both of which are bHLH-PAS (Per/Arnt/Sim) proteins. Under normoxic conditions, the HIF-1α is subjected to ubiquitination and proteasomal degradation by the interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) (9Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Google Scholar, 10Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Google Scholar, 11Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Google Scholar). Under hypoxic conditions, the HIF-1α is released from pVHL and translocates into the nucleus, where it heterodimerizes with HIF-1β, which is expressed constitutively (11Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Google Scholar, 12Kallio P.J. Pongratz I. Gradin K. McGuire J. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5667-5672Google Scholar, 13Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Google Scholar). The HIF-1 complex binds to the HIF-1 binding site (HBS) in the hypoxia response element (HRE) in the hypoxia-inducible target genes, such as erythropoeitin (14Jiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; 271: 17771-17778Google Scholar), transferrin (15Rolfs A. Kvietikova I. Gassmann M. Wenger R.H. J. Biol. Chem. 1997; 272: 20055-20062Google Scholar), vascular endothelial growth factor (16Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Google Scholar, 17Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Google Scholar, 18Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Keshet E. Nature. 1998; 394: 485-490Google Scholar), and glucose transporter-3 (16Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Google Scholar) genes. In this process, the contribution of the HIF-1 complex is determined mainly by protein stabilization and the activation of HIF-1α. A recent study (19Ivanova A.V. Ivanov S.V. Danilkovitch-Miagkova A. Lerman M.I. J. Biol. Chem. 2001; 276: 15306-15315Google Scholar) showed that DEC1 was down-regulated by pVHL and that the expression of DEC1 mRNA in glioma cells was hypoxia-sensitive. Although the pVHL/HIF-1 pathway described above was thought to regulate the expression of DEC1 mRNA under hypoxic conditions, the underlying mechanism has not been dissected fully, and the relation between hypoxia and the DEC2 mRNA expression has not yet been investigated.In this study, we examined the effects of hypoxia on the DEC2 and DEC1 mRNA expression in chondrogenic ATDC5, 293T, and HeLa cells. Because the DEC2 and DEC1 mRNA expression was induced by CoCl2 mimicking the hypoxic condition in ATDC5 cells and by hypoxia in 293T and HeLa cells, we investigated the transcriptional activities of the 5′-flanking regions in these genes. We determined the HRE in the DEC1 and DEC2 promoters and found that the DEC1 and DEC2 genes are direct targets of HIF-1.DISCUSSIONIn this study, we found that, like DEC1, DEC2 is induced by hypoxia on the transcriptional level, and we revealed the underlying molecular mechanism by identifying the functional HIF-1 binding sites in the DEC1 and DEC2 promoters. Recent studies with cartilage-specific HIF-1α-deficient mice have shown that internal chondrocytes of the growth plate cartilage are under hypoxic conditions and that HIF-1α is essential for survival of the hypoxic chondrocytes and further, for the induction of growth arrest of the chondrocytes before hypertrophic differentiation (32Schipani E. Ryan H.E. Didrickson S. Kobayashi T. Knight M. Johnson R.S. Genes Dev. 2001; 15: 2865-2876Crossref Google Scholar). The lack of HIF-1α in the chondrocytes caused a massive cell death in the center of the cartilaginous elements and a subtle delay in the process of the hypertrophic differentiation at its periphery because of the lack of growth arrest. In the mutant mice, the expression of type II and type X collagens was absent in the central portion of the proliferative and the upper hypertrophic zones in the growth plate, suggesting the requirement of HIF-1α for the expression of the cartilage-specific collagens. In the present study, the treatment for 24 h with CoCl2 mimicking a hypoxic condition had little effect on type II collagen, aggrecan, or type X collagen mRNA expression in the chondrogenic ATDC5 cell cultures (Fig. 1 A). 24 h of exposure to CoCl2 might be too brief to detect the effect of CoCl2 on the expression of these genes. On the other hand, the mRNA expression of DEC2 and DEC1 was enhanced by CoCl2 within 24 h throughout all of the stages of the chondrocyte differentiation. The reason for the enhancement by CoCl2 within 24 h is thought to be that the DEC2 and DEC1 gene expression is regulated directly by HIF-1.Because the DEC1 and DEC2 modulate transcription of various genes (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar,6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar, 33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar), DEC1 and DEC2 could be involved in the adaptation of chondrocytes to hypoxic conditions. DEC1 and DEC2 may also affect the expression of regulatory molecules involved in growth arrest, and the Stra13/DEC1 overexpression did indeed suppress the proliferation of the NIH3T3 cells (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar). The up-regulation of the Stra13/DEC1 expression was concomitant with the down-regulation of the cell proliferation-associated protein c-Myc expression levels in the NIH3T3 cells, and the Stra13/DEC1 expression strongly inhibited the promoter activity of the c-Myc gene (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar).Recent studies have shown that hypoxia inhibits adipogenesis and the expression of a key transcription factor in adipogenesis, PPARγ2, via HIF-1, and that the DEC1 overexpression inhibits the PPARγ2 gene expression (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar). Mesenchymal stem cells can differentiate to chondryocytes, osteoblasts, adipocytes, and myoblasts, and an overexpression of DEC1 in mesenchymal stem cells promoted chondrogenic differentiation.2 Therefore, DEC1 induced by hypoxia may promote chondrogenesis by inhibiting mesenchymal stem cells from going to some other lineage, such as the adipogenic lineage.We demonstrated here that the gene expression of DEC1 and DEC2 is regulated by the HIF-1 binding to the functional HBSs in their promoters under hypoxic conditions. An HBS consensus sequence 5′-RCGTG-3′ exists in the hypoxia response region of the DEC1 promoter (DEC1-HBS), and two consensus sequences exist in that of the DEC2 promoter (DEC2-HBS1 and -HBS2) (Fig 4 A). However, only DEC1-HBS and DEC2-HBS1 were functional in the luciferase reporter gene assays. In the DEC1, but not the DEC2 promoter, we found a CRE-like sequence adjacent to the functional HBS (Fig. 4 B). In the LDH-A promoter, a multiprotein complex of HIF-1, CREB-1/ATF-1, and p300/CREB-binding protein bound to the HRE under hypoxic conditions (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). The complex formation of HIF-1, CREB-1, and p300 was necessary to induce the hypoxic response in the LDH-A gene (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). As to the HRE containing the functional HBS in the DEC1 gene, the results obtained with various mutated HRE sequences showed that a disruption of the CRE-like sequence alone did not abolish the induction by HIF-1α, although it decreased the basal luciferase activity. On the other hand, the mutations of the overlapping nucleotides between the CRE-like and the CACAG sequences decreased the basal luciferase activity and almost abolished the induction. These findings suggest that the CRE-like sequence is required for the maintenance of the transcription in the absence and presence of HIF-1 and that both of the CRE-like sequence and the CACAG sequence may be required for the induction in response to hypoxia. In the supershift assays, we could not detect a supershift of the mobility of the DEC1-HREwt probe and HIF-1 complex using these antibodies, suggesting that this complex did not contain CREB-1 or ATF-1 (Fig. 5 B). Although CREB-1 or ATF-1 may bind to the CRE-like sequence of DEC1-HRE, no CREB-1·DNA or ATF-1·DNA complex was detected under these conditions. However, CREB-1 or ATF-1 could bind to DEC1-HRE with a very low affinity. The CRE-like sequence (TGGAGTCA) in DEC1-HRE differs from that in the HRE of the LDH-A gene. This may explain the low affinity of CREB-1 or ATF-1 for the sequence. In any case, the requirement of the CRE-like or CACAG sequence for the response to HIF-1 in DEC1-HRE is consistent with the previous findings that the LDH-A and Epo promoters require the CRE and CACAG sequences, respectively, to respond to hypoxia or HIF-1 (31Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Google Scholar, 35Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Google Scholar). In the HRE of the human transferrin receptor gene, a CRE-like sequence overlaps with the CACAG sequence. However, the contribution of these sequences to the HIF-1 induction has not been determined (25Lok C.N. Ponka P. J. Biol. Chem. 1999; 274: 24147-24152Google Scholar). We found that DEC2-HRE has a higher affinity for HIF-1 than DEC1-HRE. DEC2-HRE may not require a CRE or CACAG sequence because of the higher affinity for HIF-1.The protein products of genes up-regulated by HIF-1 play key roles in angiogenesis, vascular reactivity and remodeling, glucose and energy metabolism, proliferation, survival, erythropoiesis, and iron homeostasis (36Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Google Scholar). Interestingly, DEC1 and DEC2 are inducible by HIF-1 within 24 h in various cells. There have been few studies on the action of HIF-1 on transcription factors; HIF-1 enhanced transcription from the Ets promoter (37Oikawa M. Abe M. Kurosawa H. Hida W. Shirato K. Sato Y. Biochem. Biophys. Res. Commun. 2001; 289: 39-43Google Scholar). DEC1 and DEC2 may suppress the expression of some genes under hypoxic conditions, because they work as the transcriptional suppressors in vitro (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar, 6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). Previous studies have shown that the oncodevelopmental α-fetoprotein gene expression is repressed by hypoxia in hepatoma cells and that the repression is regulated by HIF-1 via the HRE containing a CACGTG sequence in the α-fetoprotein promoter (38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar). However, HIF-1 did not bind to this sequence. The CACGTG sequence is the classical E-box sequence, and the human DEC1 can bind to it (39Zawel L. Yu J. Torrance C.J. Markowitz S. Kinzler K.W. Vogelstein B. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2848-2853Google Scholar). Other researchers have reported that the hypoxia-mimicking agent CoCl2down-regulated the expression of the tumor suppressor gene p53 via the classical E-box (40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). In another study, we observed that DEC1 and DEC2 bound to the classical E-box by electrophoretic mobility shift assays. 3Kawamoto, T., et. al., manuscript in preparation. These observations raise the possibility that DEC1 and DEC2, induced by HIF-1, inhibit the transcription of genes reported to be down-regulated by hypoxia, such as the PPARγ2, α-fetoprotein, and p53 genes via the E-box (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar, 38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar,40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). Identification of the target genes for DEC1 and DEC2 should provide important insights into the molecular mechanism for the responses to hypoxia under physiological and pathological conditions. Recently we cloned the cDNA for the novel basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; HIF-1, hypoxia-inducible factor-1; HBS, HIF-1 binding site; HRE, hypoxia response element; CRE, cAMP response element; PPARγ2, peroxisome proliferator-activated receptor-γ2; RT, reverse transcriptase; wt, wild-type; CREB, CRE-binding protein; LDH, lactate dehydrogenase; Epo, erythropoeitin; TK, thymidine kinase 1The abbreviations used are: bHLH, basic helix-loop-helix; HIF-1, hypoxia-inducible factor-1; HBS, HIF-1 binding site; HRE, hypoxia response element; CRE, cAMP response element; PPARγ2, peroxisome proliferator-activated receptor-γ2; RT, reverse transcriptase; wt, wild-type; CREB, CRE-binding protein; LDH, lactate dehydrogenase; Epo, erythropoeitin; TK, thymidine kinase transcription factor DEC1 (BHLHB2), which was expressed at a higher level in human primary chondrocytes than in fibroblastic cells (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar). A mouse ortholog (Stra13) and a rat ortholog (SHARP-2) of DEC1 were identified independently by others in the P19 embryonal carcinoma cells and rat brain, respectively (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar), and the mRNA of DEC1 was found in a variety of embryonic and adult tissues (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar, 2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar). In addition, we cloned a member of the DEC subfamily of bHLH proteins, DEC2 (BHLHB3), by searching the expressed sequence tags data bank (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). DEC2 is similar to SHARP-1 (3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar), which is a truncated molecule of DEC2 produced by a sequencing error or a minor frameshift mutant (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). The bHLH regions of DEC1 and DEC2 exhibit the highest similarities to those ofDrosophila Hairy, Enhancer of split and mammal HES, which are known as transcriptional repressors with the WRPW motif, which interacts with the Groucho family members of corepressors. Although the Stra13/DEC1 and DEC2/SHARP-1 lack the WRPW motif, they also act as transcriptional repressors by a discrete mechanism (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar, 6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). Stra13/DEC1 interacts physically with the components of the basal transcription machineries, such as TATA-binding protein and TFIIB, and can recruit the histone deacetylase 1-Sin3A-NcoR corepressor complex through their carboxyl-terminal repression domain (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar). DEC1/Stra13/SHARP-2 may be involved in the control of the proliferation and/or differentiation of chondrocytes, nerve cells, fibroblasts, and T cells (1Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Google Scholar, 2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar, 3Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 10: 460-475Google Scholar, 7Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Google Scholar). The overexpression of DEC1/Stra13 promoted a chondrogenic differentiation of the mesenchymal stem cells, 2M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato, manuscript in preparation.2M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato, manuscript in preparation. and a neural differentiation of the P19 cells (2Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Google Scholar). Recently, Stra13-deficient mice were generated, and it was shown that Stra13/DEC1 is a key regulator of lymphocyte activation that is vital for the maintenance of self-tolerance and constraint of autoimmunity. Other phenotypic differences between Stra13−/− mutants and the wild-type littermates have not been reported (7Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Google Scholar). On the other hand, DEC2/SHARP-1 worked as a transcriptional repressor in vitro (6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). The mRNA of DEC2 was expressed ubiquitously, but the expression level of DEC2 was more variable tissue-dependently than that of DEC1 (4Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Google Scholar). The role of DEC2 in vivo is not known. The transcription factor, hypoxia-inducible factor-1 (HIF-1), functions as a global regulator of O2 homeostasis and the adaptation to O2 deprivation (8Semenza G.L. J. Appl. Physiol. 2000; 88: 1474-1480Google Scholar). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits, both of which are bHLH-PAS (Per/Arnt/Sim) proteins. Under normoxic conditions, the HIF-1α is subjected to ubiquitination and proteasomal degradation by the interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) (9Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Google Scholar, 10Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Google Scholar, 11Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Google Scholar). Under hypoxic conditions, the HIF-1α is released from pVHL and translocates into the nucleus, where it heterodimerizes with HIF-1β, which is expressed constitutively (11Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Google Scholar, 12Kallio P.J. Pongratz I. Gradin K. McGuire J. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5667-5672Google Scholar, 13Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Google Scholar). The HIF-1 complex binds to the HIF-1 binding site (HBS) in the hypoxia response element (HRE) in the hypoxia-inducible target genes, such as erythropoeitin (14Jiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. J. Biol. Chem. 1996; 271: 17771-17778Google Scholar), transferrin (15Rolfs A. Kvietikova I. Gassmann M. Wenger R.H. J. Biol. Chem. 1997; 272: 20055-20062Google Scholar), vascular endothelial growth factor (16Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Google Scholar, 17Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Google Scholar, 18Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Keshet E. Nature. 1998; 394: 485-490Google Scholar), and glucose transporter-3 (16Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Google Scholar) genes. In this process, the contribution of the HIF-1 complex is determined mainly by protein stabilization and the activation of HIF-1α. A recent study (19Ivanova A.V. Ivanov S.V. Danilkovitch-Miagkova A. Lerman M.I. J. Biol. Chem. 2001; 276: 15306-15315Google Scholar) showed that DEC1 was down-regulated by pVHL and that the expression of DEC1 mRNA in glioma cells was hypoxia-sensitive. Although the pVHL/HIF-1 pathway described above was thought to regulate the expression of DEC1 mRNA under hypoxic conditions, the underlying mechanism has not been dissected fully, and the relation between hypoxia and the DEC2 mRNA expression has not yet been investigated. In this study, we examined the effects of hypoxia on the DEC2 and DEC1 mRNA expression in chondrogenic ATDC5, 293T, and HeLa cells. Because the DEC2 and DEC1 mRNA expression was induced by CoCl2 mimicking the hypoxic condition in ATDC5 cells and by hypoxia in 293T and HeLa cells, we investigated the transcriptional activities of the 5′-flanking regions in these genes. We determined the HRE in the DEC1 and DEC2 promoters and found that the DEC1 and DEC2 genes are direct targets of HIF-1. DISCUSSIONIn this study, we found that, like DEC1, DEC2 is induced by hypoxia on the transcriptional level, and we revealed the underlying molecular mechanism by identifying the functional HIF-1 binding sites in the DEC1 and DEC2 promoters. Recent studies with cartilage-specific HIF-1α-deficient mice have shown that internal chondrocytes of the growth plate cartilage are under hypoxic conditions and that HIF-1α is essential for survival of the hypoxic chondrocytes and further, for the induction of growth arrest of the chondrocytes before hypertrophic differentiation (32Schipani E. Ryan H.E. Didrickson S. Kobayashi T. Knight M. Johnson R.S. Genes Dev. 2001; 15: 2865-2876Crossref Google Scholar). The lack of HIF-1α in the chondrocytes caused a massive cell death in the center of the cartilaginous elements and a subtle delay in the process of the hypertrophic differentiation at its periphery because of the lack of growth arrest. In the mutant mice, the expression of type II and type X collagens was absent in the central portion of the proliferative and the upper hypertrophic zones in the growth plate, suggesting the requirement of HIF-1α for the expression of the cartilage-specific collagens. In the present study, the treatment for 24 h with CoCl2 mimicking a hypoxic condition had little effect on type II collagen, aggrecan, or type X collagen mRNA expression in the chondrogenic ATDC5 cell cultures (Fig. 1 A). 24 h of exposure to CoCl2 might be too brief to detect the effect of CoCl2 on the expression of these genes. On the other hand, the mRNA expression of DEC2 and DEC1 was enhanced by CoCl2 within 24 h throughout all of the stages of the chondrocyte differentiation. The reason for the enhancement by CoCl2 within 24 h is thought to be that the DEC2 and DEC1 gene expression is regulated directly by HIF-1.Because the DEC1 and DEC2 modulate transcription of various genes (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar,6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar, 33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar), DEC1 and DEC2 could be involved in the adaptation of chondrocytes to hypoxic conditions. DEC1 and DEC2 may also affect the expression of regulatory molecules involved in growth arrest, and the Stra13/DEC1 overexpression did indeed suppress the proliferation of the NIH3T3 cells (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar). The up-regulation of the Stra13/DEC1 expression was concomitant with the down-regulation of the cell proliferation-associated protein c-Myc expression levels in the NIH3T3 cells, and the Stra13/DEC1 expression strongly inhibited the promoter activity of the c-Myc gene (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar).Recent studies have shown that hypoxia inhibits adipogenesis and the expression of a key transcription factor in adipogenesis, PPARγ2, via HIF-1, and that the DEC1 overexpression inhibits the PPARγ2 gene expression (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar). Mesenchymal stem cells can differentiate to chondryocytes, osteoblasts, adipocytes, and myoblasts, and an overexpression of DEC1 in mesenchymal stem cells promoted chondrogenic differentiation.2 Therefore, DEC1 induced by hypoxia may promote chondrogenesis by inhibiting mesenchymal stem cells from going to some other lineage, such as the adipogenic lineage.We demonstrated here that the gene expression of DEC1 and DEC2 is regulated by the HIF-1 binding to the functional HBSs in their promoters under hypoxic conditions. An HBS consensus sequence 5′-RCGTG-3′ exists in the hypoxia response region of the DEC1 promoter (DEC1-HBS), and two consensus sequences exist in that of the DEC2 promoter (DEC2-HBS1 and -HBS2) (Fig 4 A). However, only DEC1-HBS and DEC2-HBS1 were functional in the luciferase reporter gene assays. In the DEC1, but not the DEC2 promoter, we found a CRE-like sequence adjacent to the functional HBS (Fig. 4 B). In the LDH-A promoter, a multiprotein complex of HIF-1, CREB-1/ATF-1, and p300/CREB-binding protein bound to the HRE under hypoxic conditions (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). The complex formation of HIF-1, CREB-1, and p300 was necessary to induce the hypoxic response in the LDH-A gene (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). As to the HRE containing the functional HBS in the DEC1 gene, the results obtained with various mutated HRE sequences showed that a disruption of the CRE-like sequence alone did not abolish the induction by HIF-1α, although it decreased the basal luciferase activity. On the other hand, the mutations of the overlapping nucleotides between the CRE-like and the CACAG sequences decreased the basal luciferase activity and almost abolished the induction. These findings suggest that the CRE-like sequence is required for the maintenance of the transcription in the absence and presence of HIF-1 and that both of the CRE-like sequence and the CACAG sequence may be required for the induction in response to hypoxia. In the supershift assays, we could not detect a supershift of the mobility of the DEC1-HREwt probe and HIF-1 complex using these antibodies, suggesting that this complex did not contain CREB-1 or ATF-1 (Fig. 5 B). Although CREB-1 or ATF-1 may bind to the CRE-like sequence of DEC1-HRE, no CREB-1·DNA or ATF-1·DNA complex was detected under these conditions. However, CREB-1 or ATF-1 could bind to DEC1-HRE with a very low affinity. The CRE-like sequence (TGGAGTCA) in DEC1-HRE differs from that in the HRE of the LDH-A gene. This may explain the low affinity of CREB-1 or ATF-1 for the sequence. In any case, the requirement of the CRE-like or CACAG sequence for the response to HIF-1 in DEC1-HRE is consistent with the previous findings that the LDH-A and Epo promoters require the CRE and CACAG sequences, respectively, to respond to hypoxia or HIF-1 (31Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Google Scholar, 35Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Google Scholar). In the HRE of the human transferrin receptor gene, a CRE-like sequence overlaps with the CACAG sequence. However, the contribution of these sequences to the HIF-1 induction has not been determined (25Lok C.N. Ponka P. J. Biol. Chem. 1999; 274: 24147-24152Google Scholar). We found that DEC2-HRE has a higher affinity for HIF-1 than DEC1-HRE. DEC2-HRE may not require a CRE or CACAG sequence because of the higher affinity for HIF-1.The protein products of genes up-regulated by HIF-1 play key roles in angiogenesis, vascular reactivity and remodeling, glucose and energy metabolism, proliferation, survival, erythropoiesis, and iron homeostasis (36Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Google Scholar). Interestingly, DEC1 and DEC2 are inducible by HIF-1 within 24 h in various cells. There have been few studies on the action of HIF-1 on transcription factors; HIF-1 enhanced transcription from the Ets promoter (37Oikawa M. Abe M. Kurosawa H. Hida W. Shirato K. Sato Y. Biochem. Biophys. Res. Commun. 2001; 289: 39-43Google Scholar). DEC1 and DEC2 may suppress the expression of some genes under hypoxic conditions, because they work as the transcriptional suppressors in vitro (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar, 6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). Previous studies have shown that the oncodevelopmental α-fetoprotein gene expression is repressed by hypoxia in hepatoma cells and that the repression is regulated by HIF-1 via the HRE containing a CACGTG sequence in the α-fetoprotein promoter (38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar). However, HIF-1 did not bind to this sequence. The CACGTG sequence is the classical E-box sequence, and the human DEC1 can bind to it (39Zawel L. Yu J. Torrance C.J. Markowitz S. Kinzler K.W. Vogelstein B. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2848-2853Google Scholar). Other researchers have reported that the hypoxia-mimicking agent CoCl2down-regulated the expression of the tumor suppressor gene p53 via the classical E-box (40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). In another study, we observed that DEC1 and DEC2 bound to the classical E-box by electrophoretic mobility shift assays. 3Kawamoto, T., et. al., manuscript in preparation. These observations raise the possibility that DEC1 and DEC2, induced by HIF-1, inhibit the transcription of genes reported to be down-regulated by hypoxia, such as the PPARγ2, α-fetoprotein, and p53 genes via the E-box (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar, 38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar,40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). Identification of the target genes for DEC1 and DEC2 should provide important insights into the molecular mechanism for the responses to hypoxia under physiological and pathological conditions. In this study, we found that, like DEC1, DEC2 is induced by hypoxia on the transcriptional level, and we revealed the underlying molecular mechanism by identifying the functional HIF-1 binding sites in the DEC1 and DEC2 promoters. Recent studies with cartilage-specific HIF-1α-deficient mice have shown that internal chondrocytes of the growth plate cartilage are under hypoxic conditions and that HIF-1α is essential for survival of the hypoxic chondrocytes and further, for the induction of growth arrest of the chondrocytes before hypertrophic differentiation (32Schipani E. Ryan H.E. Didrickson S. Kobayashi T. Knight M. Johnson R.S. Genes Dev. 2001; 15: 2865-2876Crossref Google Scholar). The lack of HIF-1α in the chondrocytes caused a massive cell death in the center of the cartilaginous elements and a subtle delay in the process of the hypertrophic differentiation at its periphery because of the lack of growth arrest. In the mutant mice, the expression of type II and type X collagens was absent in the central portion of the proliferative and the upper hypertrophic zones in the growth plate, suggesting the requirement of HIF-1α for the expression of the cartilage-specific collagens. In the present study, the treatment for 24 h with CoCl2 mimicking a hypoxic condition had little effect on type II collagen, aggrecan, or type X collagen mRNA expression in the chondrogenic ATDC5 cell cultures (Fig. 1 A). 24 h of exposure to CoCl2 might be too brief to detect the effect of CoCl2 on the expression of these genes. On the other hand, the mRNA expression of DEC2 and DEC1 was enhanced by CoCl2 within 24 h throughout all of the stages of the chondrocyte differentiation. The reason for the enhancement by CoCl2 within 24 h is thought to be that the DEC2 and DEC1 gene expression is regulated directly by HIF-1. Because the DEC1 and DEC2 modulate transcription of various genes (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar,6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar, 33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar), DEC1 and DEC2 could be involved in the adaptation of chondrocytes to hypoxic conditions. DEC1 and DEC2 may also affect the expression of regulatory molecules involved in growth arrest, and the Stra13/DEC1 overexpression did indeed suppress the proliferation of the NIH3T3 cells (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar). The up-regulation of the Stra13/DEC1 expression was concomitant with the down-regulation of the cell proliferation-associated protein c-Myc expression levels in the NIH3T3 cells, and the Stra13/DEC1 expression strongly inhibited the promoter activity of the c-Myc gene (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar). Recent studies have shown that hypoxia inhibits adipogenesis and the expression of a key transcription factor in adipogenesis, PPARγ2, via HIF-1, and that the DEC1 overexpression inhibits the PPARγ2 gene expression (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar). Mesenchymal stem cells can differentiate to chondryocytes, osteoblasts, adipocytes, and myoblasts, and an overexpression of DEC1 in mesenchymal stem cells promoted chondrogenic differentiation.2 Therefore, DEC1 induced by hypoxia may promote chondrogenesis by inhibiting mesenchymal stem cells from going to some other lineage, such as the adipogenic lineage. We demonstrated here that the gene expression of DEC1 and DEC2 is regulated by the HIF-1 binding to the functional HBSs in their promoters under hypoxic conditions. An HBS consensus sequence 5′-RCGTG-3′ exists in the hypoxia response region of the DEC1 promoter (DEC1-HBS), and two consensus sequences exist in that of the DEC2 promoter (DEC2-HBS1 and -HBS2) (Fig 4 A). However, only DEC1-HBS and DEC2-HBS1 were functional in the luciferase reporter gene assays. In the DEC1, but not the DEC2 promoter, we found a CRE-like sequence adjacent to the functional HBS (Fig. 4 B). In the LDH-A promoter, a multiprotein complex of HIF-1, CREB-1/ATF-1, and p300/CREB-binding protein bound to the HRE under hypoxic conditions (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). The complex formation of HIF-1, CREB-1, and p300 was necessary to induce the hypoxic response in the LDH-A gene (34Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Google Scholar). As to the HRE containing the functional HBS in the DEC1 gene, the results obtained with various mutated HRE sequences showed that a disruption of the CRE-like sequence alone did not abolish the induction by HIF-1α, although it decreased the basal luciferase activity. On the other hand, the mutations of the overlapping nucleotides between the CRE-like and the CACAG sequences decreased the basal luciferase activity and almost abolished the induction. These findings suggest that the CRE-like sequence is required for the maintenance of the transcription in the absence and presence of HIF-1 and that both of the CRE-like sequence and the CACAG sequence may be required for the induction in response to hypoxia. In the supershift assays, we could not detect a supershift of the mobility of the DEC1-HREwt probe and HIF-1 complex using these antibodies, suggesting that this complex did not contain CREB-1 or ATF-1 (Fig. 5 B). Although CREB-1 or ATF-1 may bind to the CRE-like sequence of DEC1-HRE, no CREB-1·DNA or ATF-1·DNA complex was detected under these conditions. However, CREB-1 or ATF-1 could bind to DEC1-HRE with a very low affinity. The CRE-like sequence (TGGAGTCA) in DEC1-HRE differs from that in the HRE of the LDH-A gene. This may explain the low affinity of CREB-1 or ATF-1 for the sequence. In any case, the requirement of the CRE-like or CACAG sequence for the response to HIF-1 in DEC1-HRE is consistent with the previous findings that the LDH-A and Epo promoters require the CRE and CACAG sequences, respectively, to respond to hypoxia or HIF-1 (31Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Google Scholar, 35Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Google Scholar). In the HRE of the human transferrin receptor gene, a CRE-like sequence overlaps with the CACAG sequence. However, the contribution of these sequences to the HIF-1 induction has not been determined (25Lok C.N. Ponka P. J. Biol. Chem. 1999; 274: 24147-24152Google Scholar). We found that DEC2-HRE has a higher affinity for HIF-1 than DEC1-HRE. DEC2-HRE may not require a CRE or CACAG sequence because of the higher affinity for HIF-1. The protein products of genes up-regulated by HIF-1 play key roles in angiogenesis, vascular reactivity and remodeling, glucose and energy metabolism, proliferation, survival, erythropoiesis, and iron homeostasis (36Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Google Scholar). Interestingly, DEC1 and DEC2 are inducible by HIF-1 within 24 h in various cells. There have been few studies on the action of HIF-1 on transcription factors; HIF-1 enhanced transcription from the Ets promoter (37Oikawa M. Abe M. Kurosawa H. Hida W. Shirato K. Sato Y. Biochem. Biophys. Res. Commun. 2001; 289: 39-43Google Scholar). DEC1 and DEC2 may suppress the expression of some genes under hypoxic conditions, because they work as the transcriptional suppressors in vitro (5Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Google Scholar, 6Garriga-Canut M. Roopra A. Buckley N.J. J. Biol. Chem. 2001; 276: 14821-14888Google Scholar). Previous studies have shown that the oncodevelopmental α-fetoprotein gene expression is repressed by hypoxia in hepatoma cells and that the repression is regulated by HIF-1 via the HRE containing a CACGTG sequence in the α-fetoprotein promoter (38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar). However, HIF-1 did not bind to this sequence. The CACGTG sequence is the classical E-box sequence, and the human DEC1 can bind to it (39Zawel L. Yu J. Torrance C.J. Markowitz S. Kinzler K.W. Vogelstein B. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2848-2853Google Scholar). Other researchers have reported that the hypoxia-mimicking agent CoCl2down-regulated the expression of the tumor suppressor gene p53 via the classical E-box (40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). In another study, we observed that DEC1 and DEC2 bound to the classical E-box by electrophoretic mobility shift assays. 3Kawamoto, T., et. al., manuscript in preparation. These observations raise the possibility that DEC1 and DEC2, induced by HIF-1, inhibit the transcription of genes reported to be down-regulated by hypoxia, such as the PPARγ2, α-fetoprotein, and p53 genes via the E-box (33Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Google Scholar, 38Mazure N.M. Chauvet C. Bois-Joyeux B. Bernard M.A. Nacer-Cherif H. Danan J.L. Cancer Res. 2002; 62: 1158-1165Google Scholar,40Lee S.G. Lee H. Rho H.M. FEBS Lett. 2001; 507: 259-263Google Scholar). Identification of the target genes for DEC1 and DEC2 should provide important insights into the molecular mechanism for the responses to hypoxia under physiological and pathological conditions. We thank the Research Centre for Molecular Medicine, Hiroshima University School of Medicine for use of their facilities." @default.
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W2023279237 title "Identification of Functional Hypoxia Response Elements in the Promoter Region of the DEC1 and DEC2 Genes" @default.
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