Adrenomedullin Gene Expression Is Developmentally Regulated and Induced by Hypoxia in Rat Ventricular Cardiac Myocytes
1998; Elsevier BV; Volume: 273; Issue: 28 Linguagem: Inglês
10.1074/jbc.273.28.17787
ISSN1083-351X
AutoresStephania A. Cormier, Son Nguyen, William C. Claycomb,
Tópico(s)Receptor Mechanisms and Signaling
ResumoAdrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at –1095 and –770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5′-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at –1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter. Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at –1095 and –770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5′-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at –1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter. Adrenomedullin (Adm) 1The abbreviations used are: Adm, adrenomedullin; AdmR, adrenomedullin receptor; CGRP, calcitonin gene-related peptide; Epo, erythropoietin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; HIF-1, hypoxia-inducible factor-1; RT-PCR, reverse transcription-polymerase chain reaction; VEGF, vascular endothelial growth factor; kb, kilobase(s); bp, base pair; CMV, cytomegalovirus. 1The abbreviations used are: Adm, adrenomedullin; AdmR, adrenomedullin receptor; CGRP, calcitonin gene-related peptide; Epo, erythropoietin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; HIF-1, hypoxia-inducible factor-1; RT-PCR, reverse transcription-polymerase chain reaction; VEGF, vascular endothelial growth factor; kb, kilobase(s); bp, base pair; CMV, cytomegalovirus. is a recently discovered hypotensive peptide that was first identified in human pheochromocytoma tissue (1Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2063) Google Scholar). The Adm peptide is expressed in a variety of rat tissues including the heart, adrenal medulla, brain, kidney, pancreas, lung, spleen, thyroid, and liver (2Washimine H. Kitamura K. Ichiki Y. Yamamoto Y. Kangawa K. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1994; 202: 1081-1087Crossref PubMed Scopus (103) Google Scholar, 3Sakata J. Shimokubo T. Kitamura K. Nishizono M. Iehiki Y. Kangawa K. Matsuo H. Eto T. FEBS Lett. 1994; 352: 105-108Crossref PubMed Scopus (268) Google Scholar). The rat Adm peptide consists of 50 amino acids and shows slight structural homology to the calcitonin gene-related peptide (CGRP) family (4Muff R. Born W. Fischer J.A. Eur. J. Endocrinol. 1995; 133: 17-20Crossref PubMed Scopus (146) Google Scholar). It is capable of acting through the CGRP receptor and the recently cloned Adm receptor (AdmR) (5Kapas S. Catt K.J. Clark A.J.L. J. Biol. Chem. 1995; 270: 25344-25347Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar).The Adm peptide has been implicated as an important regulator in the renal and cardiovascular systems, where it has been observed to produce a dose-dependent increase in vasodilation (6Richards A.M. Nicholls M.G. Lewis L. Lainchbury J.G. Clin. Sci. 1996; 91: 3-16Crossref PubMed Scopus (117) Google Scholar, 7Kitamura K. Kangawa K. Matsuo H. Eto T. Drugs. 1995; 49: 485-495Crossref PubMed Scopus (60) Google Scholar). The increased vasodilation was associated with a slight increase in glomerular filtration rate and natriuresis in the renal system (8Hirata Y. Hayakawa H. Suzuki Y. Suzuki E. Ikenouchi H. Kohmoto O. Kimura K. Eto T. Kangawa K. Matsuo H. Omata M. Hypertension. 1995; 25: 790-795Crossref PubMed Google Scholar). In the cardiovascular system, Ishiyama et al. (9Ishiyama Y. Kitamura K. Ichiki Y. Sakata J. Kida O. Kangawa K. Eto T. Clin. Exp. Pharmacol. Physiol. 1995; 22: 614-618Crossref PubMed Scopus (39) Google Scholar) reported that the Adm peptide, in addition to producing a fall in blood pressure, elicited an increase in cardiac index and stroke volume without a subsequent change in heart rate.Using the differential display technique (10Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 11Liang P. Averboukh L. Pardee A.B. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar), we analyzed differential gene expression in the developing rat heart. The RNA samples analyzed were isolated from embryonic day 17; neonatal days 10, 17, and 21; and adult ventricular cardiac muscle. We observed 23 differentially expressed genes in this developmental series, and 15 were successfully reamplified and cloned. One of these clones was identified as the Adm gene.In this study, we have determined that the expression of the Adm gene increases significantly in the developing rat heart, while the AdmR mRNA levels remained relatively constant throughout development. Furthermore, Adm mRNA levels increased in cultured adult ventricular cardiac myocytes in response to hypoxia as a function of time. Studies using an Adm promoter-luciferase reporter construct indicated that the increase in Adm mRNA occurred as a result of increased transcription in response to hypoxia. These studies suggest a potential role for Adm in the development of the heart and in the response of cardiomyocytes to hypoxic stress.DISCUSSIONUsing differential display and semiquantitative RT-PCR analysis, we have determined that the expression of the Adm gene is developmentally regulated in the rat heart. Our observation is in accordance with previous immunocytochemical data presented by Montuengaet al. (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Interestingly, they show that the heart is the first organ to express the Adm peptide in both the mouse and rat, with expression first being observed as early as embryonic day eight (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Furthermore, they show that localization of the Adm peptide corresponds to the degree of cellular differentiation in several organs (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). In our studies, Adm mRNA expression increases during cardiac muscle development, indicating a possible role for the Adm peptide in growth and differentiation. Whether Adm plays a role in heart development and, if so, what that role might be remains to be determined.The increased expression of Adm in hypoxic brain tissue (28Wang X. Yue T.L. Barone F.C. White R.F. Clark R.K. Willette R.N. Sulpizio A.C. Aiyar N.V. Ruffolo Jr., R.R. Feuerstein G.Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11480-11484Crossref PubMed Scopus (179) Google Scholar) suggested, to us, a role for Adm in the hypoxic response of the heart. Recent studies have shown an increase in blood levels of Adm peptide in human congestive heart failure (29Jougasaki M. Rodeheffer R.J. Redfield M.M. Yamamoto K. Wei C.-M. McKinley L.J. Burnett Jr., J.C. J. Clin. Invest. 1996; 97: 2370-2376Crossref PubMed Scopus (170) Google Scholar, 30Kato J. Kobayashi K. Etoh T. Tanaka M. Kitamura K. Imamura T. Koiwaya Y. Kangawa K. Eto T. J. Clin. Endocrinol. Metab. 1996; 81: 180-183Crossref PubMed Scopus (173) Google Scholar) and, furthermore, that this Adm peptide secretion originates from the heart and is correlated with the severity of the heart disease (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). In addition, cardiac Adm peptide synthesis and secretion has been demonstrated to be induced in a rat heart failure model (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). Cumulatively, these studies suggest a possible involvement for Adm in the response of the heart to ischemic stress. Our preliminary studies showed that cultured adult ventricular cardiomyocytes secrete the Adm peptide in response to hypoxia.2 In addition, we show here that cardiomyocyte Adm mRNA levels increase temporally in response to hypoxia. This response reached a maximum at 12 h of hypoxia and was approximately 2.3-fold greater than the normoxic control (Fig. 4). Results from our transfection studies indicate that this increase is regulated by the Adm promoter. We found Adm promoter activity to increase 1.8-fold over control after 12 h of hypoxia, as assessed using a luciferase reporter. Although this increase in transcription in response to hypoxia appears to be modest, our data are consistent with that of other hypoxia-responsive genes such as VEGF (25Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar), Src kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), Fyn kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), and Epo (33McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), where 1.7–4.0-fold increases in transcription have been observed. In contrast, the AdmR mRNA levels decreased somewhat with increasing time of exposure to hypoxia.Our studies provide the first evidence that the Adm promoter contains HIF-1 sites that are capable of responding to hypoxia. Hypoxic regulatory elements have been identified in both the 5′- and 3′-flanking regions of numerous hypoxia-inducible genes (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar). We have demonstrated that the 5′-flanking region of the Adm gene was capable of conferring hypoxia responsiveness in transient transfection assays performed in HL-1 cells. Sequence analysis of the mouse Adm 5′-flanking region has revealed the presence of two potential HIF-1 binding sites at –1095 and –770 upstream from the transcription start site. These two sites are 100% identical to the HIF-1 core binding site, 5′-ACGT-3′ (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), and similar to the defined HIF-1 consensus binding site, 5′-BACGTGSK-3′ (23Forsythe J.A. Bing-hua J. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3165) Google Scholar, 24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar) (B = C, G, or T; S = C or G; K = G or T). Mutation analyses of the HIF-1 sites in the mouse Adm 5′-flanking region revealed that there are two functional hypoxia-responsive sites (Fig. 7) and that their disruption leads to a substantial decrease (54%) in the transcriptional response of Adm to hypoxia. We found that the HIF-1 binding site at –1095 was largely responsible for the observed hypoxic response of Adm and that the HIF-1 binding site at –770 was only responsible for a small portion of the hypoxic response. Interestingly, mutation of HIF binding sites at –1095 and –770 were not additive and failed to abolish all transcriptional activity in response to hypoxia. The lack of a complete attenuation of the transcriptional hypoxia response suggests that other as yet unidentified cis-acting elements may be responsible for the full transcriptional response of Adm. A tripartite hypoxia-inducible enhancer has recently been proposed in which elimination of one of these sites in the Epo promoter resulted in a decrease but not a total elimination of the hypoxic response (34Semenza G.L. Trends Cardiovasc. Med. 1996; 6: 151-157Crossref PubMed Scopus (110) Google Scholar). This tripartite enhancer has been proposed for the two other well characterized hypoxia-responsive genes (VEGF and lactate dehydrogenase), and further functional studies may elucidate the presence of such an enhancer in the Adm promoter.The physiological reason for the increase in cardiomyocyte Adm mRNA expression and peptide secretion in response to hypoxia is not clear, but this response may provide a compensatory mechanism for the ischemic heart. Adm is a potent hypotensive peptide (1Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2063) Google Scholar) and has been shown to be important in the regulation of both the renal and cardiovascular systems (35Ishimitsu T. Nishikimi T. Saito Y. Kitamura K. Eto T. Kangawa K. Matsuo H. Omae T. Matsuoka H. J. Clin. Invest. 1994; 94: 2158-2161Crossref PubMed Scopus (471) Google Scholar). In addition to decreasing blood pressure, the Adm peptide is capable of increasing the cardiac index and stroke volume without a subsequent change in the heart rate (9Ishiyama Y. Kitamura K. Ichiki Y. Sakata J. Kida O. Kangawa K. Eto T. Clin. Exp. Pharmacol. Physiol. 1995; 22: 614-618Crossref PubMed Scopus (39) Google Scholar). Further studies are needed to clarify the exact physiological role of the Adm peptide in the response of the heart to hypoxia. Adrenomedullin (Adm) 1The abbreviations used are: Adm, adrenomedullin; AdmR, adrenomedullin receptor; CGRP, calcitonin gene-related peptide; Epo, erythropoietin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; HIF-1, hypoxia-inducible factor-1; RT-PCR, reverse transcription-polymerase chain reaction; VEGF, vascular endothelial growth factor; kb, kilobase(s); bp, base pair; CMV, cytomegalovirus. 1The abbreviations used are: Adm, adrenomedullin; AdmR, adrenomedullin receptor; CGRP, calcitonin gene-related peptide; Epo, erythropoietin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; HIF-1, hypoxia-inducible factor-1; RT-PCR, reverse transcription-polymerase chain reaction; VEGF, vascular endothelial growth factor; kb, kilobase(s); bp, base pair; CMV, cytomegalovirus. is a recently discovered hypotensive peptide that was first identified in human pheochromocytoma tissue (1Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2063) Google Scholar). The Adm peptide is expressed in a variety of rat tissues including the heart, adrenal medulla, brain, kidney, pancreas, lung, spleen, thyroid, and liver (2Washimine H. Kitamura K. Ichiki Y. Yamamoto Y. Kangawa K. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1994; 202: 1081-1087Crossref PubMed Scopus (103) Google Scholar, 3Sakata J. Shimokubo T. Kitamura K. Nishizono M. Iehiki Y. Kangawa K. Matsuo H. Eto T. FEBS Lett. 1994; 352: 105-108Crossref PubMed Scopus (268) Google Scholar). The rat Adm peptide consists of 50 amino acids and shows slight structural homology to the calcitonin gene-related peptide (CGRP) family (4Muff R. Born W. Fischer J.A. Eur. J. Endocrinol. 1995; 133: 17-20Crossref PubMed Scopus (146) Google Scholar). It is capable of acting through the CGRP receptor and the recently cloned Adm receptor (AdmR) (5Kapas S. Catt K.J. Clark A.J.L. J. Biol. Chem. 1995; 270: 25344-25347Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The Adm peptide has been implicated as an important regulator in the renal and cardiovascular systems, where it has been observed to produce a dose-dependent increase in vasodilation (6Richards A.M. Nicholls M.G. Lewis L. Lainchbury J.G. Clin. Sci. 1996; 91: 3-16Crossref PubMed Scopus (117) Google Scholar, 7Kitamura K. Kangawa K. Matsuo H. Eto T. Drugs. 1995; 49: 485-495Crossref PubMed Scopus (60) Google Scholar). The increased vasodilation was associated with a slight increase in glomerular filtration rate and natriuresis in the renal system (8Hirata Y. Hayakawa H. Suzuki Y. Suzuki E. Ikenouchi H. Kohmoto O. Kimura K. Eto T. Kangawa K. Matsuo H. Omata M. Hypertension. 1995; 25: 790-795Crossref PubMed Google Scholar). In the cardiovascular system, Ishiyama et al. (9Ishiyama Y. Kitamura K. Ichiki Y. Sakata J. Kida O. Kangawa K. Eto T. Clin. Exp. Pharmacol. Physiol. 1995; 22: 614-618Crossref PubMed Scopus (39) Google Scholar) reported that the Adm peptide, in addition to producing a fall in blood pressure, elicited an increase in cardiac index and stroke volume without a subsequent change in heart rate. Using the differential display technique (10Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar, 11Liang P. Averboukh L. Pardee A.B. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (883) Google Scholar), we analyzed differential gene expression in the developing rat heart. The RNA samples analyzed were isolated from embryonic day 17; neonatal days 10, 17, and 21; and adult ventricular cardiac muscle. We observed 23 differentially expressed genes in this developmental series, and 15 were successfully reamplified and cloned. One of these clones was identified as the Adm gene. In this study, we have determined that the expression of the Adm gene increases significantly in the developing rat heart, while the AdmR mRNA levels remained relatively constant throughout development. Furthermore, Adm mRNA levels increased in cultured adult ventricular cardiac myocytes in response to hypoxia as a function of time. Studies using an Adm promoter-luciferase reporter construct indicated that the increase in Adm mRNA occurred as a result of increased transcription in response to hypoxia. These studies suggest a potential role for Adm in the development of the heart and in the response of cardiomyocytes to hypoxic stress. DISCUSSIONUsing differential display and semiquantitative RT-PCR analysis, we have determined that the expression of the Adm gene is developmentally regulated in the rat heart. Our observation is in accordance with previous immunocytochemical data presented by Montuengaet al. (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Interestingly, they show that the heart is the first organ to express the Adm peptide in both the mouse and rat, with expression first being observed as early as embryonic day eight (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Furthermore, they show that localization of the Adm peptide corresponds to the degree of cellular differentiation in several organs (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). In our studies, Adm mRNA expression increases during cardiac muscle development, indicating a possible role for the Adm peptide in growth and differentiation. Whether Adm plays a role in heart development and, if so, what that role might be remains to be determined.The increased expression of Adm in hypoxic brain tissue (28Wang X. Yue T.L. Barone F.C. White R.F. Clark R.K. Willette R.N. Sulpizio A.C. Aiyar N.V. Ruffolo Jr., R.R. Feuerstein G.Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11480-11484Crossref PubMed Scopus (179) Google Scholar) suggested, to us, a role for Adm in the hypoxic response of the heart. Recent studies have shown an increase in blood levels of Adm peptide in human congestive heart failure (29Jougasaki M. Rodeheffer R.J. Redfield M.M. Yamamoto K. Wei C.-M. McKinley L.J. Burnett Jr., J.C. J. Clin. Invest. 1996; 97: 2370-2376Crossref PubMed Scopus (170) Google Scholar, 30Kato J. Kobayashi K. Etoh T. Tanaka M. Kitamura K. Imamura T. Koiwaya Y. Kangawa K. Eto T. J. Clin. Endocrinol. Metab. 1996; 81: 180-183Crossref PubMed Scopus (173) Google Scholar) and, furthermore, that this Adm peptide secretion originates from the heart and is correlated with the severity of the heart disease (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). In addition, cardiac Adm peptide synthesis and secretion has been demonstrated to be induced in a rat heart failure model (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). Cumulatively, these studies suggest a possible involvement for Adm in the response of the heart to ischemic stress. Our preliminary studies showed that cultured adult ventricular cardiomyocytes secrete the Adm peptide in response to hypoxia.2 In addition, we show here that cardiomyocyte Adm mRNA levels increase temporally in response to hypoxia. This response reached a maximum at 12 h of hypoxia and was approximately 2.3-fold greater than the normoxic control (Fig. 4). Results from our transfection studies indicate that this increase is regulated by the Adm promoter. We found Adm promoter activity to increase 1.8-fold over control after 12 h of hypoxia, as assessed using a luciferase reporter. Although this increase in transcription in response to hypoxia appears to be modest, our data are consistent with that of other hypoxia-responsive genes such as VEGF (25Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar), Src kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), Fyn kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), and Epo (33McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), where 1.7–4.0-fold increases in transcription have been observed. In contrast, the AdmR mRNA levels decreased somewhat with increasing time of exposure to hypoxia.Our studies provide the first evidence that the Adm promoter contains HIF-1 sites that are capable of responding to hypoxia. Hypoxic regulatory elements have been identified in both the 5′- and 3′-flanking regions of numerous hypoxia-inducible genes (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar). We have demonstrated that the 5′-flanking region of the Adm gene was capable of conferring hypoxia responsiveness in transient transfection assays performed in HL-1 cells. Sequence analysis of the mouse Adm 5′-flanking region has revealed the presence of two potential HIF-1 binding sites at –1095 and –770 upstream from the transcription start site. These two sites are 100% identical to the HIF-1 core binding site, 5′-ACGT-3′ (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), and similar to the defined HIF-1 consensus binding site, 5′-BACGTGSK-3′ (23Forsythe J.A. Bing-hua J. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3165) Google Scholar, 24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar) (B = C, G, or T; S = C or G; K = G or T). Mutation analyses of the HIF-1 sites in the mouse Adm 5′-flanking region revealed that there are two functional hypoxia-responsive sites (Fig. 7) and that their disruption leads to a substantial decrease (54%) in the transcriptional response of Adm to hypoxia. We found that the HIF-1 binding site at –1095 was largely responsible for the observed hypoxic response of Adm and that the HIF-1 binding site at –770 was only responsible for a small portion of the hypoxic response. Interestingly, mutation of HIF binding sites at –1095 and –770 were not additive and failed to abolish all transcriptional activity in response to hypoxia. The lack of a complete attenuation of the transcriptional hypoxia response suggests that other as yet unidentified cis-acting elements may be responsible for the full transcriptional response of Adm. A tripartite hypoxia-inducible enhancer has recently been proposed in which elimination of one of these sites in the Epo promoter resulted in a decrease but not a total elimination of the hypoxic response (34Semenza G.L. Trends Cardiovasc. Med. 1996; 6: 151-157Crossref PubMed Scopus (110) Google Scholar). This tripartite enhancer has been proposed for the two other well characterized hypoxia-responsive genes (VEGF and lactate dehydrogenase), and further functional studies may elucidate the presence of such an enhancer in the Adm promoter.The physiological reason for the increase in cardiomyocyte Adm mRNA expression and peptide secretion in response to hypoxia is not clear, but this response may provide a compensatory mechanism for the ischemic heart. Adm is a potent hypotensive peptide (1Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2063) Google Scholar) and has been shown to be important in the regulation of both the renal and cardiovascular systems (35Ishimitsu T. Nishikimi T. Saito Y. Kitamura K. Eto T. Kangawa K. Matsuo H. Omae T. Matsuoka H. J. Clin. Invest. 1994; 94: 2158-2161Crossref PubMed Scopus (471) Google Scholar). In addition to decreasing blood pressure, the Adm peptide is capable of increasing the cardiac index and stroke volume without a subsequent change in the heart rate (9Ishiyama Y. Kitamura K. Ichiki Y. Sakata J. Kida O. Kangawa K. Eto T. Clin. Exp. Pharmacol. Physiol. 1995; 22: 614-618Crossref PubMed Scopus (39) Google Scholar). Further studies are needed to clarify the exact physiological role of the Adm peptide in the response of the heart to hypoxia. Using differential display and semiquantitative RT-PCR analysis, we have determined that the expression of the Adm gene is developmentally regulated in the rat heart. Our observation is in accordance with previous immunocytochemical data presented by Montuengaet al. (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Interestingly, they show that the heart is the first organ to express the Adm peptide in both the mouse and rat, with expression first being observed as early as embryonic day eight (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). Furthermore, they show that localization of the Adm peptide corresponds to the degree of cellular differentiation in several organs (27Montuenga L.M. Martı́nez A. Miller M.J. Unsworth E.J. Cuttitta F. Endocrinology. 1997; 138: 1-12Crossref Scopus (171) Google Scholar). In our studies, Adm mRNA expression increases during cardiac muscle development, indicating a possible role for the Adm peptide in growth and differentiation. Whether Adm plays a role in heart development and, if so, what that role might be remains to be determined. The increased expression of Adm in hypoxic brain tissue (28Wang X. Yue T.L. Barone F.C. White R.F. Clark R.K. Willette R.N. Sulpizio A.C. Aiyar N.V. Ruffolo Jr., R.R. Feuerstein G.Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11480-11484Crossref PubMed Scopus (179) Google Scholar) suggested, to us, a role for Adm in the hypoxic response of the heart. Recent studies have shown an increase in blood levels of Adm peptide in human congestive heart failure (29Jougasaki M. Rodeheffer R.J. Redfield M.M. Yamamoto K. Wei C.-M. McKinley L.J. Burnett Jr., J.C. J. Clin. Invest. 1996; 97: 2370-2376Crossref PubMed Scopus (170) Google Scholar, 30Kato J. Kobayashi K. Etoh T. Tanaka M. Kitamura K. Imamura T. Koiwaya Y. Kangawa K. Eto T. J. Clin. Endocrinol. Metab. 1996; 81: 180-183Crossref PubMed Scopus (173) Google Scholar) and, furthermore, that this Adm peptide secretion originates from the heart and is correlated with the severity of the heart disease (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). In addition, cardiac Adm peptide synthesis and secretion has been demonstrated to be induced in a rat heart failure model (31Nishikimi T. Horio T. Sasaki T. Yoshihara F. Takishita S. Miyata A. Matsuo H. Kangawa K. Hypertension. 1997; 30: 1369-1375Crossref PubMed Scopus (95) Google Scholar). Cumulatively, these studies suggest a possible involvement for Adm in the response of the heart to ischemic stress. Our preliminary studies showed that cultured adult ventricular cardiomyocytes secrete the Adm peptide in response to hypoxia.2 In addition, we show here that cardiomyocyte Adm mRNA levels increase temporally in response to hypoxia. This response reached a maximum at 12 h of hypoxia and was approximately 2.3-fold greater than the normoxic control (Fig. 4). Results from our transfection studies indicate that this increase is regulated by the Adm promoter. We found Adm promoter activity to increase 1.8-fold over control after 12 h of hypoxia, as assessed using a luciferase reporter. Although this increase in transcription in response to hypoxia appears to be modest, our data are consistent with that of other hypoxia-responsive genes such as VEGF (25Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar), Src kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), Fyn kinase (32Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (49) Google Scholar), and Epo (33McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), where 1.7–4.0-fold increases in transcription have been observed. In contrast, the AdmR mRNA levels decreased somewhat with increasing time of exposure to hypoxia. Our studies provide the first evidence that the Adm promoter contains HIF-1 sites that are capable of responding to hypoxia. Hypoxic regulatory elements have been identified in both the 5′- and 3′-flanking regions of numerous hypoxia-inducible genes (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar). We have demonstrated that the 5′-flanking region of the Adm gene was capable of conferring hypoxia responsiveness in transient transfection assays performed in HL-1 cells. Sequence analysis of the mouse Adm 5′-flanking region has revealed the presence of two potential HIF-1 binding sites at –1095 and –770 upstream from the transcription start site. These two sites are 100% identical to the HIF-1 core binding site, 5′-ACGT-3′ (24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), and similar to the defined HIF-1 consensus binding site, 5′-BACGTGSK-3′ (23Forsythe J.A. Bing-hua J. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3165) Google Scholar, 24Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar) (B = C, G, or T; S = C or G; K = G or T). Mutation analyses of the HIF-1 sites in the mouse Adm 5′-flanking region revealed that there are two functional hypoxia-responsive sites (Fig. 7) and that their disruption leads to a substantial decrease (54%) in the transcriptional response of Adm to hypoxia. We found that the HIF-1 binding site at –1095 was largely responsible for the observed hypoxic response of Adm and that the HIF-1 binding site at –770 was only responsible for a small portion of the hypoxic response. Interestingly, mutation of HIF binding sites at –1095 and –770 were not additive and failed to abolish all transcriptional activity in response to hypoxia. The lack of a complete attenuation of the transcriptional hypoxia response suggests that other as yet unidentified cis-acting elements may be responsible for the full transcriptional response of Adm. A tripartite hypoxia-inducible enhancer has recently been proposed in which elimination of one of these sites in the Epo promoter resulted in a decrease but not a total elimination of the hypoxic response (34Semenza G.L. Trends Cardiovasc. Med. 1996; 6: 151-157Crossref PubMed Scopus (110) Google Scholar). This tripartite enhancer has been proposed for the two other well characterized hypoxia-responsive genes (VEGF and lactate dehydrogenase), and further functional studies may elucidate the presence of such an enhancer in the Adm promoter. The physiological reason for the increase in cardiomyocyte Adm mRNA expression and peptide secretion in response to hypoxia is not clear, but this response may provide a compensatory mechanism for the ischemic heart. Adm is a potent hypotensive peptide (1Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2063) Google Scholar) and has been shown to be important in the regulation of both the renal and cardiovascular systems (35Ishimitsu T. Nishikimi T. Saito Y. Kitamura K. Eto T. Kangawa K. Matsuo H. Omae T. Matsuoka H. J. Clin. Invest. 1994; 94: 2158-2161Crossref PubMed Scopus (471) Google Scholar). In addition to decreasing blood pressure, the Adm peptide is capable of increasing the cardiac index and stroke volume without a subsequent change in the heart rate (9Ishiyama Y. Kitamura K. Ichiki Y. Sakata J. Kida O. Kangawa K. Eto T. Clin. Exp. Pharmacol. Physiol. 1995; 22: 614-618Crossref PubMed Scopus (39) Google Scholar). Further studies are needed to clarify the exact physiological role of the Adm peptide in the response of the heart to hypoxia. We thank Dr. E. J. Taparowsky of Purdue University for the pGL2BmgAM5′-3′ vector, Dr. Jawed Alam of the Alton Ochsner Medical Foundation for the pBT and the pBTmut vectors, and Chad Donaldson for assistance in sequencing the differential display clones.
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