ACE ACE Inhibitors and Other JNK
From the Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa.y[+!;4, 百拇医药
Key Words: intracellular signaling c-Jun N-terminal kinase c-Jun • angiotensin-converting enzyme angiotensin-converting enzyme inhibitorsy[+!;4, 百拇医药
The renin-angiotensin system (RAS) plays a pivotal role in the regulation of blood pressure, volume homeostasis, vascular function, and cell growth. In what is considered the classic RAS, renin is released from juxtaglomerular cells of the kidney into the circulation where it converts angiotensinogen from the liver to angiotensin I. Angiotensin I is subsequently hydrolyzed by a peptidyl dipeptidase, angiotensin-converting enzyme (ACE), from the lung to form angiotensin II. Investigators have been cognizant of renin for more than a century after its initial discovery in 1898 by Tigerstedt and Bergman (see review1). However, it was not until the middle of the 20th century that the remaining components of RAS were purified and identified by Skeggs and colleagues. These included angiotensinogen, angiotensin I, angiotensin II, and ACE, which at the time was termed "hypertensin-converting enzyme" (see review1). Since that time, many components of the RAS, especially ACE, have received considerable attention as a focal point for researchers interested in better understanding the regulation of the cardiovascular system.
Considering the rapid progress in understanding the molecular physiology of RAS and its many complexities, it should not be surprising that details would continue to emerge. It is unexpected, however, that after 100 years of research major new concepts would surface requiring scientists and clinicians to rethink the system and its role in cardiovascular regulation. Even more astounding is that over the span of 3 years, two major conceptual changes would have to be considered regarding ACE. The first surprise occurred in 2000 with the discovery of ACE2, a "homologue" of ACE capable of producing angiotensin peptides such as Ang-(1-7), which may have vasodilator properties.2,3Thus, ACE and ACE2 may ultimately have opposing physiological effects. The second new concept is described in this issue of Circulation Research.4e)ap, 百拇医药
Introduction to ACEe)ap, 百拇医药
The ACE gene yields two different protein products resulting from the use of different promoters most likely caused during evolution by genetic duplication.5 In humans, ACE resides on chromosome 17 (chromosome 11 and 10 in mouse and rats, respectively) and consists of 25 exons. Germinal ACE, expressed only in testes, arises from a promoter located within intron 12 (downstream of the gene duplication) and makes a protein with only a single domain and catalytic site.6 Its expression is critical for normal male fertility. Somatic ACE is a protein with two homologous domains and two catalytic sites (see review7). Gene-targeted deletions of somatic ACE in mice cause hypotension, improper kidney development, and reduced fertility in males.8,9
The somatic ACE isoform has been the most extensively investigated because of its importance in cardiovascular homeostasis. The two homologous domains and the amino terminus of the ACE protein are located extracellularly. The protein is anchored in the membrane and contains a short intracellular domain at the carboxy terminal (see Figure). ACE is highly expressed in the vascular endothelium as well as in the brush border membranes of the kidney. The classic biological function of ACE is to hydrolyze a dipeptide from the carboxy terminus of a protein substrate. While ACE has the potential to hydrolyze many proteins, it is most appreciated for its enzymatic processing of angiotensin I to angiotensin II, the cardiovascular effects of which have been extensively reported. In addition, ACE mediates the hydrolysis of bradykinin, which is typically considered to have blood pressure–lowering and cardioprotective effects. Interestingly, ACE more readily hydrolyzes bradykinin than it does angiotensin I. Therefore, the net physiological effect of ACE is to increase the production of a vasoconstrictor and decrease the availability of a vasodilator.
fig.ommittedj(dkftw, 百拇医药
ACE-mediated outside-in signaling pathway in endothelial cells. ACE inhibitors or substrate (bradykinin, BK) increases CK2-mediated phosphorylation of ser1270 on the carboxy terminal of ACE. Phosphorylation of ser1270 increases ACE-associated JNK activity, which phosphorylates c-Jun. c-Jun is translocated to the nucleus, and the expression of ACE and perhaps other genes is increased.j(dkftw, 百拇医药
This dual effect makes ACE a particularly appealing target for antihypertensive therapies; and indeed ACE has been an important therapeutic target for the treatment of hypertension since the development of captopril in the late 1970s. The initial benefit of ACE inhibition for patients with hypertension was thought to be due in large part to a decreased production of angiotensin II. However, it has been recognized that long-term treatment with ACE inhibitors does not necessarily lower plasma or tissue levels of angiotensin II.10,11 Moreover, the effect of ACE inhibitors to prevent the degradation of bradykinin has been recognized as making an increasingly important contribution to the cardiovascular benefits for patients taking these drugs. Therefore, the physiological functions of ACE, its substrates, and its inhibitors are still evolving.
ACE as an Intracellular Signal Transducerr#, 百拇医药
In this issue of Circulation Research, Kohlstedt et al4 skillfully elucidated a novel function for ACE as a key signal transduction molecule, thus reminding us that our understanding of the physiological role for ACE is still surfacing. These authors previously reported that a specific carboxy terminal serine residue of ACE (ser1270) was phosphorylated by protein kinase CK2. Phosphorylation of ser1270 is important for the retention of ACE in the membrane.12 In the present study, they examined the effect of ACE inhibitors (ramiprilat and perindoprilat) and ACE substrates (angiotensin I and bradykinin) on CK2-mediated phosphorylation of ser1270 and the downstream signaling that results.r#, 百拇医药
Using human umbilical vein endothelial cells or porcine aortic endothelial cells stably transfected with human somatic ACE as a model, the authors demonstrate that ramiprilat and perindoprilat increase CK2-mediated phosphorylation of ser1270. Coimmunoprecipitation studies reveal that ACE associates with CK2, and that both inhibitors can increase ACE-associated CK2 activity. Interestingly, bradykinin, but not angiotensin I, increases ser1270 phosphorylation and CK2 activity, suggesting that a naturally occurring signal transduction pathway for bradykinin may exist that does not require its classical receptor. Importantly, the possibility that the CK2-mediated phosphorylation is the result of crosstalk between signal transduction pathways was ruled out in control experiments by the use of porcine endothelial cells, which do not have angiotensin type 1 or bradykinin B2 receptors.
Subsequently, two more ACE-associated proteins, c-Jun N-terminal kinase (JNK) and MAP kinase kinase 7 (MKK7), were identified in addition to CK2. The authors not only report the association of these proteins with ACE, but they also provide convincing evidence that ACE-associated JNK activity is increased in cells treated with ACE inhibitors or bradykinin. Interestingly, JNK activation, but not its binding to ACE, is dependent on the phosphorylation of ACE ser1270. Activation of JNK by ramiprilat results in the accumulation of phosphorylated c-Jun in the nucleus, which has been reported to activate transcription of ACE.13 ACE increased in response to ramiprilat via a mechanism dependent on JNK activity in human umbilical vein endothelial cells in culture and in mouse lung in vivo.)8v, 百拇医药
By itself, the elucidation of this signaling pathway in cell culture is a novel and exciting finding that will likely alter perceptions on the physiological roles for ACE and lead to new areas of investigation regarding the beneficial effects of ACE inhibitors. What makes this study even more convincing is that the authors were able to demonstrate that the signaling pathway initiated by ACE inhibitors in cell culture can also be activated in the whole animal. This suggests that the intracellular signaling (termed "outside-in" signaling by the authors) mediated by ACE inhibitors and bradykinin may be an important physiological mechanism. Of course, understanding the full spectrum of physiological events initiated by ACE signaling will require additional studies.
With this, ACE joins other ectoenzymes, such as matrix metalloprotease-1 (MMP-1), which have been reported to link to intracellular signaling pathways.14 It is provocative to speculate that ACE may mediate intracellular signals in response to bradykinin while simultaneously destroying its ability to act as a vasodilator. Such a mechanism, although still unproven, would tightly regulate the local concentration of ACE "ligand." Presumably, this level of control would not be exerted on ACE inhibitors since they would bind but not be catalytically processed. In closing, the study by Kohlstedt et al4 reminds us of the complexities of the RAS. It is likely that additional new details of this pathway as well as novel concepts will continue to emerge as these studies progress.]][3-#, 百拇医药
Footnotes]][3-#, 百拇医药
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.]][3-#, 百拇医药
References
Hall JE. Historical perspective of the renin-angiotensin system. Mol Biotechnol. 2003; 24: 27–39.!.[1;, 百拇医药
Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem. 2000; 275: 33238–33243.!.[1;, 百拇医药
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000; 87: e1–e9.!.[1;, 百拇医药
Kohlstedt K, Brandes RP, Müller-Esterl W, Busse R, Fleming I. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res. 2004; 94: 60–67.!.[1;, 百拇医药
Kumar RS, Thekkumkara TJ, Sen GC. The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissue-specific choice of alternative transcription initiation sites. J Biol Chem. 1991; 266: 3854–3862.!.[1;, 百拇医药
Sen GC, Thekkumkara TJ, Kumar RS. Angiotensin-converting enzyme: structural relationship of the testicular and the pulmonary forms. J Cardiovasc Pharmacol. 1990; 16 (suppl 4): S14–S18.
Walder RY, Garrett MR, McClain AM, Beck GE, Brennan TMH, Kramer NA, Kanis AB, Mark AL, Rapp JP, Sheffield VC. Short tandem repeat polymorphic markers for the rat genome from marker-selected libraries. Mamm Genome. 1998; 9: 1013–1021.c\?h, 百拇医药
Esther CR Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest. 1996; 74: 953–965.c\?h, 百拇医药
Krege JH, John SWM, Langenbach LL, Hodgin JB, Hagaman JR, Bachman ES, Jennette JC, O’Brien DA, Smithies O. Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature. 1995; 375: 146–148.c\?h, 百拇医药
Farquharson CA, Struthers AD. Gradual reactivation over time of vascular tissue angiotensin I to angiotensin II conversion during chronic lisinopril therapy in chronic heart failure. J Am Coll Cardiol. 2002; 39: 767–775.c\?h, 百拇医药
Van Kats JP, Duncker DJ, Haitsma DB, Schuijt MP, Niebuur R, Stubenitsky R, Boomsma F, Schalekamp MA, Verdouw PD, Danser AH. Angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs after myocardial infarction: role of tissue angiotensin II. Circulation. 2000; 102: 1556–1563.c\?h, 百拇医药
Kohlstedt K, Shoghi F, Muller-Esterl W, Busse R, Fleming I. CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane. Circ Res. 2002; 91: 749–756.c\?h, 百拇医药
Eyries M, Agrapart M, Alonso A, Soubrier F. Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway. Circ Res. 2002; 91: 899–906.c\?h, 百拇医药
Galt SW, Lindemann S, Allen L, Medd DJ, Falk JM, McIntyre TM, Prescott SM, Kraiss LW, Zimmerman GA, Weyrich AS. Outside-in signals delivered by matrix metalloproteinase-1 regulate platelet function. Circ Res. 2002; 90: 1093–1099.(Michael J- Ryan Curt D- Sigmund)
Key Words: intracellular signaling c-Jun N-terminal kinase c-Jun • angiotensin-converting enzyme angiotensin-converting enzyme inhibitorsy[+!;4, 百拇医药
The renin-angiotensin system (RAS) plays a pivotal role in the regulation of blood pressure, volume homeostasis, vascular function, and cell growth. In what is considered the classic RAS, renin is released from juxtaglomerular cells of the kidney into the circulation where it converts angiotensinogen from the liver to angiotensin I. Angiotensin I is subsequently hydrolyzed by a peptidyl dipeptidase, angiotensin-converting enzyme (ACE), from the lung to form angiotensin II. Investigators have been cognizant of renin for more than a century after its initial discovery in 1898 by Tigerstedt and Bergman (see review1). However, it was not until the middle of the 20th century that the remaining components of RAS were purified and identified by Skeggs and colleagues. These included angiotensinogen, angiotensin I, angiotensin II, and ACE, which at the time was termed "hypertensin-converting enzyme" (see review1). Since that time, many components of the RAS, especially ACE, have received considerable attention as a focal point for researchers interested in better understanding the regulation of the cardiovascular system.
Considering the rapid progress in understanding the molecular physiology of RAS and its many complexities, it should not be surprising that details would continue to emerge. It is unexpected, however, that after 100 years of research major new concepts would surface requiring scientists and clinicians to rethink the system and its role in cardiovascular regulation. Even more astounding is that over the span of 3 years, two major conceptual changes would have to be considered regarding ACE. The first surprise occurred in 2000 with the discovery of ACE2, a "homologue" of ACE capable of producing angiotensin peptides such as Ang-(1-7), which may have vasodilator properties.2,3Thus, ACE and ACE2 may ultimately have opposing physiological effects. The second new concept is described in this issue of Circulation Research.4e)ap, 百拇医药
Introduction to ACEe)ap, 百拇医药
The ACE gene yields two different protein products resulting from the use of different promoters most likely caused during evolution by genetic duplication.5 In humans, ACE resides on chromosome 17 (chromosome 11 and 10 in mouse and rats, respectively) and consists of 25 exons. Germinal ACE, expressed only in testes, arises from a promoter located within intron 12 (downstream of the gene duplication) and makes a protein with only a single domain and catalytic site.6 Its expression is critical for normal male fertility. Somatic ACE is a protein with two homologous domains and two catalytic sites (see review7). Gene-targeted deletions of somatic ACE in mice cause hypotension, improper kidney development, and reduced fertility in males.8,9
The somatic ACE isoform has been the most extensively investigated because of its importance in cardiovascular homeostasis. The two homologous domains and the amino terminus of the ACE protein are located extracellularly. The protein is anchored in the membrane and contains a short intracellular domain at the carboxy terminal (see Figure). ACE is highly expressed in the vascular endothelium as well as in the brush border membranes of the kidney. The classic biological function of ACE is to hydrolyze a dipeptide from the carboxy terminus of a protein substrate. While ACE has the potential to hydrolyze many proteins, it is most appreciated for its enzymatic processing of angiotensin I to angiotensin II, the cardiovascular effects of which have been extensively reported. In addition, ACE mediates the hydrolysis of bradykinin, which is typically considered to have blood pressure–lowering and cardioprotective effects. Interestingly, ACE more readily hydrolyzes bradykinin than it does angiotensin I. Therefore, the net physiological effect of ACE is to increase the production of a vasoconstrictor and decrease the availability of a vasodilator.
fig.ommittedj(dkftw, 百拇医药
ACE-mediated outside-in signaling pathway in endothelial cells. ACE inhibitors or substrate (bradykinin, BK) increases CK2-mediated phosphorylation of ser1270 on the carboxy terminal of ACE. Phosphorylation of ser1270 increases ACE-associated JNK activity, which phosphorylates c-Jun. c-Jun is translocated to the nucleus, and the expression of ACE and perhaps other genes is increased.j(dkftw, 百拇医药
This dual effect makes ACE a particularly appealing target for antihypertensive therapies; and indeed ACE has been an important therapeutic target for the treatment of hypertension since the development of captopril in the late 1970s. The initial benefit of ACE inhibition for patients with hypertension was thought to be due in large part to a decreased production of angiotensin II. However, it has been recognized that long-term treatment with ACE inhibitors does not necessarily lower plasma or tissue levels of angiotensin II.10,11 Moreover, the effect of ACE inhibitors to prevent the degradation of bradykinin has been recognized as making an increasingly important contribution to the cardiovascular benefits for patients taking these drugs. Therefore, the physiological functions of ACE, its substrates, and its inhibitors are still evolving.
ACE as an Intracellular Signal Transducerr#, 百拇医药
In this issue of Circulation Research, Kohlstedt et al4 skillfully elucidated a novel function for ACE as a key signal transduction molecule, thus reminding us that our understanding of the physiological role for ACE is still surfacing. These authors previously reported that a specific carboxy terminal serine residue of ACE (ser1270) was phosphorylated by protein kinase CK2. Phosphorylation of ser1270 is important for the retention of ACE in the membrane.12 In the present study, they examined the effect of ACE inhibitors (ramiprilat and perindoprilat) and ACE substrates (angiotensin I and bradykinin) on CK2-mediated phosphorylation of ser1270 and the downstream signaling that results.r#, 百拇医药
Using human umbilical vein endothelial cells or porcine aortic endothelial cells stably transfected with human somatic ACE as a model, the authors demonstrate that ramiprilat and perindoprilat increase CK2-mediated phosphorylation of ser1270. Coimmunoprecipitation studies reveal that ACE associates with CK2, and that both inhibitors can increase ACE-associated CK2 activity. Interestingly, bradykinin, but not angiotensin I, increases ser1270 phosphorylation and CK2 activity, suggesting that a naturally occurring signal transduction pathway for bradykinin may exist that does not require its classical receptor. Importantly, the possibility that the CK2-mediated phosphorylation is the result of crosstalk between signal transduction pathways was ruled out in control experiments by the use of porcine endothelial cells, which do not have angiotensin type 1 or bradykinin B2 receptors.
Subsequently, two more ACE-associated proteins, c-Jun N-terminal kinase (JNK) and MAP kinase kinase 7 (MKK7), were identified in addition to CK2. The authors not only report the association of these proteins with ACE, but they also provide convincing evidence that ACE-associated JNK activity is increased in cells treated with ACE inhibitors or bradykinin. Interestingly, JNK activation, but not its binding to ACE, is dependent on the phosphorylation of ACE ser1270. Activation of JNK by ramiprilat results in the accumulation of phosphorylated c-Jun in the nucleus, which has been reported to activate transcription of ACE.13 ACE increased in response to ramiprilat via a mechanism dependent on JNK activity in human umbilical vein endothelial cells in culture and in mouse lung in vivo.)8v, 百拇医药
By itself, the elucidation of this signaling pathway in cell culture is a novel and exciting finding that will likely alter perceptions on the physiological roles for ACE and lead to new areas of investigation regarding the beneficial effects of ACE inhibitors. What makes this study even more convincing is that the authors were able to demonstrate that the signaling pathway initiated by ACE inhibitors in cell culture can also be activated in the whole animal. This suggests that the intracellular signaling (termed "outside-in" signaling by the authors) mediated by ACE inhibitors and bradykinin may be an important physiological mechanism. Of course, understanding the full spectrum of physiological events initiated by ACE signaling will require additional studies.
With this, ACE joins other ectoenzymes, such as matrix metalloprotease-1 (MMP-1), which have been reported to link to intracellular signaling pathways.14 It is provocative to speculate that ACE may mediate intracellular signals in response to bradykinin while simultaneously destroying its ability to act as a vasodilator. Such a mechanism, although still unproven, would tightly regulate the local concentration of ACE "ligand." Presumably, this level of control would not be exerted on ACE inhibitors since they would bind but not be catalytically processed. In closing, the study by Kohlstedt et al4 reminds us of the complexities of the RAS. It is likely that additional new details of this pathway as well as novel concepts will continue to emerge as these studies progress.]][3-#, 百拇医药
Footnotes]][3-#, 百拇医药
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.]][3-#, 百拇医药
References
Hall JE. Historical perspective of the renin-angiotensin system. Mol Biotechnol. 2003; 24: 27–39.!.[1;, 百拇医药
Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem. 2000; 275: 33238–33243.!.[1;, 百拇医药
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000; 87: e1–e9.!.[1;, 百拇医药
Kohlstedt K, Brandes RP, Müller-Esterl W, Busse R, Fleming I. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res. 2004; 94: 60–67.!.[1;, 百拇医药
Kumar RS, Thekkumkara TJ, Sen GC. The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissue-specific choice of alternative transcription initiation sites. J Biol Chem. 1991; 266: 3854–3862.!.[1;, 百拇医药
Sen GC, Thekkumkara TJ, Kumar RS. Angiotensin-converting enzyme: structural relationship of the testicular and the pulmonary forms. J Cardiovasc Pharmacol. 1990; 16 (suppl 4): S14–S18.
Walder RY, Garrett MR, McClain AM, Beck GE, Brennan TMH, Kramer NA, Kanis AB, Mark AL, Rapp JP, Sheffield VC. Short tandem repeat polymorphic markers for the rat genome from marker-selected libraries. Mamm Genome. 1998; 9: 1013–1021.c\?h, 百拇医药
Esther CR Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest. 1996; 74: 953–965.c\?h, 百拇医药
Krege JH, John SWM, Langenbach LL, Hodgin JB, Hagaman JR, Bachman ES, Jennette JC, O’Brien DA, Smithies O. Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature. 1995; 375: 146–148.c\?h, 百拇医药
Farquharson CA, Struthers AD. Gradual reactivation over time of vascular tissue angiotensin I to angiotensin II conversion during chronic lisinopril therapy in chronic heart failure. J Am Coll Cardiol. 2002; 39: 767–775.c\?h, 百拇医药
Van Kats JP, Duncker DJ, Haitsma DB, Schuijt MP, Niebuur R, Stubenitsky R, Boomsma F, Schalekamp MA, Verdouw PD, Danser AH. Angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs after myocardial infarction: role of tissue angiotensin II. Circulation. 2000; 102: 1556–1563.c\?h, 百拇医药
Kohlstedt K, Shoghi F, Muller-Esterl W, Busse R, Fleming I. CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane. Circ Res. 2002; 91: 749–756.c\?h, 百拇医药
Eyries M, Agrapart M, Alonso A, Soubrier F. Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway. Circ Res. 2002; 91: 899–906.c\?h, 百拇医药
Galt SW, Lindemann S, Allen L, Medd DJ, Falk JM, McIntyre TM, Prescott SM, Kraiss LW, Zimmerman GA, Weyrich AS. Outside-in signals delivered by matrix metalloproteinase-1 regulate platelet function. Circ Res. 2002; 90: 1093–1099.(Michael J- Ryan Curt D- Sigmund)