- Email: [email protected]
Reply: cytochrome P450-derived eicosanoids and the vascular wall
COMMENT metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolases. However, because DHETs also cause vasodilatation and inhibit CAM expression4, inhibition of EET metabolism is not a useful approach therapeutically. The expression of some cytochrome P450 isozymes is increased by xenobiotics; however, inducers of CYP2J2 have not been identified and inducers of CYP2C8 are toxic12. Xenobiotics might suppress the immune response by induction of epoxygenase(s) and an increase in EET synthesis. Future research might provide ways to upregulate endothelial epoxygenases or identify stable, orally active EET agonists and thereby take advantage of this pathway to decrease inflammation, produce vasodilatation, or both. Because of the different mechanisms of action of the EETs, it might
be possible to develop EET analogs with anti-inflammatory activity but devoid of the vasodilator or endocrine actions. 7
Selected references 1 Rosolowsky, M. and Campbell, W.B. (1996) Synthesis of hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells. Biochim. Biophys. Acta 1299, 267–277 2 Campbell, W.B. et al. (1996) Identification of epoxyeicosatrienoic acids as endotheliumderived hyperpolarizing factors. Circ. Res. 78, 415–423 3 Fisslthaler, B. et al. (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401, 493–497 4 Node, K. et al. (1999) Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science 285, 1276–1279 5 Lin, J.H-C. et al. (1996) Human umbilical vein endothelial cells express P450 2C8 mRNA: cloning of endothelial P450 epoxygenase. Endothelium 4, 219–229 6 Huang, Z.H. et al. (1997) Inhibition of stimulus-induced endothelial cell intercellular
Reply: cytochrome P450-derived eicosanoids and the vascular wall Darryl C. Zeldin and James K. Liao
Endogenous vasodilators frequently possess anti-inflammatory, anti-proliferative and anti-thrombotic properties. By linking epoxyeicosatrienoic acids (EETs) to the inhibition of the activity of the pro-inflammatory nuclear transcription factor kB (NF-kB)1, an important nonvasodilatory action of EETs is suggested, and the role of EETs in the vascular wall is broadened considerably because NF-kB is implicated in vascular inflammation and atherosclerosis2. In this respect, EETs are not unlike endotheliumderived nitric oxide (NO), which relaxes vascular smooth muscle and inhibits NF-kB (Ref. 3). The mechanisms by which EETs exert these effects, however, are distinct from that of NO, which suggests that multiple levels of vascular control by the endothelium exist. Thus, it is important to determine whether endothelium-derived EETs can also exert anti-thrombotic and antiproliferative effects. An important question that remains is which of the P450 enzymes are responsible for the biosynthesis of EETs in endothelial cells. Three laboratories,
working independently, have isolated the CYP2J2 cDNA from vascular endothelial RNA (Refs 1, 4). However, other P450 enzymes might be involved in EET production in endothelial cells. In this regard, Lin and co-workers have shown that the epoxygenase present in human umbilical vein endothelial cells is a CYP2C isoform5. More recently, Fisslthaler and co-workers showed that treatment of porcine coronary artery endothelial cells in vitro with b-naphthoflavone induces a CYP2C homolog, increases 11,12-EET biosynthesis and enhances endothelium-derived hyperpolarizing factor (EDHF)-mediated coronary artery relaxation4. b-Naphthoflavone does not induce the CYP2J subfamily enzymes6 but is known to induce CYP1A expression in endothelial cells7. Determining which of these enzymes is primarily responsible for EET production might not be an easy task given the complexity of the mammalian CYP2J and CYP2C subfamilies and the absence of isoform-specific P450 inducers, chemical inhibitors and inhibitory antibodies. Thus, future work should
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
8
9
10
11
12
adhesion molecule-1, E-selectin, and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy and hydroperoxy derivatives. Circ. Res. 80, 149–158 Karin, M. (1999) The beginning of the end: IkB kinase (IKK) and NFkB activation. J. Biol. Chem. 274, 27339–27342 Auphan, N. et al. (1995) Immunosuppression by glucocorticoids: inhibition of NF-kB activity through induction of IkB synthesis. Science 270, 286–291 Kopp, E. and Ghosh, S. (1994) Inhibition of NF-kB by sodium salicylate and aspirin. Science 265, 956–959 Stuhlmeier, K.M. et al. (1997) Arachidonic acid influences proinflammatory gene induction by stabilizing the inhibitor-kBa/nuclear factor-kB (NF-kB) complex, thus suppressing the nuclear translocation of NF-kB. J. Biol. Chem. 272, 24679–24683 Bauersachs, J. et al. (1996) Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation 94, 3341–3347 Scarborough, P.E. et al. (1999) P450 subfamily CYP2J and their role in the bioactivation of arachidonic acid in extrahepatic tissues. Drug Metab. Rev. 31, 205–234
focus on the development of reagents that can be used to manipulate the levels and/or activity of the relevant P450 isoforms and on gene overexpression and gene disruption studies. Genetic variation in human cytochrome P450 genes is well described8; however, little is known about the role of P450 genetic polymorphism in the pathogenesis of vascular disease. As part of the NIEHS (National Institute of Environmental Health Sciences) Environmental Genome Project, we have recently identified several single nucleotide polymorphisms within the CYP2J2 gene that result in non-conservative amino acid substitutions (L. King, unpublished observations). Preliminary modeling studies, based upon the crystal structure of the Bacillus megaterium cytochrome P450BM-3 (CYP102), suggest that these substitutions are likely to influence the catalytic efficiency of the enzyme. It is not yet known whether the frequency of these polymorphisms is altered in individuals with vascular disease, but this is currently the subject of intense investigation in our laboratories. The potency and regioselectivity of the EET anti-inflammatory effects suggest that the biological actions of these P450-derived eicosanoids, like those of the prostaglandins and leukotrienes, are
PII: S0165-6147(00)01454-1
TiPS – April 2000 (Vol. 21)
D.C. Zeldin, Head of Clinical Studies Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. E-mail: zeldin@ niehs.nih.gov and J.K. Liao, Director of Vascular Medicine Research, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, LMRC-322, Boston, MA 02115, USA. E-mail: jliao@ rics.bwh.harvard.edu
127
COMMENT mediated through specific cell-surface receptors; however, the identity of the putative EET receptor(s) and the details of their signaling pathways remain unknown. Identification and functional characterization of the EET receptor(s) and development of specific EET receptor agonists and antagonists would greatly aid future research in this field and might lead to the development of novel therapeutics for inflammatory conditions.
CURRENT
Selected references 1 Node, K. et al. (1999) Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science 285, 1276–1279 2 Collins, T. (1993) Endothelial nuclear factorkB and the initiation of the atherosclerotic lesion. Lab. Invest. 68, 499–508 3 De Caterina, R. et al. (1995) Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J. Clin. Invest. 96, 60–68 4 Fisslthaler, B. et al. (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401, 493–497
AWA R E N E S S
The PTH2 receptor and TIP39: a new peptide–receptor system Ted B. Usdin
The parathyroid hormone PTH2 receptor was identified in an homology-based screen for new polypeptide-recognizing receptors1. Introduction of human PTH2 receptor cDNA into tissue culture cells revealed activation of the receptor by parathyroid hormone (PTH). Recently, a distinct peptide, a tubero-infundibular peptide of 39 residues (TIP39), which is a potent and selective activator of the PTH2 receptor, was purified2. TIP39 might be the physiologically relevant PTH2 receptor ligand. Although TIP39 and the PTH2 receptor constitute a relatively uncharacterized peptide–receptor system, the anatomical distribution of the PTH2 receptor3,4 suggests that TIP39 might be involved in the modulation of processes that range from pituitary hormone release to nociception and regulation of blood pressure. Receptors that recognize PTH T.B. Usdin, Chief, Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, Building 36, Room 3D06, 36 Convent Drive, Bethesda, MD 20892-4094, USA. E-mail: usdin@ codon.nih.gov
128
Three receptors that are activated by PTH have been cloned. The PTH/ PTHrP (parathyroid hormone-related peptide) receptor, now also called the PTH1 receptor, is present in high levels in kidney and bone, where it regulates Ca21 homeostasis5. The effects of PTH and the distinct gene product PTHrP on the PTH1 receptor expressed in tissue culture cells are indistinguishable. The PTH1 receptor is widely expressed and
TiPS – April 2000 (Vol. 21)
5 Lin, J. et al. (1996) Human umbilical vein endothelial cells express P450 2C8 mRNA: cloning of endothelial P450 epoxygenase. Endothelium 4, 219–229 6 Wu, S. et al. (1997) Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J. Biol. Chem. 272, 12551–12559 7 Stegeman, J.J. et al. (1989) Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart. Mol. Pharmacol. 36, 723–729 8 Nelson, D. et al. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42
possesses a host of additional roles, including regulation of endochondral bone formation and the development and remodeling of many tissues6–8. Locally synthesized PTHrP is thought to mediate these non-classical functions of the PTH1 receptor. The PTH2 receptor has ~50% amino acid sequence identity with the PTH1 receptor and is not activated by PTHrP. Homologues of mammalian PTH1 and PTH2 receptors and a third PTH-binding receptor (PTH3) have recently been identified in zebrafish. The zebrafish PTH3 receptor has 61% and 48% amino acid identity to zebrafish PTH1 and PTH2 receptors, respectively, and possesses ligand selectivity that is very similar to that of the zebrafish PTH1 receptor9. Pharmacological studies suggest the existence of additional potentially related receptors in other species, including receptors selective for PTHrP (Refs 10, 11) and carboxyl regions of PTH or PTHrP (Refs 12, 13), but these receptors have not yet been cloned. Peptides that recognize PTH-like receptors
PTH is a classic endocrine hormone that was identified following observation of the catastrophic effects of total parathyroidectomy. PTHrP was initially identified as a factor produced by non-
parathyroid tumors that cause the syndrome of hypercalcemia of malignancy without invading bone14,15. The pathological activity of PTHrP results from activation of the PTH1 receptor in bone and kidney; the normal developmental and paracrine roles of PTHrP are now well established6–8. Several observations suggested that, despite the fact that PTH can activate the PTH2 receptor, a different peptide might normally act on the PTH2 receptor in vivo. PTH2 receptor expression is greater in the brain than in peripheral tissues1. However, only low levels of PTH have been reported in the brains of several species16 and PTH does not have a well-established role in the CNS. Furthermore, we have been unable to detect mRNA encoding the peptide PTH in rat brain17. The PTH2 receptor is present in several circumventricular organs, where neurons are accessible to circulating proteins, but it is also expressed by neurons in areas where the blood–brain barrier is impermeable to most polypeptides2,3. In most tissues, the effects of PTH and PTHrP are similar and effects caused by PTH but not by PTHrP, as would be expected from the specificity of the cloned human PTH2 receptor, have not been reported. However, most physiological studies have been performed in rodents. The rat PTH2 receptor was cloned in an attempt to reconcile the anatomical and physiological data. Surprisingly, unlike the human receptor, the rat PTH2 receptor is only weakly activated by high concentrations of PTH (Ref. 18). Thus, even if PTH is present in the rat CNS, it is unlikely to have a
0165-6147/00/$ – see front matter. Published by Elsevier Science Ltd. All rights reserved.
PII: S0165-6147(00)01455-3
be possible to develop EET analogs with anti-inflammatory activity but devoid of the vasodilator or endocrine actions. 7
Selected references 1 Rosolowsky, M. and Campbell, W.B. (1996) Synthesis of hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells. Biochim. Biophys. Acta 1299, 267–277 2 Campbell, W.B. et al. (1996) Identification of epoxyeicosatrienoic acids as endotheliumderived hyperpolarizing factors. Circ. Res. 78, 415–423 3 Fisslthaler, B. et al. (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401, 493–497 4 Node, K. et al. (1999) Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science 285, 1276–1279 5 Lin, J.H-C. et al. (1996) Human umbilical vein endothelial cells express P450 2C8 mRNA: cloning of endothelial P450 epoxygenase. Endothelium 4, 219–229 6 Huang, Z.H. et al. (1997) Inhibition of stimulus-induced endothelial cell intercellular
Reply: cytochrome P450-derived eicosanoids and the vascular wall Darryl C. Zeldin and James K. Liao
Endogenous vasodilators frequently possess anti-inflammatory, anti-proliferative and anti-thrombotic properties. By linking epoxyeicosatrienoic acids (EETs) to the inhibition of the activity of the pro-inflammatory nuclear transcription factor kB (NF-kB)1, an important nonvasodilatory action of EETs is suggested, and the role of EETs in the vascular wall is broadened considerably because NF-kB is implicated in vascular inflammation and atherosclerosis2. In this respect, EETs are not unlike endotheliumderived nitric oxide (NO), which relaxes vascular smooth muscle and inhibits NF-kB (Ref. 3). The mechanisms by which EETs exert these effects, however, are distinct from that of NO, which suggests that multiple levels of vascular control by the endothelium exist. Thus, it is important to determine whether endothelium-derived EETs can also exert anti-thrombotic and antiproliferative effects. An important question that remains is which of the P450 enzymes are responsible for the biosynthesis of EETs in endothelial cells. Three laboratories,
working independently, have isolated the CYP2J2 cDNA from vascular endothelial RNA (Refs 1, 4). However, other P450 enzymes might be involved in EET production in endothelial cells. In this regard, Lin and co-workers have shown that the epoxygenase present in human umbilical vein endothelial cells is a CYP2C isoform5. More recently, Fisslthaler and co-workers showed that treatment of porcine coronary artery endothelial cells in vitro with b-naphthoflavone induces a CYP2C homolog, increases 11,12-EET biosynthesis and enhances endothelium-derived hyperpolarizing factor (EDHF)-mediated coronary artery relaxation4. b-Naphthoflavone does not induce the CYP2J subfamily enzymes6 but is known to induce CYP1A expression in endothelial cells7. Determining which of these enzymes is primarily responsible for EET production might not be an easy task given the complexity of the mammalian CYP2J and CYP2C subfamilies and the absence of isoform-specific P450 inducers, chemical inhibitors and inhibitory antibodies. Thus, future work should
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
8
9
10
11
12
adhesion molecule-1, E-selectin, and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy and hydroperoxy derivatives. Circ. Res. 80, 149–158 Karin, M. (1999) The beginning of the end: IkB kinase (IKK) and NFkB activation. J. Biol. Chem. 274, 27339–27342 Auphan, N. et al. (1995) Immunosuppression by glucocorticoids: inhibition of NF-kB activity through induction of IkB synthesis. Science 270, 286–291 Kopp, E. and Ghosh, S. (1994) Inhibition of NF-kB by sodium salicylate and aspirin. Science 265, 956–959 Stuhlmeier, K.M. et al. (1997) Arachidonic acid influences proinflammatory gene induction by stabilizing the inhibitor-kBa/nuclear factor-kB (NF-kB) complex, thus suppressing the nuclear translocation of NF-kB. J. Biol. Chem. 272, 24679–24683 Bauersachs, J. et al. (1996) Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation 94, 3341–3347 Scarborough, P.E. et al. (1999) P450 subfamily CYP2J and their role in the bioactivation of arachidonic acid in extrahepatic tissues. Drug Metab. Rev. 31, 205–234
focus on the development of reagents that can be used to manipulate the levels and/or activity of the relevant P450 isoforms and on gene overexpression and gene disruption studies. Genetic variation in human cytochrome P450 genes is well described8; however, little is known about the role of P450 genetic polymorphism in the pathogenesis of vascular disease. As part of the NIEHS (National Institute of Environmental Health Sciences) Environmental Genome Project, we have recently identified several single nucleotide polymorphisms within the CYP2J2 gene that result in non-conservative amino acid substitutions (L. King, unpublished observations). Preliminary modeling studies, based upon the crystal structure of the Bacillus megaterium cytochrome P450BM-3 (CYP102), suggest that these substitutions are likely to influence the catalytic efficiency of the enzyme. It is not yet known whether the frequency of these polymorphisms is altered in individuals with vascular disease, but this is currently the subject of intense investigation in our laboratories. The potency and regioselectivity of the EET anti-inflammatory effects suggest that the biological actions of these P450-derived eicosanoids, like those of the prostaglandins and leukotrienes, are
PII: S0165-6147(00)01454-1
TiPS – April 2000 (Vol. 21)
D.C. Zeldin, Head of Clinical Studies Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. E-mail: zeldin@ niehs.nih.gov and J.K. Liao, Director of Vascular Medicine Research, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, LMRC-322, Boston, MA 02115, USA. E-mail: jliao@ rics.bwh.harvard.edu
127
COMMENT mediated through specific cell-surface receptors; however, the identity of the putative EET receptor(s) and the details of their signaling pathways remain unknown. Identification and functional characterization of the EET receptor(s) and development of specific EET receptor agonists and antagonists would greatly aid future research in this field and might lead to the development of novel therapeutics for inflammatory conditions.
CURRENT
Selected references 1 Node, K. et al. (1999) Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science 285, 1276–1279 2 Collins, T. (1993) Endothelial nuclear factorkB and the initiation of the atherosclerotic lesion. Lab. Invest. 68, 499–508 3 De Caterina, R. et al. (1995) Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J. Clin. Invest. 96, 60–68 4 Fisslthaler, B. et al. (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401, 493–497
AWA R E N E S S
The PTH2 receptor and TIP39: a new peptide–receptor system Ted B. Usdin
The parathyroid hormone PTH2 receptor was identified in an homology-based screen for new polypeptide-recognizing receptors1. Introduction of human PTH2 receptor cDNA into tissue culture cells revealed activation of the receptor by parathyroid hormone (PTH). Recently, a distinct peptide, a tubero-infundibular peptide of 39 residues (TIP39), which is a potent and selective activator of the PTH2 receptor, was purified2. TIP39 might be the physiologically relevant PTH2 receptor ligand. Although TIP39 and the PTH2 receptor constitute a relatively uncharacterized peptide–receptor system, the anatomical distribution of the PTH2 receptor3,4 suggests that TIP39 might be involved in the modulation of processes that range from pituitary hormone release to nociception and regulation of blood pressure. Receptors that recognize PTH T.B. Usdin, Chief, Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, Building 36, Room 3D06, 36 Convent Drive, Bethesda, MD 20892-4094, USA. E-mail: usdin@ codon.nih.gov
128
Three receptors that are activated by PTH have been cloned. The PTH/ PTHrP (parathyroid hormone-related peptide) receptor, now also called the PTH1 receptor, is present in high levels in kidney and bone, where it regulates Ca21 homeostasis5. The effects of PTH and the distinct gene product PTHrP on the PTH1 receptor expressed in tissue culture cells are indistinguishable. The PTH1 receptor is widely expressed and
TiPS – April 2000 (Vol. 21)
5 Lin, J. et al. (1996) Human umbilical vein endothelial cells express P450 2C8 mRNA: cloning of endothelial P450 epoxygenase. Endothelium 4, 219–229 6 Wu, S. et al. (1997) Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J. Biol. Chem. 272, 12551–12559 7 Stegeman, J.J. et al. (1989) Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart. Mol. Pharmacol. 36, 723–729 8 Nelson, D. et al. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42
possesses a host of additional roles, including regulation of endochondral bone formation and the development and remodeling of many tissues6–8. Locally synthesized PTHrP is thought to mediate these non-classical functions of the PTH1 receptor. The PTH2 receptor has ~50% amino acid sequence identity with the PTH1 receptor and is not activated by PTHrP. Homologues of mammalian PTH1 and PTH2 receptors and a third PTH-binding receptor (PTH3) have recently been identified in zebrafish. The zebrafish PTH3 receptor has 61% and 48% amino acid identity to zebrafish PTH1 and PTH2 receptors, respectively, and possesses ligand selectivity that is very similar to that of the zebrafish PTH1 receptor9. Pharmacological studies suggest the existence of additional potentially related receptors in other species, including receptors selective for PTHrP (Refs 10, 11) and carboxyl regions of PTH or PTHrP (Refs 12, 13), but these receptors have not yet been cloned. Peptides that recognize PTH-like receptors
PTH is a classic endocrine hormone that was identified following observation of the catastrophic effects of total parathyroidectomy. PTHrP was initially identified as a factor produced by non-
parathyroid tumors that cause the syndrome of hypercalcemia of malignancy without invading bone14,15. The pathological activity of PTHrP results from activation of the PTH1 receptor in bone and kidney; the normal developmental and paracrine roles of PTHrP are now well established6–8. Several observations suggested that, despite the fact that PTH can activate the PTH2 receptor, a different peptide might normally act on the PTH2 receptor in vivo. PTH2 receptor expression is greater in the brain than in peripheral tissues1. However, only low levels of PTH have been reported in the brains of several species16 and PTH does not have a well-established role in the CNS. Furthermore, we have been unable to detect mRNA encoding the peptide PTH in rat brain17. The PTH2 receptor is present in several circumventricular organs, where neurons are accessible to circulating proteins, but it is also expressed by neurons in areas where the blood–brain barrier is impermeable to most polypeptides2,3. In most tissues, the effects of PTH and PTHrP are similar and effects caused by PTH but not by PTHrP, as would be expected from the specificity of the cloned human PTH2 receptor, have not been reported. However, most physiological studies have been performed in rodents. The rat PTH2 receptor was cloned in an attempt to reconcile the anatomical and physiological data. Surprisingly, unlike the human receptor, the rat PTH2 receptor is only weakly activated by high concentrations of PTH (Ref. 18). Thus, even if PTH is present in the rat CNS, it is unlikely to have a
0165-6147/00/$ – see front matter. Published by Elsevier Science Ltd. All rights reserved.
PII: S0165-6147(00)01455-3