mPGES-1 : a key regulator of fever and neonatal respiratory depression
Author: Saha, Sipra
Date: 2006-06-14
Location: Samuelssonsalen, MBB, Scheeles väg 2 Karolinska Institutet
Time: 9.30
Department: Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
View/ Open:
Thesis (488.1Kb)
Abstract
Prostaglandins are potent lipid mediators, synthesized de novo from arachidonic acid (AA) upon cell activation. AA is oxidized by the cyclooxygenase isoenzymes (COX-1 and COX-2) to form PGH2, the common substrate for downstream enzymes involved in prostaglandin biosynthesis. COX-1 is constitutively expressed in most cells and regarded as housekeeping protein. In contrast, expression of COX-2 is markedly increased by pro-inflammatory cytokines at sites of inflammation. PGH2 is converted into biologically active prostanoids like PGD2, PGE2, PGF2alpha, TXA2 and PGI2 by specific enzymes in a cell-specific manner. The isomerization of PGH2 to PGE2, a potent mediator of pain and inflammation, is specifically catalyzed by human microsomal prostaglandin E synthase-1 (mPGES-1), a member of the MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) superfamily. High levels of PGE2 have been found in numerous disease states.
Human mPGES-1 was expressed as an N-terminal-histidine-tagged protein in E. coli. The membrane bound enzyme was solubilized using Triton X-100 and purified to apparent homogeneity using a combination of hydroxyapatite and immobilized metal affinity chromatography. Purified mPGES-1 exhibited high glutathione (GSH)-dependent catalytic activity for the conversion of both PGH2 to PGE2 and, PGG2 to 15-hydroperoxy-PGE2. Moreover, mPGES-1 also exhibited GSH-dependent peroxidase activity towards cumene hydroperoxide and 5-HpETE as well as low but significant glutathione transferase activity, possibly reflecting a relationship to other members of the MAPEG family. A 10 Å projection map of mPGES-1 determined using electron crystallography as well as hydrodynamic studies of mPGES-1-Triton X-100 complex, independently demonstrated the trimeric organization of mPGES-1.
The role of mPGES-1 in endotoxin-induced fever, as well as aseptic, cytokine-dependent, inflammation-induced fever was investigated. In response to intraperitoneal injection of lipopolysaccharide (LPS), wildtype DBA/1lacJ mice developed a robust fever with markedly increased PGE2 levels in the cerebrospinal fluid (CSF) and significant LPS-induced mPGES-1 activity in membrane fractions isolated from brain tissues. In contrast, the mPGES-1 knockout mice did not develop fever and, the PGE2 levels in the CSF did not differ significantly from the saline-treated wildtype mice, suggesting a critical role for mPGES-1 in the development of endotoxin-induced fever. In a cytokine-dependent fever model, subcutaneous injection of turpentine induced biphasic fever in wildtype mice, whereas mPGES-1 knockout mice displayed a core body temperature similar to the saline-treated wildtype mice, indicating that mPGES-1 activity was indispensable for the induction of cytokine-dependent fever. mPGES-1 did not, however, mediate hyperthermia induced by psychological stress.
The role of mPGES-1 in neonatal respiratory depression was investigated using 9-day old DBA/1lacj mice. Wildtype mice treated with IL-1beta exhibited a reduced respiratory frequency during normoxia as well hyperoxia compared to saline treated mice. This effect of IL-1beta was attenuated in mPGES-1 knockout mice. Moreover, IL-1beta treatment induced apneas, irregular breathing pattern and reduced the anoxic survival of the wildtype mice, and these effects were attenuated in mice lacking mPGES-1. Both IL-1beta and hypoxia treatment synergistically induced a rapid 4-fold mPGES-1 activity in the brainstem of wildtype mice compared to the saline treatment. These results suggest a central role for mPGES-1 in the regulation of neonatal breathing.
Taken together, these findings provide further support of mPGES-1 as an attractive target for the development of anti-inflammatory and anti-pyretic drugs. It is also possible that such drugs could be used in neonates at risk for respiratory suppression.
Human mPGES-1 was expressed as an N-terminal-histidine-tagged protein in E. coli. The membrane bound enzyme was solubilized using Triton X-100 and purified to apparent homogeneity using a combination of hydroxyapatite and immobilized metal affinity chromatography. Purified mPGES-1 exhibited high glutathione (GSH)-dependent catalytic activity for the conversion of both PGH2 to PGE2 and, PGG2 to 15-hydroperoxy-PGE2. Moreover, mPGES-1 also exhibited GSH-dependent peroxidase activity towards cumene hydroperoxide and 5-HpETE as well as low but significant glutathione transferase activity, possibly reflecting a relationship to other members of the MAPEG family. A 10 Å projection map of mPGES-1 determined using electron crystallography as well as hydrodynamic studies of mPGES-1-Triton X-100 complex, independently demonstrated the trimeric organization of mPGES-1.
The role of mPGES-1 in endotoxin-induced fever, as well as aseptic, cytokine-dependent, inflammation-induced fever was investigated. In response to intraperitoneal injection of lipopolysaccharide (LPS), wildtype DBA/1lacJ mice developed a robust fever with markedly increased PGE2 levels in the cerebrospinal fluid (CSF) and significant LPS-induced mPGES-1 activity in membrane fractions isolated from brain tissues. In contrast, the mPGES-1 knockout mice did not develop fever and, the PGE2 levels in the CSF did not differ significantly from the saline-treated wildtype mice, suggesting a critical role for mPGES-1 in the development of endotoxin-induced fever. In a cytokine-dependent fever model, subcutaneous injection of turpentine induced biphasic fever in wildtype mice, whereas mPGES-1 knockout mice displayed a core body temperature similar to the saline-treated wildtype mice, indicating that mPGES-1 activity was indispensable for the induction of cytokine-dependent fever. mPGES-1 did not, however, mediate hyperthermia induced by psychological stress.
The role of mPGES-1 in neonatal respiratory depression was investigated using 9-day old DBA/1lacj mice. Wildtype mice treated with IL-1beta exhibited a reduced respiratory frequency during normoxia as well hyperoxia compared to saline treated mice. This effect of IL-1beta was attenuated in mPGES-1 knockout mice. Moreover, IL-1beta treatment induced apneas, irregular breathing pattern and reduced the anoxic survival of the wildtype mice, and these effects were attenuated in mice lacking mPGES-1. Both IL-1beta and hypoxia treatment synergistically induced a rapid 4-fold mPGES-1 activity in the brainstem of wildtype mice compared to the saline treatment. These results suggest a central role for mPGES-1 in the regulation of neonatal breathing.
Taken together, these findings provide further support of mPGES-1 as an attractive target for the development of anti-inflammatory and anti-pyretic drugs. It is also possible that such drugs could be used in neonates at risk for respiratory suppression.
List of papers:
I. Thoren S, Weinander R, Saha S, Jegerschold C, Pettersson PL, Samuelsson B, Hebert H, Hamberg M, Morgenstern R, Jakobsson PJ (2003). Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination. J Biol Chem. 278(25): 22199-209. Epub 2003 Apr 2
Pubmed
II. Engblom D, Saha S, Engstrom L, Westman M, Audoly LP, Jakobsson PJ, Blomqvist A (2003). Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis. Nat Neurosci. 6(11): 1137-8. Epub 2003 Oct 19
Pubmed
III. Saha S, Engstrom L, Mackerlova L, Jakobsson PJ, Blomqvist A (2005). Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1. Am J Physiol Regul Integr Comp Physiol. 288(5): R1100-7. Epub 2005 Jan 27
Pubmed
IV. Hofstetter A, Saha S, Siljehav V, Jakobsson PJ, Herlenius E (2006). Prostaglandin E2 mediates respiratory depression induced by interleukin-1beta and hypoxia. [Manuscript]
I. Thoren S, Weinander R, Saha S, Jegerschold C, Pettersson PL, Samuelsson B, Hebert H, Hamberg M, Morgenstern R, Jakobsson PJ (2003). Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination. J Biol Chem. 278(25): 22199-209. Epub 2003 Apr 2
Pubmed
II. Engblom D, Saha S, Engstrom L, Westman M, Audoly LP, Jakobsson PJ, Blomqvist A (2003). Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis. Nat Neurosci. 6(11): 1137-8. Epub 2003 Oct 19
Pubmed
III. Saha S, Engstrom L, Mackerlova L, Jakobsson PJ, Blomqvist A (2005). Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1. Am J Physiol Regul Integr Comp Physiol. 288(5): R1100-7. Epub 2005 Jan 27
Pubmed
IV. Hofstetter A, Saha S, Siljehav V, Jakobsson PJ, Herlenius E (2006). Prostaglandin E2 mediates respiratory depression induced by interleukin-1beta and hypoxia. [Manuscript]
Issue date: 2006-05-24
Rights:
Publication year: 2006
ISBN: 91-7140-750-2
Statistics
Total Visits
Views | |
---|---|
mPGES-1 ...(legacy) | 892 |
mPGES-1 ... | 403 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
mPGES-1 ... | 9 | 27 | 14 | 15 | 14 | 22 | 23 |
File Visits
Views | |
---|---|
thesis.pdf(legacy) | 480 |
thesis.pdf | 211 |
thesis.pdf.txt(legacy) | 2 |
Top country views
Views | |
---|---|
United States | 420 |
Denmark | 134 |
Sweden | 102 |
China | 85 |
United Kingdom | 77 |
Germany | 67 |
Australia | 64 |
Ireland | 62 |
South Korea | 23 |
Russia | 13 |
Top cities views
Views | |
---|---|
Sydney | 63 |
Dublin | 61 |
Copenhagen | 50 |
Beijing | 41 |
Sunnyvale | 41 |
Ballerup | 32 |
Romeo | 29 |
Kiez | 18 |
Seoul | 17 |
Stockholm | 15 |