Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2.
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Animals↗
Biomedical subjects
W L Smith
Publications and source records attributed to W L Smith.
Prostanoid receptors of murine NIH 3T3 and RAW 264.7 cells. Structure and expression of the murine prostaglandin EP4 receptor gene.
Prostaglandin endoperoxide H synthase-1 (PGHS-1) is expressed constitutively in murine NIH 3T3 cells and RAW 264.7 cells. PGHS-2 is inducibly expressed in these cells following stimulation with serum or bacterial lipopolysaccharide (LPS), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis established that a variety of G protein-linked and peroxisomal proliferator-activated prostanoid receptors are expressed in both of these cell types. The levels of the EP2 and EP4 prostaglandin E2 (PGE2) receptors and the prostaglandin I2 receptor were changed in these cells by serum or LPS stimulation. Quantitative RT-PCR indicated that the mRNA for the murine EP4 receptor, the butaprost-insensitive PGE2 receptor that couples to Gs, increases 1.5-3-fold in response to serum (NIH 3T3) or LPS (RAW 264.7) with a time course approximating the induction of PGHS-2 expression. To study expression of the EP4 receptor we isolated the mouse EP4 receptor gene; the gene is 10 kilobase pairs (kb) in length and, like other known prostanoid receptor genes, contains three exons and two introns. The first intron is 0.5 kb and is located 16 base pairs (bp) downstream of the translational start site. This is a different location than that of the first introns of other prostanoid receptor genes. The second intron is located immediately following the sixth transmembrane domain at the same position as the second intron of the thromboxane A2 receptor, prostaglandin D2 receptor, prostaglandin I2 receptor, and one of the PGE2 (EP1) receptor genes. A major transcriptional start was detected at -142 bp upstream of the translational start. There are a variety of putative cis-acting elements within 1.5 kb upstream of the translational start site and within the first intron. Promoter analyses of the EP4 receptor gene promoter in RAW 264.7 cells indicated that there is a constitutive negative regulatory region between -992 and -928 bp, a constitutive positive region between -928 and -554 bp, and an LPS/serum-responsive region between -554 and -116 bp.
3T3 Cells↗
C-terminal Ser/Pro-Thr-Glu-Leu tetrapeptides of prostaglandin endoperoxide H synthases-1 and -2 target the enzymes to the endoplasmic reticulum.
Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are integral membrane proteins associated with the luminal surface of the endoplasmic reticulum (ER) and the nuclear envelope (NE). The C-terminal sequences of PGHSs end in -Ser/Pro-Thr-Glu-Leu (-S/PTEL), a sequence similar to one of the known ER targeting sequences, -Lys-Asp-Glu-Leu (-KDEL). Previous immunofluorescence studies from our own and another laboratory had indicated that both native ovine (o) PGHS-1 and oPGHS-1 mutants with modified or deleted -PTEL sequences were localized primarily in the ER when the proteins were expressed for 24-40 h following transient transfection of cos-1 cells. However, in characterizing an oPGHS-1 mutant lacking 15 amino acids from the C-terminus (deltaCT15 oPGHS-1), we found that when this mutant was expressed for a shorter period (18 h) following transfection, the enzyme was concentrated near the nucleus in what appeared to be the Golgi apparatus; similar results were observed when the -P/STEL mutants of oPGHS-1 prepared previously (i.e., deltaPTEL, L600N, and L600R oPGHS-1) were retested using the 18-h posttransfection expression time; in contrast, under the same conditions, the native enzyme was localized throughout the cytoplasm (i.e., in the ER). When the cos-1 cells transfected with each of the various C-terminal mutants were treated with Brefeldin A, the immunofluorescent staining was redistributed to the ER, whereas the distribution of native oPGHS-1 was unaffected. A human PGHS-2 (hPGHS-2) mutant in which the C-terminal leucine residue was replaced with an asparagine (hPGHS-2 L604N) was also found to localize to the Golgi apparatus following 18 h expression in transfected cos-1 cells. These results establish that the C-terminal-S/PTEL sequences of both PGHS-1 and PGHS-2 target the enzymes to the ER. PGHS-2 is more highly concentrated in the NE than in the ER, but apparently this isozyme traverses the Golgi apparatus and returns to the ER prior to its becoming concentrated in the NE.
Amino Acid Sequence↗
Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8,-tetrachlorodibenzo-p-dioxin.
We have examined the molecular mechanisms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated prostaglandin synthesis in Mardin Darvey canine kidney cells (MDCK). TCDD stimulates prostaglandin synthesis in these cells, at least in part, by elevating prostaglandin endoperoxide H2 synthase-2 (PGHS-2) levels. TCDD-stimulated transcription of the PGHS-2 gene was maximal (6-fold) within 2 h and resulted in a 100-fold increase in PGHS-2 mRNA and a 25-fold increase in PGHS-2 protein levels by 4 h. Transient transfection experiments using luciferase-reporter plasmids demonstrated that control element(s) responsible for TCDD activation of the murine PGHS-2 promoter in MDCK cells are located in the first 965 nucleotides upstream from the PGHS-2 transcriptional initiation site. A canonical xenobiotic response element, similar to those that control transcription of other well-known TCDD-sensitive genes, is present at position -157, but does not appear to be sufficient for halogenated aromatic hydrocarbon (HAH) activation of the PGHS-2 promoter. TCDD failed to stimulate transcription from the PGHS-2 promoter when reporter plasmids were transfected into Hepa 1c1c7 cells, a line which contains the functional aryl hydrocarbon receptor. It seems likely that inappropriate expression of PGHS-2 may contribute to the toxic effects of TCDD and other HAHs. In particular, PGHS-2 expression may affect those toxic reactions that involve inappropriate cellular growth, such as dermal hyperplasia and tumor formation. It is also likely that elevated synthesis of prostaglandins, which are potent regulators of immune function, could play a role in the immunotoxicity associated with HAH exposure.
Amino Acid Sequence↗
Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site.
Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein.
Affinity Labels↗
Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1.
Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S- flurbiprofen complex (Picot, D., Loll, P.J., and Garavito, R.M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg120; (b) that Arg120 forms an important salt bridge with Glu524; and (c) that Tyr355, which is in close proximity to Arg120, could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg120 (R120K, R120Q, R120E), Glu524 (E524D, E524Q, E524K), and Tyr355 (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The Km values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 microM, respectively, versus 4 microM for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time-dependent inhibition by flurbiprofen. These results are consistent with Arg120 binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg120 is necessary for suicide inactivation and time-dependent inhibition, respectively. The Km values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg120 and Tyr355 on the stereospecificity of inhibitor binding was determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr355 is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.
Amino Acid Sequence↗
Investigating abuse in the asymptomatic twin.
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Battered Child Syndrome↗
A Dejerine-Sottas neuropathy family with a gene mapped on chromosome 8.
The Patient is a 55-year-old black male who belongs to a large family with 9 affected relatives with autosomal dominant Dejerine-Sottas neuropathy (DSN). Onset of his condition was at 2 years of age with steppage gait followed by severe progressive weakness, atrophy, and sensory loss of his legs and hands accompanied by areflexia and thoracolumbar kyphoscoliosis. The patient became wheelchair confined at age 38. At around age 42, the left shoulder became dislocated and the humeral head underwent aseptic necrosis (Charcot joint). Nerve conduction studies showed absent motor and sensory responses for all major nerves tested. Genetic linkage suggested mapping of this DSN gene on chromosome 8qter. A younger brother with similar neurological findings also demonstrated Charcot joints with bone destruction of the joints of the fourth and fifth fingers.
Chromosome Mapping↗
Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast-like cell formation induced by interleukin-1 beta.
Interleukin-1 beta (IL-1 beta) stimulates osteoclast-like cell formation via prostaglandin E2 (PGE2) production. However, the regulatory mechanism for the production of PGE2 in bone cells is still unclear. Recently, it has been shown that two prostaglandin endoperoxide H synthase (PGHS) isozymes exist, termed PGSH-1 and PGHS-2. We report here that IL-1 beta induces PGE2 production in bone marrow culture induced by a PGHS-2-dependent mechanism. IL-1 beta stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) and the production of PGE2 in mouse bone marrow cultures. The dose response curves for the indomethacin inhibition of TRAP-positive MNC formation and PGE2 production were nearly identical. Cycloheximide (CHX) suppressed IL-1 beta-induced PGE2 production, suggesting that the production of PGE2 induced by IL-1 beta required de novo protein synthesis. Northern blot analysis determined that IL-1 beta induced PGHS-2 expression by 30 minutes and mRNA levels were maximal by 1-2 h. Cycloheximide potentiated the accumulation of PGHS-2 mRNA linearly up to 8 h. Dexamethasone, an inhibitor of the induction of PGHS-2, inhibited IL-1 beta-induced PGHS-2 mRNA expression and also suppressed IL-1 beta-stimulated formation of TRAP-positive MNC. Furthermore NS-398, as a selective PGHS-2 inhibitor, completely inhibited IL-1 beta-induced TRAP-positive MNC formation. Moreover, IL-1 beta-induced PGHS-2 mRNA expression and formation of TRAP-positive MNC were inhibited by calphostin C, a selective inhibitor of protein kinase C (PKC). These results indicate that IL-1 beta-induced formation of osteoclast-like cells requires PKC activation, induction of PGHS-2, and subsequent prostaglandin synthesis by this enzyme.
Animals↗
Prostaglandin endoperoxide H synthases-1 and -2.
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Amino Acid Sequence↗
Ignorance is bliss.
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Attitude of Health Personnel↗
How to succeed in life (and perhaps radiology).
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Attitude↗
Taking care of yourself.
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Health Behavior↗
Teleradiology services for a rural hospital: a case study.
We investigated the perceived quality of teleradiology services offered at a rural hospital in comparison with radiology provided by three other methods: 'circuit riding', an on-site radiologist, and a hybrid arrangement. The research design was a case study, with interviews of administrators, technologists and physicians at the rural hospital, followed by a structured survey of all staff physicians. Responses were analysed both qualitatively and quantitatively. Both interviews and the survey indicated that teleradiology was perceived to be as accurate as on-site film interpretation. All other aspects of the service--efficiency, reports, communications and the overall contribution to patient care--were judged to be poorer than on-site radiology. We conclude that the provision of acceptable teleradiology requires considerable attention to all aspects of the radiology service, with attention to differences in institutional culture and mission, and to communication.
Arkansas↗
Duplex Doppler sonography of changes in portal vein flow in healthy term newborn infants after feeding.
To establish reference values for changes in portal venous diameter, angle- corrected maximal flow velocity, and flow in healthy term newborn infants after formula feeding, we studied 20 subjects using duplex Doppler sonography. After feeding, portal venous diameter increased from 3.6 +/- 0.1 (mean +/- standard error of the mean) to 3.9 +/- 0.1 mm at 15 min and decreased to 3.8 +/- 0.1 at 60 min. Maximal flow velocity increased from 24.1 +/- 1.3 cm/s to a maximum of 35.9 +/- 2.4 cm/s at 15 min and decreased to 28.8 +/- 1.5 cm/s at 60 min. Flow increased from 85.0 +/- 7.5 ml/min to a maximum of 153.6 +/- 14.9 ml/min at 15 min with decrease similar to the maximal flow velocity curve. We conclude that formula feeding produces peak portal blood flows of nearly twice the fasting values at 15 min after feeding and returns almost to fasting value by 60 min.
Eating↗