Regulation of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes.
BACKGROUND
This study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).


METHODS
For COX-2 and mPGES-1 studies, human osteoarthritic chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 muM HNE to the cultures at 2-hour intervals, up to 14 hours. For 5-LOX and FLAP studies, cells were treated with a single addition of 10 muM HNE for 24 hours, 48 hours, and 72 hours in the presence or absence of naproxen (a nonspecific COX-2 inhibitor) or antibody anti-transforming growth factor-beta 1 (TGF-beta1). The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by western blot, and those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-beta1 were determined with commercial kits. The levels of mPGES-1, FLAP and 5-LOX mRNA were measured by real-time RT-PCR. Transient transfection was performed to determine promoter activities of mPGES-1 and 5-LOX.


RESULTS
Single addition of 10 muM HNE to cultured chondrocytes induced PGE2 release as well as COX-2 and mPGES-1 expression at the protein and mRNA levels, with a plateau reached respectively at 8 and 16 hours of incubation, followed by a subsequent decline. However, repeated treatments with HNE prevented the decline of COX-2 and mPGES-1 expression that occurred with a single aldehyde addition. HNE induced mPGES-1 promoter activity, possibly through transcription factor Egr-1 activation. After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation. The addition of naproxen to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that HNE significantly induced TGF-beta1 production. The addition of anti-TGF-beta1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-beta1-dependent mechanism.


CONCLUSIONS
Our data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human osteoarthritic chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-beta1 are suggested to be involved in this regulation.
Chen
Shu-Huang
SH
Orthopaedic Research Laboratory, Hôpital du Sacré-Caeur de Montréal, Department of Surgery, University of Montreal, 5400 Gouin Blvd West, Montreal, QC H4J 1C5, Canada. shu-huang.chen@umontreal.ca
Fahmi
Hassan
H
Shi
Qin
Q
Benderdour
Mohamed
M
D058927:5-Lipoxygenase-Activating Proteins; D000368:Aged; D000447:Aldehydes / Q000494:pharmacology*; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis*; D007975:Leukotriene B4 / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D010003:Osteoarthritis / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D000447:Aldehydes; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D008565:Membrane Proteins; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D007975:Leukotriene B4; D001094:Arachidonate 5-Lipoxygenase; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; C027576:4-hydroxy-2-nonenal