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Regulation of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes.

Chen SH, Fahmi H, Shi Q, Benderdour M - Arthritis Res. Ther. (2010)

Bottom Line: After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

Introduction: 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.

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Hydroxynonenal-activated 5-lipoxygenase promoter in human osteoarthritis chondrocytes. Cells were transfected with human 5-lipoxygenase (5-LOX) promoter. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. Data are means ± standard errors of the mean, n = 4, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05. VDRE: vitamin D responsive element.
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Figure 5: Hydroxynonenal-activated 5-lipoxygenase promoter in human osteoarthritis chondrocytes. Cells were transfected with human 5-lipoxygenase (5-LOX) promoter. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. Data are means ± standard errors of the mean, n = 4, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05. VDRE: vitamin D responsive element.

Mentions: To more accurately identify whether HNE induced 5-LOX at the transcription level, the 5-LOX promoter construct with luciferase gene reporter was transiently transfected into human OA chondrocytes, followed by stimulation with single addition of 10 μM HNE to all wells excepted control for different time periods (n = 4 independent experiments). Compared with unstimulated cells, HNE induced 5-LOX gene promoter activity in transfected chondrocytes. 5-LOX promoter activity increased significantly by 310% and 580% after 48 and 72 hours of incubation, respectively (P < 0.05) (Figure 5).


Regulation of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes.

Chen SH, Fahmi H, Shi Q, Benderdour M - Arthritis Res. Ther. (2010)

Hydroxynonenal-activated 5-lipoxygenase promoter in human osteoarthritis chondrocytes. Cells were transfected with human 5-lipoxygenase (5-LOX) promoter. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. Data are means ± standard errors of the mean, n = 4, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05. VDRE: vitamin D responsive element.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875653&req=5

Figure 5: Hydroxynonenal-activated 5-lipoxygenase promoter in human osteoarthritis chondrocytes. Cells were transfected with human 5-lipoxygenase (5-LOX) promoter. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. Data are means ± standard errors of the mean, n = 4, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05. VDRE: vitamin D responsive element.
Mentions: To more accurately identify whether HNE induced 5-LOX at the transcription level, the 5-LOX promoter construct with luciferase gene reporter was transiently transfected into human OA chondrocytes, followed by stimulation with single addition of 10 μM HNE to all wells excepted control for different time periods (n = 4 independent experiments). Compared with unstimulated cells, HNE induced 5-LOX gene promoter activity in transfected chondrocytes. 5-LOX promoter activity increased significantly by 310% and 580% after 48 and 72 hours of incubation, respectively (P < 0.05) (Figure 5).

Bottom Line: After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus