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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-induced leukotriene B4 biosynthesis and 5-lipoxygenase and 5-lipoxygenase-activating protein expression in human osteoarthritis chondrocytes. Cells were treated with single addition of 10 μM 4-hydroxynonenal for different incubation time periods, as indicated above. (a) Leukotriene B4 (LTB4) production was evaluated by enzyme immunoassay commercial kit. (b) 5-lipoxygenase-activating protein (FLAP) and (c) 5-lipoxygenase (5-LOX) mRNA expression was measured by real-time RT-PCR. Data are means ± standard errors of the mean, n = 5, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 4: Hydroxynonenal-induced leukotriene B4 biosynthesis and 5-lipoxygenase and 5-lipoxygenase-activating protein expression in human osteoarthritis chondrocytes. Cells were treated with single addition of 10 μM 4-hydroxynonenal for different incubation time periods, as indicated above. (a) Leukotriene B4 (LTB4) production was evaluated by enzyme immunoassay commercial kit. (b) 5-lipoxygenase-activating protein (FLAP) and (c) 5-lipoxygenase (5-LOX) mRNA expression was measured by real-time RT-PCR. Data are means ± standard errors of the mean, n = 5, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: In the next series of experiments, we further investigated whether the decrease of COX-2 expression in treated chondrocytes with single addition of 10 μM HNE to cultures lead to a switch to 5-LOX and FLAP mRNA expression, and consequently to LTB4 production. LTB4 is a potent chemoattractant and stimulator of inflammation [27] and probably contributes, along with other factors, to the upregulation of the synthesis of other catabolic factors involved in the pathophysiology of OA [28]. Human OA chondrocytes (n = 5 independent experiments) were treated for different incubation periods with single addition of 10 μM HNE to all wells except the controls. HNE-induced changes in the LTB4 level and in 5-LOX and FLAP mRNA expression and were evaluated respectively in picograms per milliliter or as a percentage of control. LTB4 release (Figure 4a) was induced only after a long incubation period. The maximum level rose significantly after 72 hours of treatment (66.6 ± 3.7 pg/105 cells, P < 0.001).


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-induced leukotriene B4 biosynthesis and 5-lipoxygenase and 5-lipoxygenase-activating protein expression in human osteoarthritis chondrocytes. Cells were treated with single addition of 10 μM 4-hydroxynonenal for different incubation time periods, as indicated above. (a) Leukotriene B4 (LTB4) production was evaluated by enzyme immunoassay commercial kit. (b) 5-lipoxygenase-activating protein (FLAP) and (c) 5-lipoxygenase (5-LOX) mRNA expression was measured by real-time RT-PCR. Data are means ± standard errors of the mean, n = 5, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Hydroxynonenal-induced leukotriene B4 biosynthesis and 5-lipoxygenase and 5-lipoxygenase-activating protein expression in human osteoarthritis chondrocytes. Cells were treated with single addition of 10 μM 4-hydroxynonenal for different incubation time periods, as indicated above. (a) Leukotriene B4 (LTB4) production was evaluated by enzyme immunoassay commercial kit. (b) 5-lipoxygenase-activating protein (FLAP) and (c) 5-lipoxygenase (5-LOX) mRNA expression was measured by real-time RT-PCR. Data are means ± standard errors of the mean, n = 5, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: In the next series of experiments, we further investigated whether the decrease of COX-2 expression in treated chondrocytes with single addition of 10 μM HNE to cultures lead to a switch to 5-LOX and FLAP mRNA expression, and consequently to LTB4 production. LTB4 is a potent chemoattractant and stimulator of inflammation [27] and probably contributes, along with other factors, to the upregulation of the synthesis of other catabolic factors involved in the pathophysiology of OA [28]. Human OA chondrocytes (n = 5 independent experiments) were treated for different incubation periods with single addition of 10 μM HNE to all wells except the controls. HNE-induced changes in the LTB4 level and in 5-LOX and FLAP mRNA expression and were evaluated respectively in picograms per milliliter or as a percentage of control. LTB4 release (Figure 4a) was induced only after a long incubation period. The maximum level rose significantly after 72 hours of treatment (66.6 ± 3.7 pg/105 cells, P < 0.001).

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