Limits...
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.

Show MeSH

Related in: MedlinePlus

Hydroxynonenal-activated microsomal prostaglandin E2 synthase-1 promoter. Chondrocytes were transfected with (a) microsomal prostaglandin E2 synthase-1 (mPGES-1)-Luc or (b) p3xEgr-1-Luc containing three early growth response factor 1 (Egr-1) binding sites. The total amount of transfected DNA was kept constant using a corresponding empty vector. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal (HNE) for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. (c) Osteoarthritis chondrocytes were treated with 10 μM HNE at increasing time of incubation and then Egr-1 protein expression was evaluated in the cellular extract by western blot. Data are means ± standard errors of the mean, n = 6, 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

License
getmorefigures.php?uid=PMC2875653&req=5

Figure 3: Hydroxynonenal-activated microsomal prostaglandin E2 synthase-1 promoter. Chondrocytes were transfected with (a) microsomal prostaglandin E2 synthase-1 (mPGES-1)-Luc or (b) p3xEgr-1-Luc containing three early growth response factor 1 (Egr-1) binding sites. The total amount of transfected DNA was kept constant using a corresponding empty vector. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal (HNE) for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. (c) Osteoarthritis chondrocytes were treated with 10 μM HNE at increasing time of incubation and then Egr-1 protein expression was evaluated in the cellular extract by western blot. Data are means ± standard errors of the mean, n = 6, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Firstly, to determine whether changes in mPGES-1 mRNA levels can be ascribed to the regulation in promoter activity, chondrocytes were transiently transfected with human mPGES-1 promoter-luciferase reporter genes (n = 6 independent experiments) and then treated with single addition of 10 μM HNE to all wells except the controls. As shown in Figure 3a, HNE induced mPGES-1 promoter activity in a time-dependent increment. Maximum promoter activity was 331% at 16 hours compared with the control (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-activated microsomal prostaglandin E2 synthase-1 promoter. Chondrocytes were transfected with (a) microsomal prostaglandin E2 synthase-1 (mPGES-1)-Luc or (b) p3xEgr-1-Luc containing three early growth response factor 1 (Egr-1) binding sites. The total amount of transfected DNA was kept constant using a corresponding empty vector. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal (HNE) for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. (c) Osteoarthritis chondrocytes were treated with 10 μM HNE at increasing time of incubation and then Egr-1 protein expression was evaluated in the cellular extract by western blot. Data are means ± standard errors of the mean, n = 6, 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

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

Figure 3: Hydroxynonenal-activated microsomal prostaglandin E2 synthase-1 promoter. Chondrocytes were transfected with (a) microsomal prostaglandin E2 synthase-1 (mPGES-1)-Luc or (b) p3xEgr-1-Luc containing three early growth response factor 1 (Egr-1) binding sites. The total amount of transfected DNA was kept constant using a corresponding empty vector. The next day, they were incubated with single addition of 10 μM 4-hydroxynonenal (HNE) for increasing incubation time periods. Luciferase activity was measured in cell extracts and normalized to β-galactosidase activity. (c) Osteoarthritis chondrocytes were treated with 10 μM HNE at increasing time of incubation and then Egr-1 protein expression was evaluated in the cellular extract by western blot. Data are means ± standard errors of the mean, n = 6, expressed as percentages of untreated cells. Student's unpaired t test: *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Firstly, to determine whether changes in mPGES-1 mRNA levels can be ascribed to the regulation in promoter activity, chondrocytes were transiently transfected with human mPGES-1 promoter-luciferase reporter genes (n = 6 independent experiments) and then treated with single addition of 10 μM HNE to all wells except the controls. As shown in Figure 3a, HNE induced mPGES-1 promoter activity in a time-dependent increment. Maximum promoter activity was 331% at 16 hours compared with the control (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