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. 2016 Jun;13(6):4569-76.
doi: 10.3892/mmr.2016.5137. Epub 2016 Apr 15.

Cannabinoid WIN‑55,212‑2 Mesylate Inhibits ADAMTS‑4 Activity in Human Osteoarthritic Articular Chondrocytes by Inhibiting Expression of syndecan‑1

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Free PMC article

Cannabinoid WIN‑55,212‑2 Mesylate Inhibits ADAMTS‑4 Activity in Human Osteoarthritic Articular Chondrocytes by Inhibiting Expression of syndecan‑1

Ying Kong et al. Mol Med Rep. .
Free PMC article

Abstract

A central feature of osteoarthritis (OA) is the loss of articular cartilage, which is primarily attributed to cartilage breakdown. A group of metalloproteinases termed the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family are reported to be important in cartilage breakdown. Recent studies have suggested that ADAMTS‑4 is a major contributor to the pathogenesis of OA and that syndecan‑1 is closely associated with activation of ADAMTS‑4 in human chondrocytes. Accumulating evidence also suggests that cannabinoids have chondroprotective effects. The current study explored the effects of synthetic cannabinoid WIN‑55,212‑2 mesylate (WIN‑55) on the expression of syndecan‑1 and ADAMTS‑4, as well as ADAMTS‑4 activity, in unstimulated and interleukin (IL)‑1β‑stimulated OA chondrocytes. Primary human OA articular chondrocytes were treated with WIN‑55 in the presence or absence of IL‑1β and cannabinoid receptor antagonists. The results of the present study demonstrated that WIN‑55 inhibited ADAMTS‑4 activity in unstimulated and IL‑1β‑stimulated primary human OA articular chondrocytes in a concentration‑dependent manner. Cannabinoid receptor type 1 (CB1) and 2 (CB2) were constitutively expressed in human OA articular chondrocytes. Furthermore, selective CB2 antagonist, JTE907, but not selective CB1 antagonist, MJ15, abolished the inhibitory effect of WIN‑55 on ADAMTS‑4 activity. WIN55 inhibited the expression of syndecan‑1 but not ADAMTS‑4, and overexpression of syndecan‑1 reversed the inhibitory effect of WIN‑55 on the ADAMTS‑4 activity in unstimulated and IL‑1β‑stimulated human OA articular chondrocytes. Despite having no significant effect on syndecan‑1 gene promoter activity, WIN‑55 markedly decreased the stability of syndecan‑1 mRNA via CB2. In conclusion, to the best of our knowledge, the present study provides the first in vitro evidence supporting that the synthetic cannabinoid WIN‑55 inhibits ADAMTS‑4 activity in unstimulated and IL‑1β‑stimulated human OA articular chondrocytes by decreasing the mRNA stability/expression of syndecan‑1 via CB2. This suggests a novel mechanism by which cannabinoids may prevent cartilage breakdown in OA. In addition, it also provides novel insights into the pharmacological effects of synthetic cannabinoids on OA.

Figures

Figure 1
Figure 1
Effect of WIN-55 on ADAMTS-4 activity in human osteoarthritis (OA) articular chondrocytes in the presence or absence IL-1β. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. The cells were subsequently treated with WIN-55 (0.5, 1.0, 2.0, 4.0 or 8.0 μM) for 24 h. ADAMTS-4 activity was measured with a fluorimetric SensoLyte 520 Aggrecanase-1 Assay and expressed as fold change to that of the unstimulated control (designated as 1). Data are presented as the mean ± standard deviation. *P<0.05 vs. control; **P<0.05 vs. WIN-55 (0.5 μM); #P<0.05 vs. WIN-55 (1.0 μM); ##P<0.05 vs. WIN-55 (2.0 μM). ADAMTS-4, A disintegrin and metalloproteinase with thrombospondin motifs-4; IL-1β, interleukin-1β; WIN-55, WIN-55,212-2 mesylate.
Figure 2
Figure 2
Expression of cannabinoid receptors in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Expression of (A) CB1 and (B) CB2 were determined with western blot analyses using whole cell lysates from two independent preparations (samples 1 and 2): Lanes 1 and 3, sample 1; lanes 2 and 4, sample 2. β-actin was used as a loading control. The density of the CB1 and CB2 blots were normalized against that of the β-actin blot to obtain a relative blot density. Three independent experiments were performed for each western blot analysis. Data are presented as the mean ± standard deviation. IL-1β, interleukin-1β; CB1/2, cannibinoid receptor type 1/2.
Figure 3
Figure 3
Effect of WIN-55 on ADAMTS-4 activity in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β and cannabinoid receptor antagonists. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. The cells were treated with or without selective cannabinoid receptor type 1 (CB1) antagonist MJ15 (1, 10 or 50 μM) or selective CB2 antagonist JTE907 (0.1, 0.3 or 0.6 μM) for 30 min prior to the addition of WIN-55 (8.0 μM) for a further 24-h incubation. ADAMTS-4 activity was measured with a fluorimetric SensoLyte 520 Aggrecanase-1 Assay and expressed as fold change to that of the unstimulated control (designated as 1). Data are presented as the mean ± standard deviation. *P<0.05 vs. control; **P<0.05 vs. WIN-55; #P<0.05 vs. WIN-55 + MJ15 (1 μM); ##P<0.05 vs. WIN-55 + MJ15 (10 μM); ***P<0.05 vs. WIN-55 + MJ15 (50 μM); ###P<0.05 vs. WIN-55 + JTE907 (0.1 μM). ADAMTS-4, A disintegrin and metalloproteinase with thrombospondin motifs-4; IL-1β, interleukin-1β; WIN-55, WIN-55,212-2 mesylate.
Figure 4
Figure 4
Expression of ADAMTS-4 in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β, WIN-55 and cannabinoid receptor antagonists. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with or without selective cannabinoid receptor type 1 (CB1) antagonist MJ15 (50 μM) or selective CB2 antagonist JTE907 (0.6 μM) for 30 min prior to the addition of WIN-55 (8.0 μM) for a further 24-h incubation. The expression of ADAMTS-4 was determined by western blot analyses: Lanes 1 and 5, control; lanes 2 and 6, WIN-55; lanes 3 and 7, WIN-55 + MJ15; lanes 4 and 8, WIN-55 + JTE907. β-actin was used as a loading control. The density of the ADAMTS-4 blot was normalized against that of the β-actin blot to obtain a relative blot density. Three independent experiments were performed for each western blot analysis. Data are presented as the mean + standard deviation. *P<0.05 vs. unstimulated control. IL-1β, interleukin-1β; ADAMTS-4, A disintegrin and metalloproteinase with thrombospondin motifs-4; WIN-55, WIN-55,212-2 mesylate.
Figure 5
Figure 5
Protein expression levels of syndecan-1 in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β, WIN-55 and cannabinoid receptor type 2 (CB2) antagonist. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with or without selective CB2 antagonist JTE907 (0.6 μM) for 30 min prior to the addition of WIN-55 (8.0 μM) for a further 24-h incubation. For syndecan-1 overexpression experiments, human OA articular chondrocytes were stably transfected with the pcDNA3.1-syndecan-1 plasmid. Cells stably transfected with the pcDNA3.1 plasmid were used as the vector control. The protein levels of syndecan-1 were determined by western blot analyses. (A) Lanes 1 and 5, control; lanes 2 and 6, WIN-55 (8.0 μM); lanes 3 and 7, JTE907 (0.6 μM); lanes 4 and 8, WIN-55 (8.0 μM) + JTE907 (0.6 μM). (B) Lanes 1 and 7, control; lanes 2 and 8, vector control; lanes 3 and 9, syndecan-1 (overexpression); lanes 4 and 10, WIN-55 (8.0 μM); lanes 5 and 11, syndecan-1 + WIN-55 (8.0 μM); lanes 6 and 12, syndecan-1 + WIN-55 (8.0 μM) + JTE907 (0.6 μM). β-actin was used as a loading control. Density of the syndecan-1 blot was normalized to that of the β-actin blot to obtain a relative blot density. Three independent experiments were performed for each western blot analysis. Data are presented as the mean ± standard deviation. For panel (A): *P<0.05 vs. control. For panel (B): *P<0.05 vs. control and vector control; **P<0.05 vs. syndecan-1; #P<0.05 vs. WIN-55; ##P<0.05 vs. syndecan-1 + WIN-55. IL-1β, interleukin-1β; WIN-55, WIN-55,212-2 mesylate.
Figure 6
Figure 6
mRNA expression levels of syndecan-1 in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β, WIN-55 and cannabinoid receptor type 2 (CB2) antagonist. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with or without selective CB2 antagonist JTE907 (0.6 μM) for 30 min prior to the addition of WIN-55 (8.0 μM) for a further 24-h incubation. For syndecan-1 overexpression experiments, human OA articular chondrocytes were stably transfected with the pcDNA3.1-syndecan-1 plasmid. Vector control cells were stably transfected with the pcDNA3.1 plasmid. The mRNA levels of syndecan-1 were determined by reverse transcription-quantitative polymerase chain reaction and expressed as fold change to that of the unstimulated control (designated as 1). Data are presented as the mean ± standard deviation. *P<0.05 vs. control and vector control; **P<0.05 vs. syndecan-1; #P<0.05 vs. WIN-55; ##P<0.05 vs. syndecan-1 + WIN-55. WIN-55, WIN-55,212-2 mesylate; IL-1β, interleukin-1β.
Figure 7
Figure 7
Effect of WIN-55 on ADAMTS-4 activity in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β, cannabinoid receptor type 2 (CB2) antagonist and overexpression of syndecan-1. Primary human OA articular chondrocytes were cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with or without selective CB2 antagonist JTE907 (0.6 μM) for 30 min prior to the addition of WIN-55 (8.0 μM) for a further 24-h incubation. For syndecan-1 overexpression experiments, human OA articular chondrocytes were stably transfected with the pcDNA3.1-syndecan1 plasmid. Vector control cells were stably transfected with the pcDNA3.1 plasmid. ADAMTS-4 activity was measured with a fluorimetric SensoLyte 520 Aggrecanase-1 Assay and expressed as fold change to that of the unstimulated control (designated as 1). Data are presented as the mean ± standard deviation. *P<0.05 vs. control and vector control; **P<0.05 vs. syndecan-1; #P<0.05 vs. WIN-55; ##P<0.05 vs. syndecan-1 + WIN-55. WIN-55, WIN-55,212-2 mesylate ADAMTS-4, A disintegrin and metalloproteinase with thrombospondin motif-4; IL-1β, interleukin-1β.
Figure 8
Figure 8
Effect of WIN-55 on human syndecan-1 gene promoter activity in human osteoarthritis (OA) articular chondrocytes in the presence or absence of IL-1β. Primary human OA articular chondrocytes were transfected with human syndecan-1 gene promoter/luciferase reporter plasmids and then cultured in the presence or absence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with WIN-55 (8.0 μM) for 24 h. Luciferase activities were determined and expressed as fold change to that of the unstimulated control (designated as 1). Data are presented as the mean ± standard deviation. WIN-55, WIN-55,212-2 mesylate; IL-1β, interleukin-1β.
Figure 9
Figure 9
Effect of WIN-55 on syndecan-1 mRNA stability in human osteoarthritis (OA) articular chondrocytes in the presence or absence of interleukin (IL)-1β and cannabinoid receptor type 2 (CB2) antagonist. Primary human OA articular chondrocytes were cultured in the (A) absence or (B) presence of 10 ng/ml IL-1β for 24 h. Then the cells were treated with or without selective CB2 antagonist JTE907 (0.6 μM) for 30 min prior to the addition of WIN-55 (1.0 or 8.0 μM) for a further 24-h incubation. Next, transcription inhibitor actinomycin D (1 mg/ml) was added and incubated with the cells for 1, 2 or 4 h. The mRNA level of syndecan-1 was determined by reverse transcription-quantitative polymerase chain reaction and expressed as fold change to that of the control cells immediately prior to actinomycin D treatment (designated as 1). *P<0.05 vs. control; **P<0.05 vs. WIN-55 (1.0 μM); #P<0.05 vs. WIN-55 (8.0 μM). WIN-55, WIN-55,212-2 mesylate.

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