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Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells.

Wei Y, Wang X, Wang L - Mol. Vis. (2009)

Bottom Line: The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage.In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.

ABSTRACT

Purpose: Cannabinoid receptors have been detected in neuron cells and proposed as potential therapeutic agents in neurodegenerative disorders because of their involvement in controlling neural cell survival and death. However, their presence and role in human retinal pigment epithelial (RPE) cells, which play a key role in initiating and developing age related macular degeneration (ARMD), have never been investigated. Here we analyzed the expression of and changes in cannabinoid receptors (CB1 and CB2) and one enzyme responsible for endocannabinoid hydrolysis, fatty acid amide hydrolase (FAAH), in RPE cell oxidative damage process, a cellular model of ARMD.

Methods: Primary human RPE cells and cells from the ARPE-19 cell line were cultured and exposed to H2O2 for 24 h to induce oxidative damage. Real time RT-PCR, immunofluorescent staining, and western blot methods were performed to study the expression of and changes in CB1 and CB2 receptors, and FAAH. Cell viability and reactive oxygen species (ROS) production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and a dichlorofluorescein (DCF) assay, respectively. PI3K/Akt and ERK1/2 protein expression and activation of signaling molecules were assessed by western blot analysis.

Results: By using real time RT-PCR, immunofluorescent staining and western blot methods, we showed that human RPE cells express CB1, CB2, and FAAH. Meanwhile, oxidative stress can upregulate CB1 and CB2 receptor expression, and downregulate FAAH expression. The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage. In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.

Conclusions: The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells. The modulation of cannabinoid receptor tone warrants consideration for future therapeutic strategies of ARMD.

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Related in: MedlinePlus

Expression of CB1, CB2, and FAAH mRNA in human primary RPE cells and changes of CB1, CB2, FAAH mRNA and protein expression in 200 μM H2O2-treated ARPE-19 cells compared to untreated ones. A: Expression of CB1, CB2, and FAAH mRNA expression in human primary RPE cells assayed by real time RT–PCR method. B: Changes of CB1, CB2, and FAAH mRNA expression in ARPE-19 cells assayed by real time RT–PCR method. Asterisk (*) represents the correlation significant at the p<0.05 level and suggest a significant increase or decrease in mRNA expression as compared to control group. C: Changes of CB1, CB2, and FAAH protein expression in ARPE-19 cells assayed by western blot method. D: Changes of CB1, CB2 protein expression in ARPE-19 cells demonstrated by immunofluorescent staining.
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f2: Expression of CB1, CB2, and FAAH mRNA in human primary RPE cells and changes of CB1, CB2, FAAH mRNA and protein expression in 200 μM H2O2-treated ARPE-19 cells compared to untreated ones. A: Expression of CB1, CB2, and FAAH mRNA expression in human primary RPE cells assayed by real time RT–PCR method. B: Changes of CB1, CB2, and FAAH mRNA expression in ARPE-19 cells assayed by real time RT–PCR method. Asterisk (*) represents the correlation significant at the p<0.05 level and suggest a significant increase or decrease in mRNA expression as compared to control group. C: Changes of CB1, CB2, and FAAH protein expression in ARPE-19 cells assayed by western blot method. D: Changes of CB1, CB2 protein expression in ARPE-19 cells demonstrated by immunofluorescent staining.

Mentions: As Figure 2A shows, real time RT–PCR revealed primary human RPE cells expressed CB1, CB2, and FAAH mRNA. ARPE-19 cells were treated with 200 μM H2O2 for 24 h, and the changes in CB1, CB2, and FAAH mRNA expression were determined by quantitative RT–PCR. The results showed that H2O2-treated ARPE-19 cells had a 7.02 fold increased CB1 mRNA expression, a 5.68 fold increased CB2 mRNA expression, and a 35.7 fold decreased FAAH mRNA expression, compared to untreated cells (Figure 2B). Consistent with the results of RT–PCR, CB1 and CB2 protein expression increased and FAAH protein expression decreased in 200 μM H2O2 -treated ARPE-19 cells (Figure 2C). Similar results were obtained with immunofluorescence assays (Figure 2D).


Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells.

Wei Y, Wang X, Wang L - Mol. Vis. (2009)

Expression of CB1, CB2, and FAAH mRNA in human primary RPE cells and changes of CB1, CB2, FAAH mRNA and protein expression in 200 μM H2O2-treated ARPE-19 cells compared to untreated ones. A: Expression of CB1, CB2, and FAAH mRNA expression in human primary RPE cells assayed by real time RT–PCR method. B: Changes of CB1, CB2, and FAAH mRNA expression in ARPE-19 cells assayed by real time RT–PCR method. Asterisk (*) represents the correlation significant at the p<0.05 level and suggest a significant increase or decrease in mRNA expression as compared to control group. C: Changes of CB1, CB2, and FAAH protein expression in ARPE-19 cells assayed by western blot method. D: Changes of CB1, CB2 protein expression in ARPE-19 cells demonstrated by immunofluorescent staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697670&req=5

f2: Expression of CB1, CB2, and FAAH mRNA in human primary RPE cells and changes of CB1, CB2, FAAH mRNA and protein expression in 200 μM H2O2-treated ARPE-19 cells compared to untreated ones. A: Expression of CB1, CB2, and FAAH mRNA expression in human primary RPE cells assayed by real time RT–PCR method. B: Changes of CB1, CB2, and FAAH mRNA expression in ARPE-19 cells assayed by real time RT–PCR method. Asterisk (*) represents the correlation significant at the p<0.05 level and suggest a significant increase or decrease in mRNA expression as compared to control group. C: Changes of CB1, CB2, and FAAH protein expression in ARPE-19 cells assayed by western blot method. D: Changes of CB1, CB2 protein expression in ARPE-19 cells demonstrated by immunofluorescent staining.
Mentions: As Figure 2A shows, real time RT–PCR revealed primary human RPE cells expressed CB1, CB2, and FAAH mRNA. ARPE-19 cells were treated with 200 μM H2O2 for 24 h, and the changes in CB1, CB2, and FAAH mRNA expression were determined by quantitative RT–PCR. The results showed that H2O2-treated ARPE-19 cells had a 7.02 fold increased CB1 mRNA expression, a 5.68 fold increased CB2 mRNA expression, and a 35.7 fold decreased FAAH mRNA expression, compared to untreated cells (Figure 2B). Consistent with the results of RT–PCR, CB1 and CB2 protein expression increased and FAAH protein expression decreased in 200 μM H2O2 -treated ARPE-19 cells (Figure 2C). Similar results were obtained with immunofluorescence assays (Figure 2D).

Bottom Line: The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage.In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.

ABSTRACT

Purpose: Cannabinoid receptors have been detected in neuron cells and proposed as potential therapeutic agents in neurodegenerative disorders because of their involvement in controlling neural cell survival and death. However, their presence and role in human retinal pigment epithelial (RPE) cells, which play a key role in initiating and developing age related macular degeneration (ARMD), have never been investigated. Here we analyzed the expression of and changes in cannabinoid receptors (CB1 and CB2) and one enzyme responsible for endocannabinoid hydrolysis, fatty acid amide hydrolase (FAAH), in RPE cell oxidative damage process, a cellular model of ARMD.

Methods: Primary human RPE cells and cells from the ARPE-19 cell line were cultured and exposed to H2O2 for 24 h to induce oxidative damage. Real time RT-PCR, immunofluorescent staining, and western blot methods were performed to study the expression of and changes in CB1 and CB2 receptors, and FAAH. Cell viability and reactive oxygen species (ROS) production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and a dichlorofluorescein (DCF) assay, respectively. PI3K/Akt and ERK1/2 protein expression and activation of signaling molecules were assessed by western blot analysis.

Results: By using real time RT-PCR, immunofluorescent staining and western blot methods, we showed that human RPE cells express CB1, CB2, and FAAH. Meanwhile, oxidative stress can upregulate CB1 and CB2 receptor expression, and downregulate FAAH expression. The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage. In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.

Conclusions: The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells. The modulation of cannabinoid receptor tone warrants consideration for future therapeutic strategies of ARMD.

Show MeSH
Related in: MedlinePlus