Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Abstract

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.