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Culture duration and PUFA treatment influence expression of endocannabinoid proteins in MC3T3‐E1 osteoblast‐like cells
Author(s) -
Hutchins Heather L,
Zhao Dongjiao,
Han Kevin M,
Li Yong,
Watkins Bruce A
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.543.15
Subject(s) - arachidonic acid , eicosapentaenoic acid , docosahexaenoic acid , endocannabinoid system , polyunsaturated fatty acid , chemistry , phospholipase a2 , osteoblast , cell culture , lysophosphatidic acid , fatty acid , medicine , endocrinology , biochemistry , receptor , in vitro , biology , enzyme , genetics
Anadamide (AEA) and 2‐arachidonoylglycerol (2‐AG) are derived from arachidonic acid (AA). Recently, the endocannabinoid (EC) system has been linked to bone remodeling with in vitro and in vivo models. The purpose of this study was to examine the effects of culture time and n‐3 or n‐6 PUFA treatment on the EC system in the MC3T3‐E1 osteoblast‐like cell line. Cells (passage 7‐8) seeded at 6 x 10 4 in 6‐well plates were cultured in osteogenic medium up to 30 d, and at the last 72 h of incubation treated with AA, or eicosapentaenoic (EPA) plus docosahexaenoic (DHA) acids (10 µM each) with BSA as a vehicle control. Cells were harvested at 10, 25 and 30 d for fatty acid analysis and quantitative RT‐PCR. All cultures reflected enrichment of the respective fatty acid treatment; however, a greater magnitude of increase for EPA and DHA was achieved compared to AA. CB2 (the EC receptor) mRNA level was greatest in cells after 30 d compared to 10 d for the vehicle control; however, AA treatment decreased CB2 mRNA expression over time. Expression of the AEA synthesis enzyme, n‐acyl phosphatidylethanolamine phospholipase D (NAPE‐PLD), increased with AA treatment and culture time. These results show that developmental stage and PUFA alters the expression of CB2 and NAPE‐PLD, which suggests that maturation stage and PUFA treatments may influence the EC system in MC3T3‐E1 cells. Supported by USDA funds.