Formation of mineralized bone nodules by rat calvarial osteoblasts decreases with donor age due to a reduction in signaling through EP1 subtype of prostaglandin E-2 receptor

The effects of prostaglandin E-2 (PGE(2)) on the parameters for proliferati on and differentiation were studied in calvarial osteoblast-like cells isol ated from rats of various ages. In cells not treated with PGE(2), it was fo und that mineralized bone nodule (BN) formation, alkaline phosphatase (ALP) activity, and the incorporation rate of [H-3]thymidine into the cells shar ply decreased with the age of the cell donor at 6-50 weeks and then remaine d at a relatively constant level up to 120 weeks. Before studying the effec ts of PGE2 on these parameters, we determined the change in the levels of P GE(2): produced by the untreated cells during the culture period and found that the endogenous PGE(2) reached a maximum on the 4th day of the culture, regardless of the cell donor age, followed by a sharp decrease. The endoge nous production was blocked by pretreatment with a cyclooxygenase-2 (COX-2) inhibitor, NS-398, indicating the generation of PGE(2) through a COX-2 pat hway. The area of BN was effectively suppressed by NS-398 in the cells from 10- to 35-week-old rats, whereas it was enhanced in the cells from 90- to 120-week-old rats. Treatment with PGE(2) markedly increased the BN formatio n and the ALP activity in the cells from 4- to 35-week-old rats (defined as you ng rats). By contrast, PGE(2) decreased [H-3]thymidine incorporation i nto the cells from young rats. The area of BN and the ALP activity decrease d significantly, whereas [H-3]thymidine incorporation into the cells increa sed by 60-80% in the cells of 80- to 120-week-old rats (defined as aged rat s). The stimulatory effects on the cell differentiation and the inhibitory effect on the proliferation in the cells from young rats was mimicked by an EP1 agonist, 17-phenyl-omega-trinor PGE(2), while an EP2/EP4 agonist, 11-d eoxy-PGE(1) and an adenylate cyclase activator, forskolin suppressed the di fferentiation and enhanced the proliferation regardless of. the cell donor age. PGE(2), 11-deoxy-PGE(1) and forskolin, but not 17-phenyl-omega-trinor PGE(2) increased cyclic adenosine monophosphate (cAMP) production. Generati on of inositol 1,4,5-triphosphate (IP3) was stimulated by 17-phenyl-omega-t rinor PGE(2) or PGE(2), but not by 11-deoxy-PGE(1) or forskolin increased c AMP production in the cells from young rats. By contrast, PGE(2) had little effect on IP3 generation in aged rats. From the overall results, we conclu ded that PGE(2) exerts stimulatory and inhibitory effects on differentiatio n through the EP1-IP3 pathway and EP2/EP4-cAMP pathway, respectively, in th e cells from young rats. The EP1-IP3 pathway seems to be inactive in the ce lls from aged rats. J. Cell. Biochem. 75:275-225, 1999. (C) 1999 Wiley-Liss , Inc.