Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Abstract

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE2 was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE2, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGF beta and tumor necrosis factor-alpha(TNF). The production of 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) and of PGF2 alpha were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGF beta and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE2 biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6-12 h, and levels had appreciably diminished by 24 h after treatment. Both TGF beta and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE2 resulting from TNF stimulation was blocked by the addition of an interleukin-1 beta (IL-1 beta) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1 beta production. TGF beta regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines.

Estrogen Potentiates the Combined Effects of Transforming Growth Factor-β and Tumor Necrosis Factor-α on Adult Human Osteoblast-like Cell Prostaglandin E2 Biosynthesis

“…Because arachidonic acid metabolism by female hOB cells and male hOB cells exhibits sexually dimorphic characteristics [25], the responses of the cells of each sex were assessed independently in this experiment. The application of TGFβ, TNF, or both cytokines simultaneously significantly stimulated hOB cell PGE 2 production (p ≤ 0.01), agreeing with previous observations [12]. Pretreatment with 17 β-E 2 did not alter PGE 2 release under basal conditions, nor in specimens stimulated singly with either TGF or with TNF.…”

“…In those experiments, 1-[ 14 C]arachidonic acid conversion into 1-[ 14 C]PGE 2 did vary according to the stimulation of the cells with TGFβ, TNF, or with both used in combination, results in accord with previously published work [12,46]. However, estrogen pretreatment did not further modify the cytokine-induced increases in product formation from substrate.…”

“…4), although an overall increase in total radioactivity release in those experiments could not be demonstrated. The sensitivity of the assay methods may have been inadequate to detect a difference that might [12,46]. However, estrogen pretreatment did not further modify the cytokine-induced increases in product formation from substrate.…”

“…The present report examines the effects of estrogen on the elaboration of prostaglandin E 2 (PGE 2 ) by adult human osteoblast-like (hOB) cell cultures variously stimulated by TGFβ, tumor necrosis factor-α (TNF), or IL-1β, cytokines that do increase prostaglandin biosynthesis by hOB cells [12]. Each of these cytokines are products of osteoblastic cells that mediate bone biology in an autocrine/paracrine manner [13,14,15].…”

“…In some experiments, IL-1β at 300 nM was used in lieu of either TGFβ or TNF. These cytokine concentrations elicit their maximal effects on hOB cell PGE 2 production [12]. During the 30 minutes preceding the cytokine additions, some specimens were pretreated with 50 μM ibuprofen to inhibit prostaglandin production.…”