title: Inhibition of osteoblast apoptosis by thrombin.
abstract: The multifunctional serine protease thrombin has been shown to be a specific agonist for a variety of functional responses of cells including osteoblasts. The current study was conducted to determine if thrombin was capable of inhibiting apoptosis in osteoblasts, and if so, to examine the mechanism by which this occurred. Thrombin (20-100 nM) significantly inhibited apoptosis in serum-starved cultures of the human osteoblast-like Saos-2 cell line and cultures of primary osteoblasts isolated from mouse calvariae, as well as dexamethasone-treated primary mouse osteoblasts. Inhibition of serum deprivation-induced apoptosis was shown to require thrombin's specific proteolytic activity. Primary mouse osteoblasts were found to express two functional thrombin receptors, PAR-1 and PAR-4. Thrombin inhibited serum deprivation-induced apoptosis in osteoblasts isolated from PAR-1 null mice to the same degree as in osteoblasts isolated from wild-type mice. Treatment of serum-deprived osteoblasts, isolated from either PAR-1 null or wild-type mice, with a PAR-4-activating peptide failed to significantly inhibit apoptosis compared to the relevant control. Medium conditioned by thrombin-treated osteoblasts, in which thrombin had been inactivated, was able to inhibit serum deprivation-induced osteoblast apoptosis almost as well as thrombin itself. Blocking protein synthesis, by cycloheximide pretreatment of the conditioning cells, prevented this action. The ability of known osteoblast survival factors, such as transforming growth factor beta1, fibroblast growth factor-2, insulin-like growth factor-II, and interleukin-6, to inhibit serum deprivation-induced osteoblast apoptosis was also tested. None of these factors was able to inhibit serum deprivation-induced osteoblast apoptosis to the same extent as thrombin. The results presented here demonstrate that thrombin treatment of osteoblasts inhibits apoptosis induced either by dexamethasone or by serum deprivation. Furthermore, it does so independently of the known thrombin receptors by bringing about the synthesis and/or secretion of an unknown survival factor or factors, which then act in an autocrine fashion to inhibit apoptosis.
authors.affiliation: School of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia. cpagel@myraid.unimelb.edu.au
authors.forename: Charles N
authors.initials: CN
authors.lastname: Pagel
authors.forename: Michael R
authors.initials: MR
authors.lastname: de Niese
authors.forename: Linda A
authors.initials: LA
authors.lastname: Abraham
authors.forename: Carla
authors.initials: C
authors.lastname: Chinni
authors.forename: Shu Jun
authors.initials: SJ
authors.lastname: Song
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Mackie
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D012333:RNA, Messenger; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D044462:Receptors, Proteinase-Activated; C481941:protease-activated receptor 3, mouse; C481942:protease-activated receptor 4, mouse; D004247:DNA; D013917:Thrombin