title: Determination and expression of platelet-derived growth factor-AA in bone cell cultures.
abstract: We describe a sensitive technique for the extraction and quantitation of platelet-derived growth factor (PDGF)-AA in serum-free culture medium conditioned by fetal rat osteoblast-enriched (Ob) cells and demonstrate the expression of PDGF-A mRNA in Ob cells. Using C18 Sep-Pak chromatography with a methanol step gradient, we extracted immunoreactive PDGF-AA from the culture medium. A RIA protocol employing a recombinant human PDGF-AA standard enabled us to measure picomolar equivalents of PDGF-AA in Ob cell culture medium. The recovery of recombinant human PDGF-AA was 50 +/- 4%, and the coefficient of variation, including chromatographic extraction, was 15% for intraassay and 8.3% for interassay variability. The polyclonal antibody to recombinant human PDGF-AA displayed approximately 20-30% cross-reactivity with PDGF-AB, but did not bind PDGF-BB or other growth factors and cytokines known to be secreted by bone cells. Medium from Ob cells cultured for 24 h contained 0.9-1.3 pM human PDGF-AA equivalents, and exposure to cycloheximide (3.6 microM) decreased those levels by 65%. Treatment of cells with recombinant human transforming growth factor-beta 1 at 0.04-4 nM for 24 h increased PDGF-AA levels by up to 3.5-fold. Northern blot analysis of RNA from Ob cells revealed the expression of PDGF-A, but not PDGF-B, transcripts, and transforming growth factor-beta 1 at 0.04 and 0.2 nM increased steady state PDGF-A mRNA by 3- to 6-fold. Our studies describe a sensitive and reproducible technique for monitoring PDGF-AA levels in cell-conditioned medium and demonstrate that Ob cells synthesize PDGF-AA.
authors.affiliation: Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut 6105.
authors.forename: S
authors.initials: S
authors.lastname: Rydziel
authors.forename: C
authors.initials: C
authors.lastname: Ladd
authors.forename: T L
authors.initials: TL
authors.lastname: McCarthy
authors.forename: M
authors.initials: M
authors.lastname: Centrella
authors.forename: E
authors.initials: E
authors.lastname: Canalis
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000737:chemistry*; D002478:Cells, Cultured; D010982:Platelet-Derived Growth Factor / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011863:Radioimmunoassay; D051381:Rats
chemical_list: D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger