Biomedical subjects

S Rydziel

Publications and source records attributed to S Rydziel.

22 records · Page 2Linked to original sources

PubMed1994

Platelet-derived growth factor-AA and -BB (PDGF-AA and -BB) enhance the synthesis of PDGF-AA in bone cell cultures.

Platelet-derived growth factor (PDGF), an agent with important mitogenic effects for bone cells, exists in three isoforms, PDGF-AA, -BB, and -AB. PDGF-AB and -BB are the prevalent circulating isoforms, whereas normal unstimulated cells of the osteoblast lineage synthesize primarily PDGF-AA. We examined the effects of PDGF-BB on PDGF-A mRNA expression and PDGF-AA polypeptide concentrations in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). In a selected number of experiments we compared the effects of PDGF-BB with those of PDGF-AA on PDGF-A mRNA levels. Steady state PDGF-A mRNA levels were determined by Northern blot analysis, and PDGF-AA concentrations were determined in acidified and fractionated culture medium by a specific RIA for PDGF-A chains. Treatment of Ob cells with PDGF-AA or -BB at 0.3-3.3 nM caused a dose-dependent increase in steady state PDGF-A mRNA, an effect that was initially observed after 2 h. Treatment with PDGF-BB at 1-3.3 nM for 24 h increased PDGF-AA polypeptide concentrations by 2- to 5-fold. The effects of PDGF on PDGF-A mRNA and polypeptide levels were prevented by the protein synthesis inhibitor cycloheximide at 3.6 microM. Phorbol 12-myristate 13-acetate at 1 microM increased PDGF-A mRNA after 2-6 h and PDGF-AA polypeptide levels after 24 h by 2-fold. However, the protein kinase-C inhibitor staurosporine at 50 nM did not modify basal PDGF-A mRNA levels and did not prevent the stimulatory effect of PDGF-AA or -BB on PDGF-A mRNA or PDGF-AA polypeptide levels. In conclusion, PDGF-BB and -AA increase skeletal PDGF-A synthesis, an effect that reveals autoinduction of PDGF in bone cells.

Alkaloids↗

PubMed1993

Growth factors regulate the synthesis of insulin-like growth factor-I in bone cell cultures.

Insulin-like growth factor-I (IGF-I), a prevalent growth factor secreted by bone cells, has important effects on bone remodeling. Hormones are known to regulate the synthesis of skeletal IGF-I, but there is limited information about the actions of growth factors on IGF-I synthesis. We tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF beta 1), and platelet-derived growth factors (PDGF) AA and BB on IGF-I mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (Ob) cells from 22-day-old fetal rat calvariae. Steady state IGF-I mRNA levels were determined by Northern blot analysis, and IGF-I concentrations were determined in acidified and fractionated culture medium by a specific RIA. Treatment of Ob cells with bFGF at 0.06-6 nM, TGF beta 1 at 0.04-4 nM, and PDGF BB at 0.3-3.3 nM caused a dose-dependent decrease in steady state IGF-I mRNA. A smaller effect was observed with PDGF AA. The effect was initially observed after 6-8 h of treatment and was maximal after 16 h. Treatment with bFGF at 0.6-6 nM, TGF beta 1 at 0.4-4 nM, and PDGF BB at 0.3-3.3 nM for 24 h decreased IGF-I polypeptide concentrations by 40-80%. The effects of bFGF, TGF beta 1, and PDGF BB and AA on IGF-I mRNA were independent of protein synthesis and cell division, as they were observed in the presence and absence of cycloheximide at 3.6 microM or hydroxyurea at 1 mM. Similarly, their inhibitory actions on immunoreactive IGF-I were not prevented by hydroxyurea. In conclusion, bFGF, TGF beta 1, PDGF BB, and, to a lesser extent, PDGF AA decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect may contribute to their actions on selected aspects of Ob cell function.

Animals↗

PubMed1992

Determination and expression of platelet-derived growth factor-AA in bone cell cultures.

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.

Animals↗

PubMed1989

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures.

We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C18 column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was less than 8%. [3H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [3H]proline and "chased" in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24-48 hour "chase" period increased the release of acid-soluble [3H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.

Animals↗