The Ca2(+)-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells.
SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca2(+)-binding protein associated with cellular populations undergoing migration, proliferation, and/or differentiation. Active preparations of SPARC bind to specific components of the extracellular matrix and cause mesenchymal cells to assume a rounded phenotype. In this study we show that SPARC modulates the progression of bovine aortic endothelial cells through the cell cycle. At a concentration of 20 micrograms/ml, SPARC inhibited the incorporation of [3H]thymidine into newly synthesized DNA by approximately 70%, as compared to control cultures within 24 hr after the release from G0 phase. The effect was dose-dependent and reached greater than 90% inhibition at 30 micrograms of SPARC per ml after 24 hr. A 20-residue synthetic peptide (termed 2.1) from a non-Ca2(+)-binding, disulfide-rich domain of SPARC also exhibited a dose-dependent inhibition of [3H]thymidine uptake in endothelial cells within 24 hr after release from G0 phase. An inhibition of 50% was seen with peptide 2.1 at a 0.4 mM concentration. Peptides from other regions of the SPARC protein did not produce this effect. Maximum inhibition of [3H]thymidine uptake by SPARC and peptide 2.1 occurred during the early-to-middle G1 phase of the endothelial-cell cycle. From 0-12 hr after release from G0 phase, cells exhibited delayed entry into S phase, which normally occurred at 24 +/- 2 hr. These results were further corroborated by flow cytometry. In the presence of SPARC at 20 micrograms/ml, 72% fewer cells were in S phase after a 24-hr period; a similar, but less marked, reduction was seen with peptide 2.1. Peptide 2.1 did not cause cell rounding, whereas peptide 1.1, a highly efficient inhibitor of endothelial-cell spreading, exhibited essentially no activity with respect to cell-cycle progression. It therefore appears that the transient, inhibitory effect of SPARC on the entry of endothelial cells into S phase does not depend on the overt changes in cell shape mediated through cytoskeletal rearrangement.
Funk
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Department of Biological Structure, University of Washington, Seattle 98195.
Sage
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D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D005434:Flow Cytometry; D007700:Kinetics; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000235:genetics / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D002135:Calcium-Binding Proteins; D015676:Osteonectin; D010455:Peptides; D014316:Tritium; D013936:Thymidine