IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production.
IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.
Rodan
S B
SB
Department of Bone Biology and Osteoporosis, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Wesolowski
G
G
Chin
J
J
Limjuco
G A
GA
Schmidt
J A
JA
Rodan
G A
GA
D000262:Adenylyl Cyclases / Q000032:analysis; D001862:Bone Resorption / Q000139:chemically induced; D000242:Cyclic AMP / Q000096:biosynthesis*; D015232:Dinoprostone / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D012516:Osteosarcoma / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000032:analysis*; D017472:Receptors, Interleukin-1; D014407:Tumor Cells, Cultured
D007375:Interleukin-1; D007457:Iodine Radioisotopes; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015232:Dinoprostone