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Locally derived growth factors and cytokines in bone play a crucial role in the regulation of bone remodeling, i.e., bone formation and bone resorption processes. We studied the effect of interleukin (IL)-1α, tumor necrosis factor (TNF)-α, and Escherichia coli lipopolysaccharide (LPS) on the hormone-activated Ca2+ message system in the osteoblastic cell line UMR-106 and in osteoblastic cultures derived from neonatal rat calvariae. In both cell preparations, IL-1α, TNF-α, and LPS did not alter basal intracellular Ca2+ concentration ([Ca2+]i) but attenuated Ca2+ transients evoked by parathyroid hormone (PTH) and PGE2 in a dose (1–100 ng/ml)- and time (8–24 h)-dependent fashion. The cytokines modulated hormonally induced Ca2+ influx (estimated by using Mn2+ as a surrogate for Ca2+) as well as Ca2+ mobilization from intracellular stores. The latter was linked to suppressed production of hormonally induced inositol 1,4,5-trisphosphate. The effect of cytokines on [Ca2+]iwas abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml). The cytokine’s effect was, however, independent of nitric oxide (NO) production, since NO donors (sodium nitroprusside) as well as permeable cGMP analogs augment, rather than attenuate, hormonally induced Ca2+ transients in osteoblasts. Given the stimulatory role of cytokines on NO production in osteoblasts, the disparate effects of cytokines and NO on the Ca2+ signaling pathway may serve an autocrine/paracrine mechanism for modulating the effect of calciotropic hormones on bone metabolism.

The interaction of cells with the surrounding extracellular matrix (ECM) or basement membrane (BM) brings about profound changes in cellular biological responses, such as cell differentiation, proliferation, and gene expression. We studied the effect of ECM on PTH receptor binding and on biological responses mediated by PTH, in two cell preparations: 1) the proximal tubular OK opossum kidney cell line; and 2) MC3T3-E1 cells, a clonal line of nontransformed murine osteoblasts. Cells were plated on either plastic surfaces or on tissue culture dishes coated with specific ECM components. In both cell types plated on collagen-type IV (Col-IV), PTH receptor binding, on day 4 of culture, was markedly diminished, when compared with cells on plastic (approximately 45% inhibition, P < 0.01). In addition, Col-IV dose dependently inhibited cAMP generation stimulated by PTH (P < 0.001 vs. plastic), whereas cAMP generation by PGE2, cholera toxin, and forskolin was not altered. In Northern blot analysis, a PTH/PTH-related-protein receptor messenger RNA transcript was detected in both the kidney and bone cells. However, only OK cells manifested a decreased abundance of receptor messenger RNA when plated on Col-IV, compared with plastic. The physiological significance of inhibited cAMP production by Col-IV was evaluated by measuring the influence of different matrices on the activity of Na+/H+ exchanger (NHE) in OK cells and cell mitogenic activity in MC3T3-E1 cells (both responses are negatively modulated by cAMP). OK cells plated on Col-IV showed 70% inhibition of NHE, compared with cells plated on plastic (P < 0.01). PTH inhibits NHE activity in cells on plastic but stimulates exchanger activity by 40% in cells plated on Col-IV. In MC3T3-E1 cells grown on plastic, PTH exerts a dose-dependent antiproliferative effect, which is mediated by cAMP. This effect is mitigated when cells are grown on Col-IV (40-50% less antiproliferative effect). In summary, Col-IV, a maj or BM constituent, has a profound inhibitory effect on PTH binding and PTH-mediated biological responses in both kidney tubular cells and osteoblasts. Altered cellular function by Col-IV may be of physiological relevance in states associated with altered composition of BM or expansion of ECM (e.g. diabetes mellitus and interstitial fibrosis).