Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism and collagen synthesis in clonal rat osteoblastic (ROS 17/2.8) cells

Abstract

EGF, a single-chain polypeptide growth factor important for many cellular functions including glycolysis and protein phosphorylation, is known to modulate calcium metabolism in several cell systems. EGF causes an increase in Ca 2+ influx and accumulation of inositol triphosphate and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; therefore, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if lead impairs collagen synthesis, which is controlled by EGF. To characterize 45Ca kinetics, cells were labelled with 45Ca (1.87 m m Ca) for 20 hr in the presence of 5 μ m Pb, 50 ng/ml EGF, or 5 μ m Pb and 50 ng/ml EGF. Kinetic parameters were determined from 45Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45Ca; 5 μ m Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast-exchanging, quantitatively small calcium pools S 1 and S 2 to the slow-exchanging, quantitatively large S 3. There was also a 50% increase in total cell calcium in cells treated with 5 μ m Pb and 50 ng/ml EGF over calcium in cells treated with 50 ng/ml EGF alone. Because EGF and phorbol 12-myristate 13-acetate (PMA) have similar effects on protein kinase C (PKC) and collagen metabolism, the transient effects of EGF and PMA on 45Ca and 210Pb were also characterized. EGF caused a rapid transient increase in efflux of both isotopes, which was further increased by the addition of PMA. In contrast, PMA pretreatment, which depletes PKC, significantly attenuated the latter effect of EGF, suggesting that downregulation by PKC of EGF-induced increases in 45Ca and 210Pb efflux. Moreover, collagen synthesis was decreased by lead, EGF, and PMA in a similar manner, further suggesting PKC as the common modulator of these effects. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis and collagen synthesis by EGF. Furthermore, these results provide additional support to the postulate that an early and discrete effect of lead involves perturbation of the calcium messenger system at one or several loci.