1,25(OH)2D3 INCREASES CALCIUM AND PHOSPHATIDYLINOSITOL METABOLISM IN DIFFERENTIATING CULTURED HUMAN KERATINOCYTES

The effect of 1,25(OH)2D3 on the intracellular calcium, (Ca+2)i, in both cultured human keratinocytes and in cultured human dermal fibroblasts was investigated. When the intracellular calcium (Ca+2)i in cultured human keratinocytes, grown in a serum-free medium containing 1.8 mM calcium, was measured by the fluorescent calcium-indicator, Furu-2, the (Ca+2i increased 154%, 202%, and 409% over the control value after incubation with 1,25(OH)2D3 at 10-10 m, 10-8 m, and 10-6 m, respectively. This response was immediate (15 seconds), specific (no effect with either 25(OH)D3 at 10-8 m or vitamin D3 at 10-8 m), and occurred with or without EGTA in the medium. In contrast, 1,25(OH)2D3 did not increase the (Ca2+)i in either cultured human keratinocytes that were grown in low calcium (0.05 mm), serum-free medium or in cultured human dermal fibroblasts that were grown in medium containing 0.05 mm calcium and 1% serum. The effect of 1,25(OH)2D3 on the the turnover of phosphatidylinositol was investigated as a possible cause for the observed increase in (Ca+2i. Cultured human keratinocytes that were incubated with 3H-inositol demonstrated a 50% ± 10% increase in the triphosphated, plasma membrane-bound metabolite of phosphatidylinositol, PIP2, by 15 seconds, followed by a rapid decrease at 30 seconds, then a return toward basal levels by 1 minute. Lysophosphatidylinositol, which results from the sn-2 deacylation of phosphatidylinositol by phospholipase A2, decreased 20% ± 8% within 30 seconds, then increased to 200% ± 10% of the control value by 5 minutes. The accumulation of IP3 was increased 50% to 100% above the control value within 30 seconds and this increase was substained during the 5-minute incubation period. Stimulation of phosphatidylinositol turnover by 1,25(OH)2D3 was not detected in either cultured human keratinocytes that were grown in serum-free, low calcium medium or in cultured human dermal fibroblasts that were grown in 1% serum. © 1990.