Thapsigargin Raises Intracellular Free Calcium Levels in Human Keratinocytes and Inhibits the Coordinated Expression of Differentiation Markers

Thapsigargin raises intracellular free calcium ([Ca 2+] i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca 2+] i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 n M thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca 2+] i rise in calcium-free or 70 μ M calcium medium but a more prolonged rise in 2 m M calcium. For keratinocytes cultured in 70 μ M calcium medium a late [Ca 2+] i rise was also observed, after 6 h, similar to the effect of known differentiation stimuli. However, immunohistochemical techniques did not show any expression of the differentiation-specific protein involucrin, a component of the cornified envelope. When keratinocyte differentiation was induced by an increase in the extracellular calcium from 70 μ M to 2 m M abundant involucrin and desmoplakin, a component of desmosomes, were synthesised. Both proteins gave staining patterns which suggested incorporation into structural proteins, but thapsigargin disrupted the calcium-induced pattern of involucrin and desmoplakin synthesis. Thapsigargin did not induce differentiation, possibly due to its inability to activate protein kinase C and raise inositol trisphosphate levels. We conclude that a rise in [Ca 2+] i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.