Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers.
Thapsigargin raises intracellular free calcium ([Ca2+]i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca2+]i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 nM thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca2+]i rise in calcium-free or 70 microM calcium medium but a more prolonged rise in 2 mM calcium. For keratinocytes cultured in 70 microM calcium medium a late [Ca2+]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 microM to 2 mM 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 [Ca2+]i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.
Jones
K T
KT
Department of Dermatology, University of Liverpool, United Kingdom.
Sharpe
G R
GR
D015415:Biomarkers; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D051180:Desmoplakins; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013997:Time Factors
D015415:Biomarkers; D003598:Cytoskeletal Proteins; C495294:DSP protein, human; D051180:Desmoplakins; D011498:Protein Precursors; D013729:Terpenes; C032253:involucrin; D019284:Thapsigargin; D002118:Calcium