thapsigargin and involucrin

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

Topics: Base Sequence; Binding Sites; CCAAT-Enhancer-Binding Protein-alpha; DNA; DNA Primers; Gene Expression Regulation; Promoter Regions, Genetic; Protein Precursors; Tetradecanoylphorbol Acetate; Thapsigargin

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.

Topics: Biomarkers; Calcium; Cell Differentiation; Cell Division; Cells, Cultured; Cytoskeletal Proteins; Desmoplakins; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Keratinocytes; Protein Precursors; Terpenes; Thapsigargin; Time Factors