Evidence for Separate Control Mechanisms at the Message, Protein, and Enzyme Activation Levels for Transglutaminase During Calcium-Induced Differentiation of Normal and Transformed Human Keratinocytes

Abstract

We analyzed the effects of three different calcium concentrations on the RNA and functional protein levels of transglutaminase (TGase) and involucrin (INV) over time in culture. We compared the results in normal human keratinocytes with those in a squamous cell carcinoma, SCC4. The highest calcium concentration (1.2 mM) induced the greatest levels of INV and TGase message, INV protein, and rates of CE formation, but not maximal levels of TGase protein. By examining cytosol and membrane fractions of keratinocytes, we found that after synthesis, TGase protein shifts, under the influence of calcium (both 0.1 mM and 1.2 mM), from the cytosol into the membrane in postconfluent cells. However, only 1.2 mM calcium induced significant amounts of TGase activity. These data indicate that elevated calcium (1.2 mM) achieves the expected induction in keratinocyte differentiation by regulation of not only INV and TGase message levels, but also the translation and activation of TGase protein. Our data suggest that this calcium-induced activation of TGase protein occurs while the protein is anchored in the membrane. In contrast, despite ample INV and TGase message levels within SCC4 cells, these RNA levels are not regulated by calcium or translated into protein, suggesting that the transformed phenotype of SCC4 cells results not only in a failure of calcium to regulate gene transcription, but also in a defect within the translation machinery of these differentiation-specific proteins.

Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinomas

“…Interestingly, Gibson et al observed a distinction between welldifferentiated keratinocytes and poorly differentiated SCC4 cells with respect to calcium ion-induced involucrin expression. In particular, high calcium concentration induced involucrin mRNA and protein in keratinocytes but not in poorly differentiated SCC4 cells (Gibson et al, 1996). This similarity between calcium and hypoxia regulation might be important for understanding how hypoxia induces involucrin.…”

“…Induction occurs over a period of 2 h and is, therefore, relatively rapid. In contrast to SCC9 cells, hypoxia induces little or no involucrin in poorly differentiated SCC4 cells in spite of the fact that the involucrin gene is reported to be functional with ample quantities of involucrin mRNA present in these cells (Gibson et al, 1996). Interestingly, Gibson et al observed a distinction between welldifferentiated keratinocytes and poorly differentiated SCC4 cells with respect to calcium ion-induced involucrin expression.…”

Differentiation-Specific Factors Modulate Epidermal CYP1–4 Gene Expression in Human Skin in Response to Retinoic Acid and Classic Aryl Hydrocarbon Receptor Ligands

“…Hyperkeratinization is most pronounced in more terminally differentiated keratinocyte cultures, consistent with the ability of TCDD to activate AhR more effectively in differentiating keratinocytes (Swanson, 2004). During differentiation, keratinocytes acquire regulatory cofactors (e.g., calcium, nucleotides) in their cellular milieu essential for post-translational processing and catalytic activation of expressed TGM proteins; this occurs mainly in the outer epidermal layers (Gibson et al, 1996).…”

Phospholipase D Signaling and Extracellular Signal-Regulated Kinase-1 and -2 Phosphorylation (Activation) Are Required for Maximal Phorbol Ester-Induced Transglutaminase Activity, a Marker of Keratinocyte Differentiation

“…In this study, we also demonstrate a correlation be- tween the inhibition of PLD activation and of increased transglutaminase activity in response to TPA, suggesting a role for PLD in this process. It should be noted that transglutaminase activity is considered a marker of late keratinocyte differentiation, with enzymatic activity regulated at multiple levels, including transcriptionally, translationally, and post-translationally (Gibson et al, 1996). Therefore, an ability of the agents used in this study to inhibit TPAinduced transglutaminase activity suggests a more general effect on keratinocyte differentiation.…”