Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice

Abstract

The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear.

Regulation of Protein Kinase D During Differentiation and Proliferation of Primary Mouse Keratinocytes

“…The changes in PKD protein levels and serine 916 phosphorylation were measured at the time points of 8, 16, and 24 h because these time points encompass the rising phase (8 h), the maximal observed transglutaminase activity (16 h), and the plateau phase (24 h) after stimulation with 1 mM CaCl 2 (Fig 5). These time points also correspond to the previous report that elevated [Ca 2+ ] e (1 mM) treatment inhibits the proliferative response of keratinocytes as shown by the significant and maximal inhibition of DNA sythesis after 8 and 24‐h calcium treatments, respectively (Bollag et al , 1994). The data indicate that PKD protein levels and serine 916 phosphorylation were significantly downmodulated after 8, 16, and 24 h of elevated [Ca 2+ ] e treatment when compared with control (Fig 5).…”

Calcium regulation of keratinocyte differentiation

“…Studies of the role of PKC in keratinocyte differentiation utilize phorbol esters [54,56,69–73]. These compounds are capable of stimulating differentiation of keratinocytes at least in vitro even in low Cao conditions, although their effects can be potentiated by Cao [69–73].…”

“…As alluded to previously, activation of PKC leads to activation of transcription factors in the Fos/Jun families that probably mediate the effects of calcium, phorbol esters and DAG on keratinocyte differentiation [53,54,89–93]. These transcription factors bind to AP-1 sites in the regulatory regions of the genes that they regulate [94].…”

Biphasic effect of 1,25‐dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation

“…We found that low concentrations of 1,25(OH),D3 (1 pM to 1 nM) actually stimulated cell growth, whereas at higher concentrations (10-1,000 nM) proliferation was inhibited, and we suggest that these may represent physiological and pharmacological effects of the hormone, respectively. We also investigated the ability of 1,25(OH),D3 to elicit rapid signal transduction events associated with keratinocyte differentiation-an elevation of intracellular calcium levels (Hennings et al, 1989;Sharpe et al, 1989;Pillai and Bikle, 1991) and expression of Fos (Fisher et al, 1991;Smeyne et al, 1992;Basset-Seguin et al, 1994;Bollag et al, 1994). We also determined the extent of growth inhibition after short exposures to the hormone.…”

“…After washing to remove the paraformaldehyde, the cells were solubilized with 0.5% Triton XlOO for 5 min a t room temperature. p-galactosidase activity was then measured essentially as described by MacGregor et al (1991), as detailed by Bollag et al (1994). Briefly, fixed cells were placed in Z buffer (60 mM Na,HPO,, 40 mM NaH,PO,, 10 mM KC1, and 1 mM MgSO,, pH 7.0) containing 0.1% Triton X100.…”