Heterogeneity of [3H]phorbol 12,13-dibutyrate binding in primary mouse keratinocytes at different stages of maturation.

Mouse keratinocytes respond heterogeneously to phorbol esters with distinct subpopulations stimulated to proliferate or induced to differentiate. The maturation state of the epidermal cell at the time of exposure may determine its response. The binding of phorbol esters to primary mouse keratinocytes was studied under culture conditions selecting for proliferating cells or differentiating cells. [20-3H]-12-Deoxyphorbol 13-isobutyrate ([3H]-DPB) bound to both types of cells at one class of binding sites. The dissociation constant (Kd) for [3H]DPB in the proliferative cells [epidermal cells grown in Eagle's minimal essential medium containing 8% fetal calf serum (Chelex treated) and 0.05 mM CaCl2] was 69 nM and the binding at saturation (Bmax) was 1.3 pmol/mg of protein. The corresponding values in the differentiative cells (epidermal cells grown in Eagle's minimal essential medium containing 8% fetal calf serum and 1.2 mM CaCl2) were 96 nM and 1.5 pmol/mg of protein, respectively. In contrast to the results obtained with [3H]DPB, [20-3H]phorbol 12,13-dibutyrate ([3H]PDBU) bound to both cell types in a heterogeneous fashion. Analyzed by the Ligand Program with a two-site model, the Kd values for the two binding sites in the cells grown in the medium containing 0.05 mM CaCl2 were 5.5 and 100 nM and the Bmax values were 0.9 and 1.7 pmol/mg of protein, respectively. The site for [3H]DPB binding seemed to correspond to the higher affinity [3H]PDBU binding site. The major difference in the cells grown in the medium containing 1.2 mM CaCl2 was an increase (approximately two-fold, depending on details of the analysis) in the Bmax of the lower affinity binding site with the other three parameters remaining similar. The state of epidermal differentiation thus appears to modulate the amount of the lower affinity binding sites for phorbol esters. Three Ca2+-resistant cell lines have been developed from carcinogen-treated primary keratinocytes. These cells are not tumorigenic and demonstrate characteristics consistent with their being initiated cells. In the medium containing 1.2 mM CaCl2, [3H]PDBU bound to these cells at one class of binding sites with affinities similar to that of the higher affinity sites for [3H]PDBU in cells grown in the medium containing either 0.05 or 1.2 mM CaCl2.