Calmodulin-antagonism inhibits human keratinocyte proliferation

Abstract

Calcium is a major regulator on intracellular metabolism. It is now established that its effects are mediated by calmodulin (CAM), a specific receptor protein [10]. The calcium-calmodulin complex influences enzymes such as adenylate cyclase, phosphodiesterase, protein kinase, and phosholipase A2 [10]. Calmodulin-dependent processes have been implicated as being important regulatory components of cell proliferation or cell-cycle progression, especially at the G1 S-boundary [1]. As keratinocytes have been shown to contain calmodulin in significant amounts [5, 12], the protein is accepted as a regulatory component of keratinocyte proliferation and differentiation [9]. Recent works have studied the role of calmodulin in the hyperproliferation of epidermal disorders, e.g., psoriasis; calmodulin levels are grossly elevated in both lesional [3, 8, 11, 14, 15] and nonlesional epidermis [4, 8, 11, 14] in psoriasis. Clearance of psoriatic plaques is accompanied by a significant overall reduction in epidermal calmodulin, irrespective of the treatment regime used [13]. Therefore we tested the effects of fendiline (Fen), a drug with potent calmodulin-antagonistic activities [6] in a system of cultured human keratinocytes; keratinocytes were removed from human skin by trypsinization (0.5% trypsin, Sigma, in PBS, 24 h at 4~ Cells were washed and placed into microtiter plates (2 x 105 cells/ml). Culture medium was RPMI 1640 (Seromed) completed with 1% nonessential amino acids, 1% L-glutamine, hydrocortisone 0.4 gg/ml, 10% fetal calf serum, 1% penicillin/streptomycin 10 E/10,000 pg/ml. The cells were incubated for 48 h (37~ 5% CO2) either in the presence or in the absence of fendiline 10 8 1 0 -5 M. In some series the tumor promotor PMA (4-beta-phorbol, 12-beta-myristate 13-alphaacetate, Sigma) was added. After a further incubation of 24 h with 3H-thymidine (Amersham, 0.5 I.tgCi/ sample) the cells were removed and filtered. Solubilized radioactivity was counted in a beta-scintillation counter.