K6PC-5, a direct activator of sphingosine kinase 1, promotes epidermal differentiation through intracellular CA²⁺ signaling

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca²⁺ signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca²⁺ signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca²⁺ concentration ([Ca ²⁺]_i) oscillations in HaCaT cells and Ca²⁺ mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca²⁺]_i. The K6PC-5-induced [Ca²⁺]_i oscillations were dependent on thapsigargin-sensitive Ca²⁺ stores and Ca²⁺ entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca²⁺]_i responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.