K6PC-5, a sphingosine kinase activator, induces anti-aging effects in intrinsically aged skin through intracellular Ca2+ signaling

Background: Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca2+ signaling, growth, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK. Objective: Here we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca2+ signaling. Methods: Fibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca2+ signaling and fibroblast proliferation. Changes in intracellular Ca2+ were detected by fluorescence with fura-2/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old). Results: K6PC-5 promoted fibroblast proliferation and procottagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca2+ concentration ([Ca2+](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca2+](i), an effect independent of the classical PLC/IP3-mediated pathway. The K6PC-5-induced [Ca2+](i) oscillations were dependent on thapsigargin-sensitive Ca2+ stores and Ca2+ entry. Topical application of K6PC-5 for 2 weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function. Conclusion: These results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging. (C) 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.