The role of sphingosine kinases and SKAM1 in cutaneous wound healing

Abstract

Sphingosine kinases (SKs) phosphorylate sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). SKs and S1P regulate a diverse range of cellular processes, including cell proliferation, survival, differentiation, migration, smooth muscle cell contraction, inflammation, cytoskeleton reorganisation and angiogenesis, mainly via the engagement of S1P to a family of five S1P-specific G protein-coupled receptors (GPCRs). As such, the SKs and S1P are involved in regulating a plethora of cellular processes that are known to be fundamental to wound healing. The role of SK and S1P in cancer and other diseases including asthma, hypertension, atherosclerosis and allergy are well established. Notably, however, the direct role of the SKs and S1P in wound healing has not been previously examined in any detail. My studies sought to fill this gap in knowledge. Using a well-established mouse model of incisional wound healing, I have shown that SK1⁻ʹ⁻, SK2⁻ʹ⁻, SK1⁻ʹ⁻ SK2⁺ʹ⁻ mice healed at a slower rate compared to wildtype mice. This may be attributed to a decrease in cellular proliferation in the early steps of wound repair. These studies highlight the importance of SKs in the very complex process of wound healing. My studies also examined the role of a relatively uncharacterised protein, fibroblast growth factor receptor-1 oncogenic partner 2 (FGFR1OP2), in wound healing. FGFR1OP2 is a protein that was identified from a yeast two-hybrid screen for SK1 interacting proteins. Unpublished work performed from the Pitson laboratory has shown that FGFR1OP2 can interact and activate SK1 in cells and in vitro. As such, we have more appropriately named this protein SKAM1 (Sphingosine Kinase Activating Molecule 1). SKAM1 has previously been reported to be upregulated following tooth extraction in rat oral mucosa. The Pitson laboratory has shown that overexpression of SKAM1 induced collagen matrix contraction, an in vitro model of wound contraction, was mediated by SK1 and the S1P receptors, S1P₁ ̷ ₃. Using a number of classical in vitro models of wound healing, I found that overexpression of SKAM1 in NIH3T3 fibroblasts did not affect cellular migration and proliferation. Notably, however, NIH3T3 fibroblasts overexpressing SKAM1 were resistant to serum deprivation-induced apoptosis. We also generated SKAM1 transgenic mice, where SKAM1 was ubiquitously expressed, to study the role of this protein in wound healing in vivo. We found no observable phenotypical difference between SKAM1 transgenic and wildtype mice at 12 and 48 weeks of age. Primary mouse embryonic fibroblasts (MEFs) isolated from SKAM1 transgenic embryos showed enhanced ability to contract collagen matrix compared with the wildtype. Somewhat surprisingly, the rate of wound healing following incisional wounding was similar between SKAM1A transgenic and wildtype mice. Notably, however, SKAM1A transgenic mice showed enhanced wound resolution compared with the wildtype following full-thickness excisional wounding. In addition, SKAM1 gene-trap mice with conditional potential have also been successfully generated and provide a tool for the study of the effect of SKAM1A knockout in wound healing in vivo. The Pitson laboratory previously showed that a 35 amino acid peptide of SKAM1, SKAM1⁷¹⁻¹⁰⁵, can surprisingly still activate SK1 and enhance collagen matrix contraction. Further to this I have shown that a 30 amino acid cell-permeable peptide of SKAM1, TAT-SKAM1⁷⁶⁻¹⁰⁵, was able to directly activate recombinant SK1 in vitro and when applied to cells. Notably, this effect was blocked by a mutant version of this peptide Tyr104→Phe mutation. Furthermore, NIH3T3 fibroblasts treated with TAT-SKAM1⁷⁶⁻¹⁰⁵ showed enhanced collagen contraction, and more importantly, intradermal injection of TAT-SKAM1A⁷⁶⁻¹⁰⁵ into full-thickness excisional wounds resulted in enhanced wound resolution in mice. Neither of these effects was observed with the mutant peptide. Taken together, my findings suggest a potential therapeutic use of this peptide for the enhancement of wound repair. In summary, my findings have demonstrated for the first time a novel role of SK and SKAM1 in wound healing. Knowledge gained from this study will be valuable for the development of potential new therapeutics for the improvement of wound healing.