Sphingosine-1-Phosphate and Its Potentially Paradoxical Effects on Critical Parameters of Cutaneous Wound Healing

Abstract

The sphingolipid metabolite sphingosine-1-phosphate has emerged as a new bioactive molecule involved in the regulation of cell growth, differentiation, survival, and chemotaxis as well as angiogenesis and embryogenesis. These effects are mediated either via G-protein-coupled receptors or through intracellular actions. The most prominent sources of sphingosine-1-phosphate are human platelets suggesting its potential role in wound healing. In agreement with a positive function on reconstruction of wounded skin, we identified sphingosine-1-phosphate as a potent chemoattractant for keratinocytes as well as an activator of extracellular matrix production by fibroblasts. An unexpected finding is a strong cell growth arrest of keratinocytes after exposure to sphingosine-1-phosphate, as keratinocyte proliferation is critical for re-epithelialization of the wound. Most interestingly, the anti-proliferative effect of sphingosine-1-phosphate is not a result of cytotoxicity or apoptosis as sphingosine-1-phosphate even protects these cells from programmed cell death. Moreover, sphingosine-1-phosphate enhances differentiation of keratinocytes. To investigate further by which signaling pathway cell growth inhibition is mediated expression of the mRNA of all sphingosine-1-phosphate receptors (S1P1-5) was identified. 1 (Edg 1), 2 (Edg 5), 3 (Edg 3), 4 (Edg 6), and 5 (Edg 8) mRNA in keratinocytes was identified. As demonstrated in guanosine 5-[gamma-35S] triphosphate-gammaS binding assays, these G-protein-coupled receptors are functional at nanomolar concentrations. As the anti-proliferative effect of sphingosine-1-phosphate is only partially inhibited in the presence of pertussis toxin, it was investigated if intracellular actions are also involved. Microinjections of sphingosine-1-phosphate in keratinocytes also reduce proliferation suggesting that both sphingosine-1-phosphate receptors as well as intracellular actions mediate sphingosine-1-phosphate- induced cell growth arrest.

Involvement of Smad Signaling in Sphingosine 1-Phosphate-mediated Biological Responses of Keratinocytes

“…Cell Culture-Human keratinocytes were isolated from juvenile foreskin from surgery as described recently (10). Briefly, skin was incubated at 4°C in a solution of 0.25% trypsin and 0.2% EDTA for 20 h, and trypsinization was terminated by the addition of ice-cold Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.…”

“…Migration Assays-Migration of human keratinocytes was measured using a modified Boyden chamber as recently described (10). Therefore, the lower chamber, separated by a fibronectin-coated membrane, was supplemented with S1P, serum, or TGF-␤ in the indicated concentrations.…”

“…Keratinocytes-Based on the similarity between the actions of S1P and TGF-␤ on keratinocytes (10,11) and the requirement for Smad3 to mediate the chemotactic as well as the antiproliferative properties of TGF-␤ (19), we investigated whether Smad3 might also be a direct or indirect mediator of S1P on keratinocyte actions. To eliminate possible artifacts based on overexpression systems using transformed cells, we examined effects of TGF-␤ and S1P on Smad3 activation in primary human keratinocytes.…”

“…Among the cell types responsive to S1P, it has been shown to be a potent chemoattractant for keratinocytes (10). Indeed, migration of this cell type is crucial for reepithelialization suggesting a positive role of S1P on cutaneous wound closure.…”

“…However, whereas S1P is generally known as an important mitogenic stimulus for many cell types, it strongly inhibits the proliferation and induces differentiation of human epidermal cells. Growth inhibition of keratinocytes forestalls wound closure and implies a more complex regulatory role of S1P in preventing excessive epidermal proliferation (10).…”