The Role of Phospholipase D2 and Its Interaction with Aquaporin 3 in Primary

Abstract

Regulated keratinocyte proliferation and differentiation is necessary for normal skin function. In mouse keratinocytes phospholipase D2 (PLD2) colocalizes with aquaporin 3 (AQP3), probably via a direct protein-protein interaction as seen in Sf9 cells. Since AQP3 can transport glycerol, a substrate of PLD2, we hypothesized that AQP3 and PLD2 function together to form phosphatidylglycerol (PG), a lipid second messenger which inhibits keratinocyte proliferation and promote differentiation. First, adenovirusmediated PLD2 overexpression enhanced keratinocyte proliferation under control conditions and inhibited differentiation induced by a moderately elevated calcium level. However, PG synthesis was inhibited with PLD2 overexpression; this decrease may result from disruption of the endogenous PLD2 and AQP3 interaction, and/or reduced AQP3 activity following overexpression of PLD2. Next, AQP3 was overexpressed. Overexpression of either PLD2 or AQP3 inhibited the activity of transglutaminase (TGase), a marker of keratinocyte differentiation. However, co-overexpression of AQP3 and PLD2 returned TGase activity to control levels, under both control and calciumstimulated conditions. Similarly, PG synthesis was inhibited by either PLD2 or AQP3 overexpression, but PG levels were returned to control values with co-overexpression. These results are consistent with our hypothesis that PG is a differentiation signal: less PG leads to proliferation and inhibition of differentiation. The caveolin-1 scaffolding domain peptide has been found to interact functionally with PLD2 in low-density membrane microdomains. We propose that reduced AQP3 and PLD2 interaction resulting from disruption of lipid rafts by the caveolin-1 scaffolding domain peptide results in less PG synthesis and the inhibition of calcium-induced keratinocyte differentiation. Mouse keratinocytes were treated with cell-permeable caveolin-1 scaffolding domain peptide (CSDP) and cell differentiation was stimulated using a moderately elevated extracellular calcium concentration. The CSDP had no effect itself on PG synthesis, differentiation or proliferation, but it prevented the changes induced by a moderate calcium concentration, whereas a negative control peptide did not. The CSDP altered PLD2 distribution within membrane microdomains, but had little or no effect on AQP3 distribution. Finally, we showed that the CSDP disrupted lipid rafts in cell membranes by itself, while when applied with calcium simultaneously it prevented the changes induced by moderate calcium. We conclude that the CSDP regulates both calcium-inhibited proliferation and -stimulated differentiation, at least in part, through effects on PG production.