View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE A Potential Role for the Phospholipase D2-Aquaporin-3 Signaling Module in Early Keratinocyte Differentiation: Production of a Phosphatidylglycerol Signaling Lipid Wendy B. Bollag1,2, Ding Xie2, Xiangjian Zheng2 and Xiaofeng Zhong2 In keratinocytes aquaporin-3 (AQP3), an efficient glycerol transporter, is associated with phospholipase D2 (PLD2) in caveolin-rich membrane microdomains. PLD catalyzes both phospholipid hydrolysis to produce phosphatidate and a transphosphatidylation reaction using primary alcohols to generate phosphatidylalcohols. As PLD2 can utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG), we hypothesized that AQP3 provides glycerol to PLD2 for PG synthesis, which then modulates keratinocyte function. Acidic medium inhibits AQP3 transport activity; both glycerol uptake and PG synthesis were inhibited by low versus physiological pH. Co-transfection experiments were performed in which AQP3 or empty vector was introduced into keratinocytes simultaneously with reporter constructs in which differentiation or proliferation promoters directed expression of a luciferase reporter gene. AQP3 coexpression decreased the promoter activity of keratin 5, increased that of keratin 10 and enhanced the effect of a differentiating agent on the promoter activity of involucrin, consistent with promotion of early differentiation. Glycerol inhibited DNA synthesis, whereas equivalent concentrations of xylitol or sorbitol, as osmotic controls, had no effect. Direct provision of PG, but not phosphatidylpropanol, inhibited DNA synthesis in proliferative cells. Thus, our results support the idea that AQP3 supplies PLD2 with glycerol for synthesizing PG, a lipid signal that promotes early keratinocyte differentiation. Journal of Investigative Dermatology (2007) 127, 2823–2831; doi:10.1038/sj.jid.5700921; published online 28 June 2007 INTRODUCTION phatidylalcohols (reviewed by Morris et al., 1997). This Phospholipase D (PLD) is a lipolytic enzyme that has been characteristic has been utilized to specifically track the implicated in multiple cellular processes including growth, activity of PLD, but the question arises: is there a differentiation, vesicle trafficking and cytoskeletal rearrange- physiological primary alcohol substrate for which PLD has ment (reviewed by Bollag and Zheng, 2005). In the presence retained this transphosphatidylation capacity throughout the of water, PLD hydrolyzes phosphatidylcholine to produce evolutionary process? phosphatidic acid, which serves as a second messenger itself In keratinocytes, PLD activation in response to several or can, in turn, be converted to the lipid messengers, agents has been associated with differentiation. Indeed, the diacylglycerol or lysophosphatidic acid. However, in the keratinocyte-differentiating agent 1,25-dihydroxyvitamin D3, presence of small amounts of a primary alcohol, PLD can also increases the expression and activity of one isoform of PLD, catalyze a transphosphatidylation reaction to produce phos- PLD-1, without affecting the other, PLD-2 (Griner et al., 1999). Phorbol esters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) also activate PLD (e.g., Jung et al., 1999), 1Departments of Medicine (Dermatology), Orthopaedic Surgery and Cell and we have shown that 1-butanol, which is used Biology and Anatomy, Medical College of Georgia, Augusta, Georgia, USA by PLD in the transphosphatidylation reaction to divert and 2Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA production away from phosphatidic acid, inhibits phorbol Correspondence: Professor Wendy B. Bollag, Program in Regenerative ester-induced transglutaminase activity, a marker of late Medicine, Institute of Molecular Medicine and Genetics, CB-2803, Medical keratinocyte differentiation (Bollag et al., 2005). On the other College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA. hand, tert-butanol, an organic alcohol and 1-butanol analog E-mail: [email protected] that is not readily utilized by PLD in the transphosphatidyla- Abbreviations: AQP3, aquaporin-3; PG, phosphatidylglycerol tion reaction, does not inhibit transglutaminase activity, PLD, phospholipase D; PP, phosphatidylpropanol; SFKM, serum-free keratinocyte medium suggesting that the inhibitory effect of 1-butanol is related to Received 10 July 2006; revised 24 March 2007; accepted 17 April 2007; its ability to inhibit PLD-mediated lipid signal generation published online 28 June 2007 (Bollag et al., 2005). & 2007 The Society for Investigative Dermatology www.jidonline.org 2823 WB Bollag et al. The PLD2-AQP3 Signaling Module in Keratinocytes In other experiments, we have shown that PLD2 can involucrin, a marker of intermediate differentiation. Glycerol utilize glycerol in vitro in the transphosphatidylation reaction and 1,2-propylene glycol (glycerol missing one hydroxyl to generate phosphatidylglycerol (PG), and this production group on the number 3 terminal carbon) inhibited DNA can occur at the expense of both phosphatidic acid and the synthesis in a dose-dependent manner both in a low (25 mM) phosphatidylalcohol, phosphatidylethanol (Zheng et al., and an intermediate (125 mM) calcium concentration, whereas 2003). Furthermore, in intact cells, elevated extracellular equivalent concentrations of the osmotically active agents, xylitol calcium levels, a treatment that triggers differentiation of and sorbitol, had little or no effect. Direct provision of PG keratinocytes, could both activate PLD and increase PG liposomes also inhibited DNA synthesis in a dose-dependent synthesis. This enhanced PG synthesis could be inhibited by fashion in rapidly dividing keratinocytes, although in growth- ethanol, indicating that it was mediated by PLD (Zheng et al., inhibited cells PG liposomes dose dependently enhanced 2003). In addition, PG production exhibited a dose depen- [3H]-thymidine incorporation into DNA. These data support the dence, with maximal synthesis observed at a concentration of idea of a signaling module consisting of AQP3, PLD2, glycerol extracellular calcium reported to be essentially maximal for and PG and involved in promoting growth inhibition and/or early expression of keratins 1 and 10, markers of early keratinocyte differentiation of proliferating keratinocytes. differentiation (Zheng et al., 2003). How might the glycerol have access to the intracellular RESULTS PLD enzyme? The aquaglyceroporin, aquaporin-3 (AQP3) is Inhibition of glycerol uptake with acidic medium inhibits PG expressed in the epidermis (Matsuzaki et al., 1999; Sugiyama synthesis et al., 2001; Sougrat et al., 2002) and is known to be an We have previously shown that PLD2 and AQP3 colocalize in efficient transporter of glycerol, but relatively inefficient in its caveolin-rich membrane microdomains in keratinocytes transport of water (Yang and Verkman, 1997). We have (Zheng and Bollag, 2003). In addition, PLD-mediated PG previously shown that, in keratinocytes, AQP3 and PLD2 synthesis is stimulated by elevated extracellular calcium levels associate in caveolin-rich membrane microdomains (Zheng in keratinocytes (Zheng et al., 2003), and we hypothesized and Bollag, 2003). Furthermore, the presence of the AQP3 that AQP3 provides glycerol to PLD2 for the transpho- glycerol channel is required for normal epidermal function. sphatidylation reaction to produce PG (reviewed by Bollag Thus, AQP3 null mutant mice have an epidermal phenotype and Zheng, 2005). Because in lung cells AQP3 is inhibited by characterized by decreased epidermal glycerol content, dry acidic medium (Zelenina et al., 2003), we investigated skin, delayed wound healing and barrier recovery and whether a medium of low pH would inhibit glycerol uptake decreased elasticity and water-holding capacity (Hara et al., and PG synthesis. Keratinocytes were incubated for 24 hours 2002; Ma et al., 2002; Hara and Verkman, 2003). This with control serum-free keratinocyte medium (SFKM) (25 mM 2 þ 2 þ phenotype can be corrected by topical or oral application of Ca ) or SFKM containing 125 mM Ca before measurement glycerol but not other osmotically active molecules (Hara and of [3H]-glycerol uptake and [14C]PG production in SFKM of 2 þ Verkman, 2003), suggesting that the effect is not simply a pH 4 or 7.4. As shown in Figure 1a, 125 mM Ca significantly function of glycerol’s hydrophilic properties. Asebia mice, stimulated glycerol uptake in control medium. Low-pH mice possessing a stearoyl-CoA desaturase-1 mutation that medium significantly inhibited glycerol uptake both under results in a lack of sebaceous glands, also show a reduced basal conditions and upon stimulation with the intermediate epidermal glycerol content and a phenotype of dry skin and calcium concentration (Figure 1a). Similarly, pH 4 medium hyperproliferation (Fluhr et al., 2003). In these mice also, the significantly inhibited radiolabeled PG synthesis after a phenotype can be corrected by topical application of glycerol 10-minute incubation with [14C]glycerol, both in cells 2 þ but not other humectants (Fluhr et al., 2003). Thus, we have incubated with control medium and 125 mM Ca medium hypothesized that AQP3 transports glycerol to PLD2 for use (Figure 1b). To ensure that the inhibition of glycerol uptake in the transphosphatidylation reaction to produce PG and that and/or PG production by pH 4 medium was not related to PG, in turn, acts as