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.

Aquaporin 3 (AQP3) is an aquaglyceroporin that transports water and glycerol and is expressed in the epidermis, among other epithelial tissues. We have recently shown that there is an association between this glycerol channel and phospholipase D2 (PLD2) in caveolin-rich membrane microdomains. While PLD2 is able to hydrolyze membrane phospholipids to generate phosphatidic acid, this enzyme also catalyzes, in the presence of primary alcohols, a transphosphatidylation reaction to produce a phosphatidylalcohol. We have proposed that AQP3 associated with PLD2 provides the physiological primary alcohol glycerol to PLD2 for use in the transphosphatidylation reaction to generate phosphatidylglycerol (PG). Further, we have proposed that PG functions as a signaling molecule to mediate early epidermal keratinocyte differentiation, and manipulation of this signaling module inhibits keratinocyte proliferation and enhances differentiation. In contrast, other investigators have suggested a proliferative role for AQP3 in keratinocytes. In addition, AQP3 knockout mice exhibit an epidermal phenotype, characterized by dry skin, decreased elasticity and delayed barrier repair and wound healing, which can be corrected by glycerol but not other humectants. AQP3 levels have also been found to be altered in human skin diseases. In this article the evidence supporting a role for AQP3 in the epidermis will be discussed.

Journal of Lipid Research Volume 54, 2013 581characterized by growth arrest and expression of the mature keratins 1 and 10 in the fi rst differentiated layer of the epidermis, the spinous layer. Early differentiation in the spinous layer is followed by further differentiation in the granular layer, which is accompanied by expression of proteins that are essential for the formation of the cornifi ed envelope and corneocytes. The corneocytes constitute the outer layer of the epidermis, the stratum corneum, and give skin its resilience to mechanical stress (as reviewed in Ref. 1 ). Defi ciencies in the mechanical barrier function of the epidermis result in skin diseases. For example, epidermolysis bullosa simplex and epidermolytic hyperkeratosis arise through mutations in keratins comprising the intermediate fi laments and are characterized by extensive blistering and epidermal sloughing as a result of the mechanical stresses encountered by routine interactions with the environment (as reviewed in Ref.2 ). Many tissues of the body in addition to the skin are exposed to mechanical stresses that result in tearing, or disrupting, the plasma membrane of the constituent cells. These disruptions will result in cell death if left unrepaired. However, cells possess an active plasma membrane repair process that can restore plasma membrane integrity if the disruption is not too extensive (as reviewed in Ref.3 ). For example, intestinal cells in the gastrointestinal tract are subjected to mechanical perturbations during the transit of a food bolus; these plasma membrane disruptions can be repaired to allow cell survival ( 4-6 ). Similarly, eccentric contraction of skeletal muscle as a result of downhill treadmill running induces plasma membrane disruptions that are largely repaired ( 7 ). Routine ambulation also appears to lead to plasma membrane disruptions in the epidermis of the digits ( 8 ). Therefore, it is critical that cells in these mechanically active tissues be able to repair membrane Keratinocytes form the epithelium of the skin, the epidermis, and comprise several cell layers. As keratinocytes migrate up from the stratum basale, they undergo a distinct pattern of differentiation that is essential for the function of the skin as a protective barrier. This pattern is Abbreviations: 1,25(OH) 2 D 3 , 1,25-dihydroxyvitamin D 3 ; AQP3, aquaporin 3; FIPI, 5-fl uoro-2-indolyl des-chlorohalopemide; HBSS, Hank's buffered salt solution; K-SFM, keratinocyte serum-free medium; PEt, phosphatidylethanol; PG, phosphatidylglycerol; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; SFKM, serum-free keratinocyte medium; TPA, 12-O -tetradecanoylphorbol 13-acetate.

Protein kinase C (PKC)-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates phospholipase D (PLD) activity in primary mouse epidermal keratinocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), which can be dephosphorylated to produce PKC-activating diacylglycerol. In the presence of small amounts of a primary alcohol, PLD can instead produce novel phosphatidylalcohols at the expense of PA and diacylglycerol. Here, we have demonstrated that inhibiting PLD signal generation with 1-butanol reduced TPA-stimulated transglutaminase activity, a marker of keratinocyte differentiation. On the other hand, the structurally related tertiary alcohol tert-butanol, which cannot be used by PLD, had no effect on TPA-induced transglutaminase activity. Since TPA activates all conventional and novel PKC isoforms directly, yet cannot overcome 1-butanol-mediated inhibition, this result suggests that PLD mediates its effects on transglutaminase activity (and keratinocyte differentiation) through an effector enzyme system distinct from the conventional or novel PKC isoenzymes. Data in the literature suggest that PA can recruit Raf-1 to the membrane, where it can be activated and initiate the mitogen-activated protein kinase cascade that culminates in activation of extracellular signal-regulated kinase (ERK)-1 and -2. Indeed, we found that inhibition of ERK-1/2 phosphorylation (activation) inhibited TPA-induced transglutaminase activity. However, inhibition of PLD-mediated signal generation had only a small effect on TPA-elicited ERK-1/2 phosphorylation (activation), whereas inhibition of ERK-1/2 did not affect PLD activation, suggesting that these two pathways likely operate largely in parallel. Thus, our results suggest the independent involvement of the PLD and ERK-1/2 pathways in mediating transglutaminase activity and keratinocyte differentiation.Phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), are known to promote the formation of tumors in epidermis initiated with carcinogens (for review, see Rubin, 2002). The mechanism of this tumor promotion is unknown, but the identification of protein kinase C (PKC) as the predominant phorbol ester binding protein in cells (for review, see Nishizuka, 1995) suggests an involvement of this enzyme family in the process. However, the acute effect of TPA both in vitro and in vivo is to induce keratinocyte differentiation (discussed in Bollag et al., 1993), a response seemingly inconsistent with its tumor-promoting action. The mechanism and downstream pathways activated in this biphasic TPA response are still at present unclear.As mentioned above, the primary target of TPA in cells is thought to be the PKC family. PKC isoenzymes can be divided into the classical members PKC-␣, -␤I and -␤II, and -␥, which are phospholipid-dependent, calcium-sensitive, and activated by phorbol ester, or the physiological activator diacylglycerol. The novel isoforms PKC-␦, -⑀, -, and -are also phospholipid-dependent and activated by phorb...

Background-The serine/threonine kinase protein kinase D (PKD) has been proposed to be a proproliferative, anti-differentiative signal in epidermal keratinocytes. Indeed, the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces biphasic PKD activation, which mirrors the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes in vitro and epidermis in vivo.