Aquaporin-3 in keratinocytes and skin: its role and interaction with phospholipase D2

Abstract

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.

Figure 1. AQP3 Glycosylation is Regulated by Extracellular Ca²⁺ Concentration

Keratinocytes were pre-treated with low 25 μM Ca²⁺- (control), 125 μM Ca²⁺- or 1 mM Ca²⁺-containing medium for 24 hours and then lysed using sodium carbonate. Light membrane fractions (fractions 4 and 5 [25]) were isolated by sucrose gradient ultracentrifugation, analyzed by western blotting with anti-AQP3 and visualized by enhanced chemiluminescence. The results shown are representative of three separate experiments. Note that unglycosylated AQP3 is partially down-regulated by elevated Ca²⁺, but glycosylated AQP3 levels are increased.

Figure 2. The Construct Used to Generate a Mouse Model Transgenic for Aquaporin 3 Overexpression in the Suprabasal Epidermis

Shown is the construct used to generate the HK1-AQP3 transgenic mouse model, in which AQP3 is overexpressed in the suprabasal epidermis under the control of the human keratin 1 promoter. Also illustrated are the primers used to genotype the mice.

Figure 3. HK1-AQP3 Transgenic Mice Exhibit Enhanced Aquaporin 3 Immunoreactivity in Epidermal Sections and in Isolated Primary Epidermal Keratinocytes

Skin from non-transgenic (Panel A) and transgenic (Panel B) neonatal littermates was dissected, placed in OCT and frozen in dimethylpentane cooled with liquid nitrogen. Cryosections were cut and incubated with hydrogen peroxide (to eliminate endogenous peroxidase activity) and blocking buffer. All sections were then stained using the ABC immunohistochemical staining kit from Santa Cruz Biotech and a primary antibody to AQP3 (Alomone Labs, Israel). Panel C illustrates a negative control in which the primary antibody was omitted. An additional control in which primary antibody was preabsorbed with antigen prior to incubation with the sections also showed minimal staining (data not shown). These results are representative of a total of 7 mice (5 transgenic newborn mice and 2 non-transgenic littermate controls). In Panel D is shown a western blot analysis of AQP3 immunoprecipitated from lysates (equal amounts of protein) of primary epidermal keratinocytes cultured from non-transgenic (Non-TG) and transgenic (AQP3 TG) littermates and treated with control medium (25 μM Ca²⁺) or medium containing 125 μM Ca²⁺ (to increase keratin 1 promoter-driven expression of AQP3) as indicated. Results are representative of three experiments. An additional experiment in which AQP3 was immunoprecipitated from lysates of whole transgenic and non-transgenic epidermis showed similarly enhanced AQP3 levels in the transgenic epidermis by subsequent western analysis (data not shown).

Figure 4. HK1-AQP3 Transgenic Mice Exhibit Accelerated Permeability Barrier Repair following Disruption by Tape-Stripping

HK1-AQP3 mice and non-transgenic littermates were subjected to tape-stripping to disrupt the epidermal permeability barrier and increase trans-epidermal water loss (TEWL) as in [69]. Barrier recovery was then measured (by monitoring TEWL) at 3 to 3.5 hours. Values represent the means of 9 (non-transgenic) and 15 (transgenic) separate sites on 5 transgenic mice and 3 non-transgenic matched littermates (male mice were used); *p<0.05 versus the non-transgenic littermates. We observed that the HK1-AQP3 male mice exhibited significantly accelerated barrier recovery relative to matched non-transgenic littermates.

Figure 5. Phospholipase D Catalyzes Both a Hydrolysis and a Transphosphatidylation Reaction

Phospholipase D (PLD) hydrolyzes phosphatidylcholine in the presence of water to yield phosphatidic acid (PA). In the presence of a primary alcohol, such as glycerol, ethanol or 1-butanol, this enzyme can also catalyze a transphosphatidylation reaction to form the corresponding phosphatidylalcohol (e.g., phosphatidylglycerol, phosphatidylethanol or phosphatidylbutanol).