The expression of differentiation markers in aquaporin-3 deficient epidermis

Abstract

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca(2+) or 1alpha,25-dihydroxyvitamin D(3) (VD(3)) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.

Fig 1

Comparable cell differentiation induced by high concentrated Ca²⁺ and VD₃ in wildtype and AQP3-deficient mouse primary cultured keratinocytes. a Immunoblot analysis using antibodies against involucrin, keratin 10, keratin 5, and β-actin. Keratinocytes from wildtype and AQP3 null mouse epidermis were cultured with 0.15 mM Ca²⁺ for 4 days (control). After confluence cells were treated with 1.5 mM Ca²⁺ for 4 days (high Ca) or 1 mM VD₃ for 3 days (VD₃). b Immunoblots were quantified as the ratio to β-actin using NIH image. The values shown are the mean ± SEM of 3–4 separate experiments performed in duplicate (*P < 0.01). c CE formation and d TGase activity. Keratinocytes were cultured with control or high Ca²⁺ medium described in (a). Data are expressed as the ratio to wildtype control (n = 4, *P < 0.01)

Fig 2

Cell differentiation induced by Ca²⁺ in AQP3-knock-down human keratinocytes. a AQP3 immunostaining of neonatal human keratinocytes (control) and AQP3-siRNA-treated keratinocytes (AQP3 RNAi). Keratinocytes were cultured with 0.15 mM Ca²⁺ for 4 days (low Ca), and replaced with 1.5 mM Ca²⁺ and cultured for 6 days (high Ca). Bar 20 μm. b Immunoblot analysis using antibodies against involucrin, loricrin, keratin 10, keratin 5, and β-actin in RISC-free siRNA-treated (control) and AQP3-siRNA-treated (RNAi) keratinocyte. c Immunoblots were quantified as the ratio to β-actin using NIH image. The values shown are the mean ± SEM of 4–5 separate experiments performed in three separate sets of experiments (*P < 0.01)

Fig 3

Retinoic acid-induced cell proliferation and differentiation in AQP3 null epidermis. Dorsal skin of wildtype and AQP3 null mice were treated topically with vehicle or four applications of retinoic acid (RA). a Hematoxylin and eosin staining of skin treated with RA. Bar 50 μm. b (left) BrdU staining of epidermis following four RA applications. (right) Percentage of BrdU positive cells in epidermal basal layer (SE n = 3, *P < 0.05). c Immunoblot analysis using antibodies against involucrin, loricrin, filaggrin, keratin14, keratin 5 and β-actin. Epidermis was excised at 24 h after final treatment with vehicle or RA. d AQP3 immunostaining of epidermis treated with vehicle or four RA applications. Bar 20 μm. e Immunostaining of AQP3 (red) and keratin 14, 10 or 6 (green) in RA-treated epidermis of wildtype and AQP3 null mice. Bar 20 μm

Fig 4

AQP3 expression in normal human epidermis. a Histology of human skin from abdomen stained with hematoxylin and eosin. Bar 100 μm. b Immunostaining of AQP3 (red) and keratin 14, keratin 10 or involucrin (green). Bar 50 μm