Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption

Abstract

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy.

FIG. 1.

AQP3 expression in human SCC and protection against cutaneous papillomas in AQP3-null mice. (A) (left) AQP3 immunostaining in human SCC (male, age 58 years). Bars, 200 μm (×10) and 20 μm (×200). (Right) Immunostaining of AQP3 and keratin-14 in SCC. (B) Dorsal skin of mice was treated with DMBA and TPA as described in Materials and Methods. Representative photographs showing multiple papillomas in AQP3^+/+ mice (left, SKH background; right, CD1) but no papillomas in AQP3^−/− mice. (C) (top) Percentages of mice with papillomas. (Bottom) Average numbers of papillomas per mouse (9 to 12 mice per group). (D) Histology of papillomas stained with hematoxylin and eosin. Bars, 500 μm (×25) and 100 μm (×100). (E) Immunostaining of AQP3 and keratin-14 or filaggrin in papilloma of AQP3^+/+ mouse. (F) RT-PCR analysis of AQPs 1 to 9 in papilloma isolated from AQP3^+/+ mouse. Control amplifications were done using a mixture of cDNAs from brain, lung, liver, and kidney.

FIG. 2.

Impaired epidermal cell proliferation in the AQP3^−/− epidermis. (A) (left) TUNEL staining of an epidermis treated with DMBA. The positive control was DNase I treatment. Arrowhead, TUNEL-positive cell. Bar, 20 μm. (Right) Numbers of TUNEL-positive cells in the epidermis, within 1 cm (error bars indicate standard errors; n = 4). (B) (left) Hematoxylin and eosin staining of an epidermis treated topically with vehicle or one or four applications of TPA (1 TPA or 4 TPA). Bar, 20 μm. (Right) Epidermal thickness in AQP3^+/+ and AQP3^−/− mice (error bars indicate standard errors; n = 5; asterisk, P < 0.05; two asterisks, P < 0.01). (C) (left) BrdU staining of the epidermis following treatment with vehicle or one or four TPA applications. (Right) Percentages of BrdU-positive cells in the epidermal basal layer (error bars indicate standard errors; n = 4; two asterisks, P < 0.01). (D) Immunostaining of filaggrin and keratin-10 in the epidermis after four TPA applications. Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). (E) [³H]TPA penetration in the epidermis after a single application (error bars indicate standard errors; n = 4 or 5).

FIG. 3.

Reduced cellular ATP content in the AQP3-deficient epidermis. (A) (left) AQP3 immunostaining of an epidermis treated with vehicle or four TPA applications (4 TPA). (Center) AQP3 immunoblot of the epidermis 24 h after one TPA application (1 TPA). (Right) Time course of AQP3 protein expression (as AQP3/β-actin ratios) (error bars indicate standard errors; n = 3 to 5; asterisk, P < 0.01). (B) [¹⁴C]glycerol incorporation into epidermal aqueous (top) and lipid (middle) phases. (Bottom) ¹⁴CO₂ release from the epidermis, normalized to total ¹⁴C incorporation. Measurements were done with organ cultured skin after 16 h of treatment with vehicle or TPA (error bars indicate standard errors; n = 4; two asterisks, P < 0.01; asterisk, P < 0.05). (C) Glycerol, glucose, and ATP content and ADP/ATP ratios in AQP3^+/+ and AQP3^−/− epidermises (error bars indicate standard errors; n = 5; asterisk, P < 0.01).

FIG. 4.

Decreased cellular ATP content in AQP3-deficient mouse keratinocytes. Keratinocytes were cultured from AQP3^+/+ and AQP3^−/− mouse epidermis. (A) (left) Osmotic water permeability as measured by calcein fluorescence quenching. The change in calcein fluorescence following an increase in perfusate osmolality from 300 (PBS) to 600 (PBS containing 300 mM mannitol) mosM was monitored. (Right) Reciprocal exponential time constants (τ⁻¹, proportional to osmotic water permeability) in five separate sets of measurements (error bars indicate standard errors; n = 5; asterisk, P < 0.01). (B) Glycerol permeability as measured by [¹⁴C]glycerol uptake for 3 min (error bars indicate standard errors; n = 5, asterisk, P < 0.01). (C) Glycerol, glucose, and ATP content in AQP3^+/+ and AQP3^−/− keratinocytes. ¹⁴CO₂ release from keratinocytes, normalized to total ¹⁴C incorporation (error bars indicate standard errors; n = 5; asterisk, P < 0.01).

FIG. 5.

Involvement of AQP3-mediated glycerol uptake in ATP generation and cell proliferation. (A) Intracellular ATP content in cultured AQP3^+/+ and AQP3^−/− keratinocytes incubated with glycerol (0.1, 1, or 10 mM, 24 h) or 2-dOG (5 mM, 1 h) (error bars indicate standard errors; n = 5; asterisk, P < 0.05 for comparison with control). (B) Correlation between cellular glycerol and ATP content in cultured AQP3^+/+ and AQP3^−/− keratinocytes. (C) G3P content in AQP3^+/+ and AQP3^−/− epidermis (error bars indicate standard errors; n = 5; asterisk, P < 0.01). (D) G3P-induced ATP production in a mitochondrial fraction from an AQP3^+/+ epidermis. (Left) ATP content in the mitochondrion-rich fraction incubated with G3P or glycerol (10 mM). (Right) ATP production in the mitochondrial fraction after incubation with 1 or 10 mM G3P for 5 min (error bars indicate standard errors; n = 5; asterisk, P < 0.01). (E) ATP production in the mitochondrial fractions from AQP3^+/+ and AQP3^−/− epidermis incubated with 10 mM G3P (error bars indicate standard errors; n = 5). (F) Correlation between cellular ATP content and cell proliferation as detected by a BrdU enzyme-linked immunosorbent assay. Ratios are expressed as BrdU incorporation per cell number.

FIG. 6.

Glycerol administration corrects impaired proliferation in the AQP3^−/− mouse epidermis. Mice were administrated glycerol orally for 3 days prior to TPA treatment. (A) BrdU staining of the epidermis at 24 h after TPA treatment. (B and C) Epidermal thickness (B) and percentages of BrdU-positive cells (C) in the basal epidermal layer (error bars indicate standard errors; n = 4).

FIG. 7.

Proposed mechanism of tumor promotion by AQP3-dependent cell proliferation during skin tumorigenesis.