Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing
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- Hara-Chikuma, M. & Verkman, A.S. J Mol Med (2008) 86: 221. doi:10.1007/s00109-007-0272-4
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Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/glycerol-transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was ~80% complete in wild-type mice at 5 days vs ~50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation.
KeywordsAQP3Wound healingCell migrationCell proliferationGlycerol transport
mitogen-activated protein kinase
Cutaneous wound healing is a multi-step process that involves several cell types, including epidermal keratinocytes, fibroblasts, endothelial cells, and peripheral nerve cells. Re-epithelialization is a crucial step during wound healing, which involves the migration and proliferation of keratinocytes from the surrounding epidermis and appendages such as hair follicles and sweat glands (reviewed in [1, 2]). After injury, keratinocytes at the wound margin begin to proliferate behind actively migrating cells, resulting in a dense, hyperproliferative epithelium seen as migrating cell sheets at the wound margins. These events are thought to be regulated mainly by growth factor receptors, integrins, extracellular matrix, and matrix metalloproteases (reviewed in (3–5]).
The aquaporins (AQPs) are a family of small, integral membrane proteins that transport water and, in some cases, water and glycerol (‘aquaglyceroporins’). Phenotype analysis of mice lacking individual AQPs has proven their anticipated involvement in the urinary concentrating mechanism and exocrine glandular fluid secretion (reviewed in ) and has elucidated various unanticipated roles of AQPs, such as in brain edema , neural signal transduction , and fat metabolism . The mechanisms underlying each of these phenomena were attributed to AQP water or glycerol transporting functions. The newly described AQP11 may be functionally distinct from other water and/or glycerol transporting AQPs, as AQP11 gene disruption produces a severe phenotype with renal vacuolization and cyst formation .
Recently, we reported a novel cellular role for AQPs in cell migration, which was initially demonstrated in endothelial cells and transfected cells  and subsequently in brain astroglial cells , kidney proximal tubule cells , tumor cells , and corneal epithelium cells . We suggested that AQP-facilitated water influx into dynamic cellular protrusions (lamellapodia) at the leading edge of migrating cells occurs in AQP-dependent cell migration.
In skin, AQP3 is expressed in plasma membranes of the basal epidermal cell layer [16–18]. Compared to wild-type mice, AQP3 null mice have relatively dry skin, reduced skin elasticity, and delayed recovery of barrier function after removal of the stratum corneum [17, 19]. We suggested that these defects were consequent to the absence of AQP3-facilitated glycerol transport, resulting in reduced stratum corneum and epidermal cell glycerol content [19, 20]. We also described delayed wound repair in AQP3 null mice in a hairless genetic background, although no information was available at that time about possible cellular mechanisms.
The goals of this study were to characterize and investigate possible mechanisms of AQP3-dependent wound healing in skin. We tested the hypothesis that AQP3-facilitated water and/or glycerol transport is involved in epidermal cell migration and proliferation during repair of skin wounds. For these studies, we utilized AQP3 null mice and primary keratinocyte cultures derived from mouse skin and siRNA-treated human keratinocyte cultures. Our results provide strong evidence for distinct defects in AQP3-dependent epidermal cell migration and proliferation, which account for impaired in vivo would healing in the AQP3 null mice. Mechanistic analysis provided evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation. Our data suggest pharmacological modulation of AQP3 as a possible therapy to accelerate wound healing in traumatic, burn, and other forms of injury.
Materials and methods
AQP3 null mice
AQP3 null mice (CD1 genetic background) were generated by targeted gene disruption as described . Protocols were approved by the UCSF Committee on Animal Research.
Human keratinocyte cell cultures
Neonatal human keratinocytes (NHK, Cascade Biologics, Portland, OR, USA) were grown in keratinocyte basal medium (KBM) supplemented with 0.2% bovine pituitary extract, 5 μg/ml insulin, 0.18 μg/ml hydrocortisone, 5 μg/ml transferrin, and 0.2 ng/ml epidermal growth factor (EGF). NHK were transfected with AQP3 siRNA or RNA-induced-silencing-complex-free siRNA (Dharmacon, Lafayette, CO, USA) at 40–50% confluence using Lipofectamine 2000™ (Invitrogen Life Technologies, Carlsbad, CA, USA). AQP3 expression was confirmed by immunofluorecence and immunoblotting using a commercial polyclonal AQP3 antibody (Chemicon, Temecula, CA, USA). Experiments were done at 48–72 h after transfections.
Mouse keratinocyte cell cultures
Full-thickness skin from 1- to 3-day-old mice was incubated in dispase II (5 U/ml, Roche, Nutley, NJ, USA) for 7 h at 4°C. The epidermis was separated from the dermis, cut into fragments, and treated with 0.25% trypsin and 0.1% ethylenediaminetetraacetic acid (EDTA) for 10 min. Cells were seeded at a density of 105 per cm2 on collagen type I plates (BD Biosciences, Bedford, MA, USA) and cultured in keratinocyte growth medium (Cambrex, East Rutherford, NJ, USA) at 37°C under 5% CO2. Recombinant adenoviruses encoding rat AQP1 and AQP3 (AQP1-Ad and AQP3-Ad) were custom generated by ViraQuest (North Liberty, IA, USA). Keratinocytes from AQP3 null mice were infected with AQP1-Ad or AQP3-Ad at 1,000 pfu/cell. Experiments were done at 48–72 h after viral infection.
Water and glycerol permeability measurements
Water permeability was measured by calcein fluorescence quenching, as described . Calcein fluorescence was monitored in response to changing perfusate osmolalities from 300 (phosphate-buffered saline, PBS) to 450 mosM (PBS containing 150 mM mannitol). Glycerol permeability was measured by 14C-glycerol uptake for 90 s or 3 min, as described .
In vivo wound healing
Two full-thickness punch biopsies extending through the epidermis and dermis (diameter, 5 mm) were done on the backs of wild-type and AQP3 null mice. Wound repair, which was expressed as the percentage of initial wound area, was monitored daily. In some experiments, mice were given water containing glycerol ad libitum (5% for wild-type and 1% for AQP3 null mice) for 3 days before wounding, along with standard solid mouse chow as described .
In vitro wound healing assay
In vitro wound healing was assayed in confluent cell monolayers as described . Cells were stained with phallotoxins conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) to visualize the F-actin and quantify lamellapodia. In some experiments, BrdU was incorporated for 8 h after wounding, and cells were stained with BrdU antibody. BrdU-positive cells were counted within 250 μm from the wound margin and expressed as the percentage of BrdU-positive cells [with all cells identified by 4′,6-diamidino-2-phenylindole (DAPI) staining].
Transwell migration assay
Cell migration was assayed using a modified Boyden chamber containing a polycarbonate Transwell membrane filter (5 μm pore size for mice keratinocytes, 8 μm pore size for NHK; Corning Costar, Cambridge, MA, USA) coated with collagen type I. Cells (105) were deposited on the upper chamber in KBM containing 1% fetal bovine serum (FBS). The lower chamber contained KBM with 10 or 1% (control) FBS. Cells were incubated for 6 h at 37°C in 5% CO2/95% air. After scraping non-migrated cells from the upper surface of the membrane with a cotton swab, the migrated cells remaining on the bottom surface were stained with 0.5% crystal violet. The stained insert was washed thoroughly, dissolved with 1% Triton X-100, and absorbance (O.D.) at 595 nm was measured.
To measure cell proliferation, mice were injected intraperitoneally with BrdU (100 μg/g body weight; Sigma-Aldrich, St. Louis, MO, USA) at 1 h before sacrifice. Paraffin-embedded sections were stained with hematoxylin and eosin, BrdU antibody (Abcam, Cambridge, MA, USA), or AQP3 antibody (Chemicon). BrdU-positive cells were counted in epidermis to 500 μm from the wound edge.
Cell size and adherence
Cell proliferation was assessed as [3H-methyl]-thymidine incorporation into DNA as described . Total cellular DNA was determined using the Hoechst reagent (Sigma). DNA synthesis was expressed as 3H-thymidine incorporated per microgram DNA. BrdU incorporation into keratinocytes was detected using a BrdU Cell Proliferation Assay kit (Calbiochem).
To analyze MAPK signaling, cells were starved for >24 h and incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and lysed with ice-cold lysis buffer (Cell Signaling Technology, Boston, MA, USA). The epidermis was separated by heat-split and homogenated in lysis buffer. The supernatant (14,000×g, 10 min, 4°C) was used for immunoblotting with antibodies against phospho-ERK, ERK, phospho-p38, p38, phospho-JNK (Cell Signaling Technology), JNK (Biosource, Camarillo, CA, USA), and β-actin (Sigma).
Phospho-p38 ELISA assay
Keratinocytes were incubated for 1 h with vehicle [0.01% dimethyl sulfoxide (DMSO)], 100 nM TPA, or 20 ng/ml EGF. Phospho-p38 content was measured by cellular activation of signaling enzyme-linked immunosorbent assay (ELISA) kit (SuperArray, Ann Arbor, MI, USA).
Statistical analysis was performed using the two-tailed Student’s t test or by analysis of variance (ANOVA).
Delayed healing of cutaneous wounds in AQP3 null mice
Impaired in vitro wound healing in AQP3-deficient keratinocytes
In vitro assays of wound healing were done in confluent NHK monolayers. Cells were scraped to produce an ~500-μm wide defect in the cell layer. Figure 2d shows significantly slowed wound closure in AQP3 knock-down keratinocytes at 24 h after creation of the linear wounds. The wounded area is repopulated by adjacent cells through the combined processes of cell migration and proliferation [5, 23]. Therefore, we next measured cell migration and proliferation, separately, to determine the role(s) of AQP3 on wound repair.
Reduced cell migration in AQP3-deficient keratinocytes
Impaired cell proliferation in AQP3-deficient keratinocytes
DNA synthesis was assayed in control and AQP3 knock-down keratinocytes to determine the involvement of AQP3 in growth factor-induced cell proliferation. For these studies, we chose EGF, which is abundantly released at wound sites as one of the key regulators of keratinocyte proliferation . Figure 5c shows reduced cell proliferation in AQP3 deficient keratinocytes. Similar results were seen in mice primary keratinocyte cultures (not shown). Figure 5d shows that EGF accelerated wound repair in NHK, with lesser effect in AQP3 knock-down cells. EGF receptor expression was identical in control and AQP3-deficient keratinocytes by immunoblot analysis (not shown). These data suggest the involvement of AQP3 in cell proliferation-induced stimuli such as wounding or growth factors. Therefore, we hypothesized that AQP3 might be involved in cellular responses to stimuli leading to cell proliferation.
Impairment of MAP kinase cell signaling in AQP3-deficient keratinocytes
To determine whether AQP3-facilitated water and/or glycerol transport is responsible for the AQP3-dependent MAPK signaling, TPA-induced p38 activation was determined in mouse keratinocytes infected with adenoviruses encoding GFP, AQP1, or AQP3. Figure 6c shows that AQP3 replacement corrected the defect in p38 activation, whereas AQP1 had little effect, suggesting a link between AQP3-facilitated glycerol transport and p38 activation. To further investigate the apparent impairment of p38 activation in AQP3 deficiency, phospho-p38 was assayed in wild-type and AQP3 null keratinocyte in response to EGF and to TPA. Figure 6d shows greatly reduced TPA- and EGF-induced p38 activation in AQP3-deficient keratinocytes.
Glycerol replacement corrects impaired wound healing and cell proliferation in AQP3-deficient mice
Healing of cutaneous wounds was slowed in vivo in AQP3-deficient mice. In wild-type mice, the wound edge during the healing phase was hyperproliferative, as shown by hyperplasia and a high percentage of BrdU-positive epidermal keratinocytes. AQP3 expression was strongly elevated in these hyperproliferative cells. In contrast, much less hyperplasia and BrdU-positivity was found during wound healing in AQP3 null mice. We showed previously the selective reduction in glycerol content in stratum corneum and epidermis of AQP3-deficient mice, without differences in glycerol content in dermis and serum , which was interpreted in terms of impaired glycerol transport from dermis to more superficial layers of skin through the normally highly glycerol-permeable basal layer of epidermal keratinocytes. Glycerol supplementation, under conditions found previously to normalize glycerol content and hydration in outer layers of skin , partially corrected the defect in wound healing in AQP3 null mice. The involvement of AQP3 in cell proliferation may be a general phenomenon of importance to other AQP3-expressing cells in tissues outside of the skin. AQP3 is prominently expressed in epithelial cells at the ocular surface and in regenerating basal enterocytes in colon. We found recently that corneal surface re-epithelialization is impaired in AQP3-deficient mice in vivo  and that colonic function and survival were greatly reduced in AQP3-deficient mice in dextran sulfate and acetic acid models of colitis . Although those studies did not establish a mechanism for the phenotypes, in both cases, defects in in vivo cell proliferation were established based on relative hypoplasia and reduced BrdU positivity in AQP3 deficiency.
A recent study showed that AQP3 knock-down reduced EGF-induced migration of human skin fibroblasts in a phagokinetic track motility assay . It was suggested that EGF increased cell migration by increasing AQP3 expression via the EGF receptor and phosphoinositide 3-kinase and ERK signal transduction pathways. Studies here using keratinocyte cell cultures provided strong evidence for distinct defects in cell migration and proliferation in AQP3 deficiency. We used normal human keratinocyte cultures, in which AQP3 expression was greatly reduced by siRNA treatment, as well as keratinocytes cultured from wild-type and AQP3 null mice, and adenovirus-corrected AQP3 null keratinocytes. Defective migration of AQP3 knock-down or knock-out keratinocytes was found using in vitro wound healing and/or transwell assays. Reduction in lamellipodial surface area was seen in migrating AQP3 knock-down keratinocytes at the leading edge of a healing wound in vitro. Impaired migration of AQP3 null keratinocytes was corrected by adenovirus-mediated replacement of AQP3 or of the water-selective transporter AQP1, providing evidence that the water transporting function of AQP3 is responsible for AQP3-facilitated keratinocyte migration (Fig. 7d). These results are in agreement with an increasing body of evidence for involvement of AQPs in migration of many different types of cells, including tumor cells, as mentioned in “Introduction”. We believe that AQP-facilitated water influx in lamellipodia facilitates their dynamic formation and extension, resulting in accelerated cell migration. However, cell AQP expression is not an absolute requirement for migration because of the substantial water permeability of lipid bilayers, such that water permeability in AQP-expressing cells is generally only 5–20-fold greater than that after AQP deletion or inhibition .
During healing of cutaneous wounds, the hair follicle is thought to contribute to epidermal renewal [30, 31]. Recently, stem cells located in the bulge region of the hair follicle were found to facilitate wound repair , in which migration of bulge cells into the healing epidermis was seen during wound healing. We found strong AQP3 expression in the hair follicle, including the bulge region (data not shown). AQP3-facilitated water transport might thus also be involved in migration of bulge cells, accelerating wound repair.
After injury, accelerated cell proliferation, which is sustained by growth factors and integrins and matrix metalloprotinases, is important for re-epithelialization during wound healing . The impaired proliferation in AQP3-deficient keratinocytes in the in vivo and in vitro wound healing assays suggested that defective AQP3-dependent keratinocyte cell proliferation is responsible, at least in part, for the defective wound healing phenotype in AQP3 null mice (Fig. 7d). The in vitro proliferation assay showed remarkable impairment of EGF-induced cell proliferation in AQP3-deficient keratinocytes. Mechanistic analysis showed impaired p38 MAPK activation induced by mitogens, including EGF and TPA, in AQP3-deficient keratinocytes. The impairment in p38 activation was corrected by adenoviral delivery of exogenous AQP3 but not the water-selective transporter AQP1. p38 MAPK is one of the MAPKs thought to be involved in regulating epidermal cell proliferation, differentiation, apoptosis, and response to inflammation and stress [26, 33]. In vitro studies using NHK showed the involvement of p38 activation in wound repair [34–36]. Although further experiments are needed to investigate the link between p38 MAPK cell signaling and wound repair, our data support the involvement of AQP3 in the cell proliferative response to exogenous stimuli such as mitogens or wounding.
We determined whether AQP3-facilitated water and/or glycerol transport is responsible for cell proliferation. Oral glycerol administration corrected the impairment in wound repair and cell proliferation in AQP3 null mice. Glycerol supplementation also corrected the decreased cell proliferation in human AQP3 knock-down keratinocytes. The detailed metabolic pathways of glycerol in the epidermis have not been established. It has been reported that about 60% of synthesized glycerol is metabolized to glycerol-3-phosphate, which is one of the key metabolic intermediates for ATP production . Although further experiments are needed to investigate the link between AQP3 and cell proliferation, our data thus support the requirement of AQP3-facilitated glycerol transport in cell proliferation during wound repair.
In conclusion, our data provide evidence that impaired wound healing in AQP3 deficiency in vivo results from distinct defects in epidermal cell migration and proliferation, which are consequent to the water and glycerol transporting functions of AQP3, respectively. Our proposed mechanism in Fig. 7d is supported by direct measurements of cell migration and proliferation in keratinocyte cell cultures. A consequence of AQP3-dependent epidermal cell proliferation is the possible involvement of AQP3 in a number of skin disorders associated with epidermal hyperproliferation, such as skin carcinogenesis, psoriasis, and atopic dermatitis and repair of burn and other wounds. Our findings suggest that pharmacological modulation of AQP3 expression or function may be of benefit in increasing epidermal proliferation during wound healing.
We thank Liman Qian for mouse breeding and genotype analysis. This work was supported by NIH grants DK35124, EY13574, EB00415, HL59198, HL73856, and DK72517 and Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation.