Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function

The calcium-sensing receptor (CaR) plays an essential role in mediating Ca2+-induced keratinocyte differentiation in vitro. In this study, we generated keratinocyte-specific CaR knockout (EpidCaR-/-) mice to investigate the function of the CaR in epidermal development in vivo. EpidCaR-/- mice exhibited a delay in permeability barrier formation during embryonic development. Ion capture cytochemistry detected the loss of the epidermal Ca2+ gradient in the EpidCaR-/- mice. The expression of terminal differentiation markers and key enzymes mediating epidermal sphingolipid transport and processing in the EpidCaR-/- epidermis was significantly reduced. The EpidCaR-/- epidermis displayed a marked decrease in the number of lamellar bodies and lamellar body secretion, thinner lipid-bound cornified envelopes and a defective permeability barrier. Consistent with in vivo results, epidermal keratinocytes cultured from EpidCaR-/- mice demonstrated abnormal Ca2+I handling and diminished differentiation. The impairment in epidermal differentiation and permeability barrier in EpidCaR-/- mice maintained on a low calcium (0.02%) diet is more profound and persistent with age then in EpidCaR-/- mice maintained on a normal calcium (1.3%) diet. Deleting CaR perturbs the epidermal Ca2+ gradient and impairs keratinocyte differentiation and permeability barrier homeostasis, indicating a key role for the CaR in normal epidermal development.

Immunohistochemical staining-Mouse skins were fixed with 4% formaldehyde, embedded in paraffin, sectioned, and used for immunohistochemistry (Komuves et al., 2002). The sections were deparaffinized, rehydrated, and washed. Sections were incubated with primary antibodies to the proteins of interest and then with appropriate biotinylated secondary antibodies, followed by ABC-peroxidase (Vector, Burlingame, CA) and a DAB substrate. Secondary antibody alone was used as a control to establish the specificity of immunoreactivity.
Electron microscopy-Mouse skins were fixed with 2% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), then post-fixed with reduced osmium tetroxide, and processed for embedding in Spurrʼs resin. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined with a Zeiss 10CR transmission electron microscope. Assessment of epidermal Ca 2+ gradient-Skin samples were processed for ion capture cytochemistry as previously described (Menon et al., 1985). Skin samples were fixed with an ice-cold fixative containing 2% glutaraldehyde, 2% formaldehyde, 90 mM potassium oxalate, and 1.4% sucrose (pH 7.4) in the dark. The samples were postfixed in 1% osmium tetroxide containing 2% potassium pyroantimonate at 4 o C in the dark for 2h, and then processed, embedded, sectioned and examined with a Zeiss 10CR transmission electron microscope.
Lanthanum perfusion-Skin samples were immersed in 4% lanthanum nitrate in 0.05 M Tris buffer (pH7.4) containing 2% glutaraldehyde and 1% paraformaldehyde for 1 h at room temperature. After lanthanum perfusion, the samples were washed and processed for electron microscopy as described above.
Skin permeability assay-The development of the skin barrier in Epid CaR-/-mouse embryos and their control littermates was assessed by toluidine blue penetration assay.
Embryos of E15.5 and E16.5 were subjected to methanol dehydration and subsequent rehydration as described previously (Koch et al., 2000). They were then washed in PBS, stained in 0.0125% toluidine blue O (Sigma Chemical, St Louis, MO) in PBS for 1 min. After de-stained in PBS, embryos were photographed with a Leica M205C microscope.

Measurement of trans-epidermal water loss (TEWL)-Epid CaR-/-mice and their
control littermates were maintained with a normal Ca 2+ diet (1.3% Ca 2+ ) or switched to a low Ca 2+ (0.02%) diet after weaning. The backs of mice were shaved. Twenty-four hours later TEWL was measured using a Meeco electrolytic water analyzer to establish a basal rate. The permeability barrier was then disrupted acutely using sequential tape stripping until the rate exceeds 500ppm/cm 2 /hr, and TEWL was then measured at 0, 3 and 6 hours after tape stripping to determine the rate of barrier recovery.

Assessment of innate immune response-Epid CaR-/-mice and their control littermates
were maintained with a normal diet (1.3% Ca 2+ ) or switched to a low (0.02%) Ca 2+ diet after weaning. The backs of mice were shaved. A 1-cm long full-depth skin incision was made on the back of the animals. Twenty-four hours later, total RNAs were collected from a 2 mm-wide area along wound edges and from skin of similar size in the uninjured area. Expressions of innate immune response genes were analyzed by qPCR.
Statistical analysis-Studentʼs t-Test (for comparing two sample groups) and one-way ANOVA (for comparing more than two groups) were performed to analyze the quantitated data of various assays described in this study. Statistical difference is determined significant if P value is lower than 0.05.  Epidermal keratinocytes were isolated from newborn Epid CaR-/-mice and their control littermates and then exposed to 2 mM Ca 2+ for 20 minutes to induce formation of cell-cell junctions. Fluorescent immunostaining was performed using E-cadherin polyclonal antibody (green), and monoclonal antibodies (red) against β-catenin, Rho A and Fyn followed by the appropriate FITC-or Texas red-conjugated secondary antibody. Overlapped staining sites are visualized as yellow. Bar = 20 μm. Ca 2+ o -induced recruitment of E-cadherin, βcatenin, Rho A and Fyn to the cell-cell junctions was suppressed in Epid CaR-/keratinocytes. Figure S4. Epid CaR-/-mice exhibited normal barrier recovery when maintained on a normal (1.3%) calcium diet. Four-month-old Epid CaR-/-mice and their control littermates were maintained on a regular diet containing 1.3% Ca 2+ . Sequential cellophane tape stripping was used to disrupt the permeability barrier and increase trans-epidermal water loss (TEWL) levels until rates exceeded 500 ppm/cm 2 /hr. TEWL was measured before (a) and 0, 3, and 6 hours after (b) barrier disruption. The degree of barrier recovery was expressed as % decrease of TEWL at 3 and 6 verses 0 hour after barrier disruption. Data were presented as mean + SE, n=11. No significant difference in basal TEWL and barrier recovery between Epid CaR-/-and control mice was found.  Figure S7. Epid CaR-/-mice and control littermates have similar levels of serum calcium and phosphate when maintained on either normal (1.3%) or a low (0.02%) calcium diet for 4 months. Data were presented as mean + SD, n=9-19. # P<0.05, normal vs. low calcium diet in control mice. Values of average serum calcium and phosphate levels (mg/dL) of each group are shown above the bars. No statistically significant difference between Epid CaR-/-(CaR-KO) and control mice was observed regardless of diets.  Figure S8. Epid CaR-/-mice and control littermates have equivalent body size when maintained on either normal (1.3%) or a low (0.02%) calcium diet for 6 months. Data were presented as mean + SD, n=7-13. # P<0.05, normal vs. low calcium diet in Epid CaR-/-mice. No statistically significant difference between Epid CaR-/-(CaR-KO) and control mice was observed regardless of diets.