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Decreased Calcium-Sensing Receptor Expression Controls Calcium Signaling and Cell-To-Cell Adhesion Defects in Aged Skin

  • Anna Celli
    Affiliations
    Department of Dermatology, SFVAHCS Medical Center and University of California San Francisco, San Francisco, California, USA
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  • Chia-Ling Tu
    Affiliations
    Endocrine Unit, San Francisco VA Medical Center (SFVAMC), San Francisco, California, USA

    Department of Medicine, University of California-San Francisco (UCSF), San Francisco, California, USA
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  • Elise Lee
    Affiliations
    Department of Dermatology, SFVAHCS Medical Center and University of California San Francisco, San Francisco, California, USA
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  • Daniel D. Bikle
    Affiliations
    Departments of Medicine and Dermatology, UCSF Staff Physician, SF Department of Health Affairs Medical Center, San Francisco, California, USA
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  • Theodora M. Mauro
    Correspondence
    Correspondence: Theodora M. Mauro, Department of Dermatology, SFVAHCS Medical Center and University of California San Francisco, 4150 Clement Street, MS 190 Dermatology, San Francisco, California 94121, USA.
    Affiliations
    Department of Dermatology, SFVAHCS Medical Center and University of California San Francisco, San Francisco, California, USA
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Open ArchivePublished:April 13, 2021DOI:https://doi.org/10.1016/j.jid.2021.03.025
      The calcium-sensing receptor (CaSR) drives essential calcium ion (Ca2+) and E-cadherin‒mediated processes in the epidermis, including differentiation, cell-to-cell adhesion, and epidermal barrier homeostasis in cells and in young adult mice. We now report that decreased CaSR expression leads to impaired Ca2+ signal propagation in aged mouse (aged >22 months) epidermis and human (aged >79 years, donor age) keratinocytes. Baseline cytosolic Ca2+ concentrations were higher, and capacitive Ca2+ entry was lower in aged than in young keratinocytes. As in Casr-knockout mice (EpidCaSR–/–), decreased CaSR expression led to decreased E-cadherin and phospholipase C-γ expression and to a compensatory upregulation of STIM1. Pretreatment with the CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine normalized Ca2+ propagation and E-cadherin organization after experimental wounding. These results suggest that age-related defects in CaSR expression dysregulate normal keratinocyte and epidermal Ca2+ signaling, leading to impaired E-cadherin expression, organization, and function. These findings show an innovative mechanism whereby Ca2+- and E-cadherin‒dependent functions are impaired in aging epidermis and suggest a new therapeutic approach by restoring CaSR function.

      Abbreviations:

      AHEK (aged human epidermal keratinocyte), Ca2+ (calcium ion), CaSR (calcium-sensing receptor), ER (endoplasmic reticulum), HK (human keratinocyte), KC (keratinocyte), NHEK (neonatal human epidermal keratinocyte), NPS R-568 (N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine), PLC (phospholipase C), TG (thapsigargin)

      Introduction

      In this study, we report that the loss of calcium-sensing receptor (CaSR) leads to impaired calcium ion (Ca2+) and E-cadherin signaling in aged human epidermal keratinocytes (AHEKs) and epidermis. Ca2+ is essential for normal keratinocyte (KC) proliferation, differentiation, and migration and wound repair (
      • Cordeiro J.V.
      • Jacinto A.
      The role of transcription-independent damage signals in the initiation of epithelial wound healing.
      ). Raised extracellular Ca2+, epidermal barrier perturbation, and mechanical or laser stimulation all act through intracellular Ca2+ release and through subsequent store-operated or voltage-sensitive Ca2+ entry (
      • Numaga-Tomita T.
      • Putney J.W.
      Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation.
      ;
      • Tu C.L.
      • Chang W.
      • Bikle D.D.
      Phospholipase C gamma1 is required for activation of store-operated channels in human keratinocytes.
      ) that propagates Ca2+ signaling to neighboring KCs both laterally and vertically (
      • Kumamoto J.
      • Goto M.
      • Nagayama M.
      • Denda M.
      Real-time imaging of human epidermal calcium dynamics in response to point laser stimulation.
      ;
      • Tsutsumi M.
      • Goto M.
      • Denda M.
      Dynamics of intracellular calcium in cultured human keratinocytes after localized cell damage.
      ). Epidermal Ca2+ signaling is driven by a marked Ca2+ gradient (
      • Forslind B.
      • Werner-Linde Y.
      • Lindberg M.
      • Pallon J.
      Elemental analysis mirrors epidermal differentiation.
      ;
      • Menon G.K.
      • Elias P.M.
      Ultrastructural localization of calcium in psoriatic and normal human epidermis.
      ), with Ca2+ concentrations approximately four-fold higher in the uppermost viable KCs than in the basal cells (
      • Elias P.M.
      • Nau P.
      • Hanley K.
      • Cullander C.
      • Crumrine D.
      • Bench G.
      • et al.
      Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent.
      ;
      • Mauro T.
      • Bench G.
      • Sidderas-Haddad E.
      • Feingold K.
      • Elias P.
      • Cullander C.
      Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.
      ). Much of this Ca2+ gradient and the resulting Ca2+ signaling depend on Ca2+ sequestered within the endoplasmic reticulum (ER) by SERCA (
      • Celli A.
      • Crumrine D.
      • Meyer J.M.
      • Mauro T.M.
      Endoplasmic reticulum calcium regulates epidermal barrier response and desmosomal structure.
      ). Although raising extracellular Ca2+ levels increases KC differentiation, it also decreases lipid secretion and barrier repair in terminally differentiated stratum granulosum KCs (
      • Lee S.E.
      • Lee S.H.
      Skin barrier and calcium.
      ). Thus, an approach that enhances KC sensitivity to Ca2+ could optimize differentiation, migration, and barrier homeostasis, especially in an aging epidermis.
      The G-protein‒associated CaSR senses Ca2+ concentrations in the micromolar to the millimolar range, making it particularly useful in sensing changes in extracellular or organelle Ca2+ concentrations. In KCs, CaSR signaling activates phospholipase C (PLC)-β by Gq and leads to inositol triphosphate‒mediated acute release of calcium from intracellular calcium stores. CaSR expression is essential for epidermal differentiation and barrier function (
      • Komuves L.
      • Oda Y.
      • Tu C.L.
      • Chang W.H.
      • Ho-Pao C.L.
      • Mauro T.
      • et al.
      Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation.
      ,
      • Tu C.L.
      • Crumrine D.A.
      • Man M.Q.
      • Chang W.
      • Elalieh H.
      • You M.
      • et al.
      Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function.
      ), controlling both KCs’ ability to take up Ca2+ and store it in the ER (
      • Tu C.L.
      • Chang W.
      • Bikle D.D.
      The role of the calcium sensing receptor in regulating intracellular calcium handling in human epidermal keratinocytes.
      ). CaSR mediates the formation and stabilization of the E-cadherin signaling complex, leading to E-cadherin‒mediated adherens junction and cell-to-cell adhesion (
      • Tu C.L.
      • Chang W.
      • Xie Z.
      • Bikle D.D.
      Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.
      ,
      • Tunggal J.A.
      • Helfrich I.
      • Schmitz A.
      • Schwarz H.
      • Günzel D.
      • Fromm M.
      • et al.
      E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions.
      ). Mice with conditional knockout of the Casr in the epidermis (EpidCasr−/−) display a loss of the epidermal Ca2+ gradient, impaired KC differentiation, and defective permeability barrier (
      • Tu C.L.
      • Crumrine D.A.
      • Man M.Q.
      • Chang W.
      • Elalieh H.
      • You M.
      • et al.
      Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function.
      ). Conversely, experimental CaSR overexpression accelerates epidermal differentiation and permeability barrier formation (
      • Turksen K.
      • Troy T.C.
      Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo.
      ). Combined vitamin D receptor and CaSR deletion delay wound re-epithelization (
      • Oda Y.
      • Hu L.
      • Nguyen T.
      • Fong C.
      • Tu C.L.
      • Bikle D.D.
      Combined deletion of the vitamin D receptor and calcium-sensing receptor delays wound re-epithelialization.
      ), and deleting CaSR from young adult mice epidermis decreases E-cadherin expression and impairs Ca2+ signal propagation (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ). Aged human epidermis and human keratinocyte (HK) show similar defects in Ca2+ signaling, expression, and re-epithelialization as those seen in mice in which CaSR was experimentally ablated. These studies suggest that CaSR may be a relevant target for improving Ca2+- and E-cadherin‒mediated processes in an aged epidermis.

      Results

      Epidermal Ca2+ signaling after laser stimulation is blunted in aged mouse epidermis and aged HK monolayers

      Previous studies showed that aged HKs respond sluggishly to mechanical stimulation (
      • Denda S.
      • Takei K.
      • Kumamoto J.
      • Goto M.
      • Denda M.
      Expression level of Orai3 correlates with aging-related changes in mechanical stimulation-induced calcium signalling in keratinocytes.
      ). To assess lateral calcium signaling in aged versus that in young epidermis, we used a multiphoton excitation microscopy‒based laser stimulation assay previously developed for EpidCasr−/− studies (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ). This experimental approach selectively perturbs a selected area of 20 × 20 μm2 (corresponding roughly to one or two cells) in the stratum basale of the epidermis of mice expressing the fluorescent Ca2+ reporter GCaMP3 under the keratin 14 promoter. After laser stimulation, we monitored Ca2+ propagation by tracking epidermal fluorescence using time-lapse imaging (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ) (Figure 1a). Ca2+ propagation spread to a significantly smaller area in the aged (>22 months) than in the young (6–8 weeks) mice (Figure 1b and d and Supplementary Figure S1). The KC cytosolic Ca2+ response after perturbation also was lower in the aged than in the young mice (Figure 1c and e and Supplementary Figure S1).
      Figure thumbnail gr1
      Figure 1Calcium response to laser perturbation in aged compared with that in young EpidGCaMP mice and human keratinocytes monolayers. (a) Calcium response to laser perturbation to a 20 × 20 μm2 SB region (red box) of young and old EpidGCaMP mouse epidermis. Arrows indicate dermal collagen (blue). (b) Time traces of response area and (c) Cytosolic Ca2+ concentration increase in young (red) and aged (blue) mice. (d) Distribution of maximum response area and (e) maximum cytosolic Ca2+ concentration increase over baseline for young and aged mice. Data were normalized to young mice mean value. n = 18 (aged mice) and 19 (young mice) traces from two biopsies per mouse and from three separate mouse pairs. (f) Time traces of calcium response area after laser perturbation in NHEK (black) compared with that in AHEK (red). (g) Distribution of maximum calcium response area (n = 15 traces from three separate experiments) and (h) single-cell maximum cytosolic Ca2+ concentration increase (n = 1,370–1,552 from three experiments). NHEK is indicated in red, and AHEK is indicated in blue. f/f0 represents baseline fluorescence. Asterisks indicate P < 0.05 by a two-tailed t-test. AHEK, aged human epidermal keratinocyte; NHEK, neonatal human epidermal keratinocyte; s, second; t, time; SB, stratum basale.
      We next examined Ca2+ signaling in response to laser stimulation in the aged (>79 years) HKs (AHEKs) versus that in neonatal KC human (neonatal human epidermal KCs [NHEKs]) monolayers using the cell-permeant, calcium-sensitive fluorescent probe Calcium Green 1-AM (Thermo Fisher Scientific, Waltham, MA). In three separate experiments conducted on cells from three separate pairs of donors, we found that aged HKs monolayers responded with blunted calcium propagation (Figure 1f and g) and a lower average increase in the aged single cells’ cytosolic calcium concentration (Figure 1h).

      Ca2+ signaling in the aged versus that in the young KCs

      We next compared the response to the extracellular Ca2+ and the intracellular Ca2+ stores and the capacitive cytosolic Ca2+ response in neonatal KCs with those in KCs obtained from aged (>79 years) humans (Figure 2a and b, respectively). Fura2-loaded KCs were exposed to 1.2 mM extracellular calcium. Traces representative of six (AHEK) to seven (NHEK) experiments on cells from three separate donors are shown in Figure 2a top panel (NHEK) and bottom panel (AHEK). Whereas NHEKs responded to raised extracellular calcium with a robust and rapid increase in cytosolic Ca2+ concentration, most AHEKs had a much more limited and slower response during the experiment time frame. Overall, AHEKs cytosolic Ca2+ response to increased extracellular calcium was significantly less pronounced than the response in NHEKs (Figure 2b).
      Figure thumbnail gr2
      Figure 2Calcium signaling is impaired in aged human keratinocytes. (a) Response to high extracellular calcium of FURA2-labeled NHEK (top panel) and AHEK (bottom panel) monolayers. Representative traces of six to seven separate experiments on cells cultured from three neonatal and three aged donors. (b) Distribution of single-cell cytosolic Ca2+ variation after calcium switch reported as ΔR. n = 220‒410 cells per group from six (AHEK) to seven (NHEK) separate experiments on cells cultured from three neonatal and three aged donors. Asterisk denotes P < 0.05. (c–f) Representative traces of cytosolic Ca2+ concentrations in keratinocytes at baseline and in response to 1 μM TG followed by 1.2 mM [Ca2+]. (c) NHEK and (d) AHEK in 0.07 mM Ca2+. (e) NHEK versus (f) AHEK cultured in 1.2 mM [Ca2+] for 24 hours. Data are reported as the ratio R of the fluorescence intensity at 340 nm excitation (fbound) over the fluorescence intensity at 390 nm excitation (ffree). n = 102–380 cells per group from three to seven separate experiments on cells cultured from three neonatal and three aged donors. Results are summarized in . [Ca2+], intracellular calcium concentration; AHEK, aged human epidermal keratinocyte; AU, arbitrary unit; Ca2+, calcium ion; NHEK, neonatal human epidermal keratinocyte; s, second; TG, thapsigargin.
      Next, we compared intracellular Ca2+ stores and cytosolic Ca2+ capacitive influx in NHEKs (Figure 2c and d) with those in AHEKs (Figure 2e and f) in KCs monolayers cultured in low (0.07 mM) and high (1.2 mM) calcium-containing medium for 24 hours (Figure 2c–f). KCs were first placed in 0 mM extracellular Ca2+. After a brief period of equilibration, 1 μM thapsigargin (TG), a concentration that releases both the ER and Golgi Ca2+ stores, was added to the medium to assess intracellular Ca2+ stores. Extracellular Ca2+ then was raised to a final concentration of 1.2 mM to quantify capacitive Ca2+ entry (Figure 2 and Table 1).
      Table 1Response to Thapsigargin and High Calcium in NHEKs and AHEKs
      Measured QuantitiesLow CalciumHigh Calcium
      NHEKAHEKNHEKAHEK
      Baseline0.644 ± 0.0051.06 ± 0.05
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      0.704 ± 0.0060.97 ± 0.05
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      P10.33 ± 0.010.22 ± 0.04
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      0.232 ± 0.0070.25 ± 0.02
      P20.45 ± 0.020.26 ± 0.02
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      0.44 ± 0.010.34 ± 0.02
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      Percentage of cells responding to 1μM thapsigargin83 ± 844 ± 8
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      92 ± 353 ± 12
      Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      Abbreviations: [Ca2+], intracellular calcium concentration; AHEK, aged human epidermal keratinocyte; NHEK, neonatal human epidermal keratinocyte; P, peak.
      Capacitive calcium entry traces (Figure 2) were analyzed to determine the baseline intracellular calcium levels (baseline), peak calcium release from stores after exposure to 1 μM thapsigargin (P1), and peak capacitive calcium entry after medium supplementation with 1.2 mM [Ca2+] (P2). Data are reported as the ratio R of the fluorescence intensity at 340 nm excitation (fbound) over the fluorescence intensity at 390 nm excitation (ffree). The percentage of AHEKs responding to thapsigargin is reported in the last row of the table.
      1 Statistically significant difference between the NHEK and AHEK values determined by two-tailed t-test with P < 0.05. n = 102–380 single cell traces from three to seven experiments per group from three aged and three neonatal donors.
      First, we found that baseline cytosolic Ca2+ concentration was markedly elevated and heterogeneous in the aged KCs. Responses of aged KCs to both TG and calcium switch were notably more variable than those in young KCs. Whereas 82% of all NHEK cells cultured in low calcium and 93% of NHEK cells cultured in high calcium responded to both TG and calcium switch, only 44% and 53% of AHEK cells cultured in low and high calcium, respectively, responded to TG or raised extracellular Ca2+ concentration.

      CaSR and E-cadherin protein expression is downregulated in aged human epidermis

      In nonexcitable cells such as KCs, Ca2+ influx is often regulated by store-operated Ca2+ entry (
      • Numaga-Tomita T.
      • Putney J.W.
      Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation.
      ;
      • Tu C.L.
      • Chang W.
      • Bikle D.D.
      Phospholipase C gamma1 is required for activation of store-operated channels in human keratinocytes.
      ;
      • Vandenberghe M.
      • Raphaël M.
      • Lehen'kyi V.
      • Gordienko D.
      • Hastie R.
      • Oddos T.
      • et al.
      ORAI1 calcium channel orchestrates skin homeostasis.
      ), which requires PLC-mediated release of Ca2+ from intracellular stores such as the ER or Golgi and refill through STIM1 migration to the plasma membrane (
      • Numaga-Tomita T.
      • Putney J.W.
      Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation.
      ). These processes lead to adherens junction and desmosome reorganization, mediated by E-cadherin.
      To define the changes in the CaSR-dependent signaling pathway, we first compared CaSR, E-cadherin, and STIM1 protein expression in total epidermal lysate from aged mice with those from young mice (Figure 3a). We found that both CaSR and E-cadherin protein expression was consistently decreased, whereas STIM1 levels were elevated in the aged mice. A similar pattern of E-cadherin downregulation was seen in young mice in which CaSR was experimentally ablated (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ).
      Figure thumbnail gr3
      Figure 3Calcium-signaling molecules expression in aged and young mice and neonatal and aged human keratinocytes. (a) Epidermal lysate from three aged and three young mice was probed for levels of CaSR, E-cadherin, and STIM1 using western blotting, and the differences in expression levels were quantified (bar graphs). (b) Crude membrane extracts of NHEK and AHEK cultured in low or high calcium for 24 hours were probed for CaSR, E-cadherin, PLCγ1, PLCβ1, and STIM1 using western blotting, and the expression levels were quantified (bar graphs). Data are representative of three to four different sets of aged and neonatal cells. Asterisks denote P < 0.05 by a two-tailed t-test. AHEK, aged human epidermal keratinocyte; Ca2+, calcium ion; CaSR, calcium-sensing receptor; NHEK, neonatal human epidermal keratinocyte; PLC, phospholipase C.
      We then compared the expression levels of CaSR, E-cadherin, STIM1, PLCγ1, and PLCβ1 in NHEKs with the expression levels of those in AHEKs from three to four separate donors per group. We found that CaSR and E-cadherin levels were consistently downregulated in AHEKs from four separate donors (Figure 3b and Supplementary Figure S2a) compared with those in NHEKs from four separate donors. PLCγ1 and PLCβ1 levels were also consistently decreased, whereas STIM1 levels were increased in AHEKs compared with the levels in NHEKs. Exposure to high calcium appeared to reduce the difference in STIM1 level between NHEKs and AHEKs, but it did not normalize the expression levels of the other proteins. We observed a similar pattern in the EpidCasr−/− mouse, where PLCγ1 levels were downregulated and STIM1 levels upregulated (Supplementary Figure S2b) compared with the pattern in wild-type mice. PLCβ1 was upregulated in EpidCasr−/− mice.

      Aged KCs display defective E-cadherin staining, slower migration, and impaired cell-to-cell adhesion

      Previous studies (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ) showed that decreased or absent epithelial CaSR levels lead to delayed re-epithelialization both in mice and in scratch assays using HKs through defective E-cadherin reorganization or CaSR-mediated cytosolic concentration increases. Passage 2 KCs from aged or neonatal donors were plated in low calcium on plastic dishes for time-lapse imaging or multichambered glass slides for immunofluorescence microscopy until 80% confluent. Extracellular calcium levels were then raised to 1.2 mM to promote cell-to-cell adhesion and E-cadherin expression. After 24 hours in high calcium, the epithelial sheets were perturbed with a scratch assay and imaged at 100-minute intervals for 12–24 hours (Figure 4a). Time-lapse images revealed that epithelial sheets from aged KCs were slower on average than those from NHEKs at closing the defect (Figure 4b) owing to an initial delay at 100 minutes (asterisk, Figure 4b). Moreover, whereas NHEKs migrated as a sheet, aged cells appeared not to migrate in unison, but instead lost cell-to-cell adhesion and developed gaps as the sheets migrated (Figure 4a). E-cadherin immunofluorescence staining was performed 6 hours after the scratch assay and revealed decreased and irregular E-cadherin plasma membrane staining in aged (Figure 4d) compared with that in young (Figure 4c) KC monolayers. Gaps in KC cell-to-cell adhesion were colocated with absent E-cadherin staining.
      Figure thumbnail gr4
      Figure 4Impaired cell-to-cell adhesion in aged keratinocytes monolayers. (a) Brightfield time-lapse images of scratch assay of second passage keratinocytes monolayers from neonatal (top row) and aged (bottom row) donors in high calcium. The yellow lines highlight the gap area at different time points, whereas the red lines highlight the gaps occurring in the AHEK monolayers during sheet migration. Inset shows the gaps in the aged epidermal keratinocytes sheet. (b) Mean percentage gap closure as a function of time. AHEK is indicated with red bars, and NHEK is indicated with blue bars. Error bars represent the SEM. n = 4–8 wells from two experiments on cells from three to four donors per condition. (c, d) E-cadherin staining (red) of (c) NHEK and (d) AHEK monolayers 6 hours after scratching. Nuclear DAPI was used for counterstain (blue). Insets show higher detail of E-cadherin staining. White arrows in (d) show the gaps in cell-to-cell adhesion. AHEK, aged human epidermal keratinocyte; min, minute; NHEK, neonatal human epidermal keratinocyte.

      The CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine rescues Ca2+ wave propagation, intracellular calcium concentration, response to increased extracellular calcium, and E-cadherin translocation in AHEKs

      N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568) selectively (
      • Nemeth E.F.
      • Steffey M.E.
      • Hammerland L.G.
      • Hung B.C.
      • Van Wagenen B.C.
      • DelMar E.G.
      • et al.
      Calcimimetics with potent and selective activity on the parathyroid calcium receptor.
      ;
      • Tang L.
      • Jiang L.
      • McIntyre M.E.
      • Petrova E.
      • Cheng S.X.
      Calcimimetic acts on enteric neuronal CaSR to reverse cholera toxin-induced intestinal electrolyte secretion.
      ) binds to the transmembrane domain of the CaSR and increases its stability (
      • Huang Y.
      • Cavanaugh A.
      • Breitwieser G.E.
      Regulation of stability and trafficking of calcium-sensing receptors by pharmacologic chaperones.
      ), thereby increasing its Ca2+ sensitivity and enhancing the effects of extracellular Ca2+ on CaSR (
      • Fox J.
      • Lowe S.H.
      • Conklin R.L.
      • Nemeth E.F.
      The calcimimetic NPS R-568 decreases plasma PTH in rats with mild and severe renal or dietary secondary hyperparathyroidism.
      ). To test whether enhancing the CaSR response would also normalize Ca2+ wave propagation and signaling and E-cadherin translocation to the plasma membrane, we pretreated aged KCs with 0.5 or 1 μM NPS R-568 for 24 hours. Vehicle-treated aged KCs and neonatal foreskin KCs were used as controls.
      We first found that similar to the epidermal response to laser stimulation, vehicle-treated aged KCs displayed markedly diminished propagation of calcium response to mechanical ablation (Figure 5a–c). However, pretreatment with NPS R-568 restored the Ca2+ wave propagation in a dose-dependent fashion (Figure 5a–c). Pretreatment with NPS R-568 for 24 hours also restored the AHEKs cytosolic calcium response to increased extracellular calcium in a dose-dependent manner (Figure 5d and Supplementary Figure 3). CaSR stimulation with NPS R-568 also partially rescued E-cadherin plasma membrane translocation (Figure 5e and f). These findings show that enhancing CaSR activity through a pharmacological activator such as NPS R-568 can rescue the abnormal Ca2+ signaling and E-cadherin organization seen in aged KCs.
      Figure thumbnail gr5
      Figure 5NPS R-568 normalizes E-cadherin and calcium response after scratch wounding. (a) NHEK and AHEKs before and 100 s after scratch. Red hue denotes higher cytosolic Ca2+. Bar = 200 μm. (b) Time traces of percentage area with increased cytosolic Ca2+ level after scratch. Representative of five to eight experiments on cells from three neonatal and three aged donors per group. (c) Distribution of maximum calcium response area after scratch. NHEK (red) and AHEK (lilac) treated with vehicle, 0.5 μM NPS R-568 (orange), or 1 μM NPS R-568 (cyan). (d) Distribution of single-cell cytosolic Ca2+ response to increased extracellular calcium. Asterisks indicate statistical significance with P < 0.05. n = 192–409 cells per group from six to 10 experiments on cells from three neonatal and three aged donors. (e) Immunofluorescence E-cadherin staining (red) of NHEK and AHEK monolayers switched to 1.2 mM [Ca2+] media containing 0 (DMSO vehicle), 0.5, or 1 μM NPS-R568 for 15 minutes. Images are representative of three separate experiments on cells from three neonatal and three aged donors. Bar = 20 μm. (f) Quantification of E-cadherin levels at the PM. Asterisks denote statistically significant difference from NHEKs levels determined by a two-tailed t-test with P < 0.05. [Ca2+], intracellular calcium concentration; AHEK, aged human epidermal keratinocyte; AU, arbitrary unit; NHEK, neonatal human epidermal keratinocyte; NPS R-568, N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine; PM, plasma membrane; s, second; t, time.

      Discussion

      These results show that normal Ca2+ signaling and Ca2+-signaling protein expression are impaired in aged epidermis and KCs. Similar to Casr-knockout cells and epidermis (
      • Tu C.L.
      • Celli A.
      • Mauro T.
      • Chang W.
      Calcium-sensing receptor regulates epidermal intracellular Ca2+ signaling and re-epithelialization after wounding.
      ), Ca2+ propagation after perturbation in murine epidermis and cell monolayers from aged donors was significantly reduced compared with that from young controls. Moreover, CaSR expression was consistently downregulated in aged KCs. CaSR acts to release Ca2+ from the ER and Golgi through PLC-generated inositol triphosphate, which then binds to the inositol trisphosphate receptor on the ER (
      • Tu C.L.
      • Chang W.
      • Xie Z.
      • Bikle D.D.
      Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.
      ). Knockdown of CaSR in human cells causes a reduction in both Gq-mediated activation of PLCβ and E-cadherin‒mediated activation of PLCγ (
      • Tu C.L.
      • Chang W.
      • Bikle D.D.
      Phospholipase C gamma1 is required for activation of store-operated channels in human keratinocytes.
      ), which are in turn necessary for the acute and sustained KCs response to elevated extracellular calcium levels. Both PLCβ and PLCγ expression levels were consistently reduced in KCs from aged donors.
      CaSR expression also stabilizes the E-cadherin complex, which in turn regulates cell-to-cell adhesion and cell migration and enables the sustained intracellular calcium level increase needed for KC differentiation through the recruitment of PLCγ. Decreased E-cadherin expression levels and translocation to the plasma membrane were associated with decreased CaSR expression in aged HKs, consistent with previous reports in mice (
      • Tu C.L.
      • Chang W.
      • Xie Z.
      • Bikle D.D.
      Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.
      ) and human cells (
      • Tu C.L.
      • Chang W.
      • Bikle D.D.
      The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.
      ). Our data also suggest that decreased E-cadherin levels result in loss of cell-to-cell junction stability and concerted epithelial sheet migration during a scratch assay. More differentiated KCs tend to have a blunted Ca2+ response, and if aged KCs are more differentiated than young KCs, this might furnish an alternative mechanism to explain our findings. However, past reports show that aged KCs and skin are actually less differentiated both in mice (
      • Bourguignon L.Y.
      • Wong G.
      • Xia W.
      • Man M.Q.
      • Holleran W.M.
      • Elias P.M.
      Selective matrix (hyaluronan) interaction with CD44 and RhoGTPase signaling promotes keratinocyte functions and overcomes age-related epidermal dysfunction.
      ) and in humans (
      • Berge U.
      • Kristensen P.
      • Rattan S.I.
      Hormetic modulation of differentiation of normal human epidermal keratinocytes undergoing replicative senescence in vitro.
      ;
      • Diekmann J.
      • Alili L.
      • Scholz O.
      • Giesen M.
      • Holtkötter O.
      • Brenneisen P.
      A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.
      ;
      • Dos Santos M.
      • Metral E.
      • Boher A.
      • Rousselle P.
      • Thepot A.
      • Damour O.
      In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.
      ).
      Taken together, these results show that decreased CaSR expression and function contribute significantly to the impaired Ca2+ signaling response seen in aging. Although changes in each of the Ca2+-signaling components could also be expected to modify Ca2+ signaling, our finding that treatment with the CaSR agonist NPS-R568 restored normal Ca2+ signaling and E-cadherin distribution strongly suggests that decreased CaSR expression in aging drives both impaired Ca2+ signaling and downstream changes in Ca2+-signaling proteins. Although decreases in CaSR protein expression might be expected to impair NPS-R568 efficacy, this agent has been shown to increase CaSR function on mutant CaSR as well (
      • Rus R.
      • Haag C.
      • Bumke-Vogt C.
      • Bähr V.
      • Mayr B.
      • Möhlig M.
      • et al.
      Novel inactivating mutations of the calcium-sensing receptor: the calcimimetic NPS R-568 improves signal transduction of mutant receptors.
      ).
      STIM1 expression also increased in both aged mouse epidermis and aged HKs, likely as a compensatory response. STIM1 expression was found to increase to a lesser extent in a previous report (
      • Takei K.
      • Denda S.
      • Nagayama M.
      • Denda M.
      Role of STIM1-Orai1 system in intra-cellular calcium elevation induced by ATP in cultured human keratinocytes.
      ), although this report examined younger subjects (maximum age of 70 years) compared with our older subjects (aged >79 years).
      Several questions remain regarding CaSR-mediated Ca2+ signaling in aged KCs. First, although raised cytosolic baseline Ca2+ is consistent with STIM1 upregulation, it also could be explained by impaired Ca2+ uptake or extrusion mechanisms, including functional defects in organelle and plasma membrane Ca2+ adenosine triphosphatase or defects in plasma membrane sodium ion and/or Ca2+ antiporters. We do not see consistent differences in the expression of these proteins. However, more subtle differences in these proteins’ functions, along with elevated STIM levels, may become apparent in subsequent investigations. Second, although increased STIM1 levels would suggest increased store-operated Ca2+ entry, similar to what was observed in Casr-knockout cells (
      • Tu C.L.
      • Chang W.
      • Xie Z.
      • Bikle D.D.
      Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.
      ), we observe a significant defect in calcium entry after exposure to high (1.2 mM) extracellular calcium in aged compared with that in young cells in both proliferative and differentiative conditions. Further investigation into the calcium entry mechanisms, such as STIM1 translocation to the plasma membrane and Orai1 interactions, will be needed to address this defect. Moreover, KCs have also been shown to express molecules involved in noncapacitive calcium entry such as voltage-sensitive calcium channels (
      • Denda M.
      • Fujiwara S.
      • Hibino T.
      Expression of voltage-gated calcium channel subunit alpha1C in epidermal keratinocytes and effects of agonist and antagonists of the channel on skin barrier homeostasis.
      ;
      • Lee S.H.
      • Elias P.M.
      • Feingold K.R.
      • Mauro T.
      A role for ions in barrier recovery after acute perturbation.
      ), transient receptor potential channels (
      • Peier A.M.
      • Reeve A.J.
      • Andersson D.A.
      • Moqrich A.
      • Earley T.J.
      • Hergarden A.C.
      • et al.
      A heat-sensitive TRP channel expressed in keratinocytes.
      ), nonselective cation channels in undifferentiated KCs (
      • Fatherazi S.
      • Belton C.M.
      • Cai S.
      • Zarif S.
      • Goodwin P.C.
      • Lamont R.J.
      • et al.
      Calcium receptor message, expression and function decrease in differentiating keratinocytes.
      ), which could also play a role in the decreased response to raised extracellular calcium we observe in aged KCs. Finally, it is not clear what mechanisms underlie the increased variability seen in the aged KC response to extracellular Ca2+, TG, or mechanical stimulation. Previous studies show that more substantial increases in cytosolic Ca2+ are seen in less differentiated KCs, both in response to extracellular Ca2+ (
      • Kruszewski F.H.
      • Hennings H.
      • Yuspa S.H.
      • Tucker R.W.
      Regulation of intracellular free calcium in normal murine keratinocytes.
      ) and in response to mechanical stimulation (
      • Dubé J.
      • Rochette-Drouin O.
      • Lévesque P.
      • Gauvin R.
      • Roberge C.J.
      • Auger F.A.
      • et al.
      Human keratinocytes respond to direct current stimulation by increasing intracellular calcium: preferential response of poorly differentiated cells.
      ). In addition, basal cytosolic Ca2+ concentration is variable within the same colonies, depending on cell size (
      • Pillai S.
      • Menon G.K.
      • Bikle D.D.
      • Elias P.M.
      Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes.
      ). Likewise, CaSR expression and function decrease as gingival KCs terminally differentiate (
      • Fatherazi S.
      • Belton C.M.
      • Cai S.
      • Zarif S.
      • Goodwin P.C.
      • Lamont R.J.
      • et al.
      Calcium receptor message, expression and function decrease in differentiating keratinocytes.
      ). Therefore, variations in aged KC response could be due to exaggerated intrinsic aging processes, mutations in response to environmental agents such as UV, or a combination of intrinsic and extrinsic processes.
      These results suggest that CaSR plays an essential role in mediating Ca2+ signaling and E-cadherin‒mediated processes in the epidermis. Moreover, decreased CaSR expression and function contribute to impaired KC signaling and E-cadherin expression. These results also suggest that CaSR may be a different target in improving E-cadherin‒mediated processes in aged epidermis.

      Materials and Methods

      Please see Supplementary Materials and Methods for more information.

      Laser perturbation assay

      All animal procedures were approved by the Animal Studies Subcommittee (Institutional Animal Care and Use Committee) of the San Francisco Veterans Administration Medical Center (CA) and were performed in accordance with their guidelines. Live epidermal explants from GCaMP3+/+-expressing young (aged 6–8 weeks) versus old (aged 22 months) mice placed dermis side down on a 3% agar gel were secured on the heated stage of an upright Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy, New York, NY) coupled to a Ti:Saph laser (Chameleon Ultra II, Coherent, Santa Clara, CA). Ca2+ signaling in the epidermis was stimulated by irradiating a spatially defined 20 × 20 μm2 region on the basal layer of the epidermis with 800 nm (∼140 mW). Irradiation parameters (laser intensity, scanning speed, number of iterations) were kept constant for all experiments and were optimized to consistently elicit a cytosolic Ca2+ response without permanent cell damage. The resulting GCaMP fluorescence was imaged with two-photon excitation microscopy. The excitation wavelength was 900 nm (∼15 mW). Two spectral windows of 550 per 50 nm and 445 per 25 nm were used to visualize the GCaMP fluorescence in the epidermis and the second harmonic generation signal, respectively. Dermal collagen, identified with the second harmonic signal, was used as a spatial reference. Time series were analyzed in Fiji (
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • Kaynig V.
      • Longair M.
      • Pietzsch T.
      • et al.
      Fiji: an open-source platform for biological-image analysis.
      ) and Matlab (MathWorks, Natick, MA). The change in GCaMP fluorescence was expressed as the ratio of the change with respect to the baseline fluorescence (f/f0), whereas the response area was measured as the area with significantly increased cytosolic calcium (f/f0 > 1.2), reported in μm2. A total of three mice per age group was used for these experiments. Two separate skin biopsies (one per flank) per mouse were used to collect 18–19 times resolved cytosolic calcium traces per experimental group.

      Cytosolic calcium imaging in KC monolayers and cultured KC sheets

      Second to third passage KCs from newborns versus those from aged subjects (NHEK and AHEK, respectively) were cultured as described earlier to 70–90% confluence for single KC Ca2+ imaging and 100% confluence for scratch assays and laser perturbation. KCs were loaded with 10 μM Calcium Green-1AM (Life Technologies, Carlsbad, CA) for KC Ca2+ response to laser perturbation. Cells were placed on the heated stage of an upright Zeiss 780 two-photon confocal microscope, and calcium recordings before and after laser perturbation were acquired as described earlier using a dipping ×20 lens with numerical aperture = 1. For the response to calcium switch, scratch assays, and capacitive calcium entry after store depletion, KCs were loaded with 7.5 μM Fura-2 AM (Sigma-Aldrich, St. Louis, MO). Dyes were loaded for 45 minutes at 37 °C and washed three times with Hank’s Balanced Salt Solution. Phenol red‒free Hank’s Balanced Salt Solution (Thermo Fisher) containing the appropriate extracellular Ca2+ (0, 0.07, or 1.2 mM) was used during imaging. Fura2-loaded cells were secured on a Zeiss Axio Imager 2 inverted fluorescence microscope and were alternately illuminated with 340 nm and 390 nm wavelengths. The fluorescence at emission wavelength 510 nm was recorded. Scratches were made to KC sheets with a 23-gauge needle. Changes in cytosolic Ca2+ levels in cells neighboring the scratched area were imaged before and for 50–200 seconds after wounding. For response to high extracellular calcium switch experiments, after a period of equilibration to establish a baseline of 60–120 seconds, a high calcium medium was added to the wells to a final concentration of 1.2 mM. Cells were imaged every second for additional 5–15 minutes Ca2+. The response is expressed in μm2 for area; R(arbitrary unit) = f390nm/f340nm, where f390nm and f340nm are the fluorescence intensities generated by excitation at 390nm and 340nm, respectively, corresponding to calcium-bound and -free FURA2, for single KC cytosolic Ca2+ responses; and ΔR((arbitrary unit) = RhiCa – Rbl, where RhiCa is the average R value between 2 and 4 minutes after calcium switch, and Rbl is the average baseline R before a switch to high calcium. For capacitive calcium entry after store depletion, 1μM TG (Sigma-Aldrich) was added to the culture well during ratiometric imaging. After the ratiometric signal returned to baseline, cells were exposed to 1.2 mM calcium-containing media. Cell migration was assessed using brightfield time-lapse imaging. Cells were plated on 24-well plates and switched to 1.2 mM extracellular Ca2+ for 24 hours before imaging on a Zeiss Cell Observer (Carl Zeiss Microscopy) with full environmental control (37 °C and 5% carbon dioxide). A 10 μl pipette tip was used to scratch the cultures.

      NPS R 568 treatment

      For calcium imaging experiments, KCs monolayers were switched to 0.07 mM or 1.2 mM calcium and 0 (1:1,000 dilution of DMSO), 0.5, or 1 μM NPS R 568 (Sigma-Aldrich)‒containing medium 24 hours before imaging. For E-cadherin immunofluorescence staining, cells were exposed to 1.2 mM calcium and NPS R 568 (0, 0.5,1 μM)‒containing media 15 minutes before fixation.

      Data availability statement

      The data that support the findings of this study are available at https://doi.org/10.17632/s5mk3nd495.1 and from the corresponding author on reasonable request.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the National Institute of Arthritis, Musculoskeletal and Skin Diseases of the National Institutes of Health under award numbers R01 AR051930 and R01 AR061106 (principal investigator, TMM) administered by the Northern California Institute for Research and Education, with additional resources provided by the Veterans Affairs Medical Center (San Francisco, CA) (award number IO1 BX003814 [principal investigator, DDB]) and grant LF16028 from LEO Foundation, Denmark. We gratefully acknowledge Joan Wakefield for her editing assistance; the contribution of the physicians and nurses at the University of California San Francisco Benioff Children’s Hospital Newborn Nursery; and Arron, Saylor, and Yu at the San Francisco Veterans Affairs Medical Center as well as the Laboratory for Cell Analysis Core Facility at the Hellen Diller Family Comprehensive Cancer Research Center (National Institute of Health grant P30CA082103).

      Author Contributions

      Conceptualization: AC, TMM, CLT; Data Curation: AC, CLT; Formal Analysis: AC, CLT; Funding Acquisition: DDB, TMM; Investigation: AC, TMM, CLT; Methodology: AC, CLT; Project Administration: TMM; Resources: TMM, DDB; Software: AC; Supervision: TMM; Validation: AC, CLT; Visualization: AC; Writing – Original Draft Preparation: AC, TMM, CLT; Writing – Review and Editing: AC, TMM

      Disclaimer

      The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of either the National Institutes of Health or the Department of Veterans Affairs.

      Supplementary Material

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