1. Wnt5a/β-Catenin Signaling Drives Calcium-Induced Differentiation of Human Primary Keratinocytes 
Tanja Popp, Dirk Steinritz, Andreas Breit, Janina Deppe, Virginia Egea, Annette Schmidt, Thomas Gudermann, Christian Weber, Christian Ries 
Journal of Investigative Dermatology 
Volume 134, Issue 8, Pages (August 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Role of calcium-sensing receptor (CaSR) in calcium-induced differentiation of keratinocytes. (a) Normal human epidermal keratinocytes (NHEKs) were incubated in medium containing 0.1 mM Ca2+ (low Ca2+) and 2.0 mM Ca2+ (high Ca2+). After 4 days, transcription levels of the differentiation markers keratin-1 and loricrin were determined using quantitative real-time reverse-transcriptase–PCR (qRT–PCR). The values were normalized to cyclophilin B mRNA. The results are given as the percentage of change in mRNA expression relative to cells grown at low extracellular calcium concentration (ex[Ca2+]) set as 100%. Values represent the mean±SD of triplicate measurements (n=3), **P<0.01, ***P< After 4 days, protein levels of keratin-1 and loricrin were analyzed in cell extracts by immunoblotting. Cellular β-actin was used as a loading control. (b) NHEKs were transfected with small interfering RNA (siRNA) targeting CaSR (KD) or control siRNA (NC). After 4 days, mRNA expression of keratin-1 and loricrin was analyzed using qRT–PCR. The values were normalized to cyclophilin B mRNA. Results are given as mean±SD of triplicate measurements (n=3), *P<0.05. After 4 days of incubation, protein levels of keratin-1 and loricrin present in cell extracts were determined by western blotting. β-Actin served as a loading control. (c) Measurement of intracellular calcium signals in G5α-NHEKs transfected with siRNA targeting CaSR (KD) or control siRNA (NC). After 5 seconds, 0.1 mM Ca2+ (low Ca2+) and 2.0 mM Ca2+ (high Ca2+) were added to the cells. Responses were normalized by defining the first value measured (1 second) as 100%. Results are expressed as the mean±SD of three independent experiments (n=3) performed in sextuplicates.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Impact of high extracellular calcium concentration (ex[Ca2+]) on the expression of effector molecules of the Wnt/β-catenin pathway and correlation between Wnt5a and calcium-sensing receptor (CaSR). (a) Normal human epidermal keratinocytes (NHEKs) were incubated in medium containing 0.1 mM Ca2+ (low Ca2+) and 2.0 mM Ca2+ (high Ca2+). After 1 day of incubation, the mRNA expression of Wnt3a, Wnt4, Wnt5a, and Dkk-1 was quantified by quantitative real-time reverse-transcriptase–PCR (qRT–PCR). The values were normalized to cyclophilin B mRNA. Results are given as the percentage of change in mRNA expression relative to cells grown at low ex[Ca2+] set as 100%. Values represent the mean±SD of one triplicate experiment from two independent measurements. **P<0.01. Protein expression of Wnt5a was examined by immunoblotting analysis of aliquots taken from 5-day culture supernatants. Equal amounts of total protein were applied in each lane. For densitometric quantification, Wnt5a release from control cells treated with low ex[Ca2+] was set as 100% densitometric units (DU). (b) Immunoblotting analysis of Wnt5a in culture supernatants obtained from NHEKs transfected with small interfering RNA (siRNA) targeting CaSR (KD) or control siRNA (NC) on day 5 after incubation in high ex[Ca2+]. For densitometric quantification, Wnt5a release from cells transfected with control siRNA was set as 100% DU. (c) Immunoblotting analysis of CaSR expression in extracts from NHEKs incubated for 5 days without (NC) and with 250 ng ml-1 Wnt5a. Cellular β-actin was used as a loading control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Influence of Wnt5a in keratinocyte differentiation and proliferation. (a, b) Normal human epidermal keratinocytes (NHEKs) were incubated in medium containing 0.1 mM Ca2+ (low Ca2+) and 2.0 mM Ca2+ (high Ca2+) as well as 0, 50, or 250 ng ml-1 of recombinant Wnt5a. After 4 days, expression of keratin-1 and loricrin were determined on mRNA level by quantitative real-time reverse-transcriptase–PCR (qRT–PCR) (a) and on protein level by immunoblotting of aliquots from cell extracts (b). qRT–PCR values were normalized to cyclophilin B mRNA and are given as the percentage of change in mRNA expression relative to cells grown at low extracellular calcium concentration (ex[Ca2+]) set as 100%. The detection of β-actin in the cell lysates served as a control. (c) NHEKs transfected with siRNA against Wnt5a (KD) or control small interfering RNA (siRNA) (NC) were incubated in medium containing low and high ex[Ca2+]. After 4 days, the cells were analyzed for the expression of keratin-1 and loricrin using qRT–PCR and immunoblotting. For densitometric quantification, the amounts of keratin-1 and loricrin present in control cells were set as 100%. (d) Quantification of NHEKs at 1 and 5 days after incubation in low ex[Ca2+] without (set as 100%) and with different concentrations of Wnt5a using the BrdU proliferation assay. (a, c, d) Results are given as mean values±SD of triplicate measurements (n=3), *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Role of β-catenin in calcium-induced differentiation of keratinocytes. (a–c) Normal human epidermal keratinocytes (NHEKs) were incubated in medium without and with recombinant Wnt5a (250 ng ml-1) as well as medium containing 0.1 mM Ca2+ (low Ca2+) and 2.0 mM Ca2+ (high Ca2+). (a) After 1 day, the protein level of β-catenin was examined by immunoblotting analysis of the cell extracts. For densitometry, the amount of β-catenin present in cells grown without recombinant Wnt5a and at low extracellular calcium concentration (ex[Ca2+]) were set as 100%. The detection of β-actin in the cell lysates served as a control. (b) After 1 day of cultivation, the activity of secreted Gaussia luciferase, which was transcribed due to β-catenin-dependent lymphoid-enhancer-binding factor-1/T-cell factor-1 (LEF/TCF) signaling in NHEKs transiently transfected with the β-catenin-activated reporter (BAR), was quantified in the conditioned medium. Values are given as the percentage of change relative to cells grown without recombinant Wnt5a and at low ex[Ca2+] set as 100%. (c) After 5 days of cultivation, the transcription level of a Wnt/β-catenin target gene, cyclin D1, was analyzed in the cells using quantitative real-time reverse-transcriptase–PCR (qRT–PCR). The values were normalized to cyclophilin B mRNA. The results represent the percentage of change in mRNA expression relative to cells grown without exogenous Wnt5a and at low ex[Ca2+] set as 100%. (d, e) NHEKs were transfected with small interfering RNA (siRNA) against β-catenin (KD) or control siRNA (NC) and incubated in medium containing high ex[Ca2+]. (d) After 1 and 4 days, the relative transcription levels of keratin-1 and loricrin were determined using qRT–PCR (value obtained in cells grown at low ex[Ca2+] was set as 1). The values were normalized to cyclophilin B mRNA. (e) After 4 days, the protein levels of keratin-1 and loricrin were analyzed using western blotting. For densitometric quantification, the amount of protein present in the control cells (NC) were set as 100%. β-Actin was used as a loading control. (f, g) NHEKs were transfected with siRNA against axin 2 (KD) or control siRNA (NC) and cultivated at low ex[Ca2+]. (f) After 4 days, relative mRNA expression of keratin-1 and loricrin was determined using qRT–PCR (the NC was set as 100%). Values were normalized to cyclophilin B mRNA. (g) After 4 days, protein levels of keratin-1 and loricrin were analyzed using immunoblotting. For densitometry, the amount of protein present in control cells (NC) was set as 100%. β-Actin was used as a loading control. (h) NHEKs were transfected with control siRNA (NC) and siRNA against β-catenin and axin 2. After cultivation at high ex[Ca2+] for 5 days, cell proliferation was determined using the water-soluble tetrazolium salt-8 (WST-8) assay. (b, c, d, f, h) Data shown are given as mean values±SD of triplicate measurements (n=3). *P<0.05, **P<0.01, ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Proposed model for the role of Wnt5a/β-catenin signaling in calcium-induced differentiation of keratinocytes. Elevated levels of extracellular calcium concentration (ex[Ca]2+) stimulate intracellular signaling through binding to calcium-sensing receptor (CaSR). This leads to an increase in free intracellular calcium (in[Ca]2+) by its release from stores such as the endoplasmatic reticulum. As a consequence, the expression and secretion of Wnt5a are upregulated. Wnt5a then binds to members of the Frizzled (Fz) family of receptors and low-density lipoprotein receptor-related protein (LRP) 5/6 on the cell surface, thus activating the canonical Wnt/β-catenin signaling pathway through augmentation of β-catenin in the cells. Improved interaction of nuclear β-catenin with lymphoid-enhancer-binding factor-1/T-cell factor-1 (LEF/TCF) transcription factors then leads to the upregulation of typical target genes such as cyclin D1. Simultaneously, the expression of keratin-1 and loricrin are enhanced, promoting the differentiation of the keratinocytes. Blockage of axin 2, a negative feedback regulator of β-catenin stability, also promotes the production of keratin-1 and loricrin in the cells, confirming the relevance of the Wnt/β-catenin pathway in keratinocyte differentiation. Autocrine Wnt5a is important but not essential in calcium-regulated differentiation of keratinocytes through β-catenin signaling.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions