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Lithium regulates keratinocyte proliferation via glycogen synthase kinase 3 and NFAT2 (nuclear factor of activated T cells 2).

Hampton PJ, Jans R, Flockhart RJ, Parker G, Reynolds NJ - J. Cell. Physiol. (2012)

Bottom Line: Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation.Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation.Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model.

View Article: PubMed Central - PubMed

Affiliation: Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

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Lithium induces nuclear translocation of NFAT2, NFAT transcriptional activation and NFAT2 protein expression in keratinocytes. A: Human keratinocytes were treated with lithium (10 mM) for 48 h, immunostained with an anti-NFAT2 antibody and visualized by confocal microscopy. One representative of three experiments is shown. Increased nuclear NFAT2 (arrow heads) was seen in the lithium group compared with vehicle control. B: Western blotting of lysates of human keratinocytes treated with lithium (10 mM) for 48 h. Protein levels were calculated by band densitometry and adjusted for GAPDH expression. All of the NFAT2 isoforms were increased by all the treatments. Band densitometry showed the following percentage increases over control: 105 kDa band, Li+ (293%), 95 kDa band, Li+ (148%). C: Lithium (2 mM) activated NFAT-luciferase at 24, 96, and 168 h. (Two-way ANOVA P = 0.039 for lithium vs. control, three replicates per treatment in 2–3 independent experiments.)
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fig04: Lithium induces nuclear translocation of NFAT2, NFAT transcriptional activation and NFAT2 protein expression in keratinocytes. A: Human keratinocytes were treated with lithium (10 mM) for 48 h, immunostained with an anti-NFAT2 antibody and visualized by confocal microscopy. One representative of three experiments is shown. Increased nuclear NFAT2 (arrow heads) was seen in the lithium group compared with vehicle control. B: Western blotting of lysates of human keratinocytes treated with lithium (10 mM) for 48 h. Protein levels were calculated by band densitometry and adjusted for GAPDH expression. All of the NFAT2 isoforms were increased by all the treatments. Band densitometry showed the following percentage increases over control: 105 kDa band, Li+ (293%), 95 kDa band, Li+ (148%). C: Lithium (2 mM) activated NFAT-luciferase at 24, 96, and 168 h. (Two-way ANOVA P = 0.039 for lithium vs. control, three replicates per treatment in 2–3 independent experiments.)

Mentions: Primary keratinocytes were treated with lithium (10 mM) for 48 h and NFAT2 localization was assessed by immunostaining. Increased nuclear localization of NFAT2 was seen following lithium treatment (Fig. 4A). Western blotting of lysates from primary human keratinocytes treated with lithium (10 mM) showed an increase in expression of NFAT2 isoforms at 48 h (Fig. 4B). Significantly increased transcriptional activation by therapeutically relevant concentrations of lithium (2 mM) was shown between 24 and 168 h, two-way ANOVA P = 0.039 (Fig. 4C). Thus, 2 mM lithium induced prolonged NFAT transcriptional activation in keratinocytes up to 7 days and at time points where we observed lithium-induced keratinocyte proliferation (Fig. 1).


Lithium regulates keratinocyte proliferation via glycogen synthase kinase 3 and NFAT2 (nuclear factor of activated T cells 2).

Hampton PJ, Jans R, Flockhart RJ, Parker G, Reynolds NJ - J. Cell. Physiol. (2012)

Lithium induces nuclear translocation of NFAT2, NFAT transcriptional activation and NFAT2 protein expression in keratinocytes. A: Human keratinocytes were treated with lithium (10 mM) for 48 h, immunostained with an anti-NFAT2 antibody and visualized by confocal microscopy. One representative of three experiments is shown. Increased nuclear NFAT2 (arrow heads) was seen in the lithium group compared with vehicle control. B: Western blotting of lysates of human keratinocytes treated with lithium (10 mM) for 48 h. Protein levels were calculated by band densitometry and adjusted for GAPDH expression. All of the NFAT2 isoforms were increased by all the treatments. Band densitometry showed the following percentage increases over control: 105 kDa band, Li+ (293%), 95 kDa band, Li+ (148%). C: Lithium (2 mM) activated NFAT-luciferase at 24, 96, and 168 h. (Two-way ANOVA P = 0.039 for lithium vs. control, three replicates per treatment in 2–3 independent experiments.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Lithium induces nuclear translocation of NFAT2, NFAT transcriptional activation and NFAT2 protein expression in keratinocytes. A: Human keratinocytes were treated with lithium (10 mM) for 48 h, immunostained with an anti-NFAT2 antibody and visualized by confocal microscopy. One representative of three experiments is shown. Increased nuclear NFAT2 (arrow heads) was seen in the lithium group compared with vehicle control. B: Western blotting of lysates of human keratinocytes treated with lithium (10 mM) for 48 h. Protein levels were calculated by band densitometry and adjusted for GAPDH expression. All of the NFAT2 isoforms were increased by all the treatments. Band densitometry showed the following percentage increases over control: 105 kDa band, Li+ (293%), 95 kDa band, Li+ (148%). C: Lithium (2 mM) activated NFAT-luciferase at 24, 96, and 168 h. (Two-way ANOVA P = 0.039 for lithium vs. control, three replicates per treatment in 2–3 independent experiments.)
Mentions: Primary keratinocytes were treated with lithium (10 mM) for 48 h and NFAT2 localization was assessed by immunostaining. Increased nuclear localization of NFAT2 was seen following lithium treatment (Fig. 4A). Western blotting of lysates from primary human keratinocytes treated with lithium (10 mM) showed an increase in expression of NFAT2 isoforms at 48 h (Fig. 4B). Significantly increased transcriptional activation by therapeutically relevant concentrations of lithium (2 mM) was shown between 24 and 168 h, two-way ANOVA P = 0.039 (Fig. 4C). Thus, 2 mM lithium induced prolonged NFAT transcriptional activation in keratinocytes up to 7 days and at time points where we observed lithium-induced keratinocyte proliferation (Fig. 1).

Bottom Line: Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation.Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation.Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model.

View Article: PubMed Central - PubMed

Affiliation: Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

Show MeSH
Related in: MedlinePlus