Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of GSK3 by both pharmacological and genetic methods induces proliferation of keratinocytes. Keratinocytes were retrovirally transduced with pLEGFP empty vector (A) or pLEGFP GSKBP (B) and imaged by confocal microscopy. Scale bar represents 40 µm. SRB assays of K14 keratinocytes (C) or human keratinocytes (D). Transduction with pLEGFP GSKBP (BP20) induced significant proliferation compared to pLEGFP empty vector in K14 keratinocytes at 7 days (C: P < 0.005, t-test, three independent experiments, 30 wells per group per experiment) and in human keratinocytes at 4 days (D: P = 0.02, t-test, three independent experiments, 30 wells per group per experiment). E: SRB assays of K14 keratinocytes following treatment with BIO for 7 days induced significant proliferation at 5, 50, and 500 nM concentrations (P < 0.005, P < 0.006, P < 0.005, respectively, ANOVA, three independent experiments, 12 wells per group per experiment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150531&req=5

fig02: Inhibition of GSK3 by both pharmacological and genetic methods induces proliferation of keratinocytes. Keratinocytes were retrovirally transduced with pLEGFP empty vector (A) or pLEGFP GSKBP (B) and imaged by confocal microscopy. Scale bar represents 40 µm. SRB assays of K14 keratinocytes (C) or human keratinocytes (D). Transduction with pLEGFP GSKBP (BP20) induced significant proliferation compared to pLEGFP empty vector in K14 keratinocytes at 7 days (C: P < 0.005, t-test, three independent experiments, 30 wells per group per experiment) and in human keratinocytes at 4 days (D: P = 0.02, t-test, three independent experiments, 30 wells per group per experiment). E: SRB assays of K14 keratinocytes following treatment with BIO for 7 days induced significant proliferation at 5, 50, and 500 nM concentrations (P < 0.005, P < 0.006, P < 0.005, respectively, ANOVA, three independent experiments, 12 wells per group per experiment).

Mentions: Having confirmed that lithium-induced keratinocyte proliferation and inhibited GSK-3β, we further investigated the role of GSK-3 in regulating keratinocyte proliferation. To investigate whether inhibition of GSK-3 by lithium accounted for the effect of lithium on keratinocyte proliferation, we overexpressed GSKBP. GSKBP is a specific GSK-3 inhibitor, which we have shown, using Tcf/LEF-dependent TOPFLASH luciferase assays, to be functionally active in human keratinocytes (data not shown). We transduced SV-k14 keratinocytes with the empty vector, pLEGFP empty vector (Fig. 2A) or with a retroviral GFP–GSKBP fusion protein, pLEGFP-GSKBP (Fig. 2B), and showed that pLEGFP-GSKBP induced significantly greater proliferation at 7 days compared to pLEGFP empty vector (Fig. 2C, P < 0.005). Transduction of human keratinocytes with pLEGFP-GSKBP for 4 days induced increased proliferation compared to empty vector control pLEGFP empty (Fig. 2D, P = 0.02). To further confirm the role of GSK-3 in regulating keratinocyte proliferation, we next investigated pharmacological inhibition of GSK-3 using a specific GSK-3 inhibitor BIO (Meijer et al., 2003) and showed that BIO induced increased proliferation of SV-k14 keratinocytes at 7 days (Fig. 2E, P < 0.005 at 5, 50, and 500 nM).


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)

Inhibition of GSK3 by both pharmacological and genetic methods induces proliferation of keratinocytes. Keratinocytes were retrovirally transduced with pLEGFP empty vector (A) or pLEGFP GSKBP (B) and imaged by confocal microscopy. Scale bar represents 40 µm. SRB assays of K14 keratinocytes (C) or human keratinocytes (D). Transduction with pLEGFP GSKBP (BP20) induced significant proliferation compared to pLEGFP empty vector in K14 keratinocytes at 7 days (C: P < 0.005, t-test, three independent experiments, 30 wells per group per experiment) and in human keratinocytes at 4 days (D: P = 0.02, t-test, three independent experiments, 30 wells per group per experiment). E: SRB assays of K14 keratinocytes following treatment with BIO for 7 days induced significant proliferation at 5, 50, and 500 nM concentrations (P < 0.005, P < 0.006, P < 0.005, respectively, ANOVA, three independent experiments, 12 wells per group per experiment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150531&req=5

fig02: Inhibition of GSK3 by both pharmacological and genetic methods induces proliferation of keratinocytes. Keratinocytes were retrovirally transduced with pLEGFP empty vector (A) or pLEGFP GSKBP (B) and imaged by confocal microscopy. Scale bar represents 40 µm. SRB assays of K14 keratinocytes (C) or human keratinocytes (D). Transduction with pLEGFP GSKBP (BP20) induced significant proliferation compared to pLEGFP empty vector in K14 keratinocytes at 7 days (C: P < 0.005, t-test, three independent experiments, 30 wells per group per experiment) and in human keratinocytes at 4 days (D: P = 0.02, t-test, three independent experiments, 30 wells per group per experiment). E: SRB assays of K14 keratinocytes following treatment with BIO for 7 days induced significant proliferation at 5, 50, and 500 nM concentrations (P < 0.005, P < 0.006, P < 0.005, respectively, ANOVA, three independent experiments, 12 wells per group per experiment).
Mentions: Having confirmed that lithium-induced keratinocyte proliferation and inhibited GSK-3β, we further investigated the role of GSK-3 in regulating keratinocyte proliferation. To investigate whether inhibition of GSK-3 by lithium accounted for the effect of lithium on keratinocyte proliferation, we overexpressed GSKBP. GSKBP is a specific GSK-3 inhibitor, which we have shown, using Tcf/LEF-dependent TOPFLASH luciferase assays, to be functionally active in human keratinocytes (data not shown). We transduced SV-k14 keratinocytes with the empty vector, pLEGFP empty vector (Fig. 2A) or with a retroviral GFP–GSKBP fusion protein, pLEGFP-GSKBP (Fig. 2B), and showed that pLEGFP-GSKBP induced significantly greater proliferation at 7 days compared to pLEGFP empty vector (Fig. 2C, P < 0.005). Transduction of human keratinocytes with pLEGFP-GSKBP for 4 days induced increased proliferation compared to empty vector control pLEGFP empty (Fig. 2D, P = 0.02). To further confirm the role of GSK-3 in regulating keratinocyte proliferation, we next investigated pharmacological inhibition of GSK-3 using a specific GSK-3 inhibitor BIO (Meijer et al., 2003) and showed that BIO induced increased proliferation of SV-k14 keratinocytes at 7 days (Fig. 2E, P < 0.005 at 5, 50, and 500 nM).

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