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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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NFAT nuclear localization and transcriptional activation in human keratinocytes is regulated by GSK3. A: Transfection of the GSKBP (BP20) induced a fourfold increase of NFAT-luciferase activity (P < 0.005, unpaired t-test, n = 3 independent experiments, three replicates per experiment) at 24 h. B: Transfection of caGSK-3β-S9A inhibited NFAT-luciferase activity at 24 h compared with an empty vector control (P = 0.002, t-test, n = 2 independent experiments, three replicates per experiment). C: Human keratinocytes were transduced with either pLEGFP GSKBP or pLEGFP empty and were then subsequently immunostained 48 h later with anti-NFAT2 antibodies and the nuclear dye Toto-3. The rabbit anti-NFAT2 antibody was labeled with an Alexa Fluor 568 anti-rabbit secondary antibody. Increased nuclear NFAT2 was seen in the GSKBP positive cells (arrow heads), compared to the empty vector GFP positive cells. Mid z images are shown taken in sequential scanning mode to minimize cross talk. The following excitation wavelengths were used; GFP 488 nM, Alexa Fluor 543 nM, and Toto-3 633 nM. Scale bar represents 40 µm.
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fig03: NFAT nuclear localization and transcriptional activation in human keratinocytes is regulated by GSK3. A: Transfection of the GSKBP (BP20) induced a fourfold increase of NFAT-luciferase activity (P < 0.005, unpaired t-test, n = 3 independent experiments, three replicates per experiment) at 24 h. B: Transfection of caGSK-3β-S9A inhibited NFAT-luciferase activity at 24 h compared with an empty vector control (P = 0.002, t-test, n = 2 independent experiments, three replicates per experiment). C: Human keratinocytes were transduced with either pLEGFP GSKBP or pLEGFP empty and were then subsequently immunostained 48 h later with anti-NFAT2 antibodies and the nuclear dye Toto-3. The rabbit anti-NFAT2 antibody was labeled with an Alexa Fluor 568 anti-rabbit secondary antibody. Increased nuclear NFAT2 was seen in the GSKBP positive cells (arrow heads), compared to the empty vector GFP positive cells. Mid z images are shown taken in sequential scanning mode to minimize cross talk. The following excitation wavelengths were used; GFP 488 nM, Alexa Fluor 543 nM, and Toto-3 633 nM. Scale bar represents 40 µm.

Mentions: Having demonstrated that inhibition of GSK-3 induced keratinocyte proliferation, we next investigated the role of the GSK substrate NFAT in keratinocyte proliferation. Since NFAT is a negatively regulated substrate of GSK-3, we investigated the effect of inhibition of GSK-3 on NFAT nuclear localization and transcriptional activity. Primary keratinocytes were transiently transfected with an NFAT-luciferase reporter and either the GSKBP or empty vector. As expected, GSKBP induced significantly greater activation of the NFAT-luciferase reporter than empty vector (Fig. 3A, P < 0.005). A similar effect on the NFAT-luciferase reporter was seen following retroviral transduction with the fusion protein pLEGFP-GSKBP compared to empty vector, further validating that this construct was functionally active in this system (data not shown). Consistent with the effect of GSKBP, overexpression of a constitutively active GSK mutant (CaGSK-3β-S9A), reduced activation of the NFAT-luciferase reporter (Fig. 3B, P = 0.002). Retroviral transduction of pLEGFP GSKBP induced increased nuclear NFAT2 compared with transduction of pLEGFP empty (Fig. 3C).


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)

NFAT nuclear localization and transcriptional activation in human keratinocytes is regulated by GSK3. A: Transfection of the GSKBP (BP20) induced a fourfold increase of NFAT-luciferase activity (P < 0.005, unpaired t-test, n = 3 independent experiments, three replicates per experiment) at 24 h. B: Transfection of caGSK-3β-S9A inhibited NFAT-luciferase activity at 24 h compared with an empty vector control (P = 0.002, t-test, n = 2 independent experiments, three replicates per experiment). C: Human keratinocytes were transduced with either pLEGFP GSKBP or pLEGFP empty and were then subsequently immunostained 48 h later with anti-NFAT2 antibodies and the nuclear dye Toto-3. The rabbit anti-NFAT2 antibody was labeled with an Alexa Fluor 568 anti-rabbit secondary antibody. Increased nuclear NFAT2 was seen in the GSKBP positive cells (arrow heads), compared to the empty vector GFP positive cells. Mid z images are shown taken in sequential scanning mode to minimize cross talk. The following excitation wavelengths were used; GFP 488 nM, Alexa Fluor 543 nM, and Toto-3 633 nM. Scale bar represents 40 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: NFAT nuclear localization and transcriptional activation in human keratinocytes is regulated by GSK3. A: Transfection of the GSKBP (BP20) induced a fourfold increase of NFAT-luciferase activity (P < 0.005, unpaired t-test, n = 3 independent experiments, three replicates per experiment) at 24 h. B: Transfection of caGSK-3β-S9A inhibited NFAT-luciferase activity at 24 h compared with an empty vector control (P = 0.002, t-test, n = 2 independent experiments, three replicates per experiment). C: Human keratinocytes were transduced with either pLEGFP GSKBP or pLEGFP empty and were then subsequently immunostained 48 h later with anti-NFAT2 antibodies and the nuclear dye Toto-3. The rabbit anti-NFAT2 antibody was labeled with an Alexa Fluor 568 anti-rabbit secondary antibody. Increased nuclear NFAT2 was seen in the GSKBP positive cells (arrow heads), compared to the empty vector GFP positive cells. Mid z images are shown taken in sequential scanning mode to minimize cross talk. The following excitation wavelengths were used; GFP 488 nM, Alexa Fluor 543 nM, and Toto-3 633 nM. Scale bar represents 40 µm.
Mentions: Having demonstrated that inhibition of GSK-3 induced keratinocyte proliferation, we next investigated the role of the GSK substrate NFAT in keratinocyte proliferation. Since NFAT is a negatively regulated substrate of GSK-3, we investigated the effect of inhibition of GSK-3 on NFAT nuclear localization and transcriptional activity. Primary keratinocytes were transiently transfected with an NFAT-luciferase reporter and either the GSKBP or empty vector. As expected, GSKBP induced significantly greater activation of the NFAT-luciferase reporter than empty vector (Fig. 3A, P < 0.005). A similar effect on the NFAT-luciferase reporter was seen following retroviral transduction with the fusion protein pLEGFP-GSKBP compared to empty vector, further validating that this construct was functionally active in this system (data not shown). Consistent with the effect of GSKBP, overexpression of a constitutively active GSK mutant (CaGSK-3β-S9A), reduced activation of the NFAT-luciferase reporter (Fig. 3B, P = 0.002). Retroviral transduction of pLEGFP GSKBP induced increased nuclear NFAT2 compared with transduction of pLEGFP empty (Fig. 3C).

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