The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis

Abstract

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.

Fig 1. Effects of UVB radiation on the gene and protein expression levels of TGase 1.

HPKs were exposed to UVB at a dose of 80 mJ/cm² and were cultured for 24 and 48 h and then analyzed by real-time RT-PCR and Western blotting, respectively. (A) The gene expression level. RT-PCR results are normalized to GAPDH; values are means ± S.D. derived from 3 independent experiments. **: p<0.01. (B) The protein expression level. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. derived from 3 independent experiments. **: p<0.01.

Fig 2. Effects of stress signaling inhibitors on the UVB-stimulated expression of TGase 1 protein.

(A) Inhibitors of p38 (at 20 μM), MEK (at 20 μM) and JNK (at 20 μM). (B) Inhibitor of NFκB translocation (at 5 μM). HPKs were incubated with signaling inhibitors as noted for 3 h, then were exposed to UVB at a dose of 80 mJ/cm² and were cultured for 48 h after which cell lysates were subjected to Western blotting analysis. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. derived from 3 independent experiments. **: p<0.01, *: p<0.05.

Fig 3. Inhibitory effects of AX on the UVB-stimulated protein expression of TGase 1.

(A) HPKs were treated for 3 h with AX at concentrations of 1, 4 or 8 μM, exposed to UVB at a dose of 80 mJ/cm² and were then cultured for 48 h prior to Western blotting analysis. (B) Immediately after UVB irradiation at a dose of 80 mJ/cm², AX was added at concentrations of 1, 4 or 8 μM after which HPKs were cultured for 48 h and subjected to Western blotting analysis. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. derived from 3 independent experiments. *: p<0.05; **: p<0.01.

Fig 4. Time course study of the phosphorylation of several stress-activated kinases after UVB exposure.

(A) Effects on the phosphorylation of ERK/p38/JNK. (B) Effects on the phosphorylation of AKT. Human HaCaT keratinocytes were exposed to UVB at 80 mJ/cm² and were cultured for the indicated times. Whole cell lysates were prepared and were analyzed by Western blot using antibodies to phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK1/2 and JNK1/2, and to phospho-AKT and AKT. Representative data from 3 independent experiments are shown.

Fig 5. Effects of AX on the UVB-induced phosphorylation of p38/ERK/JNK/c-Jun.

(A) Effects of post-irradiation treatment with AX on the UVB-induced phosphorylation of p38, JNK and ERK in human HaCaT keratinocytes. (B) Post-irradiation treatment for the phosphorylation of c-Jun in HPKs. HPKs or human HaCaT keratinocytes were exposed to UVB at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at the indicated times after UVB irradiation and were immunoblotted with antibodies to phosphorylated or non-phosphorylated p38/ERK/JNK/c-Jun. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. **: p<0.01; *: p<0.05.

Fig 6. Effects of AX on the UVB-induced phosphorylation of c-Jun and on the UVB-induced protein expression of c-Jun and c-Fos.

(A) Western blotting for the phosphorylation of c-Jun and the protein expression of c-Jun and c-Fos. (B) Densitometric analysis for the phosphorylation of c-Jun at 0.5 h post-irradiation. (C) Densitometric analysis for the protein level of c-Jun at 3 h post-irradiation. (D) Densitometric analysis for the protein level of c-Fos at 3 h post-irradiation. Human HaCaT keratinocytes were exposed to UVB at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at the indicated times after UVB irradiation and were immunoblotted with antibodies to phosphorylated or non-phosphorylated c-Jun or c-Fos. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. **: p<0.01; *: p<0.05.

Fig 7. Effects of AX on the UVB-induced phosphorylation of ATF2.

(A) Western blotting for the phosphorylation of ATF-2 in HPKs. (B) Western blotting for the phosphorylation of ATF-2in human HaCaT keratinocytes. (C) Densitometric analysis for phosphorylation of ATF-2 at 0.5 h post-irradiation. (D) Densitometric analysis for the phosphorylation of ATF-2 at 6 h post-irradiation. HPKs or human HaCaT keratinocytes were exposed to UVB at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at the indicated times after UVB irradiation and were immunoblotted with antibodies to phosphorylated or non-phosphorylated ATF-2. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. **: p<0.01.

Fig 8. Effects of AX on the UVB-induced phosphorylation of CK2 and the degradation of I-κBα.

(A) CK2, (B) I-κBα. Human HaCaT keratinocytes were exposed to UVB at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at the indicated times after UVB irradiation and were immunoblotted with antibodies to CK2 or I-κBα. Representative immunoblots from 3 independent experiments are shown.

Fig 9. Effects of AX on the Ser276/468/536 phosphorylations of NFκBp65 in UVB-exposed human HaCaT keratinocytes.

(A) Ser276 phosphorylation of NFκBp65 at 0.5, 3 and 6 h post-irradiation. (B) Densitometric analysis for Ser276 phosphorylation of NFκBp65 at 0.5 h post-irradiation. (C) Ser536 phosphorylation of NFκBp65 at 0.5, 3 and 6 h post-irradiation. (D) Ser468 phosphorylation of NFκBp65 at 0.5 h post-irradiation. Human HaCaT keratinocytes were exposed to UVB irradiation at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at 0.5, 3 or 6 h after UVB irradiation and were immunoblotted with antibodies to NFκBp65 and phosphorylated NFκBp65Ser276/Ser536/Ser468. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. **: p<0.01.

Fig 10. Effects of UVB irradiation on the Thr581/Ser360/Ser376 phosphorylation of MSK1 and the inhibitory effect of AX.

(A) Thr581 phosphorylation of MSK1 in HPKs at 0.5 h post-irradiation. (B), (C) Ser360/ Ser376 phosphorylation of MSK1 in human HaCaT keratinocytes at 0.5 h post-irradiation. HPKs or human HaCaT keratinocytes were exposed to UVB irradiation at a dose of 80 mJ/cm² immediately after which the cells were treated with AX at the indicated concentrations. Lysates were harvested at 30 min after UVB irradiation and were immunoblotted with antibodies to Thr581/Ser376/360 phosphorylated MSK1. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. *: p<0.05, **:p<0.01.

Fig 11. Effects of AX on the Ser133 phosphorylation of CREB in UVB-exposed human HaCaT keratinocytes at 0.5 and 3 h min post-irradiation.

(A) Western blotting for the phosphorylation of CREB Ser133. (B) Densitometric analysis for Ser133 phosphorylation of CREB at 0.5 h post-irradiation. Human HaCaT keratinocytes were exposed to UVB irradiation at a dose of 80 mJ/cm² immediately after which they were treated with AX at the indicated concentrations. Lysates were harvested at 0.5 and 3 h after UVB irradiation and were immunoblotted with antibodies to phosphorylated or non-phosphorylated CREB. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. from 3 independent experiments. **: p<0.01.

Fig 12. Effects of the MSK1 inhibitor H89 on the increased phosphorylation of NFκBp65Ser276 and CREBSer133 as well as on the protein expression of TGase 1 and COX-2 in UVB-exposed human keratinocytes.

(A) Effects on the phosphorylation of NFκBp65Ser276 and CREBSer133. (B) Effects on the protein expression of TGase 1 and COX-2. HPKs were incubated with H89 at the indicated concentrations immediately after UVB radiation at a dose of 80 mJ/cm² and were subsequently cultured for 0.5 h for the phosphorylation analysis or 48 h for the protein expression analysis prior to Western blotting. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. derived from 3 independent experiments. ** p < 0.01, * p<0.05.

Fig 13. Effects of silencing of MSK1 mRNA on the UVB-stimulated expression of TGase 1 protein.

HPKs were transfected with a pre-designed siRNA against MSK1 to down-regulate MSK1 along with a negative control siRNA. Twenty-four h after transfection, HPKs were exposed to UVB irradiation at a dose of 80 mJ/cm² and were cultured for 48 h after which whole cell lysates were prepared for Western blot analysis using antibodies to TGase 1 and β-actin. Representative immunoblots from 3 independent experiments are shown; values are means ± S.D. derived from 3 independent experiments. *: p<0.05; **: p<0.01.

Fig 14. Intracellular signaling pathways leading to the UVB-induced increase in the expression of TGase 1 and the site of inhibition by the post-irradiation treatment of human keratinocytes with AX.

The encircled P’s colored yellow and pink represent UVB-increased and non- or significantly abrogated phosphorylations, respectively, in each signaling molecule by the post-irradiation treatment with AX. The blue-colored signaling molecules show an increase or a decrease in each protein level by the post-irradiation treatment with AX in UVB-exposed human keratinocytes.