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UVB-induced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes.

Ming M, Han W, Maddox J, Soltani K, Shea CR, Freeman DM, He YY - Oncogene (2009)

Bottom Line: As compared with normal skin, PTEN levels are reduced in human actinic keratosis, a precancerous skin lesion caused by solar UV.AKT activation is higher in UVB-irradiated surviving cells as compared with unirradiated cells.Our findings indicate that (1) UVB radiation suppresses PTEN expression in keratinocytes; and (2) the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation.

View Article: PubMed Central - PubMed

Affiliation: Section of Dermatology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Ultraviolet (UV) radiation in sunlight is the major environmental cause of skin cancer. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a proven critical tumor suppressor. We report here that UVB downregulates PTEN in primary human keratinocytes, human HaCaT keratinocytes and mouse skin. As compared with normal skin, PTEN levels are reduced in human actinic keratosis, a precancerous skin lesion caused by solar UV. PTEN downregulation is mediated by two mechanisms: (1) PTEN is cleaved by active caspase in apoptotic cells in which AKT activation is reduced; and (2) PTEN transcription is suppressed in surviving cells, and this suppression is independent of caspase activation and occurs in parallel with increased ERK and AKT activation. We report here that the combination of ERK and AKT activation is crucial for PTEN suppression in surviving cells following UVB irradiation. AKT activation is higher in UVB-irradiated surviving cells as compared with unirradiated cells. The ERK and AKT pathways are involved in sustaining PTEN suppression in UVB-exposed cells. Increasing PTEN expression enhances apoptosis of keratinocytes in response to UVB irradiation. Our findings indicate that (1) UVB radiation suppresses PTEN expression in keratinocytes; and (2) the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation. Our identification of PTEN as a critical molecular target of UVB provides new insights into the pathogenesis of skin cancer.

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Role of ERK and AKT pathways in PTEN down-regulation in surviving cells following UVB radiationBoth ERK and AKT pathways are critical for PTEN suppression induced by UVB in surviving cells. A, cells were treated as in FIGURE 1A and ERK phosphorylation (p-ERK) was analyzed by Western blotting. B, cells were pretreated with the specific ERK activation inhibitor PD98059 (PD, 20 μM), the specific AKT activation inhibitor LY294002 (LY) (10 μM), or the combination of both, for 1 h, and then exposed to UVB (20 mJ/cm2). Cells were then treated with PD, LY, or both and collected at 24 h. The PTEN mRNA levels were determined by real-time PCR. *, p < 0.05, significant differences from UVB-exposed cells treated vehicle alone. C, same as in B except that the PTEN protein levels were determined by Western blotting. D, quantification of the PTEN levels in C. *, p < 0.05; **, p < 0.01; significant differences from UVB-exposed cells treated vehicle alone. E, same as in B except that cells were treated with both PD (20 μM) and LY (10 μM) at 24 h after UVB radiation for three days and the PTEN protein levels were determined by Western blotting. F, HaCaT cells were infected with constitutive active AKT adenovirus (A+) or adenovirus expressing GFP only as a control. Cells were collected at 48 or 72 h after infection. G, same as in F except that cells were collected at 48 h post infection for real-time PCR analysis of the PTEN mRNA levels.
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Figure 7: Role of ERK and AKT pathways in PTEN down-regulation in surviving cells following UVB radiationBoth ERK and AKT pathways are critical for PTEN suppression induced by UVB in surviving cells. A, cells were treated as in FIGURE 1A and ERK phosphorylation (p-ERK) was analyzed by Western blotting. B, cells were pretreated with the specific ERK activation inhibitor PD98059 (PD, 20 μM), the specific AKT activation inhibitor LY294002 (LY) (10 μM), or the combination of both, for 1 h, and then exposed to UVB (20 mJ/cm2). Cells were then treated with PD, LY, or both and collected at 24 h. The PTEN mRNA levels were determined by real-time PCR. *, p < 0.05, significant differences from UVB-exposed cells treated vehicle alone. C, same as in B except that the PTEN protein levels were determined by Western blotting. D, quantification of the PTEN levels in C. *, p < 0.05; **, p < 0.01; significant differences from UVB-exposed cells treated vehicle alone. E, same as in B except that cells were treated with both PD (20 μM) and LY (10 μM) at 24 h after UVB radiation for three days and the PTEN protein levels were determined by Western blotting. F, HaCaT cells were infected with constitutive active AKT adenovirus (A+) or adenovirus expressing GFP only as a control. Cells were collected at 48 or 72 h after infection. G, same as in F except that cells were collected at 48 h post infection for real-time PCR analysis of the PTEN mRNA levels.

Mentions: We have demonstrated that following UVB radiation PTEN is suppressed for at least a week (Figure 2D), and even longer than 4 weeks, and will partially recover by 6 weeks (data not shown). Considering the critical role of PTEN as a tumor suppressor in skin homeostasis, recovering PTEN expression may prove beneficial in preventing carcinogenesis, e.g., following sunburn. We examined the role of the ERK and AKT pathways to further elucidate the molecular mechanisms of PTEN suppression by UVB irradiation. UVB activated the ERK and AKT pathways (Figure 7A). While ERK was activated at all the time points examined, AKT was activated only at 3 and 24 h post radiation, possibly due to loss of AKT protein in apoptotic cells between 3 and 24 h (Figures 3C and 3D). PD98059 is an inhibitor of ERK activation, and LY2940002 is an inhibitor of AKT activation. When cells were kept in the dark or exposed to UVB and then treated with PD, LY, or both, the suppression of PTEN expression at the mRNA level was completely blocked (Figure 7B, P < 0.05). The levels of PTEN protein were also significantly increased by PD, LY, or both (P < 0.05 for PD or LY and 0.01 for PD/LY) (Figures 7C and 7D). Interestingly, LY or PD increased the levels of PTEN protein for three days even when added 24 h post UVB radiation (Figure 7E). This indicates that ERK/AKT pathway is critical in maintaining PTEN suppression in UVB-exposed keratinocytes. To further determine the effect of AKT activation alone on PTEN expression, we infected HaCaT cells with constitutively active AKT adenovirus. At 48 and 72 h after infection, the PTEN protein levels decreased (Figure 7F). This AKT activation suppressed the PTEN mRNA levels significantly at 48 h post infection (Figure 7G, P < 0.05). These results demonstrate that AKT activation negatively regulates the PTEN levels, forming a positive feedback loop in keratinocytes. Our findings suggest that UVB-induced PTEN suppression is reversible by blocking ERK/AKT pathways.


UVB-induced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes.

Ming M, Han W, Maddox J, Soltani K, Shea CR, Freeman DM, He YY - Oncogene (2009)

Role of ERK and AKT pathways in PTEN down-regulation in surviving cells following UVB radiationBoth ERK and AKT pathways are critical for PTEN suppression induced by UVB in surviving cells. A, cells were treated as in FIGURE 1A and ERK phosphorylation (p-ERK) was analyzed by Western blotting. B, cells were pretreated with the specific ERK activation inhibitor PD98059 (PD, 20 μM), the specific AKT activation inhibitor LY294002 (LY) (10 μM), or the combination of both, for 1 h, and then exposed to UVB (20 mJ/cm2). Cells were then treated with PD, LY, or both and collected at 24 h. The PTEN mRNA levels were determined by real-time PCR. *, p < 0.05, significant differences from UVB-exposed cells treated vehicle alone. C, same as in B except that the PTEN protein levels were determined by Western blotting. D, quantification of the PTEN levels in C. *, p < 0.05; **, p < 0.01; significant differences from UVB-exposed cells treated vehicle alone. E, same as in B except that cells were treated with both PD (20 μM) and LY (10 μM) at 24 h after UVB radiation for three days and the PTEN protein levels were determined by Western blotting. F, HaCaT cells were infected with constitutive active AKT adenovirus (A+) or adenovirus expressing GFP only as a control. Cells were collected at 48 or 72 h after infection. G, same as in F except that cells were collected at 48 h post infection for real-time PCR analysis of the PTEN mRNA levels.
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Figure 7: Role of ERK and AKT pathways in PTEN down-regulation in surviving cells following UVB radiationBoth ERK and AKT pathways are critical for PTEN suppression induced by UVB in surviving cells. A, cells were treated as in FIGURE 1A and ERK phosphorylation (p-ERK) was analyzed by Western blotting. B, cells were pretreated with the specific ERK activation inhibitor PD98059 (PD, 20 μM), the specific AKT activation inhibitor LY294002 (LY) (10 μM), or the combination of both, for 1 h, and then exposed to UVB (20 mJ/cm2). Cells were then treated with PD, LY, or both and collected at 24 h. The PTEN mRNA levels were determined by real-time PCR. *, p < 0.05, significant differences from UVB-exposed cells treated vehicle alone. C, same as in B except that the PTEN protein levels were determined by Western blotting. D, quantification of the PTEN levels in C. *, p < 0.05; **, p < 0.01; significant differences from UVB-exposed cells treated vehicle alone. E, same as in B except that cells were treated with both PD (20 μM) and LY (10 μM) at 24 h after UVB radiation for three days and the PTEN protein levels were determined by Western blotting. F, HaCaT cells were infected with constitutive active AKT adenovirus (A+) or adenovirus expressing GFP only as a control. Cells were collected at 48 or 72 h after infection. G, same as in F except that cells were collected at 48 h post infection for real-time PCR analysis of the PTEN mRNA levels.
Mentions: We have demonstrated that following UVB radiation PTEN is suppressed for at least a week (Figure 2D), and even longer than 4 weeks, and will partially recover by 6 weeks (data not shown). Considering the critical role of PTEN as a tumor suppressor in skin homeostasis, recovering PTEN expression may prove beneficial in preventing carcinogenesis, e.g., following sunburn. We examined the role of the ERK and AKT pathways to further elucidate the molecular mechanisms of PTEN suppression by UVB irradiation. UVB activated the ERK and AKT pathways (Figure 7A). While ERK was activated at all the time points examined, AKT was activated only at 3 and 24 h post radiation, possibly due to loss of AKT protein in apoptotic cells between 3 and 24 h (Figures 3C and 3D). PD98059 is an inhibitor of ERK activation, and LY2940002 is an inhibitor of AKT activation. When cells were kept in the dark or exposed to UVB and then treated with PD, LY, or both, the suppression of PTEN expression at the mRNA level was completely blocked (Figure 7B, P < 0.05). The levels of PTEN protein were also significantly increased by PD, LY, or both (P < 0.05 for PD or LY and 0.01 for PD/LY) (Figures 7C and 7D). Interestingly, LY or PD increased the levels of PTEN protein for three days even when added 24 h post UVB radiation (Figure 7E). This indicates that ERK/AKT pathway is critical in maintaining PTEN suppression in UVB-exposed keratinocytes. To further determine the effect of AKT activation alone on PTEN expression, we infected HaCaT cells with constitutively active AKT adenovirus. At 48 and 72 h after infection, the PTEN protein levels decreased (Figure 7F). This AKT activation suppressed the PTEN mRNA levels significantly at 48 h post infection (Figure 7G, P < 0.05). These results demonstrate that AKT activation negatively regulates the PTEN levels, forming a positive feedback loop in keratinocytes. Our findings suggest that UVB-induced PTEN suppression is reversible by blocking ERK/AKT pathways.

Bottom Line: As compared with normal skin, PTEN levels are reduced in human actinic keratosis, a precancerous skin lesion caused by solar UV.AKT activation is higher in UVB-irradiated surviving cells as compared with unirradiated cells.Our findings indicate that (1) UVB radiation suppresses PTEN expression in keratinocytes; and (2) the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation.

View Article: PubMed Central - PubMed

Affiliation: Section of Dermatology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Ultraviolet (UV) radiation in sunlight is the major environmental cause of skin cancer. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a proven critical tumor suppressor. We report here that UVB downregulates PTEN in primary human keratinocytes, human HaCaT keratinocytes and mouse skin. As compared with normal skin, PTEN levels are reduced in human actinic keratosis, a precancerous skin lesion caused by solar UV. PTEN downregulation is mediated by two mechanisms: (1) PTEN is cleaved by active caspase in apoptotic cells in which AKT activation is reduced; and (2) PTEN transcription is suppressed in surviving cells, and this suppression is independent of caspase activation and occurs in parallel with increased ERK and AKT activation. We report here that the combination of ERK and AKT activation is crucial for PTEN suppression in surviving cells following UVB irradiation. AKT activation is higher in UVB-irradiated surviving cells as compared with unirradiated cells. The ERK and AKT pathways are involved in sustaining PTEN suppression in UVB-exposed cells. Increasing PTEN expression enhances apoptosis of keratinocytes in response to UVB irradiation. Our findings indicate that (1) UVB radiation suppresses PTEN expression in keratinocytes; and (2) the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation. Our identification of PTEN as a critical molecular target of UVB provides new insights into the pathogenesis of skin cancer.

Show MeSH
Related in: MedlinePlus