Abstract
We examined the effect of inhibiting p38 MAPK on UVA-irradiated HaCaT cells, a spontaneously immortalized human keratinocyte cell line. Recent work from our laboratory has shown that UVA (250 kJ/m2) induces a rapid phosphorylation of p38 MAPK in the HaCaT cell line. Inhibition of p38 MAPK activity through the use of a specific inhibitor, SB202190, in combination with UVA treatment induced a rapid cleavage of caspase-9, caspase-8, and caspase-3, whereas UVA irradiation alone had no effect. Similarly, cleavage of the caspase substrate poly(ADP-ribose) polymerase was observed in UVA-irradiated HaCaT cells treated with SB202190 or in cells expressing a dominant-negative p38 MAPK. No effect of p38 MAPK inhibition upon caspase cleavage was observed in mock-irradiated HaCaT cells. In addition, increases in apoptosis were observed in UVA-irradiated cells treated with SB202190 by morphological analysis with no significant apoptosis occurring from UVA irradiation alone. Similar results were obtained by using normal human epidermal keratinocytes. UVA induced expression of the anti-apoptotic Bcl-2 family member, Bcl-XL, with abrogation of expression by using the p38 MAPK inhibitor SB202190. Overexpression of Bcl-XL prevented poly(ADP-ribose) polymerase cleavage induced by the combination of UVA and p38 MAPK inhibition. UVA enhanced the stability of Bcl-XL mRNA through increases in p38 MAPK activity. We determined that increases in UVA-induced expression of Bcl-XL occur through a posttranscriptional mechanism mediated by the 3'-untranslated region (UTR). We used Bcl-XL 3'-UTR luciferase constructs to determine the mechanism by which UVA increased Bcl-XL mRNA stability. Additionally, RNA binding studies indicate that UVA increases the binding of RNA-binding proteins to Bcl-XL 3'-UTR mRNA, which can be decreased by using SB202190. In conclusion, p38 MAPK and Bcl-XL expression play critical roles in the survival of UVA-irradiated HaCaT cells.
Highlights
We examined the effect of inhibiting p38 mitogen-activated protein kinases (MAPKs) on UVAirradiated HaCaT cells, a spontaneously immortalized human keratinocyte cell line
Increases in apoptosis were observed in UVA-irradiated cells treated with SB202190 by morphological analysis with no significant apoptosis occurring from UVA irradiation alone
In the studies described here, we demonstrate that UVA-induced p38 MAPK activity plays a dramatic role in the survival of keratinocytes
Summary
We examined the effect of inhibiting p38 MAPK on UVAirradiated HaCaT cells, a spontaneously immortalized human keratinocyte cell line. Inhibition of p38 MAPK activity through the use of a specific inhibitor, SB202190, in combination with UVA treatment induced a rapid cleavage of caspase-9, caspase-8, and caspase-3, whereas UVA irradiation alone had no effect. No effect of p38 MAPK inhibition upon caspase cleavage was observed in mock-irradiated HaCaT cells. Induction of p38 MAPK activity has a variety of effects, including changes in transcription, protein synthesis, cell surface receptor expression, and cytoskeletal structure with the result leading to either cell survival or programmed cell death [10]. Our laboratory has shown that both UVA and UVB can activate p38 MAPK in the HaCaT cell line [11, 12]. The opposite effect has been reported in mouse erythroleukemia SKT6 cells exposed to osmotic shock, where early activation of p38 MAPK inhibits apoptosis and prolonged activation results in apoptosis [16]
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