In the present work, cell cycle progression in response to low dose UVA irradiation was investigated using the HaCaT human keratinocyte cell line. HaCaT cells are spontaneously immortalized through p53 mutation, a modification similar to that initiated in cells during skin carcinogenesis, thereby providing a valuable model system for the investigation of tumor promotion events associated with skin carcinogenesis (27
). Using this model, we have shown that a non-lethal dose of UVA irradiation induces cell cycle progression and cell proliferation and that this process is mediated via
cyclin D1. In this process the ADAM/EGFR/AKT pathway transduced the UVA signal to cause cyclin D1 up-regulation and cell proliferation, which might be equivalent to inducing tumor promotion in skin carcinogenesis.
AKT activation has been shown to be one of the key molecular determinants of keratinocyte transformation (28
). AKT has been shown to increase the up-regulation of cyclin D1 in AKT-transformed keratinocytes via both transcriptional and post-transcriptional processes (17
). In our study, we show that AKT activation is mediated by the EGFR pathway after UVA exposure. The accumulation of cyclin D1 by UVA exposure could be due to both transcriptional and post-transcriptional regulation by AKT activation. Knockdown of cyclin D1 expression reduced the G1 to the S transitions induced by UVA exposure, demonstrating that cyclin D1 plays an important role in this process. Regardless of the precise regulation mechanisms, it appears that for cyclin-D1-mediated cell cycle progression induced by UVA exposure, AKT activation is required. In addition to cyclin D1, other factors might be involved in UVA-induced cell cycle progression, because knockdown of cyclin D1 did not block cell cycle progression completely. Multiple mechanisms including the blockade of Chk1 by phosphorylation at Ser280 by EGFR/AKT activation (29
) may play important roles in UVA-induced cell proliferation. It is possible that AKT activation as a central signaling pathway mediates the proliferative effect of UVA through several downstream targets. We are currently in the process of identifying those targets to understand the complex mechanisms of UVA-induced cell proliferation and skin carcinogenesis.
The ERK/MAPK pathway is neither activated by low-dose UVA exposure nor involved in UVA-induced cell cycle progression although EGFR activation was observed in this study. We previously detected sustained and prolonged ERK/MAPK activation after apoptosis-inducing UVA but not low-dose UVA exposure (21
). It appears that ERK signaling is clearly UVA dose dependent, which is expected because the low dose of UVA used in this study did not induce cell death. High-dose UVA-induced ERK activation protected cells from cell death and allowed more cells to survive (21
The involvement of EGFR activation was first seen in the activation of EGFR after UVA irradiation and further confirmed by the inhibitory effect of the EGFR kinase inhibitor AG on cell cycle progression. We have previously shown that a lethally high dose (24 J/cm2
) did not induce EGFR phosphorylation (20
). EGFR activation clearly seems to be UVA dose-dependent and occurs only at a low UVA dose. The level of EGFR activation in this study did not cause EGFR down-regulation, as seen by the failure of the EGFR level to decrease after UVA exposure (data not shown). This moderate EGFR activation is sufficient to activate the downstream AKT/cyclin D1 signaling, and transduce a proliferative signal to induce cell cycle progression.
ADAM proteins are membrane-anchored metalloproteases that process and shed the ectodomains of membrane-anchored growth factors, cytokines, and receptors (23
). Tumor cells frequently produce autocrine growth factors, and are often highly proliferative, motile and invasive (30
). ADAMs can activate EGFR by shedding the EGFR ligand (31
). During EGFR transactivation, an upstream signal acts through a G-protein-coupled receptor to activate a metalloprotease to shed an EGF receptor ligand (24
). ADAM17-mediated transactivation of EGFR by amphiregulin (AR) appears to be important for tumor cell growth and migration (25
Recently it has been reported that environmental stress signaling, including oxidative and osmotic stress, is mediated by ADAM proteases and HB-EGF (heparin-binding epidermal growth factor) (32
). UVC exposure stimulated MAGF cleavage via the metalloprotease pathway (33
). In this work, we observed that UVA induces cell cycle progression and that ADAM protease is involved because the metalloprotease inhibitor GM6001 inhibits cell cycle progression after UVA exposure. Furthermore, siRNA knockdown of ADAM17 attenuated EGFR activation and the cell cycle progression induced by UVA, indicating that ADAM17 was involved in the signal transduction for UVA irradiation to the EGFR/AKT/cyclin D1 pathway and cell cycle progression. The incomplete inhibition of EGFR activation and G1-S transition by siRNA against ADAM17 compared to the metalloprotease inhibitor GM suggests that other mechanisms may contribute to UVA-induced EGFR activation. A recent study has shown that the oxidative inhibition of protein tyrosine phosphatase kappa by UV results in the activation of EGFR (34
). In addition, other ADAM members and/or metalloproteases including ADAM 10 may also be involved in UVA-induced EGFR activation and G1-S transition. The precise mechanisms of EGFR/AKT activation by ADAM17 and the potential role of other metalloproteases are under further investigation in our lab.
In summary, a non-lethal dose of UVA has been shown to induce cell cycle (the G1 to the S) progression of human HaCaT keratinocytes, mediated by increased cyclin D1. The ADAM/EGFR/AKT pathway is required for UVA-induced cyclin D1 up-regulation and cell cycle progression. Given the complexity and interactions of this signaling pathway, it is entirely possible that other pathways may also play a role in UVA-induced cell proliferation. This study provides compelling evidence that UVA alone, at low, non-lethal doses, has the potential to be a human skin tumor promoter. The acquisition of proliferation signaling in UVA-irradiated keratinocytes may be an important factor in the formation of premalignant skin lesions including actinic keratoses and malignant squamous cell carcinoma in the clinical setting. Identification of the fundamental mechanisms of the effect of UVA on tumor promotion will facilitate the development of safe and efficient chemopreventive and therapeutic strategies for skin cancer.