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Cancer Research 68, 3752, May 15, 2008. doi: 10.1158/0008-5472.CAN-07-6138
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

UVA-Induced Cell Cycle Progression Is Mediated by a Disintegrin and Metalloprotease/Epidermal Growth Factor Receptor/AKT/Cyclin D1 Pathways in Keratinocytes

Yu-Ying He1,2, Sarah E. Council1, Li Feng1 and Colin F. Chignell1

1 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina and 2 Section of Dermatology, Department of Medicine, University of Chicago, Chicago, Illinois

Requests for reprints: Yu-Ying He, Section of Dermatology, Department of Medicine, University of Chicago, Chicago, IL 60637. Phone: 773-795-4696; Fax: 773-702-8398; E-mail: yyhe{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
UVA (315–400 nm), which constitutes ~95% of the UV irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here, we show that a low, nonlethal dose of UVA induces dose-dependent cell cycle progression in human HaCaT keratinocytes. We found that UVA induced cyclin D1 accumulation, whereas siRNA knockdown of cyclin D1 blocked the UVA-induced cell cycle progression, indicating that this process is mediated by cyclin D1. UVA irradiation also induced AKT activation; when cells were incubated with phosphatidylinositol-3-OH kinase/AKT inhibitor or infected with dominant-negative AKT, cyclin D1 up-regulation, cell cycle progression, and proliferation were inhibited, suggesting that AKT activation is required for UVA-induced cell cycle progression. In contrast, extracellular signal-regulated kinase (ERK) was not activated by UVA exposure; incubation with ERK/mitogen-activated protein kinase inhibitor had no effect on UVA-induced cyclin D1 up-regulation and cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVA exposure. EGFR kinase inhibitor AG attenuated the UVA-induced AKT/cyclin D1 pathway and cell cycle progression, indicating that EGFR is upstream of AKT/cyclin D1 pathway activation. Furthermore, metalloprotease inhibitor GM6001 blocked UVA-induced cell cycle progression, and siRNA knockdown of a disintegrin and metalloprotease (ADAM)17 had a similar inhibitory effect, demonstrating that ADAM17 mediates the EGFR/AKT/cyclin D1 pathway and cell cycle progression to the S phase induced by UVA radiation. Identification of these signaling pathways in UVA-induced cell proliferation will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer. [Cancer Res 2008;68(10):3752–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
UV radiation in sunlight is clearly an important environmental factor in human skin carcinogenesis. Each year, approximately one million new cases of skin cancer are diagnosed in the United States alone, making it the most common type of cancer in this country. In animal models, UV radiation is a complete carcinogen that can both initiate and promote skin carcinogenesis, resulting in squamous cell carcinoma (SCC), basal cell carcinoma, and melanoma (1–3).

UV radiation in sunlight is composed of UVB (280–315 nm) and UVA (315–400 nm). UVA has been considered far less carcinogenic based on limited direct damage to DNA (4). However, UVA is ~20-fold more abundant than UVB in sunlight and much more UVA penetrates the epidermis and reaches the basal germinative layers (5). Recently, UVA was shown to induce p53 mutations in the basal layer of the skin, and UVA signature mutations in p53 have been detected in SCC and solar keratosis (6). Although the mechanisms may differ from those involved in carcinogenesis by UVB (7), these findings confirmed that UVA can initiate tumorigenesis in vivo. Therefore, it is possible that UVA exposure may play a greater role in the development of human skin cancers than is generally assumed. The initiation stage is a rapid process, potentially taking place within a single exposure, whereas promotion may take years to occur and is a reversible process. Therefore chemoprevention targeting signaling pathways in the promotion stage may provide higher success and feasibility (8, 9).

Cell proliferation as a consequence of cell cycle progression is the key process that leads to clonal expansion of initiated cells during tumor promotion. Cyclin D1 is a cell cycle regulatory protein that acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, particularly during the G1 phase of the cell cycle (10). Increased cyclin D1 has been associated with mouse skin transformation (11–14). However, the mechanisms of cell cycle progression in UVA-induced tumor promotion and the role of cyclin D1 remain open questions. Understanding the signal transduction pathways by which the UVA signals to cyclin D1 and identifying the critical elements in these pathways will provide targets for chemoprevention.

The epidermal growth factor receptor (EGFR), one of the receptor tyrosine kinases (RTK), plays a pivotal role in regulating cell proliferation, differentiation, and transformation. Binding of growth factors to RTKs promotes receptor dimerization and subsequent activation, which enhances autophosphorylation of RTKs, phosphorylation of numerous cellular proteins, and recruitment of adaptor molecules, thus initiating signaling cascades, including the phosphatidylinositol-3-OH kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) pathways (15, 16). Activation of the PI 3-kinase/AKT and/or ERK pathways can lead to cell proliferation and growth by regulating the cyclin D1 level (17, 18).

To identify the mechanisms in UVA irradiation–induced cell proliferation, we have studied the mechanisms in cell cycle progression induced by low, nonlethal doses of UVA in HaCaT cells, a human keratinocyte line. In this study, cell cycle progression and cell proliferation were observed after UVA exposure. Cyclin D1 was found to mediate this process, and siRNA knockdown of cyclin D1 was shown to inhibit cell cycle progression. The a disintegrin and metalloprotease (ADAM)/EGFR/AKT pathways transduced UVA signaling to cyclin D1 and induced a proliferative response in human keratinocytes. These data clearly show the mechanisms and the tumor promotional potential of UVA in human skin cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture and UVA treatment. HaCaT cells were cultured in 60-mm dishes with normal culture medium. The cell culture medium was replaced with 0.1% fetal bovine serum (FBS) DMEM and cultured for 24 to 36 h. Cells were exposed to UVA as described previously (19–21). After treatment, fresh medium containing 0.1% FBS was added and the cells were incubated at 37°C.

Cell cycle analysis. At predetermined time points after treatment, the cells were harvested and fixed with 5 mL of ice cold 70% ethanol. For cell cycle analysis, the fixed cells were then centrifuged (1,000 rpm; 5 min) and washed with PBS. Then, the cells were incubated with propidium iodide (20 µg/mL) containing RNase A (1 mg/mL; Sigma Chemical). The DNA content was determined by flow cytometry (Beckman Coulter) and ModFitLT software (Verity Software House, Inc.).

Adenovirus infection. The next day after the cells were seeded, empty-vector (a kind gift from Christopher Kontos, Duke University Medical Center, Durham, NC) or dominant-negative AKT (a kind gift from Kenneth Walsh, Boston University, Boston, MA) adenovirus vectors were added to the cells in a multiplicity of infection of 50. After 14 h of infection by incubation with the virus, cells were washed and fed with fresh medium. Twenty-four hours after the initiation of infection, cells were irradiated with UVA.

siRNA transfection. Cells at ~90% confluency were trypsinized and electroporated with siRNA against ADAM17 or cyclin D1 siRNA (Dharmacon) or cy3-labeled control siRNA (Ambion) with Nucleofector (Amaxa) according to the manufacturer's instructions. Briefly, one million HaCaT cells were electroporated in 100 µL Nucleofector Solution V containing 1.5 µg siRNA using program U-20. After transfection, cells were seeded in 2 mL prewarmed medium in a MatTek dish for confocal microscopy or 60-mm dishes for cell cycle analysis or Western blotting.

Western blotting. Western blotting was performed as described previously (19–21). Antibodies used were as follows: cyclin D1 (Cell Signal) and p-AKT473 (Cell Signal); p-ERK (Santa Cruz); p-EGFR (BD Biosciences); and ADAM17 (Calbiochem), AKT (Santa Cruz), ERK (Santa Cruz), ERFR (Neomarker), and β-actin (Sigma).

Confocal microscopy. Cells were seeded into 35-mm dishes containing a glass coverslip–covered 15-mm cutout (MatTek) for live cell microscopy measurements. The next day, cells were transfected with cy3-labeled negative siRNA as described above. Fluorescence in cells was monitored using a Zeiss 510Meta confocal microscope. For confocal microscopy, excitation at 543 nm and emission at 560 to 615 nm were used for the cy3 dye.

Statistical analyses. Data were expressed as the mean of three independent experiments and were analyzed by Student's t test. A two-sided P value of <0.05 was considered significant in all cases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Cell cycle progression and EGFR/AKT/cyclin D1 signaling induced by UVA in human HaCaT keratinocytes. To study cell cycle progression induced by low-dose UVA exposure, human HaCaT keratinocytes were exposed to different doses of UVA from 1 to 4 J/cm2. Eighteen hours after exposure, cells were harvested for cell cycle analysis. ModFit was used to quantify the cell population in the S phase (Fig. 1A ). As shown in Fig. 1B, 1 J/cm2 UVA exposure increased cell accumulation in the S phase, whereas 2.5 and 4 J/cm2 induced significantly (P < 0.05) higher proportions of cells in the S phase (Fig. 1B). It is noteworthy that at these doses, no cell death was detected (data not shown). These data suggest that UVA-induced cell cycle progression is UVA dose dependent. When later time points were examined, including 24 and 48 h after UVA exposure, the proportion of cells in the S phase decreased at each UVA dose compared with the proportion at 18 h after UVA exposure (Fig. 1B). At 48 h after UVA exposure, most of the cells went back to the G0-G1 phase. At this time point, no significant increase in the G2-M phase was observed (data not shown). This suggests that the cells were able to finish the cell cycle and go back to the G1 phase after UVA-induced progression from G1-S phase.


Figure 1
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  		Figure 1. UVA exposure induced cell cycle progression in human HaCaT cells. HaCaT cells were seeded in 60-mm dishes, starved with 0.1% FBS DMEM for 24 to 36 h, and then exposed to UVA at the doses indicated. A, cells were exposed to 1, 2.5, or 4 J/cm2 or kept in the dark. Eighteen hours after UVA exposure, cells were harvested and prepared for cell cycle analysis by flow cytometry as described in the Materials and Methods. Left arrow, G1 phase; right arrow, G2 phase; middle arrow, S phase. The histograms of dark and UVA (4 J/cm2) were shown. B, cells were exposed to UVA as in A and harvested at 18, 24, and 48 h after the exposure. Data and are expressed as percentages. Columns, means (n = 3); bars, SD. *, significant differences from control cells (P < 0.05). C, HaCaT cells were cultured in 60-mm dishes and starved for 24 to 36 h. Cells were exposed to UVA as in A. Three hours after UVA exposure, cells were harvested for Western blot analysis using specific antibodies against cyclin D1, p-AKT473, AKT, p-ERK, ERK, p-EGFR, EGFR, and β-actin (Actin, an equal loading control). D, cells were exposed to UVA (4 J/cm2) and harvested at 3, 6, and 9 h after the exposure for Western blot analysis as in C.

 
To delineate the mechanisms and pathways involved in UVA-induced cell cycle progression from the G1-S, we exposed cells to UVA at different doses, harvested them at different times, and analyzed protein samples by Western blot. Cyclin D1 expression, AKT phosphorylation, ERK phosphorylation, and EGFR phosphorylation were determined at different time points after UVA exposure or kept in the dark.

As shown in Fig. 1C, UVA exposure (1, 2.5, or 4 J/cm2) increased cyclin D1 accumulation and AKT phosphorylation at 3 hours after the exposure. In contrast, ERK activation remained unchanged after UVA exposure compared with ERK phosphorylation in the dark. These data suggest that low-dose UVA induced cyclin D1 expression and activated AKT but had no effect on the ERK/ mitogen-activated protein kinase (MAPK) pathway.

To further identify the upstream signaling, we determined the level of EGFR phosphorylation with and without UVA exposure. As shown in Fig. 1C, EGFR phosphorylation in the cells kept in the dark was low, whereas UVA exposure increased the phosphorylation of EGFR in a dose-dependent manner with 2.2-fold of EGFR phosphorylation at UVA (4 J/cm2) as much as that of dark samples. These data indicated that EGFR is activated by a low dose of UVA.

To determine whether the activation of AKT and EGFR and the increase in cyclin D1 level are time dependent, cells were exposed to UVA (4 J/cm2) or kept in the dark. At 3, 6, or 9 hours after exposure, cells were harvested for Western blot analysis. The activation of AKT and EGFR and the increase in cyclin D1 were detected at 3, 6, and 9 hours after UVA exposure to a similar extent (Fig. 1D). These findings indicate that AKT and EGFR activation and cyclin D1 accumulation upon UVA radiation are sustained.

Role of cyclin D1 in UVA-induced cell cycle progression. Cyclin D1 plays a key role in cell cycle progression, especially the G1-S progression (10). To determine whether the cyclin D1 accumulation observed in Fig. 1 is responsible for cell cycle progression, HaCaT cells were transfected with siRNA against cyclin D1 or negative control siRNA and then exposed to UVA or kept in the dark.

Although keratinocytes, including HaCaT cells, are difficult to transfect, an ~95% transfection efficiency of siRNA was achieved by electroporation as described in the Materials and Methods (data not shown). When HaCaT cells were transfected with siRNA against cyclin D1, a knockdown of ~75% was observed (Fig. 2A ), whereas transfection alone or with negative siRNA had no effect on cyclin D1 expression. A similar knockdown was seen with cells exposed to UVA (Fig. 2A, right).


Figure 2
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  		Figure 2. Knockdown of cyclin D1 by siRNA inhibited cell cycle progression induced by UVA exposure. A, HaCaT cells were transfected with siRNA against cyclin D1 (si-CD1) and cyclin D1 levels were detected by Western blotting. Mock, no electrophoration; -siRNA, cells were transfected without siRNA; neg, cells were transfected with negative control siRNA. Cells transfected with negative control siRNA were kept in the dark or exposed to UVA irradiation. B, after transfection, cells were also exposed to UVA (2.5 J/cm2) or kept in the dark. Eighteen hours after exposure, cells were collected for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD. *, significant differences from the cells treated with UVA and negative control siRNA (P < 0.05).

 
As shown in Fig. 2B, UVA irradiation at 2.5 J/cm2 induced 19% of the cells in the S phase, similar to cells without transfection (Fig. 1). In comparison, cells transfected with siRNA against cyclin D1 showed only a 12% cell proportion in the S phase after the same dose of UVA exposure (Fig. 2B), indicating that cyclin D1 up-regulation plays an important role in cell cycle progression from the G1-S phase.

Role of AKT activation in UVA-induced cell cycle progression. To determine whether AKT activation is involved in the cell cycle progression induced by UVA exposure, cells were incubated with the PI3K inhibitor LY-294002 (LY; 10 µmol/L) to block AKT activation. The presence of LY inhibited UVA-induced AKT phosphorylation and cyclin D1 accumulation (Fig. 3A ), indicating that AKT activation mediates cyclin D1 up-regulation. Compared with cells incubated with vehicle alone, the proportion of cells in the S phase decreased significantly in cells treated with LY after UVA exposure (Fig. 3B). This suggests that the AKT pathway is an essential element in UVA-induced cyclin D1 up-regulation and cell cycle progression.


Figure 3
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  		Figure 3. Blocking AKT signaling reduced cyclin D1 expression and cell cycle progression induced by UVA irradiation. A, cells were incubated with or without PI3K inhibitor to block AKT activation and then exposed to UVA radiation. Three hours after UVA exposure, cells were collected for Western blotting analysis using antibodies against p-AKT473 and cyclin D1. B, cells were treated as in A except that cells were harvested 18 h after UVA exposure for cell cycle analysis. Percentage of cells in the S phase was quantified as in Fig. 1. Data are expressed as percentages. Columns, mean (n = 3); bars, SD; *, significant differences from cells treated with UVA alone (P < 0.05). C, cells were infected with EV or A– and the AKT expression level was assessed by Western blotting. Cells were infected with EV or A– as in C and then exposed to UVA. Three hours after UVA exposure, cells were collected for Western blotting using antibodies against p-AKT473, cyclin D1, and Actin as an equal loading control. D, cells were infected with EV or A– as in C and then exposed to UVA. Eighteen hours after UVA exposure, cells were collected for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD. *, significant differences from cells treated with UVA and EV (UVA; P < 0.05).

 
We also determined cell cycle progression and cell proportion in the S phase when cells were infected with empty vector (EV) or dominant-negative AKT (A–) adenovirus (22). The dominant-negative mutant adenoviral AKT (A–) expressing the mutant AKT (T308A, S473A) construct cannot be activated by phosphorylation. Infection with A– causes overexpression of mutant AKT, which competes with endogenous wild-type AKT for activation and therefore inhibits AKT activation. As shown in Fig. 3C, infection with A–increased the AKT level, confirming the expression of viral mutant protein in the cells. Overexpression of dominant AKT inhibited UVA-induced AKT activation (Fig. 3C). Compared with cells infected with EV, infection with A– significantly (P < 0.05) reduced the cyclin D1 level and the proportion of cells in the S phase after UVA exposure (Fig. 3D). These data indicate that UVA-induced cyclin D1 accumulation and cell cycle progression require AKT activation.

Role of ERK signaling in UVA-induced cell cycle progression. To determine whether ERK signaling is involved in UVA-induced cell cycle progression, cells were incubated with the ERK inhibitor PD98059 (PD; 20 µmol/L) after UVA exposure. Pretreatment with PD blocked ERK phosphorylation, although it had no effect on cyclin D1 accumulation induced by UVA exposure (Fig. 4A ). As shown in Fig. 4B, the presence of PD had no effect on UVA-induced cyclin D1 up-regulation and cell cycle progression, implying that ERK signaling is not involved in this process.


Figure 4
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  		Figure 4. Inhibition of ERK pathway had no effect on the cyclin D1 level and cell cycle progression induced by UVA exposure. A, cells were incubated with or without PD (20 µmol/L), an ERK activation inhibitor, and then exposed to UVA radiation. Three hours after UVA exposure, cells were collected for Western blotting using antibodies against p-ERK and cyclin D1. B, cells were treated as in A except that cells were harvested for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD.

 
Role of EGFR activation in UVA-induced cell cycle progression. To determine whether EGFR activation is required for the UVA-induced AKT/cyclin D1 pathway and cell cycle progression, cells were incubated with the EGFR inhibitor AG1478 (AG; 1 µmol/L). The presence of AG blocked EGFR phosphorylation, AKT activation, and basal ERK phosphorylation (Fig. 5A ). As compared with cells incubated with vehicle alone, AG significantly (P < 0.05) reduced the cell proportion in the S phase (Fig. 5B). EGF (10 ng/mL), an EGFR ligand, induced 30% of cells in the S phase compared with 8% in control cells and increased EGFR phosphorylation, AKT phosphorylation, and cyclin D1 accumulation (data not shown). These findings indicate that EGFR activation is required for the activation of the AKT/cyclin D1 pathway and cell cycle progression from the G1-S phase induced by UVA radiation.


Figure 5
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  		Figure 5. Inhibition of EGFR pathway blocked AKT/cyclin D1 pathway and cell cycle progression induced by UVA exposure. A, cells were incubated with or without AG (1 µmol/L), an EGFR kinase inhibitor, and then exposed to UVA radiation. Three hours after UVA exposure, cells were collected for Western blotting using antibodies against p-EGFR, EGFR, p-AKT473, AKT, p-ERK, and ERK. B, cells were treated as in A and harvested 18 h after UVA exposure for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD. *, significant differences from cells treated with UVA alone (P < 0.05).

 
Role of ADAM17 in UVA-induced cell cycle progression. We have found that EGFR is activated by UVA exposure at a lower nonlethal dose. However, the mechanism of EGFR activation is unknown. The members of (ADAM) family transactivate EGFR in vivo and in vitro (23). ADAM17, a member of the ADAM family, plays an essential role in activating EGFR pathways compared with other members of the ADAM family (23–26). To determine whether metalloproteases are involved in the EGFR activation and cell cycle progression induced by low-dose UVA irradiation, we incubated cells with the metalloprotease inhibitor GM6001 (GM; 25 µmol/L) and exposed them to UVA or kept them in the dark. Pretreatment with GM inhibited EGFR phosphorylation induced by UVA exposure and reduced the basal EGFR phosphorylation level (Fig. 6A ). As compared with cells incubated without GM, the presence of GM reduced the proportion of cells in the S phase significantly (P < 0.05) after UVA exposure (Fig. 6B).


Figure 6
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  		Figure 6. ADAM17 mediated UVA-induced EGFR activation and cell cycle progression. A, cells were incubated with or without metalloprotease inhibitor GM (25 µmol/L) and then exposed to UVA irradiation. Three hours after exposure, cells were collected for Western blotting using antibodies against p-EGFR and EGFR. B, cells were treated as in A except that cells were harvested 18 h after UVA exposure for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD. *, significant differences from cells exposed to UVA in the absence of GM (P < 0.05). C, cells were transfected as in Fig. 2 except that siRNA against ADAM17 was used, and the ADAM17 expression level was assessed by Western blotting using a specific antibody against ADAM17. Cells were transfected as in C and then exposed to UVA irradiation (2.5 J/cm2). Three hours after exposure, cells were collected for Western blotting using antibodies against p-EGFR and EGFR. D, cells were treated as in C except that cells were harvested 18 h after UVA exposure for cell cycle analysis. Data are expressed as percentages. Columns, mean (n = 3); bars, SD. *, significant differences from cells exposed to UVA after transfection with negative control siRNA (P < 0.05).

 
To determine the specific involvement of ADAM17 in the observed cell cycle progression, we used siRNA against ADAM17. Transfection by siRNA reduced ADAM17 expression by ~90% (Fig. 6C) and reduced EGFR phosphorylation induced by UVA exposure (Fig. 6C). UVA exposure caused an increase in S-phase cell proportion when cells were transfected with control/negative siRNA (Fig. 6D), similar to the result without transfection (Fig. 1). As compared with negative siRNA, siRNA against ADAM17 attenuated the cell proportion in the S phase significantly (P < 0.05; Fig. 6D). These data show that ADAM17 mediates EGFR activation and the cell cycle progression induced by UVA exposure.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
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 nonlethal 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 posttranscriptional 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 posttranscriptional regulation by AKT activation. Knockdown of cyclin D1 expression reduced the G1-S transitions induced by UVA exposure, demonstrating that cyclin D1 plays an important role in this process. Regardless of the precise regulation mechanisms, it seems 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 seems 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 EGFR ligand (24). ADAM17-mediated transactivation of EGFR by amphiregulin seems 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 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 GM 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 with 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 nonlethal dose of UVA has been shown to induce cell cycle (the G1-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, nonlethal 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 SCC 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.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, and the American Skin Association.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors thank Dr. Ann Motten and Carol Trempus for critical reading of the manuscript.


   Footnotes
 
Note: Current address for S.E. Council: Oral Biology Program, University of North Carolina School of Dentistry, Chapel Hill, North Carolina.

Received 11/ 7/07. Revised 2/20/08. Accepted 2/20/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. de Gruijl FR, Sterenborg HJ, Forbes PD, et al. Wavelength dependence of skin cancer induction by ultraviolet irradiation of albino hairless mice. Cancer Res 1993;53:53–60.[Abstract/Free Full Text]
  2. Noonan FP, Recio JA, Takayama H, et al. Neonatal sunburn and melanoma in mice. Nature 2001;413:271–2.[CrossRef][Medline]
  3. Setlow RB, Grist E, Thompson K, Woodhead AD. Wavelengths effective in induction of malignant melanoma. Proc Natl Acad Sci U S A 1993;90:6666–70.[Abstract/Free Full Text]
  4. Setlow RB. The wavelengths in sunlight effective in producing skin cancer: a theoretical analysis. Proc Natl Acad Sci U S A 1974;71:3363–6.[Abstract/Free Full Text]
  5. Bruls WA, Slaper H, van der Leun JC, Berrens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 1984;40:485–94.[Medline]
  6. Agar NS, Halliday GM, Barnetson RS, Ananthaswamy HN, Wheeler M, Jones AM. The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc Natl Acad Sci U S A 2004;101:4954–9.[Abstract/Free Full Text]
  7. Dahle J, Kvam E. Induction of delayed mutations and chromosomal instability in fibroblasts after UVA-, UVB-, and X-radiation. Cancer Res 2003;63:1464–9.[Abstract/Free Full Text]
  8. Bowden GT. Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling. Nat Rev Cancer 2004;4:23–35.[CrossRef][Medline]
  9. Bode AM, Dong Z. Mitogen-activated protein kinase activation in UV-induced signal transduction. Sci STKE 2003;2003:RE2.
  10. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 1993;7:812–21.[Abstract/Free Full Text]
  11. Robles AI, Conti CJ. Early overexpression of cyclin D1 protein in mouse skin carcinogenesis. Carcinogenesis 1995;16:781–6.[Abstract/Free Full Text]
  12. Zhang SY, Liu SC, Goodrow T, Morris R, Klein-Szanto AJ. Increased expression of G1 cyclins and cyclin-dependent kinases during tumor progression of chemically induced mouse skin neoplasms. Mol Carcinog 1997;18:142–52.[CrossRef][Medline]
  13. Rodriguez-Puebla ML, LaCava M, Gimenez-Conti IB, Johnson DG, Conti CJ. Deregulated expression of cell-cycle proteins during premalignant progression in SENCAR mouse skin. Oncogene 1998;17:2251–8.[CrossRef][Medline]
  14. Rodriguez-Puebla ML, Robles AI, Conti CJ. ras activity and cyclin D1 expression: an essential mechanism of mouse skin tumor development. Mol Carcinog 1999;24:1–6.[CrossRef][Medline]
  15. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 1994;4:82–9.[CrossRef][Medline]
  16. Stephens LR, Jackson TR, Hawkins PT. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta 1993;1179:27–75.[Medline]
  17. Segrelles C, Moral M, Lara MF, et al. Molecular determinants of Akt-induced keratinocyte transformation. Oncogene 2006;25:1174–85.[CrossRef][Medline]
  18. Mirza AM, Kohn AD, Roth RA, McMahon M. Oncogenic transformation of cells by a conditionally active form of the protein kinase Akt/PKB. Cell Growth Differ 2000;11:279–92.[Abstract/Free Full Text]
  19. He YY, Huang JL, Ramirez DC, Chignell CF. Role of reduced glutathione efflux in apoptosis of immortalized human keratinocytes induced by UVA. J Biol Chem 2003;278:8058–64.[Abstract/Free Full Text]
  20. He YY, Huang JL, Gentry JB, Chignell CF. Epidermal growth factor receptor down-regulation induced by UVA in human keratinocytes does not require the receptor kinase activity. J Biol Chem 2003;278:42457–65.[Abstract/Free Full Text]
  21. He YY, Huang JL, Chignell CF. Delayed and sustained activation of extracellular signal-regulated kinase in human keratinocytes by UVA: implications in carcinogenesis. J Biol Chem 2004;279:53867–74.[Abstract/Free Full Text]
  22. Fujio Y, Guo K, Mano T, Mitsuuchi Y, Testa JR, Walsh K. Cell cycle withdrawal promotes myogenic induction of Akt, a positive modulator of myocyte survival. Mol Cell Biol 1999;19:5073–82.[Abstract/Free Full Text]
  23. Blobel CP. ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005;6:32–43.[CrossRef][Medline]
  24. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999;402:884–8.[Medline]
  25. Gschwind A, Hart S, Fischer OM, Ullrich A. TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. EMBO J 2003;22:2411–21.[CrossRef][Medline]
  26. Gschwind A, Prenzel N, Ullrich A. Lysophosphatidic acid-induced squamous cell carcinoma cell proliferation and motility involves epidermal growth factor receptor signal transactivation. Cancer Res 2002;62:6329–36.[Abstract/Free Full Text]
  27. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988;106:761–71.[Abstract/Free Full Text]
  28. Segrelles C, Ruiz S, Perez P, et al. Functional roles of Akt signaling in mouse skin tumorigenesis. Oncogene 2002;21:53–64.[CrossRef][Medline]
  29. Jean C, Hernandez-Pigeon H, Blanc A, Charveron M, Laurent G. Epidermal growth factor receptor pathway mitigates UVA-induced G2-M arrest in keratinocyte cells. J Invest Dermatol 2007;127:2418–24.[CrossRef][Medline]
  30. Seals DF, Courtneidge SA. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 2003;17:7–30.[Free Full Text]
  31. White JM. ADAMs: modulators of cell-cell and cell-matrix interactions. Curr Opin Cell Biol 2003;15:598–606.[CrossRef][Medline]
  32. Fischer OM, Hart S, Gschwind A, Prenzel N, Ullrich A. Oxidative and osmotic stress signaling in tumor cells is mediated by ADAM proteases and heparin-binding epidermal growth factor. Mol Cell Biol 2004;24:5172–83.[Abstract/Free Full Text]
  33. Montero JC, Yuste L, Diaz-Rodriguez E, Esparis-Ogando A, Pandiella A. Mitogen-activated protein kinase-dependent and -independent routes control shedding of transmembrane growth factors through multiple secretases. Biochem J 2002;363:211–21.[CrossRef][Medline]
  34. Xu Y, Shao Y, Voorhees JJ, Fisher GJ. Oxidative inhibition of receptor-type protein-tyrosine phosphatase {kappa} by ultraviolet irradiation activates epidermal growth factor receptor in human keratinocytes. J Biol Chem 2006;281:27389–97.[Abstract/Free Full Text]




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