Abstract
Exposure to the sun's UV radiation appears to be the most important environmental factor involved in the development of skin cancer. UVA is the major portion of UV radiation in sunlight and is considered to be a human carcinogen. In this study, we have investigated the delayed and sustained activation of ERK MAPK by UVA exposure. In parallel, a delayed Ras activation with a similar time course was observed after UVA exposure. The activated Ras was found to be localized in endomembranes such as the Golgi apparatus instead of plasma membranes. Expression of dominant negative Ras (N17Ras) abolished ERK activation by UVA. The presence of AG1478, an epidermal growth factor (EGF) receptor (EGFR) kinase inhibitor, had no effect on ERK or Ras activation, indicating that EGFR kinase activity is not involved in ERK activation by UVA. In contrast, protein kinase C (PKC) depletion by chronic 12-O-tetradecanoylphorbol-13-acetate treatment nearly abolished UVA-induced ERK and Ras activation. The presence of the Ca(2+)-dependent-PKC inhibitor Go6976 had a similar effect. These findings suggest that ERK activation by UVA is mediated by PKC in a Ras-dependent pathway. In addition, a gradual increase in intracellular calcium level after UVA exposure was detected by flow cytometry. The presence of the PLC inhibitor U73122 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) blocked both ERK and Ras activation, suggesting that both PLC and calcium are required for ERK activation. Our findings demonstrated that, different from UVC and UVB, UVA-induced delayed and sustained ERK activation is EGFR kinase activity-independent, but PLC/calcium/PKC-mediated. The delayed and sustained ERK activation provides a survival signal to human HaCaT keratinocytes, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure.
Highlights
From the Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
The delayed and sustained extracellular signal-regulated kinase (ERK) activation provides a survival signal to human HaCaT keratinocytes, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure
We found that ERK activation by UVA was mediated by protein kinase C (PKC) in a Ras-dependent pathway that required phospholipase C (PLC) and calcium but not the epidermal growth factor (EGF) receptor kinase activity
Summary
Membrane translocation of the Grb2SOS complex stimulates Ras-GDP to -GTP exchange, in turn calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis (acetoxymethyl ester)); EGF, epidermal growth factor; EGFR, EGF receptor; EGFR-KI, EGFR-kinase inactive; ERK, extracellular signal-regulated kinase; Go6976, calcium-dependent PKC inhibitor; MAPK, mitogen-activated protein kinase; PD98059, MEK1 inhibitor; PKC, protein kinase C; PLC, phospholipase C; RTKs, receptor tyrosine kinases; TPA, 12-O-tetradecanoylphorbol-13-acetate; U73122, PLC inhibitor; RBD, Ras-binding domain; UVB, ultraviolet B (280 –315 nm); UVC, ultraviolet C (200 –280 nm); MEK, MAPK/ERK kinase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; GFP, green fluorescent protein; ER, endoplasmic reticulum. The delayed and sustained ERK activation provides a survival signal allowing human HaCaT keratinocytes to escape from apoptosis
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