Abstract
BackgroundMutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. Information on the roles of MEK2 in these effects is still limited. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation.ResultsWe found that both constitutive MEK2 and MEK1 are able to phosphorylate the extracellular signal-regulated kinase 1 (ERK1) protein. Transient expression of constitutive MEK2 and MEK1 in the zebrafish epidermis induced papillary formation at 48 h post-fertilization, but no effects were observed due to the expression of MEK1, MEK2, or the dominant negative form of MEK2. The transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), developed skin papillomas in the epidermis within 6 days post-fertilization (dpf). The phospho-ERK signal was detected in section of skin papillomas in an immunohistochemical experiment. Treatment with 50 μM of the MEK inhibitor, U0126, had significantly decreased the skin papilloma formation in Tg(krt14:MEK2S219D-GFP) zebrafish by 6 dpf. In vitro and in vivo proliferation assay in COS-1 cells and in Tg(krt14:MEK2S219D-GFP) transgenic fish show significantly increased cell number and Ki-67 signaling.ConclusionOur data indicate that MEK2 is sufficient to induce epidermal papilloma formation through MAPK signaling in zebrafish, and this transgenic model can be used as a new platform for drug screening.
Highlights
Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described
The phosphorylated site of MEK1 was conserved (Fig. 1b, arrow). These data suggest that zebrafish MEK1 and MEK2 are highly conserved with mammalian MEK1 and MEK2
U0126 inhibits skin papilloma formation induced by MEK2S219D We further examined whether skin papilloma formation in Tg(krt14:MEK2S219D-green fluorescence protein (GFP)) zebrafish was induced by MEK2 activation by specific MEK1/2 inhibitor, U0126
Summary
Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation. The Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is the downstream pathway of Ras which transforms its effects on its targets [15]. When Ras is activated by a mutation, it was hypothesized that downstream components will be constitutively activated and corresponding abnormal gene expressions will occur that drive proliferation. No squamous skin cell tumor model has been established in zebrafish [9, 10, 16, 25, 29].
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