Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish.

BACKGROUND: Mutations 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. RESULTS: We 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. CONCLUSION: Our 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.

Impact of flavonoids on matrix metalloproteinase secretion and invadopodia formation in highly invasive A431-III cancer cells.

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.

Phosphorylation of the zebrafish M6Ab at serine 263 contributes to filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.

BACKGROUND: Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth. CONCLUSIONS/SIGNIFICANCE: Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.

Zona pellucida domain-containing protein ß-tectorin is crucial for zebrafish proper inner ear development.

BACKGROUND: The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian ß-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. ß-Tectorin is specifically expressed in the mammalian and avian inner ear. METHODOLOGY/PRINCIPAL FINDINGS: We identified and cloned the gene encoding zebrafish ß-tectorin. Through whole-mount in situ hybridization, we demonstrated that ß-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish ß-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of ß-tectorin morphants were abnormal since the development of the inner ear was compromised. CONCLUSIONS/SIGNIFICANCE: Our results reveal that zebrafish ß-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish ß-tectorin caused severe defects in inner ear formation of otoliths and function.

Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt.

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.

The zebrafish erythropoietin: functional identification and biochemical characterization.

In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.