The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors.

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Oncogenic Ras mutant causes the hyperactivation of NF-κB via acceleration of its transcriptional activation.

It is well established that nuclear factor κB (NF-κB) acts as one of the most important transcription factors for tumor initiation and progression, as it both protects cells from apoptotic/necrotic signals and accelerates angiogenesis and tumor metastasis, which is mediated via the expression of target genes. However, it has not yet been clarified how oncogenic signals accelerate the activation of NF-κB. In the current study, we utilized untransformed NIH-3T3 cells stably harboring a κB-driven luciferase gene to show that an oncogenic mutant of Ras GTPase augmented TNFα-induced NF-κB activation. Notably, enforced expression of cyclin-dependent kinase inhibitors, such as p27Kip1 and p21Cip1 , effectively canceled the accelerated activation of NF-κB, suggesting that oncogenic Ras-induced cell cycle progression is essential for the hyperactivation of NF-κB. Furthermore, we found that Ras (G12V) augmented the transcriptional activation of NF-κB, and this activation required the p38 MAP kinase. We observed that a downstream kinase of p38 MAP kinase, MSK1, was activated by Ras (G12V) and catalyzed the phosphorylation of p65/RelA at Ser-276, which is critical for its transcriptional activation. Significantly, phosphorylation of the p65/RelA subunit at Ser-276 was elevated in patient samples of colorectal cancer harboring oncogenic mutations of the K-Ras gene, and the expression levels of NF-κB target genes were drastically enhanced in several cancer tissues. These observations strongly suggest that oncogenic signal-induced acceleration of NF-κB activation is caused by activation of the p38 MAP kinase-MSK1 signaling axis and by cell cycle progression in cancer cells.

ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant.

The ST2 gene was originally identified as a primary responsive gene, and the expressions of its gene products are induced by stimulation with growth factors and by oncogenic stresses. In this study, we observed that oncogenic Ras mutant induced the expression of ST2 and ST2L proteins. Interestingly, the enforced expression of ST2 gene products in NIH-3T3 murine fibroblasts remarkably enhanced Ras (G12V)-induced cellular transformation. Furthermore, when the expression of ST2 gene products was silenced by RNA-interference technique, Ras (G12V)-induced cellular transformation was drastically suppressed. According to these observations, it was indicated that the oncogenic Ras-induced expression of ST2 gene products is required for the acceleration of cellular transformation, and this seems to be independent of the stimulation with IL-33, a ligand for ST2/ST2L. Interestingly, knockdown of ST2 gene products caused a reduction in Rb phosphorylation in transformed murine fibroblasts, suggesting the functional involvement of ST2 gene products in cell cycle progression during cellular transformation. Our current study strongly suggests the importance of ST2 gene products in cellular transformation, and the presence of novel mechanism how ST2 gene products affect the cellular transformation and cell proliferation.

STAT3 and ERK pathways are involved in cell growth stimulation of the ST2/IL1RL1 promoter.

The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters - a distal promoter and a proximal promoter. In this study, we identified a novel type of serum-responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.

Intracellular NF-HEV/IL-33 harbors essential roles in Ras-induced cellular transformation by contributing to cyclin D1 protein synthesis.

A member of the interleukin-1 family, interleukin-33 (NF-HEV/IL-33), is a ligand for the receptor, ST2L and stimulates the production of Th2 cytokines. Although IL-33 localizes to the nucleus and may be involved in the regulation of transcription independent of ST2L, its functions in the nucleus currently remain unclear. We herein demonstrated that the expression of IL-33 was markedly enhanced in NIH-3T3 cells transformed by an oncogenic H-Ras mutant (H-Ras (G12V)), and the induced IL-33 was mainly located in the nuclei of these cells. The enforced expression of IL-33 accelerated H-Ras (G12V)-induced transformation in NIH-3T3 cells, and this transforming activity was markedly reduced by the knockdown of IL-33 with shRNA. We subsequently analyzed several signaling molecules regulated by Ras in order to elucidate the mechanism by which IL-33 contributes to Ras (G12V)-induced transformation. We found that the knockdown of IL-33 effectively attenuated the Ras (G12V)-induced expression of cyclin D1. However, the knockdown of IL-33 failed to affect cyclin D1 mRNA expression levels, and epoxomicin, a proteasome inhibitor, did not cancel the IL-33 knockdown-induced down-regulation of its protein levels. We showed that Ras (G12V)-induced cyclin D1 protein synthesis was markedly suppressed by the knockdown of IL-33. Taken together, the results of the present study strongly suggest a novel role for IL-33 in cellular transformation.

Biochemical properties of Caenorhabditis elegans HMG-5, a regulator of mitochondrial DNA.

Caenorhabditis elegans HMG-5, which is encoded by F45E4.9, contains two high mobility group (HMG) box domains and shows sequence similarity with mammalian mitochondrial transcription factor A (TFAM). In this study, using soaking RNA interference, we found that knockdown of HMG-5 reduced the amount of mtDNA in P0 hermaphrodites, suggesting it as functional orthologue of mammalian TFAM. We also examined the biochemical property of HMG-5 in mammalian cells and in vitro. We found that HMG-5 localized to the mitochondria in human cultured cells and was included in the NP-40-insoluble fraction in which mtDNA and TFAM were enriched. By immunoprecipitation analysis, HMG-5 was found to associate with human mitochondrial DNA (mtDNA) in the cells. In vitro binding experiment also showed that HMG-5 binds to C. elegans mtDNA and plasmid DNA, indicating its feature as a non-specific DNA-binding protein. Furthermore, it was found that HMG-5 can interact with itself. These results demonstrate that HMG5 shares similar biochemical properties with mammalian TFAM as a nucleoid factor. HMG-5 could be a good candidate for investigating mtDNA metabolism in multicellular organisms.

Bacillus subtilis tRNA(Pro) with the anticodon mo5UGG can recognize the codon CCC.

In Bacillus subtilis, four codons, CCU, CCC, CCA, and CCG, are used for proline. There exists, however, only one proline-specific tRNA having the anticodon mo(5)UGG. Here, we found that this tRNA(Pro)(mo(5)UGG) can read not only the codons CCA, CCG and CCU but also CCC, using an in vitro assay system. This means that the first nucleoside of its anticodon, 5-methoxyuridine (mo(5)U), recognizes A, G, U and C. On the other hand, it was reported that mo(5)U at the first position of the anticodon of tRNA(Val)(mo(5)UAC) can recognize A, G, and U but not C. A comparison of the structure of the anticodon stem and loop of tRNA(Pro)(mo(5)UGG) with those of other tRNAs containing mo(5)U at the first positions of the anticodons suggests that a modification of nucleoside 32 to pseudouridine (Psi) enables tRNA(Pro)(mo(5)UGG) to read the CCC codon.

Genomic investigation of the system for selenocysteine incorporation in the bacterial domain.

The elucidation of nearly 100 bacterial genomes has made it possible to categorize them into two groups, according to the presence or absence of a selenocysteine (Sec) tRNA. In the group with the tRNA, a Sec incorporation system like that of Escherichia coli would be expected. However, for the other group, the following question has been left unsolved. Is it reasonable to assume that bacteria without the tRNA lack the entire Sec system, and do such bacteria exist commonly? To explore it experimentally, we chose Bacillus subtilis, a representative eubacterium for which a Sec tRNA has not been found. First, we reviewed the genome to search for the tRNA gene. Second, we examined the possible expression of an unknown tRNA. Third, we examined Sec-related enzymes and proteins in B. subtilis cell extracts. Fourth, the B. subtilis and E. coli seryl-tRNA synthetases were expressed, and the specificity was analyzed. Consequently, all of the data showed negative results about the existence of the Sec system in B. subtilis. Additionally, we discuss the possibility of predicting the existence or absence of the system in each bacterial organism by using the Sec tRNA and seryl-tRNA synthetase as indicators.

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons.

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.