MAB21L4 regulates the TGF-ß-induced expression of target genes in epidermal keratinocytes.

Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma.

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1.

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Identification of a phosphorylation site in c-Ski as serine 515.

c-Ski has been known to be phosphorylated at serine residue(s), which results in slower migration of c-Ski in SDS-polyacrylamide gel electrophoresis. The position(s) of phosphorylation, however, has not been determined. In the present study, we identified a phosphorylation site of c-Ski which affects its electrophoretic motility as serine 515 using MALDI-TOF mass spectrometry. A phosphorylation-resistant mutant, c-Ski S515A, did not exhibit a phosphatase-sensitive band shift. In addition, we confirmed that endogenous c-Ski is phosphorylated at serine 515, using a specific antibody. The phosphorylation status of c-Ski, however, does not appear to affect its stability or effects on TGF-ß signalling. Identification of the phosphorylation site of c-Ski would allow us further examination of physiological significance of c-Ski phosphorylation.

Binding properties of hydrophobic molecules to human serum albumin studied by fluorescence titration.

Two fluorescence modes were combined to analyze the binding properties of terminally substituted alkanes (C(n)X, X = COOH, OH, CHO, NH(2)) to human serum albumin (HSA). A competitive binding assay using an 8-anilino-1-naphthalenesulfonate (ANS) fluorescence probe provides information on all the hydrophobic binding sites in HSA. A binding assay using the intrinsic fluorescence of the tryptophan residue in HSA (Trp-HSA) provides information on the specific binding site close to the tryptophan residue. There are three fluorescence-active ANS binding sites in HSA, which can be classified into two types by their affinity for ANS. C(n)COOH bound to all three ANS binding sites including the Trp-HSA site, however, it did not quench the fluorescence of Trp-HSA. C(n)CHO bound only to the Trp-HSA site with quenching of the fluorescence of Trp-HSA. By comparing the binding affinities of HSA for C(n)OH and C(n)CHO, it was concluded that the C(n)OH binding site is different from the C(n)CHO binding site. C(n)NH(2) did not bind to any of the three ANS binding sites in HSA.

Crystallization and preliminary X-ray diffraction analysis of GCIP/HHM transcriptional regulator.

GCIP/HHM is a human nuclear protein that is implicated in regulation of cell proliferation. Its primary structure contains helix-loop-helix and leucine-zipper motifs but lacks a DNA-binding basic region. Native and selenomethionine-derivatized (SeMet) crystals of full-length GCIP/HHM were obtained using the hanging-drop vapour-diffusion method. The crystals were greatly improved by adding tris(2-carboxyethyl)phosphine as a reducing reagent and diffracted to 3.5 A resolution. Preliminary phase calculations using the data set obtained from the SeMet crystal suggested that the crystal belonged to space group P3(2)21 and contained one molecule per asymmetric unit. Structure determination by the multiple-wavelength anomalous dispersion method using the SeMet crystals is in progress.

SKI and MEL1 cooperate to inhibit transforming growth factor-beta signal in gastric cancer cells.

Chromosomal amplification occurs frequently in solid tumors and is associated with poor prognosis. Several reports demonstrated the cooperative effects of oncogenic factors in the same amplicon during cancer development. However, the functional correlation between the factors remains unclear. Transforming growth factor (TGF)-beta signaling plays important roles in cytostasis and normal epithelium differentiation, and alterations in TGF-beta signaling have been identified in many malignancies. Here, we demonstrated that transcriptional co-repressors of TGF-beta signaling, SKI and MDS1/EVI1-like gene 1 (MEL1), were aberrantly expressed in MKN28 gastric cancer cells by chromosomal co-amplification of 1p36.32. SKI and MEL1 knockdown synergistically restored TGF-beta responsiveness in MKN28 cells and reduced tumor growth in vivo. MEL1 interacted with SKI and inhibited TGF-beta signaling by stabilizing the inactive Smad3-SKI complex on the promoter of TGF-beta target genes. These findings reveal a novel mechanism where distinct transcriptional co-repressors are co-amplified and functionally interact, and provide molecular targets for gastric cancer treatment.

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells.

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.

Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth factor-beta signaling.

Transforming growth factor-beta (TGF-beta) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-beta signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-beta signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-beta signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-beta family signaling in target cells.