Analysis of Gene Expression Profiling in Meningioma: Deregulated Signaling Pathways Associated with Meningioma and EGFL6 Overexpression in Benign Meningioma Tissue and Serum

Molecular mechanisms underlying the pathogenesis of meningioma are not fully elucidated. In this study, we established differential gene expression profiles between meningiomas and brain arachnoidal tissue by using Affymetrix GeneChip Human U133 Plus 2.0 Array. KEGG pathway analysis demonstrated that PI3K/Akt and TGFβ signaling pathways were up-regulated in fibroblastic meningioma, and focal adhesion and ECM-receptor interaction pathways were activated in anaplastic meningioma. EGFL6 was one of the most up-regulated genes in fibroblastic meningioma by microarray analysis. Quantitative real-time PCR demonstrated that benign meningiomas had significantly higher levels of EGFL6 mRNA than brain arachnoidal tissue and atypical and anaplastic meningiomas (P<0.001). EGFL6 gene was also highly expressed in ovarian cancer, but expressed lowly in other investigated tumors. ELISA analysis showed that patients with benign meningiomas and ovarian cancers had the highest serum levels of EGFL6 (mean concentration: 672 pg/ml for benign meningiomas, and 616 pg/ml for ovarian cancers). Healthy people and patients with other tumors, however, had low levels of serum EGFL6. In conclusion, we proposed that activation of PI3K/Akt and integrin-mediated signaling pathways was involved in the pathogenesis of benign and anaplastic meningiomas, respectively. We also presented evidence that EGFL6 was overexpressed in benign meningioma tissues and serum.     

Thyroid Hormone Receptor Sumoylation Is Required for Preadipocyte Differentiation and Proliferation

Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRβ reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRβ resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ₂ mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially “rescued” the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/β-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRβ K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, β-catenin, LEF1, and CCND1. Additionally, the TRβ K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

Keratin 76 Is Required for Tight Junction Function and Maintenance of the Skin Barrier

Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.     

Overexpression of the cat-86 Gene Is Associated with Thermosensitivity in Bacillus subtilis

Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with an ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cm^s) and exhibited relative thermostability when heated at 47°C. Reversion to chloramphenicol resistance (Cm^r) occurred at a frequency of 5.4 × 10⁻⁸. All of the plasmid Cm^r revertants tested were thermosensitive. Similarly, wild-type pPL708C2 present in B. subtilis also rendered the bacterium thermosensitive. When a nonsense mutation is introduced at codon 141, however, this terc 141 variant of pPL708C2 failed to thermosensitize B. subtilis. Another variant of pPL708C2 that produces intact yet catalytically inactive CAT-86 has both His-16 and His-17 at the active site replaced by Pro. Nevertheless, cells of B. subtilis carrying this variant were thermosensitive. Plasmid-free and pPL708C2-bearing strains did not exhibit differences in major heat shock proteins. Electron micrographs revealed a threefold increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain. Received: 26 July 1999 / Accepted: 30 August 1999     

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.     

Rad9 Is Required for B Cell Proliferation and Immunoglobulin Class Switch Recombination

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9^−/− B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9^−/− mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9^−/− B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.     

Mycoplasma pneumoniae Protein P30 Is Required for Cytadherence and Associated with Proper Cell Development

The attachment organelle of Mycoplasma pneumoniae is a polar, tapered cell extension containing an intracytoplasmic, electron-dense core. This terminal structure is the leading end in gliding motility, and its duplication is thought to precede cell division, raising the possibility that mutations affecting cytadherence also confer a defect in motility or cell development. Mycoplasma surface protein P30 is associated with the attachment organelle, and P30 mutants II-3 and II-7 do not cytadhere. In this study, the recombinant wild-type but not the mutant II-3 p30 allele restored cytadherence when transformed into P30 mutants by recombinant transposon delivery. The mutations associated with loss of P30 in mutant II-3 and reacquisition of P30 in cytadhering revertants thereof were identified by nucleotide sequencing of the p30 gene. Morphological abnormalities that included ovoid or multilobed cells having a poorly defined tip structure were associated with loss of P30. Digital image analysis confirmed quantitatively the morphological differences noted visually. Transformation of the P30 mutants with the wild-type p30 allele restored a normal morphology, as determined both visually and by digital image analysis, suggesting that P30 plays a role in mycoplasma cell development. Finally, the P30 mutants localized the adhesin protein P1 to the terminal organelle, indicating that P30 is not involved in P1 trafficking but may be required for its receptor-binding function.     

Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.     

Mitogenic and drug-resistance mediating effects of PKCalpha require RLIP76

PKCalpha-activation is a key signaling event governing cell growth, stress-resistance, and drug-resistance. Our recent studies demonstrated that DOX-resistance mediating effects of PKCalpha require the presence of RLIP76, and their concerted action is sufficient to explain intrinsic DOX-resistance of NSCLC [S.S. Singhal, D. Wickramarachchi, J. Singhal, S. Yadav, Y.C. Awasthi, et al., Determinants of differential doxorubicin sensitivity between SCLC and NSCLC. FEBS Lett. 580 (2006) 2258-2264]. Present studies were carried out to further explore the suggestion from the previous studies that the mitogenic effects of PKCalpha also require RLIP76. RLIP76-/- MEFs were resistant to PKCalpha-depletion mediated growth inhibition, as well as to the PKCalpha-dependent mitogen, phorbol 12-myristate 13-acetate (PMA). Augmenting cellular levels of RLIP76 using purified recombinant RLIP76 increased growth rate in all cells, and restored the sensitivity of RLIP76-/- MEFs to both inhibition through PKCalpha-depletion and stimulation through PMA. These results show that RLIP76 is a necessary down-stream effector for PKCalpha-mediated mitogenesis.     

Overexpression of SATB1 Is Associated with Biologic Behavior in Human Renal Cell Carcinoma

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC.     

Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2), urokinase-type plasminogen activator(uPA) and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel mechanism underlying EMMPRIN-2 enhanced tumor progression in head and neck cancer.     

Identification of membrane-anchoring domains of RLIP76 using deletion mutant analyses

RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168]. Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains. Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides. We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity. RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol. The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted. Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0. For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione. In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity. NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76. Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface. Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression. These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.     

Trf1 Is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells

The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5–deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.