TGFβ and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation

CCN2/CTGF is an established effector of TGFß driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFß leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function.

Genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential involvement of immune-related gene loci

Converging evidence implicates immune abnormalities in schizophrenia (SCZ), and recent genome-wide association studies (GWAS) have identified immune-related single-nucleotide polymorphisms (SNPs) associated with SCZ. Using the conditional false discovery rate (FDR) approach, we evaluated pleiotropy in SNPs associated with SCZ (n=21 856) and multiple sclerosis (MS) (n=43 879), an inflammatory, demyelinating disease of the central nervous system. Because SCZ and bipolar disorder (BD) show substantial clinical and genetic overlap, we also investigated pleiotropy between BD (n=16 731) and MS. We found significant genetic overlap between SCZ and MS and identified 21 independent loci associated with SCZ, conditioned on association with MS. This enrichment was driven by the major histocompatibility complex (MHC). Importantly, we detected the involvement of the same human leukocyte antigen (HLA) alleles in both SCZ and MS, but with an opposite directionality of effect of associated HLA alleles (that is, MS risk alleles were associated with decreased SCZ risk). In contrast, we found no genetic overlap between BD and MS. Considered together, our findings demonstrate genetic pleiotropy between SCZ and MS and suggest that the MHC signals may differentiate SCZ from BD susceptibility.Molecular Psychiatry advance online publication, 28 January 2014; doi:10.1038/mp.2013.195.

Sequence and expression of complement factor H gene cluster variants and their roles in age-related macular degeneration risk

Purpose: To investigate how potentially functional genetic variants are coinherited on each of four common complement factor H (CFH) and CFH-related gene haplotypes and to measure expression of these genes in eye and liver tissues.

Methods: We sequenced the CFH region in four individuals (one homozygote for each of four common CFH region haplotypes) to identify all genetic variants. We studied associations between the haplotypes and AMD phenotypes in 2157 cases and 1150 controls. We examined RNA-seq profiles in macular and peripheral retina and retinal pigment epithelium/choroid/sclera (RCS) from eight eye donors and three liver samples.

Results: The haplotypic coinheritance of potentially functional variants (including missense variants, novel splice sites, and the CFHR3–CFHR1 deletion) was described for the four common haplotypes. Expression of the short and long CFH transcripts differed markedly between the retina and liver. We found no expression of any of the five CFH-related genes in the retina or RCS, in contrast to the liver, which is the main source of the circulating proteins.

Conclusions: We identified all genetic variants on common CFH region haplotypes and described their coinheritance. Understanding their functional effects will be key to developing and stratifying AMD therapies. The small scale of our expression study prevented us from investigating the relationships between CFH region haplotypes and their expression, and it will take time and collaboration to develop epidemiologic-scale studies. However, the striking difference between systemic and ocular expression of complement regulators shown in this study suggests important implications for the development of intraocular and systemic treatments.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

Functional interaction of E1AF and Sp1 in glioma invasion.

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

Targeted genome-wide investigation identifies novel SNP's associated with diabetic nephropathy

Loci contributing to complex disease have been identified by focusing on genome-wide scans utilising non-synonymous single nucleotide polymorphisms (nsSNPs). We employed Illumina’s HNS12 BeadChip (13,917 high-value SNPs) which was specifically designed to capture nsSNPs and ideally complements more dense genome-wide association studies that fail to consider many of these putatively functional variants. The HNS12 panel also includes 870 tag SNPs covering the major histocompatibility region. All individuals genotyped in this study were Caucasians with (cases) and without (controls) diabetic nephropathy. About 449 individuals with type 2 diabetes (203 cases, 246 controls) were genotyped in the initial study. 1,467 individuals with type 1 diabetes (718 cases, 749 controls) were genotyped in the follow up study. 11,152 SNPs were successfully analysed and ranked for association with diabetic nephropathy based on significance (P) values. The top ranked 32 SNPs were subsequently genotyped using MassARRAY iPLEX™ and TaqMan technologies to investigate association of these polymorphisms with nephropathy in individuals with type 1 diabetes. The top ranked nsSNP, rs1543547 (P = 10-5), is located in RAET1L, a major histocompatibility class I-related gene at 6q25.1. Of particular interest, multiple nsSNPs within the top ranked (0.2%) SNPs are within several plausible candidate genes for nephropathy on 3q21.3 and 6p21.3.

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system. (C) 2010 Elsevier Inc. All rights reserved.

Role of AcrR and ramA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore

The MICs of ciprofloxacin for 33 clinical isolates of K. pneumoniae resistant to extended-spectrum cephalosporins from three hospitals in Singapore ranged from 0.25 to >128 microg/ml. Nineteen of the isolates were fluoroquinolone resistant according to the NCCLS guidelines. Strains for which the ciprofloxacin MIC was >or=0.5 microg/ml harbored a mutation in DNA gyrase A (Ser83-->Tyr, Leu, or IIe), and some had a secondary Asp87-->Asn mutation. Isolates for which the MIC was 16 microg/ml possessed an additional alteration in ParC (Ser80-->IIe, Trp, or Arg). Tolerance of the organic solvent cyclohexane was observed in 10 of the 19 fluoroquinolone-resistant strains; 3 of these were also pentane tolerant. Five of the 10 organic solvent-tolerant isolates overexpressed AcrA and also showed deletions within the acrR gene. Complementation of the mutated acrR gene with the wild-type gene decreased AcrA levels and produced a two- to fourfold reduction in the fluoroquinolone MICs. None of the organic solvent-tolerant clinical isolates overexpressed another efflux-related gene, acrE. While marA and soxS were not overexpressed, another marA homologue, ramA, was overexpressed in 3 of 10 organic solvent-tolerant isolates. These findings indicate that multiple target and nontarget gene changes contribute to fluoroquinolone resistance in K. pneumoniae. Besides AcrR mutations, ramA overexpression (but not marA or soxS overexpression) was related to increased AcrAB efflux pump expression in this collection of isolates.

Neuropeptides regulate expression of angiogenic growth factors in pulpal fibroblasts

In the dental pulp angiogenesis is crucial for tooth development and a prerequisite for successful repair following injury and inflammation. The role of neuropeptides in pulpal inflammation has been well documented but their role in the regulation of angiogenesis in the dental pulp has not been elucidated. Objectives: The aim was to profile the expression of angiogenic growth factors produced by pulp fibroblasts and to study the effects of neuropeptides on their expression. Methods: Human pulp fibroblasts derived from healthy molar teeth were stimulated with neuropeptides previously identified in dental pulp, namely, Substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and calcitonin related gene peptide (CGRP) for 24 and 48 hrs. Simultaneous expression of ten growth factors was quantified using a novel human angiogenesis array (Ray Biotech, USA). Results: Pulp fibroblasts expressed human angiogenic growth factors, VEGF, bFGF, PDGF-BB, HGF, ANG2, HB-EGF, PIGF, angiogenin and leptin. Among the growth factors expressed VEGF, angiogenin and HGF were abundantly expressed compared to others. Neuropeptides induced variable effects on the expression of the angiogenic factors: CGRP potently up-regulated VEGF, bFGF, HGF and PIGF after 24 hr, while NPY tended to down regulate growth factors after 24 hr in culture but markedly up regulated ANG2, bFGF and leptin after 48 hr. SP down regulated expression of all angiogenic growth factors except for leptin, while VIP induced a small increase in expression of each growth factor, irrespective of time. Conclusion: Pulp fibroblasts express a range of angiogenic growth factors including angiogenin and leptin. Neuropeptides regulate the expression of these factors, suggesting an additional role for neuropeptides in the regulation of inflammation and healing in the dental pulp.
This work is supported by TC White Research Fund

Carboxypeptidase-mediated metabolism of calcitonin gene-related peptide in human gingival crevicular fluid - a role in periodontal inflammation?

Background: Metabolism by peptidases plays an important role in modulating the levels of biologically-active neuropeptides. The metabolism of the anti-inflammatory neuropeptide calcitonin gene-related peptide (GCRP), but not the pro-inflammatory neuropeptides substance P (SP) and neurokinin A (NKA) by components of the gingival crevicular fluid (GCF), could potentiate the inflammatory process in periodontitis.