Expanded mutational spectrum of the GLI3 gene substantiates genotype–phenotype correlations

Greig cephalopolysyndactyly syndrome (GCPS) and isolated preaxial polydactyly type IV (PPD-IV) are rare autosomal dominant disorders, both caused by mutations in the GLI3 gene. GCPS is mainly characterised by craniofacial abnormalities (macrocephaly/prominent forehead, hypertelorism) and limb malformations, such as PPD-IV, syndactyly and postaxial polydactyly type A or B (PAPA/B). Mutations in the GLI3 gene can also lead to Pallister?CHall syndrome (PHS) and isolated PAPA/B. In this study, we investigated 16 unrelated probands with the clinical diagnosis of GCPS/PPD-IV and found GLI3 mutations in 12 (75?%) of them (nine familial and three sporadic cases). We also performed a detailed clinical evaluation of all 12 GLI3-positive families, with a total of 27 patients. The hallmark triad of GCPS (preaxial polydactyly, macrocephaly/prominent forehead, hypertelorism) was present in 14 cases (52?%), whereas at least one typical dysmorphic feature was manifested in 17 patients (63?%). Upon sequencing of the GLI3 gene, we demonstrated eight novel and two previously reported heterozygous point mutations. We also performed multiplex ligation-dependent probe amplification (MLPA) to screen for intragenic copy number changes and identified heterozygous deletions in the two remaining cases (16.7?%). Our findings fully support previous genotype?Cphenotype correlations, showing that exonic deletions, missense mutations, as well as truncating variants localised out of the middle third of the GLI3 gene result in GCPS/PPD-IV and not PHS. Additionally, our study shows that intragenic GLI3 deletions may account for a significant proportion of GCPS/PPD-IV causative mutations. Therefore, we propose that MLPA or quantitative polymerase chain reaction (qPCR) should be implemented into routine molecular diagnostic of the GLI3 gene.     

Defects of protein phosphatase 2A causes corticosteroid insensitivity in severe asthma

Background

Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser²²⁶, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2A_C-Tyr³⁰⁷ phosphorylation compared with those in healthy volunteers. As the results, GR-Ser²²⁶ and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser²²⁶. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.

Conclusions/Significance

PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser²²⁶ via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.     

Mutational Analysis of Hedgehog Signaling Pathway Genes in Human Malignant Mesothelioma

Background

The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown.

Methodology/Principal Findings

Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour.

Conclusions/Significance

We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.     

Protein phosphatase 2A mediates dormancy of glioblastoma multiforme-derived tumor stem-like cells during hypoxia

Purpose

The hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.

Experimental Design

Expression of PP2A subunits and HIF-α proteins was measured in 65 high-grade astrocytoma and 18 non-neoplastic surgical brain specimens by western blotting. PP2A activity was measured by an immunoprecipitation assay. For in vitro experiments, GBM-derived tumor stem cell-like cells (TSCs) were exposed to severe hypoxia produced by either CoCl₂ or 1% O₂. PP2A activity was inhibited either by okadaic acid or by shRNA depletion of the PP2A C subunit. Effects of PP2A activity on cell cycle progression and cell survival during hypoxic conditions were assessed using flow cytometry.

Results

In our patient cohort, PP2A activity was positively correlated with HIF-1∝ protein expression (P = 0.002). Patients with PP2A activity levels above 160 pMP had significantly worse survival compared to patients with levels below this threshold (P = 0.002). PP2A activity was an independent predictor of survival on multivariable analysis (P = 0.009). In our in vitro experiments, we confirmed that severe hypoxia induces PP2A activity in TSCs 6 hours after onset of exposure. PP2A activity mediated G1/S phase growth inhibition and reduced cellular ATP consumption in hypoxic TSCs. Conversely, inhibition of PP2A activity led to increased cell proliferation, exhaustion of intracellular ATP, and accelerated P53-independent cell death of hypoxic TSCs.

Conclusions

Our results suggest that PP2A activity predicts poor survival in GBM. PP2A appears to reduce the metabolic demand of hypoxic TSCs and enhances tumor cell survival. Modulation of PP2A may be a potential target for cancer therapy.     

Gadd45a is an RNA binding protein and is localized in nuclear speckles

Background

The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids.

Principal Findings

Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function.

Significance

The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.