Unknown,Transcriptomics,Genomics,Proteomics

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HtrA1-dependent proteolysis of TGF-M-NM-2 controls both neuronal maturation and developmental survival

ABSTRACT: Transforming growth factor-M-NM-2 (TGF-M-NM-2) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-M-NM-2 accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice over-expressing a TGF-M-NM-2 responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-M-NM-2 signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-M-NM-21, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-M-NM-2 signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-M-NM-2 could be one of the critical events controlling both neuronal maturation and developmental survival. Keywords: HtrA1 / neuronal survival / PAI-1 / TGF-M-NM-2 signalling / tPA Total RNA were extracted from 3 cultures of 2 DIV Human neurons. For each stage, equal amounts of each RNA were pooled and 5M-BM-5g were reverse-transcribed, labelled and hybridized on pangenomic microarrays. Each pool was hybridized in duplicate dye-swap independent experiments.

ORGANISM(S): Homo sapiens

SUBMITTER: Benoit Roussel

PROVIDER: E-GEOD-11162 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival.

Launay S S Maubert E E Lebeurrier N N Tennstaedt A A Campioni M M Docagne F F Gabriel C C Dauphinot L L Potier M C MC Ehrmann M M Baldi A A Vivien D D

Cell death and differentiation 20080613 9

Transforming growth factor-beta (TGF-beta) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-beta accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice overexpressing a TGF-beta-responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-beta signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing ...[more]

PMID: 18551132

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Project description:Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia. For each stage (4 months and 21 months), samples were prepared by pooling an equal amount of the 3 individual RNA. Five M-BM-5g of total RNA from each pool were reverse-transcribed in the presence of 7.5M-BM-5M random hexamers (GE Healthcare), 75M-BM-5M aminoallyl-dUTP (Sigma-Aldrich) and 100U Rtases Reverse-iT Blend (Thermo scientific) overnight at 37M-BM-0C. Aminoallyl cDNAs were then labelled with Cy5 or Cy3 mono-Reactive Dye (GE Healthcare) according to the manusfacturerM-bM-^@M-^Ys instructions. Labelled cDNAs were then purified on Qiaquick PCR prurification kit (Qiagen) and hybridized to the RNG-MRC_MM25k_EVRY microarrays (Le brigand et al., NAR, 2006) according to the RNG procedures (http://www.microarray.fr:8080/merge/index). Each pool was hybridized in duplicate dye-swap independent experiments. After hybridization, median signal and median background intensities were extracted using Genepix 4.1 software (Axon Instruments). We have first evaluated the level of expression of each gene by calculating the mean ratio M-bM-^@M-^\signal/backgroundM-bM-^@M-^] for each corresponding spot from the 2 dye-swap experiments. Genes were considered to be expressed if their mean ratio was higher or equal to 1.2. Genes with mean ratio < 1.2 in the 2 conditions were not used for further analysis. These filtered data were then submitted to VARAN (http//:www. bionet. espci.fr) for normalization by lowess fit and differential expression analysis (Golfier et al., 2004). Normalized log2ratio 21 months/4 months were then used for further statistical analysis by SAM software (Tusher et al., 2001) and t-test with a p-value equal to 5% using TIGR Multiexperiment Viewer (TM4:MeV, http://www.tm4.org/mev.html). The final list of differentially expressed genes was established by comparing the results obtained with the 3 methods.

Project description:Most available CRC cell lines have lost their TGF-beta response through the acquisition of mutations either in TGF-beta type II receptor (TGFBR2) or the intracellular mediator SMAD4 (Reviewed in Markowitz et al. 2002). To study the effect of TGF-beta in CRC epithelial cells, we restored wild-type TGFBR2 expression in the CRC cell line LS174T (LS). Ls174T cells are mutant for beta-catenin and therefore exhibit constitutive Wnt activity, which results in the expression of a genetic profile similar to that of intestinal epithelial progenitor cells (van de Wetering, Sancho et al. 2002). They thus reflect tumor cells at the initial stages of tumorogenesis. In addition, Ls174T cells bear mutations in the TGF-beta receptor II (TGFBR2) sequence at the BATRII locus (Grady and Markowitz 2002). This mutation gives rise to a non-functional truncated form of the receptor lacking the transmembrane domain and the serine-threonine kinase domain (Parsons, Myeroff et al. 1995). We generated inducible clones of Ls174T cells, where the expression of TGFBR2 could be controlled at will by the presence or absence of doxycycline (LSTGFBR2 cell line). Re-expression of wild-type receptor in Ls174T cells (LSTGFBR2) restored TGF-beta signalling resulting in strong phosphorylation of SMADs and activation of SMAD-binding reporter activity. Functionally, TGF-beta signalling in LSTGFBR2 resulted in a strong cytostatic response. By genome wide expression profiling using microarrays we investigated genes regulated by TGF-beta in this cell line. LsTGFBR2 inducible cells were generated by the Invitrogen T-Rex system following manufacturers instructions. LSTGFBR2 were seeded at 60% confluence in the presence or absence of Doxycycline and treated with TGF-M-NM-21 for 16 hours. Gene expression profiles were measured in duplicate using HG-U133 plus 2.0. We used RMA background correction, quantile normalization and RMA summarization (Gautier et al., 2004). A TGF-M-NM-2 response signature was obtained by selecting genes with limma P-value < 0.05 and at least two fold up-regulation in TGF-M-NM-2 treated LSTGFBR2 cells.

Dataset Information

HtrA1-dependent proteolysis of TGF-M-NM-2 controls both neuronal maturation and developmental survival

Publications

HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival.

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