The matricellular protein Fibulin-5 regulates β-cell proliferation in an autocrine/paracrine manner
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE143633 | GEO | 2024/11/19
REPOSITORIES: GEO
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Project description:The formation of elastic fibers is active only in the perinatal period. How elastogenesis is developmentally regulated is not fully understood. Citrullination is a unique form of post-translational modification catalyzed by peptidylarginine deiminases (PADIPADs), including PADIPAD1-4. Its physiological role is largely unknown. By using an unbiased proteomic approach of lung tissues, we discovered that FBLN5 and LTBP4, two key elastogenic proteins, were temporally modified in mouse and human lungs. We further demonstrated that PADIPAD2 citrullinated FBLN5 preferentially in young lungs compared to adult lungs. Genetic ablation of PADIPAD2 resulted in attenuated elastogenesis in vitro and age-dependent emphysema in vivo. Mechanistically, citrullination protected FBLN5 from proteolysis and subsequent inactivation of its elastogenic activity. Furthermore, citrullinated but not native FBLN5 partially rescued in vitro elastogenesis in the absence of PADIPAD activity. Our data uncover a novel function of citrullination, namely promoting elastogenesis, and provide additional insights to how elastogenesis is regulated.
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Project description:To investigate the relevance and the impact of the cleaved FBLN5 variant on fibroblast’s physiology, we have employed whole genome microarrays. Human neonatal dermal fibroblasts from three different donors were seeded in duplicates on plates pre-coated with FBLN5, cleaved FBLN5 or heat denatured BSA as a control. After cultivation for 24 h, RNA was isolated and microarrays were performed. Statistical analysis of microarrays resulted in 367 genes significantly differentially expressed in cells cultured on a FBLN5 matrix and 525 genes differentially expressed in cells cultured on a cleaved FBLN5 matrix compared to control (adjusted p-value <0.05). To further validate these gene expression changes identified by microarray analysis 10 randomly choosen genes were reanalyzed by qRT-PCR (quantitative real time polymerase chain reaction). In nine cases the fold changes measured by qRT-PCR were comparable with those measured by microarray analysis, revealing reproducibility and validity of analysis.
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Project description:To investigate the relevance and the impact of the cleaved FBLN5 variant on fibroblastM-bM-^@M-^Ys physiology, we have employed whole genome microarrays. Human neonatal dermal fibroblasts from three different donors were seeded in duplicates on plates pre-coated with FBLN5, cleaved FBLN5 or heat denatured BSA as a control. After cultivation for 24 h, RNA was isolated and microarrays were performed. Statistical analysis of microarrays resulted in 367 genes significantly differentially expressed in cells cultured on a FBLN5 matrix and 525 genes differentially expressed in cells cultured on a cleaved FBLN5 matrix compared to control (adjusted p-value <0.05). To further validate these gene expression changes identified by microarray analysis 10 randomly choosen genes were reanalyzed by qRT-PCR (quantitative real time polymerase chain reaction). In nine cases the fold changes measured by qRT-PCR were comparable with those measured by microarray analysis, revealing reproducibility and validity of analysis. Fibulin-5 and cleaved FBLN5 contributed to changes in expression pattern of human neonatal dermal fibroblasts after cultivation for 24 h. Three different donors were used in technical duplicates.
2014-07-20 | E-GEOD-58635 | biostudies-arrayexpress
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Project description:TGFbi (transforming growth factor-beta-induced) is a secreted protein and is capable of binding to both extracellular matrix (ECM) and cells. It thus acts as a bifunctional molecule enhancing ECM and cell interactions, a lack of which results in dysfunction of many cell types. In this study, we investigated the role of TGFbi in the function and survival of islets. Based on DNA microarray analysis followed by qPCR confirmation, the TGFbi gene showed drastic increases in expression in islets after culture. We demonstrated that recombinant TGFbi could preserve the integrity and enhance the function of cultured islets. Such a beneficial effect was mediated via signalling through FAK. Exogenous TGFbi was capable of sustaining high-level FAK phosphorylation in isolated islets, and FAK knockdown by siRNA in islets resulted in compromised islet function. TGFbi Tg islets showed better integrity and insulin release after in vitro culture. In vivo, b-cell proliferation was detectable in Tg but not wild type pancreata. At age above 12 months, Tg pancreata contained giant islets. Tg mice displayed better glucose tolerance than the controls. Tg islets were more potent in lowering blood glucose when transplanted into syngeneic mice with streptozotocin-induced diabetes, and these transplanted islets also underwent regeneration. Our results indicate that TGFbi is a vital trophic factor promoting islet survival, function and regeneration. At least some of its beneficial effect was mediated by signalling through FAK. Six independent batches of islets were isolated. For each batch, islets from 4 C57BL/6 mice were pooled, and were divided into 3 groups, which were harvested at 0 h, 24 h and 48 h after culture. Total RNA was extracted from the islets with QIAGEN RNeasy Micro Kits. After reverse transcription, cDNA served as a template for PCR-based cRNA amplification. It was then reverse-transcribed to produce second-generation cDNA, which was employed to generate biotin-labelled cRNA probes. The probes were hybridized on GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA), which contains 39,000 transcripts. DNA microarray analysis was performed at the McGill University Genome Quebec Innovation Centre. For each time point, 6 chips were hybridized with cRNA from the 6 batches of isolated islets. For samples collected at 24 and 48 hours (6 batches for each time point), if the mean signal strength of a gene had above 4-fold difference over that of the 0 h samples, it was selected for reverse polymerase chain reaction (PCR) confirmation. Details of the experimental protocols and data analysis procedure can be found in http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf .
2011-02-28 | E-GEOD-27547 | biostudies-arrayexpress
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2015-06-18 | E-GEOD-68317 | biostudies-arrayexpress