title: Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions.
abstract: OBJECTIVE
  Osmolarity is a major biophysical regulator of chondrocyte function. Modulation of chondrocytic marker gene expression occurs at the post-transcriptional level following exposure of human articular chondrocytes (HAC) to hyperosmotic conditions. This study aims to further characterise the post-transcriptional response of HAC to hyperosmolarity.
  
  
  METHODS
  Gene expression and microRNA (miRNA) levels in freshly isolated HAC after 5h under control or hyperosmotic conditions were measured using microarrays. Regulated genes were checked for the presence of AU rich elements (AREs) in their 3' untranslated regions (3'UTR), whilst gene ontology was examined using Ingenuity Pathway Analysis (IPA). RNA decay rates of candidate ARE-containing genes were determined in HAC using actinomycin D chase experiments and the involvement of the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways were investigated using pharmacological inhibitors.
  
  
  RESULTS
  Hyperosmolarity led to the regulation of a wide variety of genes. IPA identified enrichment of genes involved with cell stress responses, cell signalling and transforming growth factor β (TGFβ) signalling. Importantly, upregulated genes were over-represented with those containing AREs, and RNA decay analysis demonstrated that many of these were regulated post-transcriptionally by hyperosmolarity in HAC. Analysis of miRNA levels in HAC indicated that they are only modestly regulated by hyperosmotic conditions, whilst inhibitor studies showed that p38 MAPK and ERK1/2 were able to block hyperosmotic induction of many of these genes.
  
  
  CONCLUSIONS
  Through microarray and bioinformatics analysis we have identified genes which are post-transcriptionally regulated in HAC following exposure to hyperosmotic conditions. These genes have a range of functions, and their regulation involves transduction through the p38 MAPK and ERK1/2 pathways. Interestingly, our results suggest that miRNA regulation is not key to the process. Overall, this work illustrates the range of processes regulated in chondrocytes by changes in their osmotic environment, and underlines the importance of post-transcriptional mRNA regulation to chondrocyte function.
authors.affiliation: Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, Cheshire, UK. tew@liverpool.ac.uk
authors.forename: S R
authors.initials: SR
authors.lastname: Tew
authors.forename: O
authors.initials: O
authors.lastname: Vasieva
authors.forename: M J
authors.initials: MJ
authors.lastname: Peffers
authors.forename: P D
authors.initials: PD
authors.lastname: Clegg
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000737:chemistry / Q000378:metabolism*; D024043:Collagen Type II / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D055758:SOX9 Transcription Factor / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C499177:COL2A1 protein, human; D024043:Collagen Type II; D035683:MicroRNAs; D011500:Protein Synthesis Inhibitors; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D016212:Transforming Growth Factor beta; D003609:Dactinomycin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases