ABSTRACT
BACKGROUND: An individual with a bicuspid aortic valve (BAV) runs a substantially higher risk of developing aneurysm in the ascending aorta compared to the normal population with tricuspid aortic valves (TAV). Aneurysm formation in patients with BAV and TAV is known to be distinct at the molecular level but the underlying mechanisms are undefined. Here, we investigated the still incompletely described role of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, in such aortic disease of patients with BAV as compared with TAV. METHODS AND RESULTS: Using a system biology approach, based on data obtained from proteomic analysis of non-dilated aortas from BAV and TAV patients, we constructed a gene-interaction network of regulatory microRNAs associated with the observed differential protein signature. The miR-200 family was the highest ranked miRNA, hence potentially having the strongest effect on the signalling network associated with BAV. Further, qRT-PCR and ChIP analyses showed lower expression of miR-200c, higher expression of miR-200 target genes, ZEB1/ZEB2 transcription factors, and higher chromatin occupancy of the miR-200c promoter by ZEB1/ZEB2 in BAV patients, indicating a miR-200c/ZEBs negative feedback loop and induction of endothelial/epithelial mesenchymal transition (EndMT/EMT). CONCLUSION: We propose that a miR-200-dependent process of EndMT/EMT is a plausible biological mechanism rendering the BAV ascending aorta more prone to aneurysm development. Although initially supported by a miR-200c/ZEB feedback loop, this process is most probably advanced by cooperation of other miRNAs.
Subject(s)
Aorta/metabolism , Aorta/pathology , Aortic Aneurysm/genetics , Aortic Valve/abnormalities , Epithelial-Mesenchymal Transition/genetics , Heart Valve Diseases/pathology , MicroRNAs/genetics , Aortic Aneurysm/pathology , Aortic Valve/pathology , Bicuspid Aortic Valve Disease , Female , Gene Expression Regulation , Humans , Male , Proteomics , Signal Transduction , Zinc Finger E-box Binding Homeobox 2/genetics , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
The activation of the growth arrest-specific (gas) p20K gene depends on the interaction of C/EBPß with two elements of a 48-bp promoter region termed the quiescence-responsive unit (QRU). Here we identify extracellular signal-related kinase 2 (ERK2) as a transcriptional repressor of the p20K QRU in cycling chicken embryo fibroblasts (CEF). ERK2 binds to repeated GAAAG sequences overlapping the C/EBPß sites of the QRU. The recruitment of ERK2 and C/EBPß is mutually exclusive and dictates the expression of p20K. C/EBP homologous protein (CHOP) was associated with C/EBPß under conditions promoting endoplasmic reticulum (ER) stress and, to a lesser extent, in cycling CEF but was not detectable when C/EBPß was immunoprecipitated from contact-inhibited cells. During ER stress, overexpression of CHOP inhibited p20K, while its downregulation promoted p20K, indicating that CHOP is also a potent inhibitor of p20K. Transcriptome analyses revealed that hypoxia-responsive genes are strongly induced in contact-inhibited but not serum-starved CEF, and elevated levels of nitroreductase activity, a marker of hypoxia, were detected at confluence. Conditions of hypoxia (2% O2) induced growth arrest in subconfluent CEF and markedly stimulated p20K expression, suggesting that the control of proliferation and gas gene expression is closely linked to limiting oxygen concentrations associated with high cell densities.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Fibroblasts/cytology , Lipocalins/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Transcription Factor CHOP/metabolism , Animals , Avian Proteins/genetics , Avian Proteins/metabolism , Cell Cycle , Cell Hypoxia , Cell Proliferation , Cells, Cultured , Chick Embryo , Endoplasmic Reticulum Stress , Fibroblasts/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation , Promoter Regions, GeneticABSTRACT
OBJECTIVE: Since the biological effect of cartilage mediators is generally studied in a non-physiologic environment of 21% O2, we investigated the effects of a chronic hypoxia on the capability of articular chondrocytes to respond to one anabolic stimulation. DESIGN: Human Articular Chondrocytes (HACs) were cultured under hypoxia and stimulated with the chondrogenic growth factor BMP-2. The phenotype of the chondrocytes was studied by RT-PCR, and the cartilage-specific type II collagen production and deposition were also examined by western immunoblot and immunofluorescence. The Bone Morphogenetic protein (BMP) signalling pathway was also analysed. RESULTS: BMP-2 is much more efficient to stimulate the expression of the cartilage-specific gene COL2A1 by HACs when cultured under hypoxia (1%O2) compared to normoxia (21%O2). Analysis of the BMP-activated signalling shows that the Smad pathway is inhibited under hypoxia, whereas p38 MAPK is activated, and is involved in a synergy between hypoxia and BMP signalling, thus contributing to the enhanced anabolic response. CONCLUSIONS: Our study shows that hypoxia interplays with a chondrogenic factor and enhances the overall anabolic activity of the HACs. Alternatively to Hypoxia-Inducible Factor (HIF) signalling, and through a cross-talk with the BMP signalling which involves the p38 pathway, hypoxic stimulation markedly increases the capability of chondrocytes to produce the cartilage-specific type II collagen. Therefore our study provides new evidences of the multilayered effects of hypoxia in the anabolic functions of chondrocytes. This understanding may help promoting the anabolic function of articular chondrocytes, and thus improving their manipulation for cell therapy.
