ABSTRACT
Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.
Subject(s)
Apoptosis/physiology , DNA-Binding Proteins/physiology , Receptors, Steroid/physiology , T-Lymphocytes/physiology , Transcription Factors/physiology , Transcriptional Activation/genetics , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA Primers , DNA-Binding Proteins/genetics , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/physiology , Female , Genotype , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Nuclear Receptor Subfamily 4, Group A, Member 1 , Oligonucleotide Array Sequence Analysis , Pregnancy , Rats , Receptors, Antigen, T-Cell/physiology , Receptors, Cytoplasmic and Nuclear , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Transcription Factors/geneticsABSTRACT
Signaling through the protein phosphatase calcineurin may play a critical role in cardiac hypertrophy. The gene for Down Syndrome Critical Region-1 (DSCR1) encodes a protein that is an endogenous calcineurin inhibitor. This study was designed to test the hypothesis that DSCR1 is directly induced by biomechanical stimuli. Neonatal rat cardiac myocytes were exposed to biaxial cyclic mechanical strain; mechanical strain upregulated DSCR1 mRNA expression in a time- and amplitude-dependent manner (3.4 +/- 0.2-fold at 8% strain for 6 h, n = 11, P < 0.01), and this induction was angiotensin II and endothelin I independent. Biomechanical induction of DSCR1 mRNA was partially blocked by calcineurin inhibition with cyclosporine A (30 +/- 5%, n = 3, P < 0.01). DSCR1 promoter-reporter experiments showed that mechanical strain induced DSCR1 promoter activity by 2.3-fold and that this induction was completely inhibited by cyclosporin A. Furthermore, DSCR1 gene expression was increased in the left ventricles of mice with pressure-overload hypertrophy induced by transverse aortic banding. These data demonstrate that biomechanical strain directly induces gene expression for the calcineurin inhibitor DSCR1 in cardiac myocytes, indicating that mechanically induced DSCR1 may regulate the hypertrophic response to mechanical overload.
Subject(s)
Muscle Proteins/metabolism , Myocardium/metabolism , Animals , Calcineurin/physiology , Calcium/physiology , Cells, Cultured , Cytokines/pharmacology , DNA-Binding Proteins , Enzyme Activation , Gene Expression Regulation/physiology , Hypertension/metabolism , Hypertension/pathology , Hypertrophy , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred Strains , Mitogen-Activated Protein Kinases/metabolism , Muscle Proteins/genetics , Myocardium/cytology , Neurotransmitter Agents/pharmacology , Promoter Regions, Genetic/physiology , Protein Kinase C/metabolism , RNA Stability , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Stress, MechanicalABSTRACT
Biomechanical strain is a stimulus for cardiomyocyte hypertrophy and heart failure, but the underlying molecular mechanisms remain incompletely understood. Using an in vivo murine model of pressure overload and an in vitro model of mechanical stimulation of primary cardiomyocytes, we identified iex-1 as a gene activated during the early response of cardiomyocytes to hypertrophic stimuli and as a gene product that inhibits hypertrophy without affecting cardiomyocyte viability. On stimulation of cardiomyocytes, iex-1 mRNA and protein expression increased and translocation of the gene product to the cardiomyocyte nucleus occurred. iex-1 has previously been proposed as a mediator of NF-kappaB-dependent cell survival and growth in tumor cells. Here, we demonstrate that the biomechanical induction of iex-1 in cardiomyocytes was NF-kappaB-dependent, as overexpression of the NF-kappaB inhibitor IkappaBalpha completely inhibited strain-mediated iex-1 mRNA accumulation. The functional role of iex-1 was investigated by overexpressing wild-type iex-1 with replication-defective adenoviral gene transfer. Overexpression of iex-1 abolished cardiomyocyte hypertrophy by mechanical strain, phenylephrine, or endothelin-1 at levels that did not affect cell viability. These studies identify iex-1 as a biomechanical stress-inducible and NF-kappaB-dependent gene in cardiac muscle cells during the acute phase of hypertrophy with negative growth regulatory effects that may counterbalance early hypertrophic responses in activated cardiomyocytes.
