Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis

Agonistic anti-beta1-adrenergic receptor autoantibodies from cardiomyopathy patients reduce the beta1-adrenergic receptor expression in neonatal rat cardiomyocytes

BACKGROUND: Autoantibodies directed against the beta1-adrenergic receptor have been described in patients with dilated cardiomyopathy. These autoantibodies exert an agonistic, chronotropic effect on spontaneously beating cultured neonatal rat cardiomyocytes. We studied the effect of such antibodies on beta1-adrenergic receptor expression. METHODS AND RESULTS: Cardiomyocytes were incubated with either the beta-adrenergic agonist isoproterenol or autoantibodies for 72 hours. beta-Adrenergic receptor expression was studied on the mRNA level with semiquantitative reverse transcription-polymerase chain reaction and on the protein level with immunoblotting. Isoproterenol downregulated both mRNA and beta1- and beta2-adrenergic receptor protein subtypes, whereas the anti-beta1-adrenergic receptor autoantibodies decreased only the beta1-adrenergic receptor mRNA and protein. Long-term incubation of cultured cardiomyocytes with isoproterenol or the anti-beta1-adrenergic receptor autoantibodies reduced the acute stimulatory effect of isoproterenol on the myocytes. These effects were prevented by incubating the cells with isoproterenol in the presence of propranolol or with anti-beta1-adrenergic receptor autoantibodies in the presence of bisoprolol. Bisoprolol also abolished the reduction of the beta1-adrenergic receptor expression caused by longer-term incubation with isoproterenol and the autoantibodies. CONCLUSIONS: We conclude that after longer-term treatment with the anti-beta1-adrenergic receptor autoantibodies, the rat cardiomyocytes showed a beta-adrenergic receptor expression similar to that observed in failing hearts from patients with dilated cardiomyopathy

Cardiac troponin I sense-antisense RNA duplexes in the myocardium

Natural antisense RNA is now thought to regulate, at least in part, a growing number of eukaryotic genes. It is becoming increasingly apparent that such endogenous antisense RNA molecules may modulate gene expression in a manner analogous to synthetic oligomers. Here, we report the detection of antisense-orientated RNA transcripts of cardiac specific troponin I in rat and human myocardium. Interestingly, the different sizes of the rat and human antisense cTNI transcripts suggest species-specific reverse transcription initiation sites. Moreover, for the first time in cardiomyocytes, we could demonstrate in vivo duplex formation between sense and antisense transcripts. The existence of antisense-sense duplexes represents compelling evidence and a potential mechanism for endogenous antisense transcript-mediated modulation of mRNA translation. The potential effect of attenuating translation was illustrated by in vitro and in vivo model systems. Testing several oligonucleotides based on the natural antisense sequences, the optimal region for inhibition of translation was identified as being close to the translational start codon

Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the {beta} 1-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the {beta} 1,-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the {beta} 1-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-{beta} 1-adrenoceptor antibodies in the induction of human cardiomyopath

Expression of alpha1-adrenergic receptor subtypes in heart cell culture

Three {alpha}1-AR subtypes have been cloned so far and are designated as {alpha}1a, {alpha}1b, and {alpha}1d. Organ-specific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of {alpha}1-AR subtypes on RNA- and protein-level in heart tissue, cultured cardiomyocytes and non-myocytes of the rat. Each {alpha}1-AR-subtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the {alpha}1a-AR subtype is preferentially expressed in adult cardiomyocytes, the {alpha}1b-AR subtype was preferentially expressed in the non-myocyte cell fraction. The RT-PCR results were confirmed by Western-blotting ({alpha}1b) and immunocytochemical studies. Incubation with an {alpha}1-agonist (phenylephrine) for 72 h led to a significant reduction of the {alpha}1b-AR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an {alpha}1-antagonist (prazosin) induced a 1.6 fold upregulation of the {alpha}1a-AR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the α1-AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents