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Cardiac expression of Brn-3a and Brn-3b POU transcription factors and regulation of Hsp27 gene expression.

ABSTRACT: The Brn-3 family of transcription factors play a critical role in regulating expression of genes that control cell fate, including the small heat shock protein Hsp27. The aim of this study was to investigate the relationship between Brn-3a and Brn-3b and Hsp27 expression in the developing rodent heart. Brn-3a and Brn-3b were detected from embryonic days 9.5-10.5 (E9.5-E10.5) in the mouse heart, with significant increases seen later during development. Two isoforms (long and short) of each protein were detected during embryogenesis and postnatally. Brn-3a messenger RNA (mRNA) and protein were localized by E13.0 to the atrio-ventricular (AV) valve cushions and leaflets, outflow tract (OFT), epicardium and cardiac ganglia. By E14.5, Brn-3a was also localised to the septa and compact ventricular myocardium. An increase in expression of the long Brn-3a(l) isoform between E17 and adult coincided with a decrease in expression of Brn-3b(l) and a marked increase in expression of Hsp27. Hearts from Brn-3a-/- mice displayed a partially penetrant phenotype marked by thickening of the endocardial cushions and AV valve leaflets and hypoplastic ventricular myocardium. Loss of Brn-3a was correlated with a compensatory increase in Brn-3b and GATA3 mRNA but no change in Hsp27 mRNA. Reporter assays in isolated cardiomyocytes demonstrated that both Brn-3a and Brn-3b activate the hsp27 promoter via a consensus Brn-3-binding site. Therefore, Brn-3 POU factors may play an important role in the development and maintenance of critical cell types and structures within the heart, in part via developmental regulation of myocardial Hsp27 expression. Furthermore, Brn-3a may be necessary for correct valve and myocardial remodelling and maturation.

SUBMITTER: Farooqui-Kabir SR

PROVIDER: S-EPMC2673938 | biostudies-other | 2008 Sep

REPOSITORIES: biostudies-other

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Project description:In cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.Bioinformatics analysis was used to identify the regulatory promoter region and map transcription start site as well as transcription factor binding sites. Polymerase chain reaction (PCR) cloning was used to generate promoter constructs for reporter assays. Chromatin immunoprecipitation and site-directed mutagenesis were used to confirm the transcription start site and autoregulation. MCF-7 and Cos-7 breast cancer cells were used. Cells grown in culture were transfected with Brn-3b promoter and treated with growth factors or estradiol to test for effects on promoter activity. Quantitative reverse transcriptase PCR assays and immunoblotting were used to confirm changes in gene and protein expression.We cloned the Brn-3b promoter, mapped the transcription start site and showed stimulation by estradiol and growth factors, nerve growth factor and epidermal growth factor, which are implicated in breast cancer initiation and/or progression. The effects of growth factors are mediated through the mitogen-activated protein kinase pathway, whereas hormone effects act via oestrogen receptor ? (ER?). Brn-3b also autoregulates its expression and cooperates with ER? to further enhance levels.Key regulators of growth in cancer cells, for example, oestrogens and growth factors, can stimulate Brn-3b expression, and autoregulation also contributes to increasing Brn-3b in breast cancers. Since increasing Brn-3b profoundly enhances growth in these cells, understanding how Brn-3b is increased in breast cancers will help to identify strategies for reducing its expression and thus its effects on target genes, thereby reversing its effects in breast cancer cells.

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Cardiac expression of Brn-3a and Brn-3b POU transcription factors and regulation of Hsp27 gene expression.

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