Relevance of truncating titin mutations in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), a genetically heterogeneous cardiac disease characterized by left ventricular dilatation and systolic dysfunction, is caused majorly by truncations of titin (TTN), especially in A-band region. Clinical interpretation of TTN-truncating variants (TTNtv) has been challenged by the existing inaccurate variant assessment strategies and uncertainty in the true frequency of TTNtv across the general population. We aggregated TTNtv identified in 1788 DCM patients and compared the variants with those reported in over 60,000 Exome Aggregation Consortium reference population. We implemented our current variant assessment strategy that prioritizes TTNtv affecting all transcripts of the gene, and observed a decline in the prevalence of TTNtv in DCM. Despite this decline, TTNtv are more prevalent in DCM patients compared with reference population (p = 4.1 × 10(-295) ). Moreover, our extended analyses confirmed the enrichment of TTNtv not only in the A-band but also in the I/A-band junction of TTN. We estimated the probability of pathogenicity of TTNtv affecting all transcripts of TTN, identified in unselected DCM patients to be 97.8% (likelihood ratio (LR) = 42.2). We emphasize that identifying a TTNtv, especially in the A-band region, has a higher risk of being disease-causing than previously anticipated, and recommend prioritizing TTNtv affecting at least five transcripts of the gene.

Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.

Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

Genomic organization and promoter analysis of the human heat shock factor 2 gene.

Heat shock factor 2 (HSF2) is a member of the heat shock transcription factor family, which appears to be activated during differentiation and development rather than on cellular stress. Here we report the isolation and characterization of the human hsf2 gene and its 5'-flanking region. The transcription unit of the human hsf2 gene consists of 13 exons dispersed over 33 kbp of genomic DNA on chromosome 6. The hsf2 mRNA is transcribed from multiple start sites, and initiation from the major site results in a transcript of 2.45 kb. A functional promoter, as determined by the ability to direct expression of a transiently transfected luciferase reporter gene, resides in a 950-bp upstream region of the human hsf2 gene. Examination of the core promoter sequence revealed a high GC content and lack of a canonical TATA box. This feature seems to be common among various species, as comparison of the hsf2 proximal promoter sequences from human, mouse, and rat showed distinct conserved regions. Moreover, the overall architecture of the human hsf2 gene is similar to its mouse counterpart. A comparison between human hsf2 gene and other hsf genes showed striking similarities in exon size. However, the exons are assembled in an hsf-specific manner.

Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.

Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356-362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091-5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264-268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.

Differentiation lineage-specific expression of human heat shock transcription factor 2.

Differentiation of multipotential hematopoietic cells into lineage-committed precursors involves the selection and maintenance of appropriate programs of gene expression, regulated by specific transcription factors. Using human K562 erythroleukemia cells capable of differentiating along erythroid and megakaryocytic lineages, we explore the differentiation-related role of heat shock transcription factor 2 (HSF2), which belongs to a family of transcription factors generally known to regulate heat shock gene expression. We demonstrate that enhanced HSF2 expression and the acquisition of HSF2 DNA binding activity are strictly specific for erythroid characteristics of K562 cells. Our results reveal a multistep regulatory process of HSF2 gene expression. In K562 cells undergoing hemin-mediated erythroid differentiation, the increase in HSF2 protein levels is preceded by transcriptional induction of the HSF2 gene, accompanied by increased HSF2 mRNA stability. In contrast, during megakaryocytic differentiation induced by the phorbol ester TPA, expression of HSF2 is rapidly down-regulated, leading to a complete loss of the HSF2 protein. These results indicate that the determination of HSF2 expression occurs at the early stages of lineage commitment. Taken together, our data suggest that HSF2 could function as a lineage-restricted transcription factor during differentiation of K562 cells along either the erythroid or the megakaryocytic pathway.

Stage-specific expression and cellular localization of the heat shock factor 2 isoforms in the rat seminiferous epithelium.

Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, we examined HSF2 expression at specific stages of the rat seminiferous epithelial cycle. We found that expression of the alternatively spliced HSF2-alpha and HSF2-beta isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrated that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I-IV and in the nuclei of round spermatids at stages V-VIIab. In contrast a strong HSF2 immunoreactivity was detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis revealed that these structures are mainly cytoplasmic bridges between germ cells. Our results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells.

Hormonal and developmental regulation of DAX-1 expression in Sertoli cells.

Mutations in the human DAX-1 gene lead to X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. DAX-1 has been proposed to play a role in steroidogenesis because it is highly expressed in adrenocortical and testicular Leydig cells and because loss-of-function mutations lead to low serum levels of steroid hormones. Recent reports of DAX-1 expression in hypothalamus and pituitary, however, suggest additional functions for this protein. Here we demonstrate that DAX-1 is expressed in Sertoli cells of rat testis. This expression is regulated during spermatogenesis and peaks during the androgen-sensitive phase of the spermatogenic cycle. In addition, we show that DAX-1 expression in Sertoli cells is regulated developmentally. Maximum levels are present in the rat between postnatal days 20 and 30, during the first spermatogenic wave. Moreover, we show that activation of the cAMP-signaling pathway by the pituitary hormone FSH leads to a potent down-regulation of DAX-1 expression in cultured Sertoli cells. This down-regulation requires transcription and de novo protein synthesis. Taken together, these data indicate that DAX-1 expression in Sertoli cells may influence the development of spermatogenic cells in response to steroid and pituitary hormones.