Faquan Liang’s research while affiliated with University of California, San Francisco and other places

Atrial natriuretic peptide (ANP) is a hormone that has both antihypertrophic and antifibrotic properties in the heart. We hypothesized that myocyte-derived ANP inhibits endothelin (ET) gene expression in fibroblasts. We have investigated the mechanism(s) involved in the antiproliferative effect of ANP on cardiac fibroblasts in a cell culture model. We found that cardiac myocytes inhibited DNA synthesis in co-cultured cardiac fibroblasts as did treatment with the ET-1 antagonist BQ610. The effect of co-culture was reversed by antibody directed against ANP or the ANP receptor antagonist HS-142-1. ANP inhibited the expression of the ET-1 gene and ET-1 gene promoter activity in cultured fibroblasts. The site of the inhibition was localized to a GATA-binding site positioned between -132 and -135 upstream from the transcription start site. GATA4 expression was demonstrated in cardiac fibroblasts, GATA4 bound the ET-1 promoter both in vitro and in vivo, and siRNA-mediated knockdown of GATA4 inhibited ET-1 expression. ET-1 treatment resulted in increased levels of phospho-serine(105) GATA4 in cardiac fibroblasts and this induction was partially suppressed by co-treatment with ANP. Collectively, these findings suggest that locally produced ET-1 serves as an autocrine stimulator of fibroblast proliferation, that ANP produced in neighbouring myocytes serves as a paracrine inhibitor of this proliferation, and that the latter effect operates through a reduction in GATA4 phosphorylation and coincident reduction in GATA4-dependent transcriptional activity.

The peroxisome proliferator activated receptors (PPARs) appear to have beneficial effects in the cardiovascular system. PPAR gamma has been shown previously to exert an inhibitory effect on cardiac myocyte hypertrophy in vivo and in vitro. Using endothelin to activate the hypertrophic program in neonatal rat cardiac myocytes, we demonstrate that PPAR alpha ligands (fenofibrate and WY14,643) suppress hypertrophy-dependent increases in protein synthesis, cell surface area, and sarcomeric organization in vitro. This was accompanied by a decrease in brain natriuretic peptide gene expression, a marker of transcriptional activation in hypertrophy. These effects were equivalent to or greater than those seen with the PPAR gamma agonist rosiglitazone. Fenofibrate and rosiglitazone suppressed endothelin stimulation of human brain natriuretic peptide gene promoter activity, and this effect was amplified by cotransfection of PPAR alpha and PPAR gamma expression vectors, respectively. The fenofibrate-dependent suppression of endothelin's stimulatory activity was dependent upon promoter sequence positioned between -904 and -40 relative to the transcription start site and did not appear to involve a number of positive and negative regulatory elements that are known to govern transcription of this gene. These findings suggest that PPAR alpha ligands could prove to be useful in the management of disorders associated with hypertrophy and remodeling of the myocardium.

Brain natriuretic peptide (BNP) gene expression is a well documented marker of hypertrophy in the cardiac myocyte. Triiodothyronine (T3), the bioactive form of thyroid hormone, triggers a unique form of hypertrophy in cardiac myocytes that accompanies the selective activation or suppression of specific gene targets. In this study, we show that the BNP gene is a target of T3 action. BNP secretion was increased 6-fold, BNP mRNA levels 3-fold, and BNP promoter activity 3–5-fold following T3 treatment. This was accompanied by an increase in myocyte size, sarcomeric organization, and protein synthesis. Of note, several of the responses to T3 synergized with those to the conventional hypertrophic agonist endothelin. The response to the liganded thyroid hormone receptor (TR) was mediated by an unusual thyroid hormone response element located between −1000 and −987 relative to the transcription start site. Both TR homodimers and TR·retinoid X receptor heterodimers associated with this element in an electrophoretic mobility shift assay. Protein fragments harboring the LXXLL motifs of the coactivators GRIP1 and SRC1 or TRAP220 interacted predominantly with the TR·retinoid X receptor heterodimeric pair in a ligand-dependent fashion. Both TR homodimers and heterodimers in the unliganded state selectively associated with glutathione S-transferase-nuclear receptor corepressor fragments harboring one of three receptor interaction domains containing the sequence (I/L)XX(I/V)I. These interactions were dissociated following the addition of T3. Collectively, these findings identify the BNP gene as a potential model for the investigation of TR-dependent gene regulation in the heart.

Activation of brain natriuretic peptide (BNP) gene promoter activity represents one of the earliest and most reliable markers of ventricular cardiac myocyte hypertrophy. We recently demonstrated that mechanical strain increases immunoreactive BNP secretion, steady-state BNP mRNA levels and BNP gene transcriptional activity in neonatal rat myocyte cultures. We have also shown that strain-dependent BNP gene transcription is critically dependent on the functional integrity of a number of integrins (specfically beta1, beta3, and alpha(v)beta5 integrins) present on the surface of cardiac myocytes. When used alone, each of these antibodies resulted in a significant reduction in strain-dependent activation of a transfected hBNP-luciferase reporter and inhibition of a number of signaling pathways that have been linked to stimulation of this reporter (e.g., extracellular signal regulated kinase and c-Jun amino terminal kinase). The present study shows that combinations of these antibodies resulted in further reductions in hBNP gene promoter activity and inhibition of the relevant signaling cascades. These studies provide further support for the importance of integrin-matrix interactions in promoting strain-dependent changes in cardiac myocyte gene transcription.

Application of mechanical strain to neonatal rat ventricular myocytes in culture evokes changes in gene expression reminiscent of those that occur with hypertrophy in vivo, such as stimulation of brain natriuretic peptide (BNP) gene expression. Here, we show that a major component of strain-dependent BNP promoter activation results from stimulation of p38 mitogen-activated protein kinase (MAPK) in the cardiac myocyte. Strain increased p38 activity in a time-dependent fashion. The p38 inhibitor SB203580 led to a reduction of approximately 60% in strain-activated human BNP (hBNP) promoter activity. Cotransfection of wild-type p38 increased both basal and strain-dependent promoter activity, while cotransfection with MKK6AL, a dominant-negative inhibitor of p38 MAPK kinase, resulted in partial inhibition of either p38- or strain-activated hBNP promoter activity. p38 MAPK increased hBNP promoter activity through activation of the transcription factor NF-kappaB. Activation of the hBNP promoter by either p38 or strain was mediated by DNA elements present in the 5' flanking sequence of the gene. Mechanical strain promoted assembly of NF-kappaB components on these DNA elements in vitro. Thus, induction of the hBNP promoter by mechanical strain depends, at least in part, on stimulation of p38 and subsequent activation of NF-kappaB. This activation may play an important role in signaling the increased BNP gene expression that accompanies hemodynamic overload and cardiac hypertrophy in vivo.

The atrial natriuretic peptide receptor (NPR-A) Is expressed in smooth muscle cells of the vasculature, where it is thought to signal the vasodilatory properties of the peptide. Despite its important role as a regulator of cardiovascular homeostasis, relatively little is known of the genomic factors governing expression of this gene. We show here that NPR-A promoter activity is reduced by 50–75% when any of three GC-rich sites are mutated. Simultaneous mutation of all three leads to a >90% reduction in NPR-A promoter activity. Transfection of wild-type, but not mutant, decoy oliogonucleotides encoding any one of the sites reduces NPR-A activity, presumably reflecting competition for a common transcription factor. Gel shift analyses show that each of the wild-type, but not the mutant, sites interferes with the formation of selected DNA-protein complexes on the other sites. These complexes share similar electrophoretic mobility. Immunoperturbation studies show that one of these shared complexes contains Sp1, whereas two others contain Sp3. Overexpression of either Sp1 or Sp3 in a cell type containing very low levels of these transcription factors (i.e. Drosophila Schneider cells) leads to induction of the wild-type, but not the mutant, NPR-A promoter. The data suggest that the Sp1 family of transcription factors plays a central role in NPR-A gene transcription. The association of Sp1 family members with transcriptional regulation of a number of genes involved in hemodynamic control will be discussed.

In 1981 DeBold et al. (1) made a seminal observation that opened an entirely new field of investigation in cardiovascular research. They found that injection of atrial, but not ventricular, extracts into test animals resulted in a natriuretic diuresis and reduction in intravascular volume. This activity was originally termed atrial natriuretic factor, and later, atrial natriuretic peptide (ANP), following its isolation and characterization. ANP is a member of a family of peptides, each encoded by a different gene (Fig. 1). The sites of production of these peptides and, to some degree, their functional activity are typically unique for each member of the group. This chapter will focus on the molecular and cellular mechanisms that govern the production and activity of the natriuretic peptides (NP), with particular emphasis on the heart.

Using a device that applies cyclical strain (1 Hz) to ventricular cardiocytes cultured on collagen-coated silicone elastomer surfaces, we have demonstrated strain-dependent increases in brain natriuretic peptide (BNP) secretion, BNP mRNA levels, and expression of a transiently transfected −1595 human BNP-luciferase reporter. When actinomycin D (10 μm) was introduced concomitantly with the strain stimulus, the strain-induced increase in BNP mRNA was eliminated, and the decay of transcripts was identical in the control and strained cells, indicating the lack of independent effects on transcript stability. Strain-dependent −1595 human BNP-luciferase activity was completely inhibited by chelerythrine, 2-aminopurine, genistein, and W-7 and only partially or not at all by KN-62, wortmannin, and H-89. The effects of these individual agents paralleled their effects on mitogen-activated protein kinase (MAPK) activity, but not c-Jun N-terminal kinase (JNK) activity, in the cells. Overexpression of wild-type MAPK and, to a lesser extent, JNK increased strain-dependent BNP promoter activity, whereas dominant-negative mutants of MAPK kinase, JNK kinase, or Ras completely blocked strain-dependent reporter activity. These findings provide the first demonstration that mechanical strain can increase myocardial gene expression through a transcriptional mechanism and suggest important roles for MAPK and JNK in mediating this effect.