Differential Effects of Protein Kinase C, Ras, and Raf-1 Kinase on the Induction of the Cardiac B-type Natriuretic Peptide Gene through a Critical Promoter-proximal M-CAT Element

Abstract

The cardiac genes for the A-and B-type natriuretic peptides (ANP and BNP) are coordinately induced by growth promoters, such as ␣ 1 -adrenergic receptor agonists (e.g. phenylephrine (PE)). Although inducible elements in the ANP gene have been identified, responsible elements in the BNP gene are unknown. In this study, reporter constructs transfected into neonatal rat ventricular myocytes showed that in the context of 2.5 kilobase pairs of native BNP 5-flanking sequences, a 2-base pair mutation in a promoter-proximal M-CAT site (CAT-TCT) disrupted basal and PE-inducible transcription by more than 98%. Expression of constitutively active forms of Ras, Raf-1 kinase, and protein kinase C, all of which are activated by PE in cardiac myocytes, strongly stimulated BNP reporter expression. Isolated M-CAT elements conferred PE, protein kinase C, and Ras inducibility to a minimal BNP promoter, however, they did not confer Raf-1 inducibility. These results show that M-CAT elements can serve as targets for Ras-dependent, Raf-1-independent pathways, implying the involvement of c-Jun N-terminal kinase and/or p38 mitogen-activated protein kinases, but not extracellular signal-regulated protein kinase/mitogen-activated protein kinase. Moreover, the essential M-CAT element distinguishes the BNP gene from the ANP gene, which utilizes serum response elements and an Sp1-like sequence.The A-, B-, and C-type natriuretic peptides (NPs) 1 are structurally related cardiac-derived peptides with vasorelaxant, diuretic, and natriuretic effects (1-7). Upon treatment with stimuli that can eventually lead to the hypertrophic growth of ventricular myocytes, several embryonic cardiac genes are reactivated, including those for ANP, BNP, ␤-myosin heavy chain, and skeletal ␣-actin (6 -18). While the precise function of this recapitulation of embryonic cardiac gene expression is unclear, it can be speculated that increased NP production represents a compensatory endocrine response to stimuli that often increased blood pressure. Accordingly, a knowledge of the mechanisms by which the NPs are induced during the hypertrophic growth program will provide a better framework upon which to understand how the expression levels of the hormones are regulated under less severe, but nonetheless hemodynamically challenging physiological conditions.A variety of studies have addressed the mechanisms responsible for ANP induction in primary neonatal rat cardiac myocytes (11,12,15,17). Recent studies have demonstrated the importance of serum response elements (SREs) (18) as well as SP-1-like elements (16) in the transcriptional activation of ANP in response to ␣ 1 -adrenergic agonists. Earlier reports have also indicated the probable involvement of AP-1-binding cis-sequences, also known as 12-O-tetradecanoylphorbol-13-acetate response elements (TREs), in regulating transcription of human ANP (19). Accordingly, it is believed that ␣ 1 -adrenergic agonists, and perhaps other ANP inducers, stimulate myocardial cell signaling pathways, which eventually lead to the act...

p38 Mitogen-activated Protein Kinase Mediates the Transcriptional Induction of the Atrial Natriuretic Factor Gene through a Serum Response Element

“…Cell Culture-Primary ventricular myocytes, prepared from 1-4-day-old neonatal rats as described previously (9,15,17), were transfected (see below), plated onto fibronectin-coated plastic dishes, and then maintained for about 18 h in DMEM, 10% fetal bovine serum. The cultures were then washed briefly with medium, refed with serum-free, hormone-free DMEM containing 1 mg/ml bovine serum albumin, maintained for an additional 48 h and then extracted for reporter enzyme assays.…”

“…Transfections-After isolation, myocardial cells were resuspended at a density of 30 million cells/ml minimal medium (DMEM (Sigma) containing 1 mg/ml bovine serum albumin) and transfections were carried out as described previously (9,15,17). Briefly, for each transfection, 300 l, or 9 million cells, were mixed with 15 to 30 g of a reporter construct (e.g.…”

“…The levels of plasmid used in each culture within an experiment were equalized using empty vector DNA, such as pCMV6. Each 300-l aliquot was then electroporated in a Bio-Rad Gene Pulser at 500 V, 25 microfarads, 100 ⍀ in a 0.2-cm gap cuvette, a protocol that allows for the selective transfection of only cardiac myocytes (9,15,17). This procedure results in an approximate 30% viability (9); accordingly, the 3 million viable cells were plated into fibronectin-coated 35-mm wells.…”

“…Panel B, effects of MKK6 (Glu): the constructs diagrammed in Panel A, and SV40-␤-galactosidase, were each co-transfected into primary myocardial cells Ϯ MKK6 (Glu). After the 18-h cell plating period, the media were replaced with serum-free media and the MKK6 (Glu)-transfected cells were treated Ϯ SB 203580 (5 M) for 48 h, extracted and luciferase and ␤-galactosidase reporter assays carried out as described (9,15,17). Luciferase values were normalized with ␤-galactosidase values and plotted as relative luciferase compared with the control for each construct.…”

Natriuretic Peptides in the Regulation of Cardiovascular Physiology and Metabolic Events

“…In addition to GATA4, a number of other transcriptional regulators, such as nuclear factor of activated T‐cells (NFAT), NKX‐2.5, Elk‐1, activator protein 1, myocardin, serum response factor, and nuclear factor kappa B, have been shown to participate in transcriptional regulation of BNP 20, 27, 28, 29, 30, 31, 32. Several studies have also indicated a central role for M‐CAT element, a thyroid‐responsive element and shear stress‐responsive element on the BNP promoter regulating BNP transcriptional activity 33, 34, 35, 36. In conclusion, the factors regulating cardiac hypertrophy, mainly MAPKs and GATA4 transcription factor activity, are also associated with regulation of NP expression levels.…”