Quan He

Interleukin-1β Regulation of the Human Brain Natriuretic Peptide Promoter Involves Ras-, Rac-, and p38 Kinase–Dependent Pathways in Cardiac Myocytes

Because both the brain natriuretic peptide (BNP) gene and the cytokine interleukin-1beta (IL-1beta) are induced in the infarcted myocardium, localized production of IL-1beta may regulate the BNP gene. We tested whether (1) IL-1beta regulates the human BNP promoter, (2) cis elements in the proximal promoter respond to IL-1beta, and (3) mitogen-activated protein kinase (MAPK) signaling pathways [p42/44, c-jun (JNK) and p38 kinase] are involved. We transferred the hBNP promoter coupled to a luciferase reporter gene or constructs with mutations in the proximal promoter GATA and M-CAT elements into neonatal rat ventricular myocytes and treated the cells with IL-1beta for 24 hours. IL-1beta-stimulated hBNP luciferase activity was eliminated by pretreatment with the transcription inhibitor actinomycin D. Both the p38 kinase inhibitor SB205380 (SB) and cotransfection of a dominant-negative mutant of p38 kinase reduced IL-1beta stimulation of the hBNP promoter. Dominant-negative mutants of Ras and Rac inhibited IL-1beta-stimulated hBNP luciferase activity by 64% and 90%, respectively. Constitutively active forms of Rac and MKK6, the immediate upstream activator of p38, were stimulatory; however, only the effect of MKK6 was inhibited by SB. Neither the p42/44 nor the JNK pathway was involved in the action of IL-1beta. Both IL-1beta and MKK6 activation of the hBNP promoter were partially reduced when the promoter contained a mutated M-CAT element. In summary, (1) IL-1beta is a transcriptional activator of the hBNP promoter; (2) IL-1beta acts through a Ras-dependent pathway not coupled to activation of p42/44 MAPK or JNK; (3) IL-1beta acts through a Rac-dependent pathway, but the downstream effector is not known; and (4) IL-1beta activation of p38 kinase is partially involved in regulation of the hBNP promoter, targeting the proximal M-CAT element.

Src and Rac Mediate Endothelin-1 and Lysophosphatidic Acid Stimulation of the Human Brain Natriuretic Peptide Promoter

Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET) may be involved in the development of these diseases. ET has also been shown to activate phospholipase A2 (PLA2), and the resulting metabolites are important second messengers. We studied how ET and PLA2 metabolites regulate BNP gene expression. The human BNP (hBNP) promoter (from −1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVMs), and luciferase activity was measured as an index of promoter activity. ET induced BNP mRNA in NVMs as assessed by Northern blot. It also stimulated the hBNP promoter, an effect completely inhibited by actinomycin D. To test the involvement of different PLA2 isoforms, transfected cells were treated with various PLA2 inhibitors before stimulation with ET. Only Ca2+-independent PLA2 blockade prevented ET-stimulated hBNP promoter activity. The PLA2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter, but arachidonic acid itself did not. ET regulation of the hBNP promoter is pertussis toxin–sensitive. The nonreceptor tyrosine kinase Src and the small GTPase Rac mediate the effects of both ET and LPA in stimulation of the hBNP promoter. We studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from −1818 to −408 and from −408 to −40 reduced the effect of ET by 60% and 80%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 50% when the proximal GATA element was mutated. These data suggest that (1) ET activates the hBNP promoter through a transcriptional mechanism; (2) LPA, perhaps generated by iPLA2, is involved in the effect of ET; (3) Src and Rac mediate ET and LPA stimulation of the hBNP promoter; and (4) ET regulation of the hBNP promoter targets both distal and proximal cis elements.

PKA, Rap1, ERK1/2, and p90RSK mediate PGE2 and EP4 signaling in neonatal ventricular myocytes

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE(2) production reduces hypertrophy after myocardial infarction in mice and 2) PGE(2) acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE(2) activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE(2) activation of ERK1/2. PGE(2) activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE(2) regulation of gene expression. PGE(2) stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE(2) stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE(2) regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.

Interleukin-1β Regulates the Human Brain Natriuretic Peptide Promoter via Ca2+-Dependent Protein Kinase Pathways

Abstract —We have shown that interleukin-1β (IL-1β) activates the human brain natriuretic peptide (hBNP) promoter via a transcriptional mechanism. Others have reported that changes in intracellular calcium (Ca2+) mediate the action of IL-1β. We questioned whether Ca2+ and Ca2+-dependent pathways mediate IL-1β regulation of the hBNP promoter in cardiac myocytes. The hBNP promoter (−1818 to +100) coupled to a luciferase cDNA reporter gene was transferred into neonatal cardiac myocytes. Cells were then treated with agents that modify Ca2+ levels or inhibit Ca2+-dependent kinases, and luciferase activity was measured as an index of hBNP promoter activity. The Ca2+ ionophore A23187 increased hBNP promoter activity; however, neither EGTA nor nifedipine reduced IL-1β–stimulated promoter activity. Long-term treatment with thapsigargin, which depletes intracellular Ca2+ stores, decreased basal promoter activity and blocked the effect of IL-1β. Inhibition of protein kinase C completely blocked IL-1β–stimulated hBNP promoter activity, whereas inhibition of Ca2+/calmodulin-dependent kinase II decreased promoter activity by 40%. In contrast, inhibition of the Ca2+-regulated phosphatase calcineurin by cyclosporin A had no effect. These data suggest that (1) Ca2+ activates the hBNP promoter; (2) release of Ca2+ from intracellular stores is important to IL-1β regulation of the hBNP promoter, but transport via voltage-sensitive Ca2+ channels is not; (3) protein kinase C and Ca2+/calmodulin-dependent kinase II mediate the action of IL-1β; and (4) the phosphatase calcineurin is not involved in IL-1β regulation of the hBNP promoter. Thus, Ca2+ and Ca2+-dependent pathways are critical to IL-1β regulation of the hBNP promoter.

Tafazzin knockdown interrupts cell cycle progression in cultured neonatal ventricular fibroblasts

Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. Previous studies have shown that tafazzin knockdown promotes hypertrophy of neonatal cardiac myocytes. The current investigation was designed to show whether tafazzin knockdown affects cardiac fibroblast proliferation and collagen secretion, which contribute to fibrosis in dilated cardiomyopathy. In primary cultures of neonatal ventricular fibroblasts (NVFs) transduced with a tafazzin short hairpin RNA adenovirus, tafazzin knockdown increased production of reactive oxygen species and activation of mitogen-activated protein kinases and induced protein and DNA synthesis via cell cycle regulators. It also reduced intracellular ATP, activated AMPK, and caused multinucleation, hypertrophy, and enhanced collagen secretion. We concluded that tafazzin knockdown interrupts the NVF cell cycle and this in turn may contribute to fibrosis and dilated cardiomyopathy in Barth syndrome.

Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress.