No abstract

Cardiac-directed expression of adenylyl cyclase type VI (AC VI ) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with AC VI expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by AC VI in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of AC VI down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca 2؉ -ATPase. We determined that the cAMPresponsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by AC VI . Overexpression of AC VI did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of AC VI increased expression of ATF3 protein, a suppressor of transcription. Following AC VI gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then showed that AC VI -induced ATF3 protein binds to the cAMP-responsive-like element on the PLB promoter and that overexpression of ATF3 in cardiac myocytes inhibits PLB promoter activity. These findings indicate that AC VI has effects on gene transcription that are not directly dependent on cAMP generation. Adenylyl cyclase (AC)1 is the effector molecule in the ␤-adrenergic receptor-G-protein-AC signaling pathway in cardiac myocytes and other cells. Previous studies showed that the amount of AC sets a limit on the ability of cardiac myocytes to generate cAMP (1), and cardiac-directed expression of AC type VI (AC VI ) has pronounced favorable effects on cardiovascular function in normal and failing hearts (2-6).The mechanisms explaining these favorable effects of AC VI are not precisely known. The most direct explanation is that the benefits stem from increased intracellular levels of cAMP; this explanation is contrary to current dogma in heart failure which asserts that inotropic agents that increase cAMP are bad for the heart. Indeed, pharmacological agents that stimulate the ␤-adrenergic receptor or decrease the breakdown of cAMP increase cardiac function but do not appear to prolong life (7-9). In contrast, AC VI , a dominant AC isoform in mammalian cardiac myocytes, improves global cardiac function, attenuates myocardial hypertrophy, and increases survival in murine cardiomyopathy (3, 4). However, when cardiac-directed ␤-adrenergic receptor expression is used to treat this same model, life is shortened (10), underscoring the marked differences evoked by these signaling elements, both of which are associated w...

Pulmonary fibroblasts regulate extracellular matrix production and degradation; thus, they are critical for maintenance of lung structure, function, and repair. In pulmonary fibrosis, fibroblasts produce excess collagen and form fibrotic foci that eventually impair lung function, but the mechanisms responsible for these alterations are not known. Receptors coupled to the stimulation of cAMP production can inhibit activation of fibroblasts and thereby are antifibrotic. To test whether this signaling pathway is altered in pulmonary fibrosis, we compared the ability of normal adult human pulmonary fibroblasts to generate and respond to cAMP with that of cells isolated from lungs with idiopathic pulmonary fibrosis. Serum-and transforming growth factor (TGF)-␤-stimulated cell proliferation was inhibited ϳ50% by forskolin and ϳ100% by prostaglandin (PG) E 2 in the normal cells but substantially less in the diseased cells. Collagen synthesis was also inhibited Ͼ50% by the same drugs in the normal cells but significantly less so in the diseased cells, despite responding with similar increases in cAMP production. Although expression of protein kinase A (PKA) and cAMPstimulated PKA activity were similar in both the normal and diseased cell types, forskolin-and PGE 2 -stimulated cAMP response element-binding protein (CREB) phosphorylation was decreased in the diseased cell lines compared with the normal cells. cAMP-mediated activation and TGF-␤-mediated inhibition of CREB DNA binding was also diminished in the diseased cells. Thus, pulmonary fibroblasts derived from patients with pulmonary fibrosis are refractory to the inhibition by cAMP due to altered activity of components distal to the activity of PKA, in particular the phosphorylation of CREB.

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP2 (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP2 receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.

Previous data has shown that adenylyl cyclase type 6 (AC6) is expressed principally in lipid rafts or caveolae of cardiac myocytes and other cell types while certain other isoforms of AC are excluded from these microdomains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we show AC6 is localized in lipid rafts of COS-7 cells (expressing caveolin-1) and in HEK-293 cells or cardiac fibroblasts isolated from caveolin-1 knock-out mice (both of which lack prototypical caveolins). To determine the region of AC6 that confers raft localization, we independently expressed each of the major intracellular domains, the N-terminus, C1 and C2 domains, and examined their localization with various approaches. The N-terminus did not associate with lipid rafts or caveolae of either COS-7 or HEK-293 cells nor did it immunoprecipitate with caveolin-1 when expressed in COS-7 cells. By contrast, the C1 and C2 domains each associated with lipid rafts to varying degrees and was present in caveolin-1 immunoprecipitates. There were no differences in the pattern of localization of either the C1 or C2 domains between COS-7 and HEK-293 cells. Further dissection of the C1 domain into four individual proteins indicated that the N-terminal half of this domain is responsible for its raft localization. To probe for a role of a putative palmitoylation motif in the C-terminal portion of the C2 domain, we expressed various truncated forms of AC6 lacking most or all of the C-terminal 41 amino acids. These truncated AC6 proteins were not altered in terms of their localization in lipid rafts or their catalytic activity, implying that this C-terminal region is not required for lipid raft targeting of AC6. We conclude that while the C1 domain may be most important, both the C1 and C2 domains of AC6 play a role in targeting AC6 to lipid rafts.