Recent advances in cardiac beta(2)-adrenergic signal transduction

Recent studies have added complexities to the conceptual framework of cardiac beta-adrenergic receptor (beta-AR) signal transduction. Whereas the classical linear G(s)-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling cascade has been corroborated for beta(1)-AR stimulation, the beta(2)-AR signaling pathway bifurcates at the very first postreceptor step, the G protein level. In addition to G(s), beta(2)-AR couples to pertussis toxin-sensitive G(i) proteins, G(i2) and G(i3). The coupling of beta(2)-AR to G(i) proteins mediates, to a large extent, the differential actions of the beta-AR subtypes on cardiac Ca(2+) handling, contractility, cAMP accumulation, and PKA-mediated protein phosphorylation. The extent of G(i) coupling in ventricular myocytes appears to be the basis of the substantial species-to-species diversity in beta(2)-AR-mediated cardiac responses. There is an apparent dissociation of beta(2)-AR-induced augmentations of the intracellular Ca(2+) (Ca(i)) transient and contractility from cAMP production and PKA-dependent cytoplasmic protein phosphorylation. This can be largely explained by G(i)-dependent functional compartmentalization of the beta(2)-AR-directed cAMP/PKA signaling to the sarcolemmal microdomain. This compartmentalization allows the common second messenger, cAMP, to perform selective functions during beta-AR subtype stimulation. Emerging evidence also points to distinctly different roles of these beta-AR subtypes in modulating noncontractile cellular processes. These recent findings not only reveal the diversity and specificity of beta-AR and G protein interactions but also provide new insights for understanding the differential regulation and functionality of beta-AR subtypes in healthy and diseased hearts.     

Deactivation of the sympathetic nervous system in patients with chronic congestive heart failure

In this article, we review the basic biology, signal transduction pathways, and clinical pharmacology associated with cardiac β-adrenergic receptors (β-ARs) in the context of the use of β-blocking agents in patients with chronic congestive heart failure. Adrenergic receptors, particularly the β-AR subtypes (β₁-AR and β₂-AR), are known to play a critical role in the modulation of cardiac function, providing for both "adaptive" and "maladaptive" compensatory changes. In the context of exercise or self-preservation, the adrenergic nervous system, acting via β-ARs permits an appropriately rapid, highly-dynamic increase in cardiac function. Conversely, in individuals with chronic congestive heart failure, the sustained, heightened activation of adrenergic nervous system, as manifested by increases in circulating catecholamines, results in downregulation and desensitization of myocardial β-ARs, and potentially, significant myocardial damage. A number of recent clinical trials have demonstrated a marked mortality benefit from using β-blocking agents such as metoprolol and carvedilol in patients with heart failure. The pharmacologic properties of several of these drugs and some of the specifics of their usefulness and limitations are discussed herein.     

Expression of the cytoplasmic domain of β1 integrin induces apoptosis in adult rat ventricular myocytes (ARVM) via the involvement of caspase-8 and mitochondrial death pathway

Abstract Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β_1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.     

G-protein and Membrane Signaling in Cardiovascular Disease

Guanine nucleotide regulatory proteins (G-proteins) play a key role in the regulation of various signal transduction systems, which are implicated in the modulation of a variety of physiological functions such as platelet functions, including platelet aggregation, secretion, and clot formation, and cardiovascular functions, including arterial tone and reactivity. Several abnormalities in adenylyl cyclase activity, cAMP levels, and G-protein levels have shown to be responsible for the altered cardiac performance and vascular functions observed in cardiovascular disease states. The enhanced or unaltered levels of inhibitory G-proteins(Giα-2 and Giα-3) and mRNA have been reported in different models of hypertension, whereas Gsα levels were shown to be unaltered. These changes in G-protein were associated with functions. The enhanced levels of Giα proteins precede the development of blood pressure and suggest that over expression of Gi proteins may be one of the contributing factors for the pathogenesis of hypertension. On the other hand, the levels of Gsα and not of Giα proteins were decreased in volume- or pressure-overload hypertrophy. The responsiveness of adenylyl cyclase to β-adrenergic agonists was also attenuated. Similarly, is chemia was shown to be associated with decreased, increased,or unaltered levels of Gsα, with decreased levels of Giα, and with decreased responsiveness of adenylyl cyclase to various stimuli such as β-adrenergic agonists, guanine nucleotides, forskolin, etc. Thus,the altered levels of G-proteins and cAMP levels may be responsible for the impaired cardiovascular functions observed in hypertension, hypertrophy,and cardiac failure. This revised version was published online in August 2006 with corrections to the Cover Date.     

Tumor necrosis factor receptor 2 signaling limits β-adrenergic receptor-mediated cardiac hypertrophy in vivo

The in vivo role of TNF signaling in the genesis of β-adrenergic receptor (β-AR)-mediated cardiac hypertrophy is unknown. Wild-type (WT), TNF receptor 1 (TNFR1)-/- and TNFR2-/- mice were given isoproterenol (ISO, 12.5 μg/kg/h) or saline (SAL) for 1 or 7 days. In WT mice, 7 days of ISO yielded chamber/myocyte hypertrophy and hyperdynamic function without hypertension or fibrosis. WT ISO hearts exhibited an early (1 day) pro-inflammatory response with significant (p < 0.05) activation of nuclear factor (NF)-κB and activator protein 1 (AP-1) and upregulation of TNF, interleukin (IL)-1β and IL-6, inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1), together with increased anti-inflammatory IL-10. This response diminished markedly by 7 days. As compared with WT ISO mice, TNFR1-/- ISO mice exhibited significantly (p < 0.05) less NF-κB and AP-1 activation, less IL-1β, TNF, iNOS and MCP-1 upregulation, but greater IL-10 at 1 day. However, there were no differences in hypertrophy or contractility at 7 days. In contrast, TNFR2-/- ISO mice exhibited augmented NF-κB and AP-1 activation, increased IL-1β and diminished IL-10 expression at 1 day, and significant exaggeration of hypertrophy and less contractile augmentation at 7 days. Moreover, TNFR2-/- mice exposed to tenfold higher ISO doses displayed significant mortality. TNF signaling contributes to β-AR-mediated cardiac remodeling in vivo in a receptor-specific manner. Unopposed TNFR1 activation is pro-inflammatory, pro-hypertrophic and promotes functional decline. However, co-activation of TNFR2 during β-AR stress is anti-inflammatory and counterbalances these deleterious effects. TNF modulatory strategies that maintain TNFR2 signaling may help prevent the detrimental long-term effects of β-AR stimulation in the heart.     

PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation

Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (β-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed ≥50% by the PDE5A inhibitor sildenafil (SIL, 1 μM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of β3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in β3-AR knockout (β3-AR^−/−) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not β3-AR^−/− myocytes. These data support a cascade involving β3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial β-adrenergic stimulation.     

A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.     

βARKct: A Therapeutic Approach for Improved Adrenergic Signaling and Function in Heart Disease

One of the most powerful regulators of cardiovascular function is catecholamine-stimulated adrenergic receptor (AR) signaling. The failing heart is characterized by desensitization and impaired β-AR responsiveness as a result of upregulated G protein-coupled receptor kinase-2 (GRK2) present in injured myocardium. Deterioration of cardiac function is progressively enhanced by chronic adrenergic over-stimulation due to increased levels of circulating catecholamines. Increased GRK2 activity contributes to this pathological cycle of over-stimulation but lowered responsiveness. Over the past two decades the GRK2 inhibitory peptide βARKct has been identified as a potential therapy that is able to break this vicious cycle of self-perpetuating deregulation of the β-AR system and subsequent myocardial malfunction, thus halting development of cardiac failure. The βARKct has been shown to interfere with GRK2 binding to the βγ subunits of the heterotrimeric G protein, therefore inhibiting its recruitment to the plasma membrane that normally leads to phosphory-lation and internalization of the receptor. In this article we summarize the current data on the therapeutic effects of βARKct in cardiovascular disease and report on recent and ongoing studies that may pave the way for this peptide towards therapeutic application in heart failure and other states of cardiovascular disease.     

Polymorphisms of the Beta Adrenergic Receptor Predict Left Ventricular Remodeling Following Acute Myocardial Infarction

Purpose  

Prior studies demonstrate an association between specific beta-adrenergic receptor (β-AR) polymorphisms and clinical outcomes in patients with chronic heart failure and following acute coronary syndromes. The underlying mechanism may be due to differences in left ventricular remodeling. This study was undertaken to explore the relationship between LV remodeling after myocardial infarction and polymorphisms in the cardiac β1-AR and β2-AR genes.     

Compartmentalization of beta-adrenergic signals in cardiomyocytes

Activation of adrenergic receptors (AR) represents the primary mechanism to increase cardiac performance under stress. Activated βAR couple to Gs protein, leading to adenylyl cyclase-dependent increases in secondary-messenger cyclic adenosine monophosphate (cAMP) to activate protein kinase A. The increased protein kinase A activities promote phosphorylation of diversified substrates, ranging from the receptor and its associated partners to proteins involved in increases in contractility and heart rate. Recent progress with live-cell imaging has drastically advanced our understanding of the βAR-induced cAMP and protein kinase A activities that are precisely regulated in a spatiotemporal fashion in highly differentiated myocytes. Several features stand out: membrane location of βAR and its associated complexes dictates the cellular compartmentalization of signaling; βAR agonist dose-dependent equilibrium between cAMP production and cAMP degradation shapes persistent increases in cAMP signals for sustained cardiac contraction response; and arrestin acts as an agonist dose-dependent master switch to promote cAMP diffusion and propagation into intracellular compartments by sequestrating phosphodiesterase isoforms associated with the βAR signaling cascades. These features and the underlying molecular mechanisms of dynamic regulation of βAR complexes with adenylyl cyclase and phosphodiesterase enzymes and the implication in heart failure are discussed.     

Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues

Estrogens regulate various biological processes in a diverse range of reproductive and nonreproductive tissues through two genetically distinct but structurally related high affinity nuclear receptors, the estrogen receptor alpha and beta (ERα and ERβ). The physiological significance of the presence of two ERs that have redundant functions is not known. Several unique properties of ERβ together with its distinct expression patterns are considered to be, in part, the basis for diverse functional actions of estrogens and opposing actions of selective estrogen receptor modulators (SERMs) in different tissues. To understand how relative expression levels of two ERs correlate to seemingly dissimilar actions of estrogens and SERMs, quantitative methods are required that can precisely measure the levels of every isoform. Previously, methods to quantify eight ERα isoforms have been described [Poola I. (2003) Anal. Biochem. 314, 217–226]. In this article, real-time PCR-based molecular assays are described that can distinguish and quantify as low as 100 copies of 10 ERβ isoform mRNAs, the ERβ1, ERβ2, ERβ4, ERβ5, and ERβ exon 2Δ, exon 3Δ, exon 4Δ, exon 5Δ, exon 6Δ, and exons 5–6Δ. Each isoform mRNA is quantified using a specific primer pair and a 5′FAM (carboxy-fluorescein)- and 3′TAMARA (6-carboxy tetraethyl-rhodamine)-labeled probe and in comparison with a standard curve constructed with known copy numbers of its respective reverse transcribed cRNA. The devised assays were applied to profile 10 ERβ isoforms in four estrogen-sensitive tissues—ovary, breast, uterus, and bone. The sensitivity of detection of each isoform in these tissues varied from picograms to nanograms of reverse-transcribed total RNA depending on the isoform and the tissue. The results presented also show that each tissue has a distinct profile of 10 isoform mRNAs. Interestingly, ERα-negative breast cancer cell lines and tumors expressed significant amounts of ERβ isoforms suggesting that mitogenic stimulation by estrogen exists in these tissues. Bone tissues expressed several isoforms, although wild type was not present. In addition to the assay development, evidence is presented to demonstrate for the first time that ERβ4 and ERβ5 are full length receptors, contrary to previous reports that they are short receptors of exon 7–8. It is expected that the methods described here will significantly contribute to delineating the functional roles of various ERβ isoforms and in conjunction with ERα isoform profiling, will highly facilitate designing of individualized tissue specific therapies to treat estrogen-related pathologies.     

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca²⁺) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca²⁺ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects.     

Inhibition of rat granulosa cell differentiation by overexpression of Galphaq

Activation of FSH and LH receptors in undifferentiated granulosa cells (i.e., no prior exposure to FSH) results in comparable induction of progesterone production, but activation of the LH receptor is less effective than FSH in inducing aromatase and the native LH receptor. Because the LH receptor can also activate the Gαq signaling pathway, we investigated whether activation of this pathway could be responsible for these differences. Overexpression of Gαq inhibited FSH induction of both the estradiol and progesterone biosynthetic pathways as well as mRNA levels for cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), LH receptor (LHr), and P450aromatase (aromatase). This suppression was associated with a reduction (P < 0.05) in FSH-stimulated cAMP production. Lower cAMP levels were not due to reduced FSH receptor (FSHr) mRNA levels or reduced levels of Gαs. Phosphodiesterase (PDE) activity and regulator of G-protein signaling 2 (RGS2) mRNA levels were significantly (P < 0.05) increased by Gαq, both of which could account for diminished cAMP levels. We conclude that Gαq signaling pathway inhibits both estradiol and progesterone production comparably and thus activation of this pathway does not seem to account for differences between FSH and LH in the regulation of aromatase and the LH receptor.     

Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart

Cardiac fibroblasts become activated and differentiate to smooth muscle-like myofibroblasts in response to hypertension and myocardial infarction (MI), resulting in extracellular matrix (ECM) remodeling, scar formation and impaired cardiac function. cAMP and cGMP-dependent signaling have been implicated in cardiac fibroblast activation and ECM synthesis. Dysregulation of cyclic nucleotide phosphodiesterase (PDE) activity/expression is also associated with various diseases and several PDE inhibitors are currently available or in development for treating these pathological conditions. The objective of this study is to define and characterize the specific PDE isoform that is altered during cardiac fibroblast activation and functionally important for regulating myofibroblast activation and ECM synthesis. We have found that Ca²⁺/calmodulin-stimulated PDE1A isoform is specifically induced in activated cardiac myofibroblasts stimulated by Ang II and TGF-β in vitro as well as in vivo within fibrotic regions of mouse, rat, and human diseased hearts. Inhibition of PDE1A function via PDE1-selective inhibitor or PDE1A shRNA significantly reduced Ang II or TGF-β-induced myofibroblast activation, ECM synthesis, and pro-fibrotic gene expression in rat cardiac fibroblasts. Moreover, the PDE1 inhibitor attenuated isoproterenol-induced interstitial fibrosis in mice. Mechanistic studies revealed that PDE1A modulates unique pools of cAMP and cGMP, predominantly in perinuclear and nuclear regions of cardiac fibroblasts. Further, both cAMP-Epac-Rap1 and cGMP-PKG signaling was involved in PDE1A-mediated regulation of collagen synthesis. These results suggest that induction of PDE1A plays a critical role in cardiac fibroblast activation and cardiac fibrosis, and targeting PDE1A may lead to regression of the adverse cardiac remodeling associated with various cardiac diseases.     

Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β-adrenergic signaling defects including down-regulation of myocardial β-adrenergic receptors (β-ARs), functional β-AR uncoupling, and an up-regulation of the β-AR kinase (βARK1). Adenoviral-mediated gene transfer of the human β₂-AR or an inhibitor of βARK1 to these failing myocytes led to the restoration of β-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of βARK1 activity in the heart.     

Amelioration of Cardiac Remodeling in Congestive Heart Failure by β-Adrenoceptor Blockade is Associated with Depression in Sympathetic Activity

This study investigated whether improvement in cardiac function and attenuation of cardiac remodeling by some β-adrenoceptor (β-AR) antagonists were associated with a depression in sympathetic activity in congestive heart failure (CHF) due to myocardial infarction (MI). Although cardiac dysfunction, hypertrophy and dilatation as well as increased plasma level of catecholamines are known to occur in CHF, the relationship between these parameters is poorly understood. Three weeks after occlusion of the coronary artery, rats were treated daily with 20 and 75 mg/kg of either atenolol or propranolol for 5 weeks. Sham-operated rats served as controls. Both atenolol and propranolol at 20 and 75 mg/kg doses attenuated the MI-induced cardiac hypertrophy, increases in left ventricular (LV) end-diastolic pressure, LV end-systolic volume and LV end-diastolic volume as well as depressions in LV systolic pressure, LV fractional shortening and cardiac output. PR interval was decreased and QT _c interval was increased in CHF; these alterations were ameliorated by both atenolol and propranolol. The increased level of plasma epinephrine in CHF was also depressed by both low and high doses of atenolol and propranolol whereas the increased level of plasma norepinephrine was reduced by high but not low doses of these drugs. The results indicate that the beneficial effects of β-AR antagonists on cardiac remodeling and heart dysfunction in CHF may be due to the blockade of β-ARs in the myocardium and a depression in the sympathetic activity.     

Beta1-adrenergic receptors promote focal adhesion signaling downregulation and myocyte apoptosis in acute volume overload

Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (β(1)-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk(2) and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. β(1)-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with β(1)-AR agonists or adenoviral expression of β(1)-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by β(1)-ARs. Collectively, these data show that β(1)-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.     

Role of the β1 integrin molecule in T-cell activation and migration

β1 integrins play crucial roles in a variety of cell processes such as adhesion, migration, proliferation, and differentiation of lymphocytes. To understand the molecular mechanisms of these various biological effects, it is particularly important to analyze cell signaling through the β1 integrins. Our previous study showed that PLC-γ, pp125FAK (focal adhesion kinase), pp105, paxillin, p59fyn, p56lck, and ERK1/2 are phosphorylated in their tyrosine residues upon engagement of β1 integrins. We identified pp105 as Cas (Crk-associated substrate)-related protein and successfully cloned its cDNA. pp105 is a Cas homologue predominantly expressed in the cells of lymphoid lineage, which led us to designate it Cas-L. Like p130Cas, Cas-L contains a single SH3 domain and multiple SH2-binding sites (YXXP motif), which are suggested to bind SH2 domains of Crk, Nck, and SHPTP2. Subsequent studies revealed that pp125FAK binds Cas-L on its SH3 domain and phosphorylates its tyrosine residues upon β1 integrin stimulation. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. We have shown that Cas-L is involved in the T-cell receptor (TCR)/CD3 signaling pathway as well as the β1 integrin signaling pathway. Cas-L is transiently phosphorylated following CD3 crosslinking and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant (Cas-LΔSH3), which lacks the binding site for FAK, is still tyrosine-phosphorylated upon CD3 crosslinking but not upon β1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Finally, we have identified a crucial role of Cas-L in β1 integrin-mediated T-cell co-stimulation. We have found that this co-stimulatory pathway is impaired in the Jurkat T-cell line, and that the expression level of Cas-L is reduced in the Jurkat cells compared to peripheral T-cells. The transfection of Cas-L cDNA into Jurkat cells restored the β1 integrin-mediated co-stimulation, while the transfection of Cas-LΔSH3 mutant failed to do so, which contrasts with the case of CD3-mediated signaling. These results indicate that Cas-L plays a key role, through the association and phosphorylation by FAK, in β1 integrin-mediated T-cell co-stimulation. Moreover, tyrosine phosphorylation of Cas-L is critical for T-cell receptor and β1 integrin-induced T-lymphocyte migration. Taken together, Cas-L might be the bi-modal docking protein which assembles the signals through β1 integrins and TCR/CD3, and which participates in a variety of T-cell functions. Received: August 24, 1999 / Accepted: August 31, 1999     

Regulation of adenohypophyseal pyroglutamyl aminopeptidase II activity by thyrotropin-releasing hormone and phorbol esters

Thyrotropin-releasing hormone (TRH) is inactivated by a narrow specificity ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in the proximity of target cells. In adenohypophysis, PPII is present on lactotrophs. Its activity is regulated by thyroid hormones and 17β-estradiol. Studies with female rat adenohypophyseal cell cultures treated with 3,3′,5′-triiodo-l-thyronine (T₃) showed that hypothalamic/paracrine factors, including TRH, can also regulate PPII activity. Some of the transduction pathways involve protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP). The purpose of this study was to determine whether T₃ levels or gender of animals used to propagate the culture determine the effects of TRH or PKC. PPII activity was lower in cultures from male rats. In cultures from both sexes, T₃ induced the activity. The percentages of decrease due to TRH or PKC were independent of T₃ or gender; the percentage of decrease due to cAMP may also be independent of gender. These results suggest that T₃ and hypothalamic/paracrine factors may independently control PPII activity in adenohypophysis, in either male or female animals.