Raymond B. Penn

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

Asthma is characterized by airway inflammation, remodeling, and hyperresponsiveness to contractile stimuli that promote airway constriction and wheezing. Here we present evidence that sphingosine 1‐phosphate (SPP) is a potentially important inflammatory mediator implicated in the pathogenesis of airway inflammation and asthma. SPP levels were elevated in the airways of asthmatic (but not control) subjects following segmental antigen challenge, and this increase was correlated with a concomitant increase in airway inflammation. Because human airway smooth muscle (ASM) cells expressed EDG receptors for SPP (EDG‐1, ‐3, ‐5, and ‐6), we examined whether SPP may play a role in airway inflammation and remodeling, by affecting ASM cell growth, contraction, and cytokine secretion. SPP is mitogenic and augments EGF‐ and thrombin‐induced DNA proliferation by increasing G1/S progression. SPP increased phosphoinositide turnover and intracellular calcium mobilization, the acute signaling events that affect ASM contraction. By modulating adenylate cyclase activity and cAMP accumulation, SPP had potent effects on cytokine secretion. Although SPP inhibited TNF‐α–induced RANTES release, it induced substantial IL‐6 secretion alone and augmented production of IL‐6 induced by TNF‐α. These studies are the first to associate SPP with airway inflammation and to identify SPP as an effective regulator of ASM growth, contraction and synthetic functions.

Regulation of G Protein-Coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) specifically interact with the agonist-activated form of G protein-coupled receptors (GPCRs) to effect receptor phosphorylation and desensitization. Recent studies demonstrate that GRK function is a highly regulated process, and it is perhaps in this manner that a handful of GRKs (7 have been identified to date) are able to regulate the responsiveness of numerous GPCRs in a given cell type in a coordinated manner. The mechanisms by which GRK activity is regulated can be divided into 3 categories: 1) subcellular localization; 2) alterations in intrinsic kinase activity; and 3) alterations in GRK expression levels. This review will summarize our current understanding of each of these regulatory processes, and offer explanations as to how such mechanisms influence GPCR regulation under various physiologic conditions.

Visualization of Agonist-induced Sequestration and Down-regulation of a Green Fluorescent Protein-tagged β2-Adrenergic Receptor

To date, the visualization of β2-adrenergic receptor (β2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a β2AR conjugated with green fluorescent protein (β2AR-GFP) provides the opportunity for a more extensive optical analysis of β2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of β2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different β-agonists revealed time courses of β2AR-GFP redistribution reflective of the intrinsic activity of each agonist.

MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation.

Asthma is frequently associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness to contractile agents. Although numerous hormones and cytokines have been shown to induce human ASM (HASM) proliferation, the cellular and molecular mechanisms underlying HASM hyperplasia are largely unknown. Here we characterize the roles of the mitogen-activated protein kinase (MAPK) superfamily [p42/p44 MAPK, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38] in mediating hormone- and cytokine-induced HASM proliferation. Significant enhancement of [3H]thymidine incorporation in HASM cultures was observed only by treatment with agents (epidermal growth factor, platelet-derived growth factor, thrombin, and phorbol 12-myristate 13-acetate) that promoted a strong and sustained activation of p42/p44 MAPK. Significant activation of the JNK/SAPK and p38 pathways was only observed on stimulation with interleukin (IL)-1β and tumor necrosis factor-α, agents that did not appreciably stimulate HASM proliferation. Two different inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), PD-98059 and U-0126, inhibited mitogen-induced [3H]thymidine incorporation in a manner consistent with their ability to inhibit p42/p44 activation. Elk-1 and activator protein-1 reporter activation by mitogens was similarly inhibited by inhibition of MEK, suggesting a linkage between p42/p44 activation, transcription factor activation, and HASM proliferation. These findings establish a fundamental role for p42/p44 activation in regulating HASM proliferation and provide insight into species-specific differences observed among studies in ASM mitogenesis.

Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation.

Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC(50) approximately 10 nM and approximately 3 microM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70(S6k)) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70(S6k), whereas a dominant-negative PtdIns 3-kinase (Deltap85) blocked EGF- and thrombin-stimulated pp70(S6k) activity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC50 ∼10 nM and ∼3 μM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70S6k) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a dominant-negative PtdIns 3-kinase (Δp85) blocked EGF- and thrombin-stimulated pp70S6kactivity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.

Epinephrine Protects Cancer Cells from Apoptosis via Activation of cAMP-dependent Protein Kinase and BAD Phosphorylation

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with β2-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.

Hybrid Transgenic Mice Reveal In Vivo Specificity of G Protein–Coupled Receptor Kinases in the Heart

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

β-Arrestins specifically constrain β2-adrenergic receptor signaling and function in airway smooth muscle

Chronic use of inhaled beta‐agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta‐agonist‐promoted desensitization of airway smooth muscle beta‐2‐adrenergic receptors, mediated by G protein‐coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta‐arrestin‐2 gene ablation augments beta‐agonist‐mediated airway smooth muscle relaxation, while augmenting beta‐agonist‐stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA‐mediated knockdown of arrestins also augments beta‐agonist‐stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E2 receptors were not affected by either beta‐arrestin‐2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta‐2‐adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.—Deshpande, D. A., Theriot, B. S., Penn, R. B., Walker, J. K. L. β‐Arrestins specifically constrain β2‐adrenergic receptor signaling and function in airway smooth muscle. FASEB J. 22, 2134–2141 (2008)

Cooperative mitogenic signaling by G protein-coupled receptors and growth factors is dependent on Gq/11

Previously we reported that the G protein‐coupled receptor (GPCR) agonist thrombin potentiated the mitogenic effect of epidermal growth factor (EGF) on human airway smooth muscle (ASM) by promoting sustained late‐phase activation of PI3K and p70S6K via a pathway dependent on Gβγ subunits of heterotrimeric G proteins. Here, we provide additional mechanistic insight and reveal the robustness of this phenomenon by demonstrating that H1 histamine and thromboxane receptors utilize the same mechanism to augment ASM growth via specific activation of the heterotrimeric G protein Gq/11. Thrombin, histamine, and U46619 all enhanced EGF‐stimulated [3H]‐thymidine incorporation as well as late‐phase Akt and p70S6K phosphorylation in ASM cultures. Heterologous expression of Gβγ sequestrants (GRK2CT‐GFP or GαiG203A), as well as GRK2NT‐GFP (an RGS protein for Gq/11) but neither p115RhoGEFRGS‐GFP (an RGS for G12/13) nor pertussis toxin pretreatment (inactivating Gi/o), attenuated the effects on both signaling and growth. Inhibition of Rho, Rho kinase, or Src, or modulation of arrestin expression did not significantly affect the cooperative signaling by EGF and any of the GPCR agonists. Thus, Gq/11‐coupled receptors are the principal GPCR subfamily mediating cooperative mitogenic signaling in ASM, acting through Gβγ‐dependent, and Src/arrestin‐independent activation of PI3K and p70S6K.—Kong, K. C., Billington, C. K., Gandhi, U., Panettieri, R. A., Penn, R. B. Cooperative mitogenic signaling by G protein‐coupled receptors and growth factors is dependent on Gq/11. FASEB J. 20, E880–E887 (2006)