Brian P. Head

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.

Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

Background:Exposure to anesthetics during synaptogenesis results in apoptosis and subsequent cognitive dysfunction in adulthood. Probrain-derived neurotrophic factor (proBDNF) is involved in synaptogenesis and can induce neuronal apoptosis via p75 neurotrophic receptors (p75NTR). proBDNF is cleaved into mature BDNF (mBDNF) by plasmin, a protease converted from plasminogen by tissue plasminogen activator (tPA) that is released with neuronal activity; mBDNF supports survival and stabilizes synapses through tropomyosin receptor kinase B. The authors hypothesized that anesthetics suppress tPA release from neurons, enhance p75NTR signaling, and reduce synapses, resulting in apoptosis. Methods:Primary neurons (DIV5) and postnatal day 5-7 (PND5-7) mice were exposed to isoflurane (1.4%, 4 h) in 5% CO2, 95% air. Apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Dendritic spine changes were evaluated with the neuronal spine marker, drebrin. Changes in synapses in PND5-7 mouse hippocampi were assessed by electron microscopy. Primary neurons were exposed to tPA, plasmin, or pharmacologic inhibitors of p75NTR (Fc-p75NTR or TAT-Pep5) 15 min before isoflurane. TAT-Pep5 was administered by intraperitoneal injection to PND5-7 mice 15 min before isoflurane. Results:Exposure of neurons in vitro (DIV5) to isoflurane decreased tPA in the culture medium, reduced drebrin expression (marker of dendritic filopodial spines), and enhanced Cl-Csp3. tPA, plasmin, or TAT-Pep5 stabilized dendritic filopodial spines and decreased Cl-Csp3 in neurons. TAT-Pep5 blocked isoflurane-mediated increase in Cl-Csp3 and reduced synapses in PND5-7 mouse hippocampi. Conclusion:tPA, plasmin, or p75NTR inhibition blocked isoflurane-mediated reduction in dendritic filopodial spines and neuronal apoptosis in vitro. Isoflurane reduced synapses and enhanced Cl-Csp3 in the hippocampus of PND5-7 mice, the latter effect being mitigated by p75NTR inhibition in vivo. These data support the hypothesis that isoflurane neurotoxicity in the developing rodent brain is mediated by reduced synaptic tPA release and enhanced proBDNF/p75NTR-mediated apoptosis.

Caveolae and Lipid Rafts G Protein-Coupled Receptor Signaling Microdomains in Cardiac Myocytes

Abstract: A growing body of data indicates that multiple signal transduction events in the heart occur via plasma membrane receptors located in signaling microdomains. Lipid rafts, enriched in cholesterol and sphingolipids, form one such microdomain along with a subset of lipid rafts, caveolae, enriched in the protein caveolin. In the heart, a key caveolin is caveolin‐3, whose scaffolding domain is thought to serve as an anchor for other proteins. In spite of the original morphologic definition of caveolae (“little caves”), most work related to their role in compartmenting signal transduction molecules has involved subcellular fractionation or immunoprecipitation with anti‐caveolin antibodies. Use of such approaches has documented that several G protein‐coupled receptors (GPCR), and their cognate heterotrimeric G proteins and effectors, localize to lipid rafts/caveolae in neonatal cardiac myocytes. Our recent findings support the view that adult cardiac myocytes appear to have different patterns of localization of such components compared to neonatal myocytes and cardiac fibroblasts. Such results imply the existence of multiple subcellular microdomains for GPCR‐mediated signal transduction in cardiac myocytes, in particular adult myocytes, and raise a major unanswered question: what are the precise mechanism(s) that determine co‐localization of GPCR and post‐receptor components with lipid rafts/caveolae in cardiac myocytes and other cell types?

Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1

Caveolae, small invaginations in the plasma membrane, contain caveolins (Cav) that scaffold signaling molecules including the tyrosine kinase Src. We tested the hypothesis that cardiac protection involves a caveolin‐dependent mechanism. We used in vitro and in vivo models of ischemia‐reperfusion injury, electron microscopy (EM), transgenic mice, and biochemical assays to address this hypothesis. We found that Cav‐1 mRNA and protein were expressed in mouse adult cardiac myocytes (ACM). The volatile anesthetic, isoflurane, protected ACM from hypoxia‐induced cell death and increased sarcolemmal caveolae. Hearts of wild‐type (WT) mice showed rapid phosphorylation of Src and Cav‐1 after isoflurane and ischemic preconditioning. The Src inhibitor PP2 reduced phosphorylation of Src (Y416) and Cav‐1 in the heart and abolished isoflurane‐induced cardiac protection in WT mice. Infarct size (percent area at risk) was reduced by isoflurane in WT (30.5±4 vs. 44.2±3, n=7, P<0.05) but not Cav‐1−/− mice (46.6±5 vs. 41.7±3, n=7). Cav‐1−/−mice exposed to isoflurane showed significant alterations in Src phosphorylation and recruitment of C‐terminal Src kinase, a negative regulator of Src, when compared to WT mice. The results indicate that isoflurane modifies cardiac myocyte sarcolemmal membrane structure and composition and that activation of Src and phosphorylation of Cav‐1 contribute to cardiac protection. Accordingly, therapies targeted to post‐translational modification of Src and Cav‐1 may provide a novel approach for such protection.—Patel, H. H., Tsutsumi, Y. M., Head, B. P., Niesman, I. R., Jennings, M., Horikawa, Y. Huang, D., Moreno, A. L., Patel, P. M., Insel, P. A., Roth, D. M. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin‐1. FASEB J. 21, 1565–1574 (2007)

Intracoronary Delivery of Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function and cAMP-Generating Capacity

BackgroundWe tested the hypothesis that the intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (ACVI) would increase cardiac function in pigs. Methods and ResultsLeft ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after the intracoronary delivery of histamine followed by the intracoronary delivery of an adenovirus encoding lacZ (control) or ACVI (1.4×1012 vp). Animals that had received ACVI gene transfer showed increases in peak LV dP/dt (average increase of 1267±807 mm Hg/s;P =0.0002) and cardiac output (average increase of 39±20 mL · kg−1 · min−1;P <0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving ACVI gene transfer showed increased ACVI protein content (P =0.0007) and stimulated cAMP production (P =0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks (P <0.0002). ConclusionsIntracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning

Background— Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte–specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. Methods and Results— Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3β phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3β and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. Conclusions— Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension

Vasoconstriction and vascular medial hypertrophy, resulting from increased intracellular [Ca2+] in pulmonary artery smooth muscle cells (PASMC), contribute to elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Caveolae, microdomains within the plasma membrane, contain the protein caveolin, which binds certain signaling molecules. We tested the hy‐pothesis that PASMC from IPAH patients express more caveolin‐1 (Cav‐1) and caveolae, which contribute to increased capacitative Ca2+ entry (CCE) and DNA synthesis. Immunohistochemistry showed increased expression of Cav‐1 in smooth muscle cells but not endothelial cells of pulmonary arteries from patients with IPAH. Subcellular fractionation and electron microscopy confirmed the increase in Cav‐1 and caveolae expression in IPAH‐PASMC. Treatment of IPAH‐PASMC with agents that deplete membrane cholesterol (methyl‐β‐cyclodextrin or lovastatin) disrupted caveo‐lae, attenuated CCE, and inhibited DNA synthesis of IPAH‐PASMC. Increasing Cav‐1 expression of normal PASMC with a Cav‐1‐encoding adenovirus increased caveolae formation, CCE, and DNA synthesis;treatment of IPAH‐PASMC with siRNA targeted to Cav‐1 produced the opposite effects. Treatments that down‐regulate caveolin/caveolae expression, including cho‐lesterol‐lowering drugs, reversed the increased CCE and DNA synthesis in IPAH‐PASMC. Increased caveolin and caveolae expression thus contribute to IPAH‐PASMC pathophysiology. The close relationship between caveolin/caveolae expression and altered cell physiology in IPAH contrast with previous results obtained in various animal models, including caveolin‐knockout mice, thus emphasizing unique features of the human disease. The results imply that disruption of caveolae in PASMC may provide a novel therapeutic approach to attenuate disease manifestations of IPAH.—Patel H. H., Zhang, S., Murray, F., Suda, R. Y. S., Head, B. P., Yokoyama, U., Swaney, J. S., Niesman, I. R., Schermuly, R. T., Savai Pullamsetti, S., Thistlethwaite, P. A., Miyanohara, A., Farquhar M. G., Yuan J. X.‐J., Insel P. A. Increased smooth muscle cell expression of caveolin‐1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension. FASEB J. 21, 2970–2979 (2007)

Propofol Neurotoxicity Is Mediated by p75 Neurotrophin Receptor Activation

Background: Propofol exposure to neurons during synaptogenesis results in apoptosis, leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75NTR) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75NTR. Methods: Days in vitro 5–7 neurons were exposed to propofol (3 &mgr;M) for 6 h and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75NTR−/− mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 &mgr;M, 15 min), a specific p75NTR inhibitor. P75NTR−/− neurons were transfected for 72 h with a lentiviral vector containing the synapsin-driven p75NTR gene (Syn-p75NTR) or control vector (Syn–green fluorescent protein) before propofol. To confirm our in vitro findings, wild-type mice and p75NTR−/− mice (PND5) were pretreated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. Results: Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75NTR−/− neurons. In p75NTR−/− neurons transfected with Syn-p75NTR, propofol significantly increased Cl-Csp3 in comparison with Syn–green fluorescent protein–transfected p75NTR−/− neurons. Wild-type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75NTR−/− mice. Conclusion: These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75NTR and the downstream effector RhoA kinase.