Andrew J. Danielsen

The EGF/ErbB receptor family and apoptosis.

The ErbB receptor family can activate a multitude of cell signaling pathways that involve many aspects of cellular function. The four members of the ErbB receptor family interact with diverse ligands and substrates, as well as with each other through cell surface heterodimerization. The sum of these diverse interactions is a signaling network that is complex but also finely regulated. Among the cellular functions influenced by ErbB signaling is cell survival. ErbB receptor signaling has been demonstrated to interact with all of the major mechanisms of cell death signaling in a manner that promotes cell survival. Survival factors such as Ras, PI3-K, Akt, and Bcl-x/-2 all have been shown to be activated by ErbB signaling (Fig. 5). ErbB abrogation of apoptotic signals has been shown to play an important role during embryonic tissue development, in normal adult tissue maintenance (e.g. mammary tissue, wound healing), and also in tumor development and progression. Although the majority of studies suggest that ErbB receptor family members are mediators of cell survival, there have been occasional reports suggesting that ErbB receptors can induce cell death under selected experimental conditions. While this apparent discrepancy remains unresolved, in many of these reports, cell death may be the result of anoikis in response to changes in the cytoskeleton associated with hyperstimulation of ErbB signaling. The notion that ErbB receptor family members function to promote cell survival is not a recent observation. However, how this family functions to prevent apoptosis is an area that only recently has been considered. The understanding of ErbB receptor signaling as it relates to the avoidance of apoptosis had profound implications for the treatment of solid tumors originating in multiple tissues, as well as for the treatment of neurodegenerative disease. Further elucidation of the complex relationships between ErbB receptor signaling networks and the apoptotic machinery is certain to yield biologically important and potentially life-saving information.

Ligand-independent oncogenic signaling by the epidermal growth factor receptor: v-ErbB as a paradigm

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While all of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth.

An Oncogenic Epidermal Growth Factor Receptor Signals via a p21-activated Kinase-Caldesmon-Myosin Phosphotyrosine Complex

Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth.

Intramyocardial pacing and sensing for the enhancement of cardiac stimulation and sensing specificity

Background: Intracardiac electrodes create an “antenna” capable of unintentionally recording and stimulating tissue beyond the chamber in which they are positioned, resulting in far‐field R wave oversensing in pacemakers and inappropriate detection in defibrillators. This feasibility study sought to determine whether a specially constructed lead with two distal totally intramyocardial electrodes could overcome these limitations.

Synchronous Ventricular Pacing without Crossing the Tricuspid Valve or Entering the Coronary Sinus—Preliminary Results

Background: Right ventricular apical (RVA) pacing promotes tricuspid regurgitation (TR), electromechanical dyssynchrony, and ventricular dysfunction. We tested a novel intramyocardial bipolar lead to assess whether stimulation of the atrioventricular septum (AVS) produces synchronous ventricular activation without crossing the tricuspid valve (TV).

Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

Mutations within members of the EGF/ErbB receptor family frequently release the oncogenic potential of these receptors, resulting in the activation of downstream signaling events independent of ligand regulatory constraints. We previously have demonstrated that the signal transduction events originating from S3-v-ErbB, a ligand-independent, oncogenic EGF receptor mutant, are qualitatively distinct from the ligand-dependent mitogenic signaling pathways associated with the wild-type EGF receptor. Specifically, expression of S3-v-ErbB in primary fibroblasts results in anchorage-independent growth, increased invasive potential, and the formation of a transformation-specific phosphoprotein signaling complex, all in a Ras-independent manner. Here we demonstrate the transformation-specific interaction between two components of this complex: the adaptor protein Grb2 and the cytoskeletal regulatory protein caldesmon. This interaction is mediated via both the amino-terminal SH3 and central SH2 domains of Grb2, and the amino-terminal (myosin-binding) domain of caldesmon. Expression of a dominant-negative Grb2 deletion mutant, which lacks the carboxy-terminal SH3 domain, in fibroblasts expressing S3-v-ErbB results in a reduction in phosphoprotein complex formation, the loss of anchorage-independent growth, and a reduction in invasive potential. Together, these results demonstrate a Ras-independent role for Grb2 in modulating cytoskeletal function during ligand-independent EGF receptor-mediated transformation, and provide further support for the hypothesis that ligand-independent oncogenic signaling is qualitatively distinct from ligand-dependent mitogenic signaling by the EGF receptor.

Tyrosine phosphorylation of caldesmon is required for binding to the Shc.Grb2 complex.

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, transforming mutant of the epidermal growth factor receptor. This oncogene has been shown to be sarcomagenicin vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M. J., Lingle, W. L., Salisbury, J. L., and Maihle, N. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11351–11356) showed that expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protein. This phosphorylation is transformation-associated, and the phosphorylated form of CaD is associated with a signaling complex consisting of Shc, Grb2, and Sos in transformed fibroblasts. To identify the tyrosine phosphorylation site(s) in the CaD molecule and to further elucidate the functional role of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we have generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cells (an immortalized chicken embryo fibroblast cell line), and transient transfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major sites of tyrosine phosphorylation on CaD. Interestingly, Tyr-27 is located within the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies also show that the tyrosine phosphorylation of CaD enhances its binding to the Shc·Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-165 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc·Grb2 complex. Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc·Grb2 complex.

Transvenous Stimulation of the Renal Sympathetic Nerves Increases Systemic Blood Pressure: A Potential New Treatment Option for Neurocardiogenic Syncope

Neurocardiogenic syncope (NCS) is a common and sometimes debilitating disorder, with no consistently effective treatment. NCS is due to a combination of bradycardia and vasodilation leading to syncope. Although pacemaker devices have been tried in treating the bradycardic aspect of NCS, no device‐based therapy exists to treat the coexistent vasodilation that occurs. The renal sympathetic innervation has been the target of denervation to treat hypertension. We hypothesized that stimulation of the renal sympathetic nerves can increase blood pressure and counteract vasodilation in NCS.

Novel balloon catheter device with pacing, ablating, electroporation, and drug-eluting capabilities for atrial fibrillation treatment--preliminary efficacy and safety studies in a canine model.

Pulmonary vein isolation is an established therapeutic procedure for symptomatic atrial fibrillation (AF). This approach involves ablation of atrial tissue just outside the pulmonary veins. However, patient outcomes are limited because of a high rate of arrhythmia recurrence. Ablation of electrically active tissue inside the pulmonary vein may improve procedural success, but is currently avoided because of the complication of postablation stenosis. An innovative device that can ablate inside pulmonary veins and prevent stenosis is a viable strategy to increase long-term efficacy. We have developed a prototypical balloon catheter device capable of nonthermal pulmonary vein ablation along with elution of an antifibrotic agent intended to eliminate arrhythmogenic substrate without the risk of stenosis and have demonstrated its functionality in 4 acute canine experiments. Further optimization of this device may provide an innovative means to simultaneously ablate and prevent pulmonary vein stenosis for improved AF treatment in humans.

Novel Percutaneous Epicardial Autonomic Modulation in the Canine for Atrial Fibrillation: Results of an Efficacy and Safety Study

Endocardial ablation of atrial ganglionated plexi (GP) has been described for treatment of atrial fibrillation (AF). Our objective in this study was to develop percutaneous epicardial GP ablation in a canine model using novel energy sources and catheters.