RESUMEN
CAIR-1/BAG-3 forms an EGF-regulated ternary complex with Hsp70/Hsc70 and latent phospholipase C-gamma (PLC-gamma). The expression of CAIR-1, CAI stressed-1, was induced in A2058 human melanoma cells by continuous exposure to CAI, an inhibitor of nonvoltage-gated calcium influx. CAIR-1 sequence is identical, save 2 amino acids, to BAG-3 also cloned recently as Bis, a member of the bcl-2-associated athanogene family. We show that CAIR-1/BAG-3 binds to Hsp70/Hsc70 in intact cells and this binding is increased by short term exposure to CAI (P<0.007). CAIR-1/BAG-3 is phosphorylated in vivo in the absence of stimulation. Basal phosphorylation is inhibited by treatment with d-erythrosphingosine (d-ES), a broad inhibitor of the protein kinase C family. CAIR-1/BAG-3 contains several PXXP SH3 binding domains leading to the hypothesis that it is a partner protein of phospholipase C-gamma. PLC-gamma is bound to CAIR-1/BAG-3 in unstimulated cells. It is increased by CAI or d-ES (P=0.05) treatment, and abrogated by EGF (r2=0.99); d-ES treatment blocks the EGF-mediated dissociation. We show that CAIR-1/BAG-3 binds to PLC-gamma and Hsp70/Hsc70 through separate and distinct domains. Hsp70/Hsc70 binds to the BAG domain of BAGs-1 and -3. CAIR-1/BAG-3 from control and EGF-treated cell lysates bound selectively to the SH3 domain of PLC-gamma, but not its N-SH2 or C-SH2 domains. Confirming the SH3 interaction, PLC-gamma was pulled down by CAIR-1/BAG-3 PXXP-GST fusions, but GST-PXXP constructs confronted with lysates from EGF-treated cells did not bind PLC-gamma as was seen in intact cells. Hsp70/Hsc70 was brought down by the PLC-gamma SH3 construct equally from native and EGF-treated cells, but did not bind the PXXP construct under either condition. We propose that CAIR-1/BAG-3 may act as a multifunctional signaling protein linking the Hsp70/Hsc70 pathway with those necessary for activation of the EGF receptor tyrosine kinase signaling pathways.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Isoenzimas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Bloqueadores de los Canales de Calcio/farmacología , Proteínas Portadoras/efectos de los fármacos , Línea Celular/efectos de los fármacos , Clonación Molecular , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Datos de Secuencia Molecular , Fosfolipasa C gamma , Fosforilación , Triazoles/farmacologíaRESUMEN
Fibroblast cytomorphology is tightly coupled to phenotypic expression, particularly as it relates to extracellular matrix protein synthesis and degradation. We have observed that calcium antagonists, such as verapamil and trifluoperazine, depolymerize actin filaments and alter fibroblast cell shape from bipolar to spherical. Characteristically, the depolymerization of actin filaments, which mediates the cell shape change, turns on procollagenase gene expression in normal human skin fibroblasts. We have found the same effects of calcium antagonists on cell shape, cytoskeletal components, and induction of procollagenase in the keloid fibroblasts of three cell lines, CB792, CW792, and WT949. Rounded cells were seen in 74.8% of verapamil-treated and 86.7% of trifluoperazine-treated cells, whereas only 1.1% of the control cells were spherical. The percentage of cells that synthesized collagenase in the control, verapamil-treated, and trifluoperazine-treated groups was 3.8%, 42.8%, and 53.4%, respectively. Approximately 60% of rounded cells exhibited increased collagenase synthesis when the cells were treated with a calcium antagonist. These results indicate considerable heterogeneity in the phenotypic response to morphologic change. The amount of procollagenase synthesized in a cell was estimated by the fluorescence intensity of the fluorescein-labeled antibody. The normalized fluorescence intensity of procollagenase in the control cells was about 2 to 2.6 times that of background. In contrast, the normalized fluorescence intensity of procollagenase in the calcium antagonist-treated cells was about 2.4 to 12 times that of background. This high intensity level indicates an increase in procollagenase production in the calcium antagonist-treated cells. Calcium green dye used to study cytosolic calcium revealed that after cells were treated with verapamil, the cytosolic calcium ion concentration first increased and then decreased. The change of cytosolic calcium ion concentration may be related to the depolymerization of actin filaments and the alteration of cell shape.
Asunto(s)
Distinciones y Premios , Bloqueadores de los Canales de Calcio/farmacología , Colagenasas/biosíntesis , Precursores Enzimáticos/biosíntesis , Fibroblastos/efectos de los fármacos , Queloide/patología , Trifluoperazina/farmacología , Verapamilo/farmacología , Calcio/análisis , Bloqueadores de los Canales de Calcio/administración & dosificación , Células Cultivadas , Colagenasas/efectos de los fármacos , Precursores Enzimáticos/efectos de los fármacos , Fibroblastos/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queloide/metabolismo , Valores de Referencia , Trifluoperazina/administración & dosificación , Verapamilo/administración & dosificaciónRESUMEN
Postburn dermal scars can be aesthetically disfiguring and severely disabling. Existing medical and surgical strategies to prevent or to treat scars are all too often disappointing. More effective strategies are needed. It has been postulated that cell membrane calcium channel blockers could potentially trigger extracellular matrix degradation in dermal scars, resulting in scar volume reduction. To test this hypothesis, we studied the effects of intralesional verapamil hydrochloride therapy, 0.1 to 0.5 mmol/L, on scar volume and color in patients with burns. Our study chronicles five reports on arbitrarily selected clinical cases of hypertrophic burn scarring and the effect of therapy with intralesional verapamil. The cellular functions and processes explaining this effect are discussed. There were no significant side effects or complications from the verapamil therapy. It is now clear that regulation of fibroblast calcium metabolism is a safe and often effective strategy to treat hypertrophic scars. To our knowledge, this is the first clinical report of control of burn scar with calcium channel blockers.
Asunto(s)
Quemaduras/complicaciones , Cicatriz Hipertrófica/tratamiento farmacológico , Verapamilo/uso terapéutico , Adolescente , Adulto , Cicatriz Hipertrófica/etiología , Femenino , Humanos , Inyecciones Intralesiones , Masculino , Persona de Mediana EdadRESUMEN
Interest in the role of electrical interactions as epigenetic regulators of wound healing had its beginnings nearly 40 years ago. Because the mechanisms of action are not understood (which obviates rational therapy), the empiric application of fields to wounds has produced mixed results. However, taken collectively, clinical trials have demonstrated some beneficial effects. Tests on soft tissues of animals have shown that electric stimulation can influence the rate of wound healing and scar strength. Natural epithelial-derived sodium currents have been discovered in the wounds of invertebrates and mammals. It is theorized that these currents may be a normal controlling factor in wound healing. Therefore, perturbation of these signals is important to understand. The purpose of this review is to put into proper perspective the biophysical, physiological, and clinical data pertaining to use of electricity to control wound healing, with the goal of minimizing much of the prevailing confusion.