RESUMEN
Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.
Asunto(s)
Carcinoma Pulmonar de Lewis/genética , Endotelio Vascular/patología , Neovascularización Patológica/genética , Canales Catiónicos TRPV/genética , Animales , Señalización del Calcio/genética , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucina/administración & dosificación , Leucina/análogos & derivados , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Sulfonamidas/administración & dosificación , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Madin-Darby canine kidney (MDCK)-D1 cells, a canine renal epithelial cell line, co-express at least three different P2Y receptor subtypes: P2Y(1), P2Y(2), and P2Y(11) (24). Stimulation of P2Y receptors in these cells results in the release of arachidonic acid (AA) and metabolites and the elevation of intracellular cAMP. To define in more precise terms the signaling contributed by the MDCK-D1 P2Y(2) (cP2Y(2)) receptor, we have cloned and heterologously expressed it in CF2Th (canine thymocyte) cells, a P2Y(2)-null cell. Analysis by RT-PCR indicated that canine P2Y(2) receptors are expressed in skeletal muscle, spleen, kidney, lung, and liver. When expressed in CF2Th cells, cP2Y(2) receptors promoted phospholipase C-mediated phosphatidylinositol (PI) hydrolysis [uridine 5'-triphosphate > or = ATP > adenosine 5'-diphosphate > 2MT-ATP] and mobilization of intracellular Ca(2+). In contrast to their actions in MDCK-D1 cells, cP2Y(2) receptors did not stimulate formation of cAMP or AA release when expressed in CF2Th cells. The data indicate that cell setting plays an essential role in the ability of P2Y receptors to regulate AA release and cAMP formation. In particular, renal epithelial cells preferentially express components critical for cP2Y(2)-induced cAMP formation, including the expression of enzymes involved in the generation and metabolism of AA and receptors that respond to PGE(2).
Asunto(s)
Riñón/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Bovinos , Línea Celular , Clonación Molecular , AMP Cíclico/biosíntesis , ADN/biosíntesis , ADN/genética , Humanos , Ratones , Datos de Secuencia Molecular , Fosfatidilinositoles/metabolismo , ARN/biosíntesis , ARN/genética , Ratas , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2Y2 , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Linfocitos T/metabolismoRESUMEN
We have studied G(q)-linked ANG II signaling [inositol phosphate (IP) accumulation, Ca(2+) mobilization] in primary cultures of rat cardiac fibroblasts (CFs) and have found that ANG II initiates a protein kinase C (PKC)-mediated negative feedback loop that rapidly terminates the ANG II response. Pharmacological inhibition of PKC by staurosporine and GF-109203X doubled IP production over that achieved in response to ANG II alone. Inhibition of PKC also led to larger Ca(2+) transients in response to ANG II, suggesting that Ca(2+) mobilization was proportional to G(q)-phospholipase C-IP(3) activity under the conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMA halved IP formation, with an EC(50) approximately 1 nM; 4alpha-PMA was inactive. Time course data demonstrated that ANG II-mediated IP production fully desensitized within 30 s; PKC inhibition reduced the rate and extent of this desensitization. In cells desensitized to ANG II, a purinergic agonist still mobilized intracellular Ca(2+), indicating that desensitization was homologous. The ANG II-induced Ca(2+) signal was fully resensitized within 30 min. The data demonstrate that a large portion of the IP-Ca(2+) responses of rat CFs to ANG II are short-lived because of rapid, PKC-mediated desensitization.
Asunto(s)
Angiotensina II/farmacología , Ácido Egtácico/análogos & derivados , Miocardio/citología , Miocardio/enzimología , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Vasoconstrictores/farmacología , Factores de Edad , Animales , Tampones (Química) , Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Ácido Egtácico/farmacología , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/enzimología , Fosfatos de Inositol/metabolismo , Masculino , Miocardio/química , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Receptores Purinérgicos/metabolismo , Transducción de Señal/efectos de los fármacos , Uridina Trifosfato/farmacologíaRESUMEN
Cardiac fibroblasts (CFs) are an important cellular component of myocardial responses to injury and to hypertrophic stimuli. We studied G protein-coupled receptors to understand how CFs integrate signals that activate G(q), G(s), and G(i). We predicted that the second messenger pathways present in CFs were distinct from those in cardiac myocytes and that unique signaling interactions existed in the CFs. ANG II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP) production 2.2- to 7-fold. Each of these agonists elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) via release from the intracellular Ca(2+) storage compartment. Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase either IP production or [Ca(2+)](i). Although agonists that activated IP and Ca(2+) transients had no effect on cAMP production when administered alone, these agents potentiated the beta(2)-adrenergic response two- to fourfold. Hormones known to inhibit adenylyl cyclase activity in cardiac myocytes, such as ET-1 and carbachol, failed to lower the beta-adrenergic response in fibroblasts. Order of potency and inhibitor data indicate that the functional receptor subtypes in these cells are beta(2), P2Y(2), and AT(1) for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G protein-linked receptors that couple to G(q) and G(s), with little or no coupling to G(i). The expression of receptors and their coupling to G(q)- but not to G(i)-linked responses distinguishes the signaling in CFs from that in myocytes. Furthermore, agonists that activate G(q) in CFs potentiate stimulation of G(s), an example of signaling cross talk not observed in adult myocytes. These data suggest that G protein-mediated signaling in CFs is unique and may contribute to the specificity of hormone and drug action on individual cell types within the heart.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP/metabolismo , Miocardio/enzimología , Receptor Cross-Talk/fisiología , Transducción de Señal/fisiología , Adenosina Trifosfato/farmacología , Agonistas Adrenérgicos beta/farmacología , Angiotensina II/farmacología , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Carbacol/farmacología , Cardiotónicos/farmacología , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Inositol 1,4,5-Trifosfato/metabolismo , Isoproterenol/farmacología , Masculino , Miocardio/citología , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Simpatomiméticos/farmacología , Fosfolipasas de Tipo C/metabolismo , Uridina Trifosfato/farmacología , Vasoconstrictores/farmacologíaAsunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Osteoblastos/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Células 3T3 , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Radioisótopos de Calcio , Técnicas de Cultivo de Célula/métodos , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo , Colorantes Fluorescentes , Fura-2 , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Osteoblastos/efectos de los fármacos , Técnicas de Placa-Clamp , Técnica de Dilución de Radioisótopos , Ratas , Espectrometría de Fluorescencia/métodos , Células Tumorales CultivadasRESUMEN
Osteoblast Ca2+ channels play a fundamental role in controlling intracellular and systemic Ca2+ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the alpha1C-a and the alpha1C-d isoforms. The ability of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and structural analogs to modulate expression of voltage-sensitive calcium channel mRNA transcripts was then investigated. ROS 17/2.8 cells were cultured for 48 h in the presence of either 1,25(OH)2D3,1,24-dihydroxy-22-ene-24-cyclopropyl D3 (analog BT) or 25-hydroxy-16-ene-23-yne-D3 (analog AT), and the levels of mRNA encoding alpha1C were quantitated using a competitive reverse transcription-polymerase chain reaction assay. We found that 1, 25(OH)2D3 and analog BT reduced steady state levels of alpha1C mRNA. Conversely, the Ca2+-mobilizing analog AT did not alter steady state levels of voltage-sensitive calcium channel mRNA. Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor.
Asunto(s)
Calcitriol/fisiología , Canales de Calcio/genética , Osteoblastos/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/fisiología , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Cráneo/metabolismo , Células Tumorales CultivadasRESUMEN
Voltage-Sensitive Calcium Channels (VSCCs) are important cell surface regulators of membrane permeability to Ca2+. In non-excitable cells such as osteoblasts, VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Effort focused on the large membrane-spanning alpha 1 subunit that is responsible for ion translocation. We designed primers using conserved alpha 1 subunit sequences common to many channel isoforms from various tissues and species. The generation of several RT-PCR products from various osteoblastic cell lines and subsequent sequencing of these products indicated that osteoblasts express at least two variants of the L-type VSCC which share regions of identity to the CaCh2 isoform first identified in cardiac myocytes. Our work provides the first molecular evidence for the existence and identity of VSCCs present in osteoblasts.
Asunto(s)
Canales de Calcio/genética , Osteoblastos/metabolismo , Células 3T3 , Animales , Membrana Celular/metabolismo , Clonación Molecular , Ratones , Reacción en Cadena de la Polimerasa , Conejos , Ratas , Transducción de Señal , Células Tumorales CultivadasRESUMEN
While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calcio/metabolismo , Retículo Endoplásmico/enzimología , Osteoblastos/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Antioxidantes/farmacología , Neoplasias Óseas/patología , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Fura-2/química , Hidroquinonas/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Nifedipino/farmacología , Osteoblastos/metabolismo , Osteosarcoma/patología , Terpenos/farmacología , Tapsigargina , Trombina/farmacología , Células Tumorales CultivadasRESUMEN
We report here that osteoblasts and osteoblast-like osteosarcoma cells express PMCA1b, an alternatively spliced transcript of plasma membrane Ca(2+)-ATPase. Synthetic oligonucleotide pairs were designed based upon unique regions of the cDNA encoding known PMCA isoforms (PMCA1-3) and used as primers in PCR-mediated amplification of cDNA synthesized from ROS 17/2.8 osteosarcoma cell RNA. A product was observed only when PMCA1-specific primers were present; no products were seen with PMCA2 or PMCA3 primers unless cDNA synthesized from rat brain RNA was present. Examination of the cDNA encoding the C terminus of PMCA1 from ROS 17/2.8 cells revealed that the mRNA is spliced to yield the PMCA1b isoform, a Ca(2+)-ATPase containing a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. PMCA1b was also detected in UMR-106-01 osteosarcoma cells and unpassaged primary rat calvarial osteoblasts. These results suggest that the regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca(2+)-ATPase.
Asunto(s)
ATPasas Transportadoras de Calcio/biosíntesis , Isoenzimas/biosíntesis , Osteoblastos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/genética , Membrana Celular/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Osteosarcoma/enzimología , Fosforilación , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificación , Empalme del ARN , Ratas , Homología de Secuencia de Aminoácido , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The endothelial cell is recognized as a critical modulator of blood vessel tone and reactivity. This regulatory function of endothelial cells occurs via synthesis and release of diffusible paracrine substances which induce contraction or relaxation of adjacent vascular smooth muscle. In response to stimulation by blood-borne agonists such as bradykinin or histamine, the endothelial cell utilizes cytosolic ionic Ca2+ as a trigger in the transduction of the stimulatory signal into a paracrine response. Considerable evidence has accumulated to indicate that various forms of biologically important oxidant stress alter vascular function in an endothelium-dependent manner. Further, oxidant stress is known to alter the mechanisms which govern Ca2+ homeostasis in the endothelial cell. Recently, we have described a model in which the oxidant tert-butylhydroperoxide is utilized to examine the effects of oxidant stress on Ca(2+)-dependent signal transduction in vascular endothelial cells. In this model, three temporal phases are evident and consist of (1) inhibition of the agonist-stimulated Ca2+ influx pathway, (2) inhibition of receptor-activated release of Ca2+ from internal stores and elevation of resting cytosolic free Ca2+ concentration, and (3) progressive increase in resting cytosolic Ca2+ concentration and loss of responsiveness to agonist stimulation. In this review, the mechanisms which characterize agonist-stimulated Ca2+ signaling in vascular endothelial cells, and the effects of oxidant stress on signal transduction will be described. The mechanisms potentially responsible for oxidant-induced inhibition of Ca2+ signaling will be considered.