RESUMEN
Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth.
Asunto(s)
Antineoplásicos/farmacología , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Oligopéptidos/farmacología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Microambiente Tumoral , Secuencia de Aminoácidos , Línea Celular Tumoral , Endopeptidasas , Células Endoteliales/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Gelatinasas/antagonistas & inhibidores , Gelatinasas/química , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Células Madre Mesenquimatosas/enzimología , Microvasos/patología , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Prolil Oligopeptidasas , Serina Endopeptidasas/químicaRESUMEN
Although several protein-protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1-specific properties. TRPP2/TRPC1 is activated in response to G-protein-coupled receptor activation and shows a pattern of single-channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain-of-function and loss-of-function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co-immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium-based Ca(2+) signalling.
Asunto(s)
Canales Iónicos/biosíntesis , Subunidades de Proteína/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Canales Catiónicos TRPP/química , Amilorida/farmacología , Animales , Línea Celular , Células Cultivadas , Cilios/metabolismo , Riñón/citología , Células LLC-PK1 , Elementos de la Serie de los Lantanoides/farmacología , Ratones , Agonistas Muscarínicos/farmacología , Neuronas/citología , Oxotremorina/análogos & derivados , Oxotremorina/farmacología , Ratas , Bloqueadores de los Canales de Sodio/farmacología , Porcinos , Canales Catiónicos TRPP/metabolismoRESUMEN
Depletion of intracellular Ca2+ stores leads to the activation of Ca2+ inflow through store-operated Ca2+ channels. Although the identity of these channels is unknown, there is considerable evidence that the transient receptor potential channel 1 (TRPC1) participates in the formation of these channels. We show that TRPC1 physically interacts with the a-isoform of the inhibitor of the myogenic family (I-mfa), a known inhibitor of basic helix-loop-helix transcription factors, in vitro and in vivo. The interaction is mediated by the C-terminal cytoplasmic tail of TRPC1 and the C-terminal cysteine-rich domain of I-mfa. Using the whole cell configuration of the patch clamp technique, we show that ectopic expression of I-mfa in CHO-K1 cells reduces native store-activated Ca2+ currents, whereas knock-down of endogenous I-mfa in A431 cells by RNA interference enhances these currents. Pipette perfusion of purified recombinant I-mfa rescues the effect of I-mfa knock-down on store-operated conductance. Finally, cell dialysis with a monoclonal antibody specific to TRPC1 results in the suppression of store-activated conductance in cells lacking I-mfa, but not in I-mfa expressing cells. We propose that I-mfa functions as a molecular switch to suppress the store dependence of TRPC1.
Asunto(s)
Canales de Calcio/metabolismo , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Calcio/metabolismo , Línea Celular , Cricetinae , Cisteína/química , Citoplasma/metabolismo , Electrofisiología , Femenino , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Miogenina/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPC , Factores de Transcripción/química , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80 kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92 kDa or 120 kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated beta-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.