RESUMEN
We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4ß1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4ß1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC(50) values of 138 and 279 µm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.
Asunto(s)
Movimiento Celular , Precursores Enzimáticos/metabolismo , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Antineoplásicos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/genética , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Humanos , Integrina alfa4beta1/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Persona de Mediana Edad , Mutagénesis , Mutación Missense , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Mapeo Peptídico , Fosforilación/efectos de los fármacos , Fosforilación/genética , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Regulación hacia Arriba , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Células Endoteliales/citología , Humanos , ARN Interferente Pequeño , Factor de Transcripción STAT1/genética , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti-MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and alpha4beta1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to alpha4beta1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of alpha4beta1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. alpha4beta1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-alpha4, anti-beta1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that alpha4beta1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified alpha4beta1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.
Asunto(s)
Receptores de Hialuranos/metabolismo , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Isoformas de Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Adhesión Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Movimiento Celular , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Femenino , Silenciador del Gen , Variación Genética , Humanos , Receptores de Hialuranos/química , Receptores de Hialuranos/genética , Integrina alfa4beta1/antagonistas & inhibidores , Integrina alfa4beta1/química , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Metaloproteinasa 9 de la Matriz/química , Persona de Mediana Edad , Peso Molecular , Complejos Multiproteicos , Invasividad Neoplásica , Isoformas de Proteínas/química , ARN Interferente Pequeño/genéticaRESUMEN
The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL+ cells. We have previously reported that VHL(-) cells fail to form beta1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.