Interacción de la metaloproteinasa de matriz 9 con células de leucemia linfocítica crónica: caracterización bioquímica funcional

• Autores: Estefanía Ugarte Berzal
• Directores de la Tesis: Ángeles García Pardo (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2014
• Idioma: español
• Tribunal Calificador de la Tesis: Francisco Sánchez Madrid (presid.), Miguel Campanero Garcia (secret.), Alicia García Arroyo (voc.), Cecilia Muñoz Calleja (voc.), Joaquín Teixidó Calvo (voc.)
• Materias:
  • Ciencias médicas
    • Ciencias clínicas
      • Oncología
      • Genética clínica
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood of CD5+ B lymphocytes and their progressive infiltration of the bone marrow and secondary lymphoid organs. These migration processes are mediated, among other molecules, by matrix metalloproteinases (MMPs). We and others have reported that proMMP (92 kDa) is the major MMP in CLL cells and plays an important role in CLL migration and invasion. Moreover, in contrast with normal B cells, proMMP¿9 is present at the CLL cell surface. Using immunoprecipitation, cell fractionation and confocal microscopy we have demonstrated that ¿4ß1 integrin and 190 kDa CD44v constitute a docking complex for proMMP¿9 in CLL cells. Binding of proMMP¿9 to this complex inhibited cell migration and induced cell survival, thus contributing to CLL progression. Using proMMP¿9 recombinant proteins containing deletions of various domains, we show that the proMMP¿9 hemopexin domain (PEX9) is required for its interaction with CLL cells. Since this interaction may constitute a therapeutic target in CLL, and to help designing specific inhibitors, we have addressed its detailed study. CLL cells bound to the GST¿PEX9 fusion protein generated here, but not to GST, and this binding was primarily mediated by ¿4ß1 integrin. Upon the preparation of truncated GSTPEX9 forms containing the structural blades D1D2 or D3D4, we have demonstrated that CLL cells also efficiently bound to the GST¿D1D2 and GST¿D3D4 proteins and this binding involved two different receptors: GST¿D3D4 primarily bound to ¿4ß1 integrin, while GST¿D1D2 bound to CD44. Furthermore, both regions, D1D2 and D3D4 inhibited CLL cell migration. By preparing overlapping synthetic peptides spanning the entire PEX9 sequence, we identified two specific cell binding sites: The FPGVPLDHDVFQYREKAYFC sequence, located in D1 and contained in peptide P6 and the FDAIAEWIGNQLYFKDGKYW sequence, located in D4 and contained in peptide P3. Both peptides inhibited cell adhesion to proMMP¿9 and GST¿PEX9 as well as transendothelial migration. Moreover, combination of P3 and P6 synergistically increased the inhibitory effect of the individual peptides. Therefore, P3 and P6 could constitute excellent targets to prevent proMMP¿9 contribution to CLL pathogenesis. Vascular endothelial growth factor (VEGF) is also involved in the extravasation and survival of CLL cells. VEGF is a proangiogenic factor also produced by CLL cells and whose elevated levels correlate with bad prognosis. Our results have demonstrated that addition of VEGF to CLL cells significantly reduced proMMP¿9 expression and in a dose¿dependent manner. Inhibition of VEGFR2 blocked this effect, indicating that VEGF signaled through this receptor. Reduction of proMMP¿9 resulted in inhibition of CLL migration through endothelium or Matrigel, confirming the important role of proMMP¿9 in these processes. RT¿PCR analyses indicated that VEGF regulation of proMMP¿9 was at the transcriptional level. Indeed, VEGFVEGFR2 interaction induced phosphorylation of the transcription factor STAT1 and STAT1 silencing restored proMMP¿9 production and CLL migration. As both, VEGF and proMMP¿9, are abundant components of the LLC microenvironment we have also studied the possible regulation of VEGF by proMMP¿9. CLL cell incubation with proMMP¿9 transcriptionally upregulated VEGF levels. In contrast, cell incubation with GST¿PEX9 reduced VEGF expression. These results were confirmed in vasculogénesis assays, where CLL cells preincubated with proMMP¿9 increased tube formation in co¿cultures with HUVEC cells, while preincubation with GST¿PEX9 inhibited vasculogénesis Additionally, immunoprecipitation analyses indicated a possible association between the receptors for proMMP¿9 (¿4ß1/CD44 ) and VEGF (VEGFR2), that could potentiate intracellular signaling and CLL cell expansion. Altogether, our results provide novel evidences for the pathogenic role of proMMP¿9 and VEGF in CLL and strongly suggest that both proteins could be good therapeutic targets in this malignancy.