Hypoxia induces metalloproteinase-9 activation and human vascular smooth muscle cell migration through low-density lipoprotein receptor-related protein 1-mediated Pyk2 phosphorylation.
OBJECTIVE
Hypoxia disturbs vascular function by promoting extracellular matrix remodeling. Extracellular matrix integrity and composition are modulated by metalloproteinases (MMPs). Our aim was to investigate the role of low-density lipoprotein receptor-related protein 1 (LRP1) in regulating MMP-9/MMP-2 activation and vascular smooth muscle cells (VSMCs) migration in response to hypoxia, and to elucidate the LRP1-signaling pathways involved in this process.


RESULTS
Western blot analysis showed that hypoxia induced a sustained phosphorylation of proline-rich tyrosine kinase 2 concomitantly with LRP1 overexpression in human VSMCs (hVSMCs). Deletion of LRP1 using small-interfering RNA technology or treatment of hVSMCs with the Src family kinase inhibitor PP2 impaired hypoxia-induced phosphorylation of proline-rich tyrosine kinase 2 levels. Coimmunoprecipitation experiments showed that the higher amounts of phosphorylation of proline-rich tyrosine kinase 2/LRP1β immunoprecipitates in hypoxic hVSMCs were abolished in PP2-treated hVSMCs. Both LRP1 silencing and PP2 treatment were highly effective in the prevention of hypoxia-induced MMP-9 activation and hVSMC migration. Cellular subfractionation experiments revealed that PP2 effects may be caused by impairment of hypoxia-induced nuclear factor-κβ translocation to the nucleus. ELISA measurements showed that LRP1 silencing but not PP2 treatment increased interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 secretion by hypoxic hVSMCs.


CONCLUSIONS
Our findings determine a crucial role of LRP1-mediated Pyk2 phosphorylation on hypoxia-induced MMP-9 activation and hVSMC migration and therefore in hypoxia-induced vascular remodeling. Both LRP1 silencing and PP2 treatments also influence hypoxia-induced proinflammatory effects in hVSMCs. Therefore, further studies are required to establish therapeutical strategies that efficiently modulate vascular remodeling and inflammation associated with hypoxia-vascular diseases.
From the Cardiovascular Research Center, CSIC-ICCC, IIB-Sant Pau, Barcelona, Spain (E.R.-L., J.C., L.N., L.B.); ICREC Research Program, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Spain (S.R., C.G.-M., A.B.-G.); and Cardiovascular Biochemistry Group, Biomedical Research Institute Sant Pau, IIB-Sant Pau, Barcelona, Spain (S.B.).
Elena
E
Revuelta-López
José
J
Castellano
Santiago
S
Roura
Carolina
C
Gálvez-Montón
Laura
L
Nasarre
Sonia
S
Benitez
Antoni
A
Bayes-Genis
Lina
L
Badimon
Vicenta
V
Llorente-Cortés
D021581:Active Transport, Cell Nucleus; D015687:Cell Hypoxia; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D016328:NF-kappa B / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction; D014162:Transfection
C111835:CCL2 protein, human; D018932:Chemokine CCL2; C508600:IL6 protein, human; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D051418:Focal Adhesion Kinase 2; C103547:PTK2B protein, human; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
LRP1 protein, human; hypoxia; matrix metalloproteinase 1; protein tyrosine kinase Pyk2