The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth.
Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.
From the Departments of Pharmacology and.
Jacqueline
J
Sayyah
Pathology, University of California at San Diego, La Jolla, California 92093 and.
Alena
A
Bartakova
From the Departments of Pharmacology and.
Nekeisha
N
Nogal
the Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, Indiana 46202.
Lawrence A
LA
Quilliam
Pathology, University of California at San Diego, La Jolla, California 92093 and.
Dwayne G
DG
Stupack
From the Departments of Pharmacology and jhbrown@ucsd.edu.
Joan Heller
JH
Brown
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism*; D064593:Heterografts; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009368:Neoplasm Transplantation; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D019012:Integrin beta1; D009363:Neoplasm Proteins; C430803:RAP1A protein, human; C066737:RHOA protein, human; D013917:Thrombin; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
Cell Proliferation; Cell Signaling; G-protein-coupled Receptor (GPCR); Glioblastoma; Integrin; Rap1; Small GTPase; Thrombin