A novel integrin alpha5beta1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells.
BACKGROUND
Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin alpha5beta1 on pancreatic stellate cells (PSC) in which it stimulates fibrogenesis, adhesion, migration, and proliferation.


OBJECTIVE
To determine the structural domain(s) in CCN2 that interact with integrin alpha5beta1 to regulation PSC functions.


METHODS
Primary activated rat PSC were tested for their adherence to isoforms of CCN2 comprising modules 1-4 (CCN2(1-4)), modules 3-4 (CCN2(3-4)), module 3 alone (CCN2(3)), or module 4 alone (CCN2(4)). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin alpha5beta1 antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin alpha5beta1 binding was analysed in cell free systems. The ability of CCN2(1-4), CCN2(3-4), or CCN2(4) to stimulate PSC migration was evaluated in the presence of anti-integrin alpha5beta1 or heparin.


RESULTS
PSC adhesion was stimulated by CCN2(1-4), CCN2(3-4), or CCN2(4) and supported by Mg2+ but not Ca2+. CCN2(4) supported PSC adhesion or migration were blocked by anti-integrin alpha5beta1 antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2(4) and integrin alpha5beta1 was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2(4) and integrin alpha5beta1 as well as CCN2(4) mediated PSC adhesion and migration.


CONCLUSIONS
A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha5beta1 binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.
Gao
R
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Center for Cell and Vascular Biology, Children's Research Institute, Columbus, OH 43205, USA.
Brigstock
D R
DR
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002474:Cell-Free System; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D008297:Male; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship
C526316:CCN2 protein, rat; D017077:Culture Media, Conditioned; D017874:Immediate-Early Proteins; D039081:Integrin alpha5beta1; D036341:Intercellular Signaling Peptides and Proteins; D055513:Connective Tissue Growth Factor