Domain 1 of the urokinase-type plasminogen activator receptor is required for its morphologic and functional, beta2 integrin-mediated connection with actin cytoskeleton in human microvascular endothelial cells: failure of association in systemic sclerosis endothelial cells.
OBJECTIVE
In systemic sclerosis (SSc) microvascular endothelial cells (MVECs), angiogenesis is blocked by matrix metalloproteinase 12-dependent cleavage of domain 1 of the urokinase-type plasminogen activator receptor (uPAR). Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and truncated uPAR are differentially associated with integrins and with motor components of the cytoskeleton.


METHODS
SSc and normal MVECs were isolated from human skin biopsy specimens and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of truncated and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by reverse transcription-polymerasechain reaction. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting.


RESULTS
Only full-size uPAR showed a connection with the actin cytoskeleton in ECs. This connection was mediated by the uPAR-associated alphaMu- and alphaX-subunits of beta2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with beta2 integrins or with actin. beta3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of Rac and Cdc42 (thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility) and blocked the integrin-engagement-delivered signals to the actin cytoskeleton. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that ligation of uPAR by uPA empowers the beta2/beta3 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc MVECs.


CONCLUSIONS
The reduced angiogenic properties of SSc MVECs can be explained by the effects of uPAR truncation and the subsequent loss of the beta2 integrin-mediated connection of uPAR with the actin cytoskeleton in these ECs.
Margheri
Francesca
F
University of Florence, Florence, Italy.
Manetti
Mirko
M
Serratì
Simona
S
Nosi
Daniele
D
Pucci
Marco
M
Matucci-Cerinic
Marco
M
Kahaleh
Bashar
B
Bazzichi
Laura
L
Fibbi
Gabriella
G
Ibba-Manneschi
Lidia
L
Del Rosso
Mario
M
D000199:Actins / Q000378:metabolism*; D018821:CD18 Antigens / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008833:Microcirculation / Q000166:cytology / Q000187:drug effects; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000503:physiopathology; D012867:Skin / Q000098:blood supply; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
C470196:ARHGAP32 protein, human; D000199:Actins; D018821:CD18 Antigens; D020690:GTPase-Activating Proteins; C404106:MRC2 protein, human; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D014568:Urokinase-Type Plasminogen Activator; D020764:cdc42 GTP-Binding Protein