Here we report that PAI-1, in a VN-dependent manner, prevents the cooperation of avb3 integrin with insulin signaling in NIH3T3 fibroblasts, resulting in a decrease in insulin-induced pro
Trang 1P R I O R I T Y P A P E R
Plasminogen activator inhibitor type-1 inhibits insulin signaling
by competing with avb3 integrin for vitronectin binding
Roser Lo´pez-Alemany1, Juan M Redondo2, Yoshikuni Nagamine3* and Pura Mun˜oz-Ca´noves1,4*
1
Institut de Recerca Oncolo`gica (IRO), Centre d’Oncologia Molecular, L’Hospitalet de Llobregat, Barcelona, Spain;
2
C.B.M Severo Ochoa, C.S.I.C., U.A.M., Facultat de Ciencias, Madrid, Spain;3Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland;4Centre de Regulacio´ Geno`mica (CRG),
Programa de Diferenciacio´ i Cancer, Barcelona, Spain
Functional cooperation between integrins and growth factor
receptors has been reported for several systems, one of which
is the modulation of insulin signaling by avb3 integrin
Plasminogen activator inhibitor type-1 (PAI-1), competes
with avb3 integrin for vitronectin (VN) binding Here we
report that PAI-1, in a VN-dependent manner, prevents the
cooperation of avb3 integrin with insulin signaling in
NIH3T3 fibroblasts, resulting in a decrease in
insulin-induced protein kinase B (PKB) phosphorylation, vascular
endothelial growth factor (VEGF) expression and cell
migration Insulin-induced HUVEC migration and
angio-tube formation was also enhanced in the presence of VN and
this enhancement is inhibited by PAI-1 By using specific
PAI-1 mutants with either VN binding or plasminogen activator (PA) inhibiting activities ablated, we have shown that the PAI-1-mediated interference with insulin signaling occurs through its direct interaction with VN, and not through its PA neutralizing activity Moreover, using cells deficient for uPA receptor (uPAR) we have demonstrated that the inhibition of PAI-1 on insulin signaling is inde-pendent of uPAR-VN binding These results constitute the first demonstration of the interaction of PAI-1 with the insulin response
Keywords: plasminogen activator inhibitor type-1; vitro-nectin; insulin; angiogenesis; HUVEC
Insulin plays a central role in regulating metabolic pathways
associated with energy storage and utilization Its action is
initiated by receptor-mediated tyrosine phosphorylation of
ShcA and insulin receptor substrates (IRSs), and
recruite-ment of these molecules to the intracellular receptor domain
[1] ShcA is linked to the Ras/Erk signaling pathway while
IRS provides docking sites for several other signaling
molecules including phosphatidylinositol 3-kinase (PI3-K),
phospholipase Cc and Grb2, conveying insulin signals to
various cellular events IRS proteins couple the insulin
receptor to both the PI3-K and MAPK pathways, and ShcA (through Gab1) also couples the insulin receptor to both pathways (reviewed in [2]) Perturbations of insulin-induced metabolic responses are associated with severe health complications, such as type 2 diabetes and obesity [3] Under these pathological conditions, sensitivity of the insulin receptor to insulin is significantly reduced Insulin stimulates endothelial cell survival [4] and promotes angio-genesis in vivo [5,6] It is therefore noteworthy that type 2 diabetes is very often accompanied by cardiovascular complications, of which endothelial dysfunction is an early event [7]
Several lines of evidence indicate that integrin-mediated signaling processes synergize with growth factor responses [8,9] In particular, integrin avb3, the main receptor of vitronectin (VN), potentiates platelet-derived growth factor (PDGF) and insulin/insulin-like growth factor (IGF) recep-tor signaling responses [10,11] Smooth muscle cell migration
in response to IGF1 is dependent on VN occupancy of avb3 intregrin [12,13] IRS-1 binds to avb3 after insulin receptor activation in Rat1 fibroblasts, providing a potential mech-anism for the synergistic action between growth factors and extracellular matrix receptors [11] Furthermore, Persad
et al have recently shown that the integrin-linked kinase (ILK) can directly phosphorylate PKB, suggesting a role as
an upstream regulator for PKB [14]
VN occurs in the circulation as a complex with plasmi-nogen activator inhibitor-1 (PAI-1), the primary physiolo-gical inhibitor of fibrinolysis [15] PAI-1 is synthesized in an active form but is rapidly converted into an inactive form [16] Association with VN stabilizes PAI-1 and targets it to
Correspondence to P Mun˜oz-Ca´noves, Centre de Regulacio´
Geno`mica (CRG), Programa de Diferenciacio´ i Ca´ncer,
Passeig Maritim 37-49, E-08003 Barcelona, Spain.
Fax: + 34 93 224 08 99, Tel.: + 34 93 224 09 33,
E-mail: pura.munoz@crg.es
Y Nagamine, Friedrich Miescher Institute for Biomedical Research,
Maubeleerstrasse 66, CH-4058 Basel, Switzerland.
Fax: + 41 61 697 3976, Tel.: + 41 61 697 6669,
E-mail: nagamine@fmi.ch
Abbreviations: CsA, cyclosporin A; HUVEC, human umbilical vein
endothelial cells; IGF, insulin-like growth factor; IRS, insulin receptor
substrate; PAI-1, plasminogen activator inhibitor type-1; PI3-K,
phosphatidylinositol 3-kinase; PKB, protein kinase B; tPA, tissue-type
plasminogen activator; uPA, urokinase-type plasminogen activator;
uPAR, urokinase-type plasminogen activator receptor; VEGF,
vascular endothelial growth factor; VN, vitronectin.
*Both authors contributed equally to this work.
(Received 24 September 2002, revised 9 December 2002,
accepted 7 January 2003)
Trang 2specific sites in the extracellular matrix PAI-1 binds to the
N-terminal domain of VN, adjacent to the RGD sequence
(i.e the integrin binding site), preventing VN from binding
to avb3 [17] VN interacts not only with PAI-1 but also with
the urokinase receptor (uPAR) [18] PAI-1 and uPAR share
binding regions on the VN molecule and thus bind
competitively [19]
In this work, we have tested whether PAI-1 might be
modifying the cellular response to insulin We demonstrate
for the first time that PAI-1 modulates insulin signaling
by preventing the binding of VN to avb3 This results in
a decrease in insulin-induced PKB phosphorylation,
VEGF expression, endothelial cell migration and angiotube
formation
Experimental procedures
Cells
NIH3T3 cells were obtained from the American Type
Culture Collection (ATCC, Rockville, MD, USA)
HUVEC were isolated from umbilical veins and cultured
as described previously [20] All experiments were
per-formed using HUVEC between passages 3 and 6 Mouse
embryo fibroblasts (MEFs) from mice lacking
urokinase-type plasminogen receptor gene (uPAR-/–) were gently
provided by F Blasi and M Resnati (DIBIT, Milan, Italy)
Proteins
Murine vitronectin and recombinant murine PAI-1 in an
active conformation were from Molecular Innovations
(Royal Oak, MI) Mutants of PAI-1 were gently provided
by D Lawrence (American Red Cross, Rockville, MD,
USA): PAI-1 containing a mutation of Gln123 to Lys
(PAI-1K) has a specific defect in VN binding; PAI-1 containing
a mutation of Arg340 to Ala (PAI-1A) binds VN with
wild-type affinity but does not inhibit plasminogen activation
[21] Insulin from bovine pancreas was from Sigma
Western-blot analysis
Cells were grown to subconfluency and then kept overnight
in serum-free medium After treatments, cells were lysed in
20 mMTris HCl pH 8, 150 mMNaCl, 2 mMEDTA, 100 lM
Na3VO4, 10 mM NaF, 25 lM b-glycerophosphate, 1%
Triton X-100 and 100 mMphenylmethanesulfonyl fluoride
Phosphorylated PKB was detected with an
anti-(phospho-PKB Ser473) polyclonal Ig (New England Biolabs) Cell
lysates and conditioned media were collected and tested, for
intracellular VEGF and secreted VEGF, respectively, with a
polyclonal anti-VEGF Ig (sc-152, Santa Cruz
Biotech-nology, Santa Cruz, CA, USA)
Migration assay
Cell migration assays were performed on Transwells (8-lm
pore size), coated with Matrigel (Beckton and Dickinson,
Bedford, MA, USA; 1 : 20 dilution in serum free medium)
After blocking with 1% (w/v) BSA, Transwells were
incubated with 5 lgÆmL)1murine VN (Molecular
Innova-tions, Royal Oak, MI, USA) or 5 lgÆmL)1collagen type IV
(Sigma) Cells (4· 104) were added to the upper chamber of the Transwell Insulin (170 nM), 100 nMPAI-1, echistatin (100 nM) or decorsin (100 nM) were added to the media in the upper and lower chamber of the Transwell After incubation at 37C, cells on the underside of the Transwell were fixed in methanol/acetic acid (75 : 25, v/v) and stained with 5% Trypan blue The number of cells per high-power field that had migrated across the Matrigel were counted, after 16 h for NIH3T3 cells and uPAR–/– MEFs, and after
4 h for HUVEC Data are presented as the mean values from at least five high-power fields
Proliferation assay Cells (1.0· 104cells per well) were seeded on 96-well plates previously coated with VN (5 lgÆmL)1) or collagen (5 lgÆmL)1) After 24 h of culture, complete medium was replaced by medium containing 1% (v/v) fetal bovine serum, 0.5% (w/v) BSA, 170 nM insulin and 0.2 lCi [3H]thymidine per well (Amersham), and cells were cultured for a further 24 h for NIH3T3 cells or for 72 h for HUVEC Cells were precipitated by the addition of 5% cold trichloroacetic acid and precipitates solubilized in 1M NaOH Radioactivity (c.p.m.) in the lysate was detected
by liquid scintillation counting Each experimental point was determined five times
In vitro angiogenesis assay HUVEC (2.0· 104 cells per well) were resuspended in OPTI-MEM (Life Technologies) supplemented with 1% (v/v) fetal bovine serum Cells were treated with 100 nM PAI-1, 30 nM VN or 200 ngÆmL)1 cyclosporin A (CsA, from Sandoz) for 2 h at 37C Matrigel was meanwhile diluted 1 : 2 in cold serum-free RPMI 1640 without growth factors and plated into 96-well plates and allowed to gel for 1–2 h at 37C before seeding The cell suspension was then stimulated with 170 nMinsulin or 50 ngÆmL)1recombinant human VEGF165(PeproTech), and plated onto the surface
of the Matrigel After 12 h at 37C, cells were photo-graphed with a ZEISS inverted phase-contrast photomicro-scope Capillary tubes were defined as cellular extensions linking cell masses or branch points, and tube formation was quantified from photographs of standardized fields from triplicate wells
Statistical analysis ANOVA test was used to determine whether there were significant (P < 0.05) differences in cell migration, cell proliferation and angiotube formation under different treatments
Results
Vitronectin induction of insulin signaling is inhibited
by PAI-1 Integrin-mediated signaling processes synergize with growth factor responses [8,9] In this work, we first examined whe-ther VN was able to potentiate insulin-mediated responses
in NIH3T3 cells As shown in Fig 1A, insulin-induced PKB
Trang 3phosphorylation was augmented when cells were
preincu-bated with VN Echistatin, a specific avb3 antagonist,
prevented VN enhancement of insulin-induced PKB
phos-phorylation In contrast, decorsin, a structurally distinct
disintegrin with high affinity for the IIb/IIIa platelet integrin
but low affinity for avb3, had no effect (Fig 1B), suggesting
that the VN enhancement of insulin signaling is dependent
on avb3 integrin
To assess the potential role of PAI-1 in the
VN-mediated induction of insulin signaling, PAI-1 was
included in preincubations with VN There was a
significant decrease in insulin/VN-induced
phosphoryl-ation of PKB in the presence of PAI-1 (Fig 1C),
suggesting that PAI-1 might be competing with avb3
for VN binding Moreover, we observed that
urokinase-type plasminogen activator (uPA), which prevents PAI-1/
VN complex formation [22], was able to reverse the
PAI-1-mediated inhibition of PKB phosphorylation in
response to insulin/VN (Fig 1C)
To determine whether the PAI-1 effect on the insulin response was dependent on its binding to VN or on its plasminogen activator inhibiting activity, two different mutants of PAI-1 were used A mutant of PAI-1 (PAI-1A) that binds to VN normally, but does not inhibit plasminogen activators, inhibited phosphorylation of PKB
in response to insulin/VN, in a manner identical to that of wild-type PAI-1 In contrast, a second PAI-1 mutant (PAI-1K) that inhibits plasminogen activators normally, but has a significantly reduced affinity for VN, did not inhibit insulin/
VN phosphorylation of PKB (Fig 1D) These results indicate that PAI-1 binding to VN is sufficient to block
VN binding to avb3, and that the inhibition of insulin signaling by PAI-1 is independent of the antiproteolytic activity of PAI-1
VN interacts not only with PAI-1 but also with the urokinase receptor (uPAR) [18] PAI-1 and uPAR share binding regions on the VN molecule and thus bind competitively [19] We hypothesized that the interference
of PAI-1 with avb3 integrins leading to the inhibition of the insulin response might occur via uPAR, in a uPA dependent
or independent manner To test this hypothesis we used mouse embryonic fibroblasts (MEFs) derived from uPAR-deficient mice As seen in Fig 1E, PAI-1 has the same inhibitory effect on PKB phosphorylation induced by insulin/VN in uPAR-devoid MEFs than in NIH3T3 fibroblast cells, indicating that uPAR does not play a role
in the inhibition of insulin/VN signaling by PAI-1
To further assess the functional relevance of PAI-1/VN interaction on insulin signaling, we analyzed the expres-sion of an insulin-inducible gene, the VEGF gene Insulin induces VEGF expression and secretion in a variety of cell types [23,24], including NIH3T3 cells, via the PI3-K/ PKB signaling cascade [25] Western analysis of cell lysates demonstrated that VN enhanced insulin-stimulated VEGF expression in NIH3T3 cells, and this induction was inhibited by pretreatment with PAI-1 (Fig 2A) Additionally, echistatin was also able to inhibit this enhancement, while decorsin had no effect Moreover, VEGF secretion to the media, which was only detectable when cells were treated with insulin in the presence of
VN, was inhibited by PAI-1 or echistatin, but not by decorsin (Fig 2B) Furthermore, when the effect of mutants of PAI-1 were tested, only PAI-1A was able to inhibit VEGF secretion, as wild-type PAI-1, while mutant PAI-1K had no inhibitory effect (Fig 2C) Taken together, these results indicate that PAI-1 can inhibit insulin signaling through a mechanism involving VN, and this inhibition is independent on its ability to inhibit plasminogen activation
PAI-1 inhibits cell migration in response to insulin/VN
It has been reported that insulin stimulates migration of different cell types including NIH3T3 and human umbi-lical vein endothelial cells (HUVEC) [4,13,26] Based on the above results, we tested the effect of PAI-1 on insulin-induced cell migration In VN-containing Matrigel, NIH3T3 cell migration in response to insulin was increased twofold, and this was blocked when PAI-1 was added to the media (Fig 3A) In contrast, PAI-1 had
no effect on insulin-stimulated NIH3T3 cell migration on
Fig 1 Inhibition of insulin/VN induced PKB phosphorylation by PAI-1.
NIH3T3 cells were incubated overnight with 100 n M echistatin (Ec) or
decorsin (Dc), or for 4 h with 30 n M VN, 100 n M PAI-1 or 200 n M uPA,
prior to 5 min treatment with 170 n M insulin (I) as indicated Cell lysates
(40 lg) were subjected to Western blotting with an anti-(phospho-PKB)
or anti-PKB Ig (loading control) (A) Insulin-induced PKB
phos-phorylation is increased by VN (B) avb3 specificity in insulin/VN
signaling (C) PAI-1 inhibits insulin/VN-induced PKB
phosphoryla-tion (D) Effect of PAI-1 mutants on insulin/VN-induced PKB
phos-phorylation (E) Effect of insulin/VN/PAI-1 system in uPAR–/– MEFs.
Results are representative of at least four independent experiments.
Trang 4collagen-containing Matrigel As expected, echistatin
exerted a similar inhibitory effect on insulin-induced cell
migration, while decorsin had no effect When the
mutants of PAI-1 were tested in a migration assay, only
PAI-1A was able to inhibit insulin-induced migration on
VN, while PAI-1K had no effect (Fig 3B) Furthermore,
the migration of uPAR-deficient MEFs on VN-containing
Matrigel was also induced by insulin and inhibited by
PAI-1 (Fig 3C) indicating that the interference of PAI-1
with insulin-induced migration occurred through its
interaction with VN/avb3, independently of uPAR
Insulin stimulated also HUVEC migration whether cells
were plated on VN- or collagen-coated Transwells
However, PAI-1 inhibited insulin-stimulated cell
migra-tion only when cells were plated on VN-coated plates
(Fig 3D) These results indicate that PAI-1 was able to
block insulin-induced cell migration on VN-containing
matrices, and this inhibition was dependent on its ability
to bind to VN
Knowing that insulin also stimulates cell proliferation, we
wondered whether PAI-1 had any effect on insulin/
VN-induced NIH3T3 and HUVEC proliferation As shown
in Fig 4, insulin induced 4- and 10-fold [3H]thymidine
incorporation in NIH3T3 cells and HUVEC, respectively In
both cell types, this induction was not affected by treatment
of cells with PA1-1, either on VN or collagen matrices,
indicating that PAI-1 has no effect on insulin-induced proliferation of NIH3T3 and HUVEC proliferation
PAI-1 inhibition of endothelial cell angiogenesis induced by insulin/VN
Recently, a role for PAI-1 in angiogenesis in vivo has been proposed [21,27], but the mechanism responsible for this action has yet to be defined On the other hand, other authors have reported a role for insulin in angiogenesis by stimulating endothelial cell growth as well as tube formation through autocrine VEGF expression [24,28] We therefore investigated the effect of PAI-1 on insulin-induced angio-tube formation of HUVEC on Matrigel, an in vitro model of angiogenesis VEGF was used as a positive angiogenesis-inducing factor, and cyclosporin A (CsA) as an inhibitor of VEGF-induced angiogenesis Consistently with results pre-viously published by our group [29], VEGF induced HUVEC to form capillary-like structures (increase of 39%), while CsA inhibited this induction (Fig 5) Under identical experimental conditions, insulin induced a 59% increase in angiotube formation, which was markedly potentiated by VN (increase of 93%) PAI-1, in contrast, was able to completely inhibit the VN-mediated augmen-tation of insulin-induced angiotube formation PAI-1A inhibited angiotube formation at the same level as wild-type PAI-1, while PAI-1K had no effect Because PAI-1 is highly expressed in endothelial cells in vitro [30,31], it is tempting
to hypothesize that PAI-1 inhibition of insulin-mediated endothelial cell migration and angiotube formation can take place in vivo
Discussion
We have demonstrated in this study that PAI-1 inhibits insulin signaling, affecting the phosphorylation of PKB, the expression of VEGF, one of key proteins in angiogenesis, and various cell activities including endothelial cell migra-tion and capillary formamigra-tion This inhibimigra-tion is exerted through the interference of PAI-1 with the VN/avb3 system The results shown here provide the first demonstration of the interaction between PAI-1 and the insulin signaling, through the ability of PAI-1 to interact with VN/avb3 system
Using different mutants of PAI-1, we have demon-strated that the inhibition of insulin-induced responses by PAI-1 is due to its ability to form a complex with VN and not to its antifibrinolytic properties Mutant PAI-1A, which binds normally to VN but lacks plasminogen activator inhibitory activity, was able to inhibit insulin/ VN-induced PKB phosphorylation, VEGF secretion, cell migration and angiotube formation, in a manner identical
to that of wild-type PAI-1 In contrast, mutant PAI-1K, which retains the ability to inhibit plasminogen activation but does not bind to VN, had no inhibitory effect in the insulin/VN induced signaling It is well known that uPAR binds VN and competes with PAI-1 for VN binding [18,19]; therefore PAI-1 could be interfering with insulin/VN through a uPAR/uPA-mediated effect Because PKB phosphorytion and cell migration in response to insulin/VN was equally inhibited by PAI-1
in uPAR-deficient fibroblasts, we concluded that uPAR
Fig 2 Inhibition of insulin/VN-induced VEGF expression by PAI-1.
After the indicated pretreatments, NIH3T3 cells were incubated or not
for 4 h with 170 n M insulin Cell lysates (A) or conditioned media
(B and C) were subjected to Western blotting with an anti-VEGF Ig.
An anti-tubulin Ig was used as loading control Results are
represen-tative of at least four independent experiments performed.
Trang 5does not play a role in the inhibition of the insulin/VN
response by PAI-1 No effect of PAI-1 was observed on
insulin/VN-induced proliferation of NIH3T3 fibroblasts
and HUVEC, suggesting that the above described effects
of PAI-1 on insulin signaling and insulin-induced
proli-feration are uncoupled As activated insulin receptor
recruits several signaling molecules, including IRS and
Shc, it is likely that insulin can activate different siganling
pathways [32] The point of divergence of the
insulin-induced intracellular responses will be deciphered in
future studies
Upon ligand binding, the insulin receptor recruits
several signaling molecules, such as IRS A number of
reports suggest that full activation of IRS-1 requires avb3
integrin, although the precise mechanism underlying this
cooperation remains to be elucidated [10,11] In type 2
diabetes, reduced insulin sensitivity is not due to the
decrease in insulin receptor number or activity, but rather
to reduced activation (tyrosine phosphorylation) of IRS-1
[1] Under these conditions, IRS-1-mediated insulin
sign-aling is impaired while IRS-1-independent signsign-aling still
functions A prominent feature of type 2 diabetic patients
is elevated blood levels of PAI-1 [3] It is therefore reasonable to propose that PAI-1 may contribute to the development of type 2 diabetes by interfering with IRS-1-dependent insulin signaling
In circulation, there is a great excess of VN over PAI-1 (estimated to be 1000–10 000-fold) VN exists both in free form and membrane-bound form; it is synthesized in the liver but it disseminates abundantly into the vessel wall from the plasma [33] Several studies have demon-strated the increase of VN and PAI-1 during the progression of the atherosclerotic plaques [34,35] Thus,
it is tempting to speculate that PAI-1 concentration could
be locally raised to a level enough to inhibit the VN/avb3 system-mediated insulin effects in the atherosclerotic plaque environment It has been reported that the concentration of active PAI-1 in the atherosclerotic vessel wall is between 25 and 50 nM [36,37], which is in the same range of concentration used in this work, and that has been able to inhibit endothelial cell migration and angiotube formation (Figs 3 and 5) Thus, the inhibition
of the insulin response by PAI-1 may well be taking place in vivo, in certain pathological conditions
Fig 3 PAI-1 inhibits insulin-induced cell migration on VN (A) Effect of PAI-1 on insulin-induced NIH3T3 cell migration Transwells were coated with Matrigel containing collagen or VN The extent of NIH3T3 cell migration through Matrigel is shown with and without 100 n M PAI-1 or disintegrins, in the presence of 170 n M insulin Cells that had crossed the Matrigel membrane were counted after 16 h migration (B) Effect of mutants of PAI-1 on insulin-induced NIH3T3 cell migration on VN (C) Effect of PAI-1 on insulin-induced uPAR–/– MEFs migration (D) Effect
of PAI-1 on insulin-induced HUVEC migration Transwells were coated with 5 lgÆmL)1collagen, 5 lgÆmL)1VN or BSA The extent of HUVEC migration is shown through Transwells induced by 50 ngÆmL)1VEGF, 170 n M insulin (± 100 n M PAI-1) and PAI-1 alone Cells that had crossed the Transwell membrane were counted after 4 h of migration The data represent the average of four experiments *P < 0.05 compared to the value
vs control **P < 0.05 compared to the value corresponding to insulin.
Trang 6The interference of PAI-1 with the VN/avb3 system and
its consequences on insulin signaling, shown in this work,
may constitute a general mechanism by which PAI-1, the
main fibrinolytic regulator, could influence insulin-induced
responses, providing a potential explanation for the
fre-quently observed positive correlation between impairment
of the fibrinolytic system and insulin resistance syndrome in
diabetes and obesity Indeed, PAI-1 is highly expressed in
diabetic and obese patients and amelioration of
insulin-resistance is achieved by diet leading to the reduction of
fat tissues, the main source of PAI-1 [38]
PAI-1 has been recognized as a marker of endothelial
dysfunction in diseases associated with impaired
angio-genesis, including atherosclerosis and diabetic
vasculo-pathy [39] Although regulation of in vitro and in vivo
angiogenesis by PAI-1 has been reported, it is not clear
whether PAI-1 affects angiogenesis via its antiproteolytic
activity or by interacting with VN [21,27] Our observa-tion that PAI-1 can inhibit insulin-induced HUVEC migration and angiotube formation by interfering with the VN/avb3 system suggests that the latter mechanism is likely Given that PAI-1 and VN are highly expressed in the atherosclerotic plaques, it is tempting to propose that
Fig 5 PAI-1 inhibits insulin/VN-induced angiotube formation HUVEC were preincubated with and without CsA (200 ngÆmL)1) or PAI-1, PAI-1A or PAI-1K (100 n M ), and then seeded in 96-well plates pre-coated with Matrigel containing or not 5 lgÆmL)1VN Next, cells were stimulated with 50 ngÆmL)1VEGF or 170 n M insulin as indicated Tube formation was quantified 6 h after plating on Matrigel by counting the number of tubular structures in four to six fields (A) Fold-increase of tubes formed in comparison with the control Each column represents the mean value ± SD of four different experiments *P < 0.05 compared to the value vs insulin; **P < 0.05 compared to the value insulin + VN (B) Representative photographs (original magnification · 100) of four different fields corresponding to the experiment showed in (A) Results are representative of at least four independent experiments performed.
Fig 4 PAI-1 has no effect on insulin/VN induced cell proliferation The
effect of PAI-1 on insulin-induced NIH3T3 cells (A) and HUVEC (B)
proliferation Cells were seeded on VN or collagen-coated plates, and
incubated in the absence or presence of 170 n M insulin or 100 n M PAI-1.
Cell proliferation was determined by [ 3 H]thymidine incorporation after
24 h of incubation for NIH3T3 cells and after 72 h of incubation for
HUVEC The data represent the average of three experiments.
Trang 7PAI-1 can contribute to the development of vascular
complications associated with diabetes, such as diabetic
retinopathy, impaired wound healing, and cardiovascular
complications
Acknowledgements
We want to thank Dr D Lawrence for the generous gift of
PAI-mutants, and Drs F Blasi and M Resnati for uPAR–/– cells This work
was supported by grants from the European Union (1999/C361/06),
Fundacio´ La Marato´-TV3, MCyT (SAF2001-0482) and FIS (01/1474),
PM 990116 and FIS01/218 We thank A Blanco for technical
assistance Y.N is supported partly by the Roche Research Foundation.
References
1 Withers, D.J & White, M (2000) Perspective: The insulin
sig-naling system – a common link in the pathogenesis of type 2
diabetes Endocrinology 141, 1917–1921.
2 Giovannone, B., Scaldaferri, M.L., Federici, M., Porzio, O.,
Lauro, D., Fusco, A., Sbraccia, P., Borboni, P., Lauro, R & Sesti,
G (2000) Insulin receptor substrate (IRS) transduction system:
distinct and overlapping signaling potential Diabetes Metab Res.
Rev 16, 434–441.
3 Juhan-Vague, I., Alessi, M.C & Vague, P (1996) Thrombogenic
and fibrinolytic factors and cardiovascular risk in
non-insulin-dependent diabetes mellitus Ann Med 28, 371–380.
4 Hermann, C., Assmus, B., Urbich, C., Zeiher, A.M & Dimmeler,
S (2000) Insulin-mediated stimulation of protein kinase Akt: a
potent survival signaling cascade for endothelial cells Arterioscler.
Thromb Vasc Biol 20, 402–409.
5 Smith, L.E., Shen, W., Perruzzi, C., Soker, S., Kinose, F., Xu, X.,
Robinson, G., Driver, S., Bischoff, J., Zhang, B., Schaeffer, J.M &
Senger, D.R (1999) Regulation of vascular endothelial growth
factor-dependent retinal neovascularization by insulin-like growth
factor-1 receptor Nature Med 5, 1390–1395.
6 Treins, C., Giorgetti-Peraldi, S., Murdaca, J & Van Obberghen,
E (2001) Regulation of vascular endothelial growth factor
expression by advanced glycation end products J Biol Chem.
276, 43836–43841.
7 McLaren, M., Elhadd, T.A., Greene, S.A & Belch, J.J (1999)
Elevated plasma vascular endothelial cell growth factor and
thrombomodulin in juvenile diabetic patients Clin Appl Thromb.
Hemost 5, 21–24.
8 Ruoslahti, E & Engvall, E (1997) Integrins and vascular
extra-cellular matrix assembly J Clin Invest 99, 1149–1152.
9 Miyamoto, S., Teramoto, H., Gutkind, J.S & Yamada, K.M.
(1996) Integrins can collaborate with growth factors for
phos-phorylation of receptor tyrosine kinases and MAP kinase
activa-tion: roles of integrin aggregation and occupancy of receptors.
J Cell Biol 135, 8856–8860.
10 Bartfield, N.S., Pasquale, E.B., Geltosky, J.E & Languino, L.R.
(1993) The alpha V beta 3 integirn associates with a 190-kDa
protein that is phosphorylated on tyrosine in response to
platelet-derived growth factor J Biol Chem 268, 17270–17276.
11 Vuori, K & Ruoslahti, E (1994) Association of insulin receptor
substrate-1 with integrins Science 266, 1576–1578.
12 Jones, J.I., Prevette, T., Gockerman, A & Clemmons, D.R (1996)
Ligand occupancy of the alphaVbeta3 integrin is necessary for
smooth muscle cells to migrate in response to insulin-like growth
factor I Proc Natl Acad Sci USA 93, 2482–2487.
13 Zheng, B & Clemmons, D.R (1998) Blocking ligand occupancy
of the alphaVbeta3 integrin inhibits insulin-like growth factor I
signaling in vascular smooth muscle cells Proc Natl Acad Sci.
USA 95, 11217–11222.
14 Persad, S., Attwell, S., Gray, V., Mawji, N., Deng, J.T., Leung, D., Yan, J., Sanghera, J., Walsh, M.P & Dedhar, S (2001) Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343 J Biol Chem 276, 27462–27469.
15 Carmeliet, P., Kieckens, L., Schoonjans, L., Ream, B., v an Nuffelen, A., Prendergast, G., Cole, M., Bronson, R., Collen, D.
& Mulligan, R.C (1993) Plasminogen activator inhibitor-1 gene-deficient mice I Generation by homologous recombination and characterization J Clin Invest 92, 2746–2755.
16 Loskutoff, D.J., Sawdey, M & Mimuro, J (1989) Type 1 plas-minogen activator inhibitor In Progress in Hemostasis and Thrombosis, Vol 9 (Coller, B.S., eds), pp 87–115 Saunders, Philadelphia, USA.
17 Deng, G., Royle, G., Wang, S., Crain, K & Loskutoff, D.J (1996) Structural and functional analysis of the plasminogen activator inhibitro-1 binding motif in the somatomedin B domain of vitronectin J Biol Chem 271, 12716–12723.
18 Wei, Y., Waltz, D.A., Rao, N., Drummond, R.J., Rosenberg, S & Chapman, H.A (1994) Identification of the urokinase receptor
as an adhesion receptor for vitronectin J Biol Chem 269, 32380–32388.
19 Deng, G., Curriden, S.A., Wang, S., Rosenberg, S & Loskutoff, D.J (1996) Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release? J Cell Biol 134, 1563–1571.
20 Munoz, C., Castellanos, M.C., Alfranca, A., Vara, A., Esteban, M.A., Redondo, J.M & de Landazuri, M.O (1996) Transcrip-tional up-regulation of intracellular adhesion molecule-1 in human endothelial cells by the antioxidant pyrrolidine dithiocarbamate involves the activation of activating protein-1 J Immunol 157, 3587–3597.
21 Stefansson, S., Petitclerc, E., Wong, M.K., McMahon, G.A., Brooks, P.C & Lawrence, D.A (2001) Inhibition of angiogenesis
in vivo by plasminogen activator inhibitor-1 J Biol Chem 276, 8135–8141.
22 Lawrence, D.A., Palaniappan, S., Stefansson, S., Olson, S.T., Francis-Chmura, A.M., Shore, J.D & Ginsburg, D (1997) Characterization of the binding of different conformational forms
of plasminogen activator inhibitor-1 to vitronectin J Biol Chem.
272, 7676–7680.
23 Zelzer, E., Levy, Y., Kahana, C., Shilo, B.Z., Rubinstein, M & Cohen, B (1998) Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT EMBO
J 17, 5085–5094.
24 Yamagishi, S., Kawakami, T., Fujimori, H., Yonekura, H., Tanaka, N., Yamamoto, Y., Urayama, H., Watanabe, Y & Yamamoto, H (1999) Insulin stimulates the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor Microvasc Res 57, 329–339.
25 Miele, C., Rochford, J.J., Filippa, N., Giorgetti-Peraldi, S & Van Obberghen, E (2000) Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways J Biol Chem 275, 21695–21702.
26 Shigematsu, S., Yamauchi, K., Nakajima, K., Iijima, S., Aizawa,
T & Hashizume, K (1999) D -Glucose and insulin stimulate migration and tubular formation of human endothelial cells
in vitro Am J Physiol 277, E433–E438.
27 Bajou, K., Masson, V., Gerard, R.D., Schmitt, P.M., Albert, V., Praus, M., Lund, L.R., Frandsen, T.L., Brunner, N., Dano, K., Fusenig, N.E., Weidle, U., Carmeliet, G., Loskutoff, D., Collen, D., Carmeliet, P., Foidart, J.M & Noel, A (2001) The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by
Trang 8interaction with proteases, not vitronectin Implications for
anti-angiogenic strategies J Cell Biol 152, 777–784.
28 Dunn, S.E (2000) Insulin-like growth factor I stimulates
angio-genesis and the production of vascular endothelial growth factor.
Growth Horm IGF Res 10, S41–S42.
29 Hernandez, G.L., Volpert, O.V., Iniguez, M.A., Lorenzo, E.,
Martinez-Martinez, S., Grau, R., Fresno, M & Redondo, J.M.
(2001) Selective inhibition of vascular endothelial growth
factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor
of activated T cells and cyclooxygenase 2 J Exp Med 193,
607–620.
30 Olofson, B., Korpelainen, E., Pepper, M.S., Mandriota, S.J., Aase,
K., Kumar, V., Gunji, Y., Jeltsch, M.M., Shibuya, M & Alitalo, K.
(1998) Vascular endothelial growth factor B (VEGF-B) binds to
VEGF receptor-1 and regulates plasminogen activator activity in
endothelial cells Proc Natl Acad Sci USA 95, 11709–11714.
31 Pepper, M.S., Belin, D., Montesano, R., Orci, L & Vassalli, J.D.
(1990) Transforming growth factor-beta 1 modulates basic
fibro-blast growth factor-induced proteolytic and angiogenic properties
of endothelial cells in vitro J Cell Biol 111, 743–755.
32 Saltiel, A.R & Pessin, J.E (2002) Insulin signaling pathways in
time and space Trends Cell Biol 12, 65–71.
33 Dufourcq, P., Louis, H., Moreau, C., Daret, D., Boisseau, M.R.,
Lamaziere, J.M & Bonnet, J (1998) Vitronectin expression and
interaction with receptors in smooth muscle cells from human
atheromatous plaque Arterioscler Thromb Vasc Biol 18, 168–176.
34 Robbie, L.A., Booth, N.A., Brown, A.J & Bennett, B (1996) Inhibitors of fibrinolysis are elevated in atherosclerotic plaque Arterioscler Thromb Vasc Biol 16, 539–545.
35 Sato, R., Komine, Y., Imanaka, T & Takano, T (1990) Monoclonal antibody EMR1a/212D recognizing site of depos-ition of extracellular lipid in atherosclerosis Isolation and char-acterization of a cDNA clone for the antigen J Biol Chem 265, 21232–21236.
36 Padro, T., Emeis, J.J., Steins, M., Schmid, K.W & Kienast, J (1995) Quantification of plasminogen activators and their inhibitors in the aortic vessel wall in relation to the presence and severity of atherosclerotic disease Arterioscler Thromb Vasc Biol.
15, 893–902.
37 Underwood, P.A., Bean, P.A & Cubeddu, L (2001) Human endothelial cells grow poorly on vitronectin: role of PAI-1 J Cell Biochem 82, 98–109.
38 Primrose, J.N., Davies, J.A., Prentice, C.R.M., Hughes, R & Johnston, D (1992) Reduction in factor VII, fibrinogen and plasminogen activator inhibitor-1 activity after surgical treatment
of morbid obesity Thrombosis Haemostasis 68, 396–399.
39 Brodsky, S., Chen, J., Lee, A., Akassoglou, K., Norman, J.
& Goligorsky, M.S (2001) Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis Am J Physiol Heart Circ Physiol 281, H1784–H1792.