Plasminogen Activator Inhibitor-1: The Expression,
Biological Functions, and Effects on Tumorigenesis
and Tumor Cell Adhesion and Migration
Chun-Chung Lee and Tze-Sing Huang1
National Cancer Research Center, National Health Research Institutes, Taipei, Taiwan
Plasminogen activator inhibitor-1 (PAI-1) is a primary regulator of urokinase-type plas-
minogen activator (uPA). It inhibits uPA by forming a covalent complex, thus blocking
uPA’s interaction with substrates. The half-life of active PAI-1 is < 1 h and it is easily
transformed to a more stable inactive latent form. By binding with vitronectin, PAI-1
can be stabilized and remained its activity. PAI-1 expression is regulated by many in-
trinsic factors (e.g. cytokines, growth factors, hormones, and lipids) and extrinsic fac-
tors (e.g. physical injury and DNA-damaging agents). PAI-1 is an essential regulator in
physiological thrombotic/fibrinolytic process in vessel. It is also in the extracellular
matrix (ECM) where it controls local proteolysis via inhibiting uPA. PAI-1 can regulate
cell adhesion via either inhibition of uPA or interference with the binding between cellu-
lar integrins or uPA receptor (uPAR) and vitronectin. In addition, PAI-1 can regulate cell
migration by inducing clearance receptor-mediated cycled attachment-detachment- Keywords:
reattachment of integrins. PAI-1 may have a role in regulating tumor invasion, angio-
genesis and metastasis. Elevated levels of both uPA and PAI-1 are associated with a PAI-1
poor prognosis in many cancers. However, most in vivo experiments revealed: (1) High uPA
level (pharmacological level) of PAI-1 prevented angiogenesis and tumorigenesis; (2)
Low level (physiological level) of PAI-1 conversely facilitated tumor growth and angio- uPAR
genesis, and (3) While in PAI-1-deficient host, tumor growth and angiogenesis could not integrin
progress. The conclusions of PAI-1 in cancer development are still controversial. Be-
cause PAI-1 may play a role in tumor cell migration and invasion through anti- cell adhesion
proteinase activity and interference with cell attachment to ECM, some strategies can cell migration
be considered to apply PAI-1 derivatives to cancer therapy.
Journal of Cancer Molecules 1(1): 25-36, 2005.
Introduction molecular weight of 45 kDa. It is expressed in many cell
types, such as fibroblasts, smooth muscle cells, endothelial
Plasminogen activator inhibitors (PAIs ) are classified as a cells, hepatocytes, inflammatory cells, let alone the platelets,
subgroup of the serine protease inhibitor (serpin) super- the main source of the circulating PAI-1 . PAI-2 was origi-
family with a common characteristic of possessing an argin- nally described in the extracts prepared from placenta and
ine in the reactive center . The members, including PAI-1, accordingly was called placental PAI . Macrophage line-
PAI-2, PAI-3 (protein C inactivator), and protease nexin 1 age cells has also been found to express PAI-2 . PAI-3
(PN-1), act as the inhibitors of tissue-type plasminogen acti- was isolated from human urine and this molecule is identical
vator (tPA), urokinase-type plasminogen activator (uPA) and with protein C inactivator found in plasma . PN-1 was
thrombin [1-4]. PAI-1 is a single chain glycoprotein with the isolated from cultured fibroblasts, but is also produced by
other cultured cell types [1-2]. Generally, PAIs are specific
Received 8/10/05; Revised 9/14/05; Accepted 9/20/05. and fast-acting inactivators with distinct biological character-
Correspondence: Dr. Tze-Sing Huang, National Cancer Research istics and are present in most body fluids, tissues and cell
Center, National Health Research Institutes, 7F, No. 161, Min-Chuan lines. They can act as a pseudo-substrate to stably couple
East Road Sec. 6, Taipei 114, Taiwan, ROC. Phone: 886-2-26534401- with target proteases by ester bond formation, and thus con-
25138; Fax: 886-2-27929654. E-mail: firstname.lastname@example.org
Abbreviations: PAI-1, plasminogen activator inhibitor-1; serpin, serine vert the target enzymes into an inactive conformation .
protease inhibitor; PN-1, protease nexin 1; tPA, tissue-type plasminogen PAI-1 is the most extensively studied serpin member as we
activator; uPA, urokinase-type plasminogen activator; uPAR, uPA know today. In this article, we will review the biological func-
receptor; ECM, extracellular matrix; MMP, matrix metalloproteinase; tion of PAI-1 and the factors that regulate PAI-1 expression.
TGF-β1, transforming growth factor-β1; bFGF, basic fibroblast growth Additionally, we will also focus on the effects of PAI-1 on
factor; VEGF, vascular endothelial growth factor; RCL, reactive center cancer cell growth, adhesion, migration and invasion. The
loop; LRP, lipoprotein receptor-related protein; SMB, somatomedin B;
possible role of PAI-1 for cancer therapeutics will be dis-
LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; PMA, phor-
bol 12-myristate 13-acetate; HRE, hypoxia-responsive element. cussed.
2005 MedUnion Press 25
Lee et al. J. Cancer Mol. 1(1): 25-36, 2005
Figure1: Plasminogen activator inhibitor-
1 is a regulator of plasminogen activators.
There are two types of plasminogen
activator, i.e. tissue-type plasminogen
activator (tPA) and urokinase-type plasmi-
nogen activator (uPA). In the vessel, tPA
binds to fibrin and converts plasminogen
into plasmin for proteolytic degradation of
the clot. uPA is the major plasminogen
activator for migrating cells, and it is
activated by binding with uPA receptor
(uPAR) and subsequently initiates a
proteinase cascade. Plasmin is a pro-
teinase with a wide range of substrates. It
is able to degrade fibrin or other extracel-
lular matrix (ECM) components, to cleave
and activate other proteinases sush as
matrix metalloproteinases, and to activate
latent transforming growth factor-β1 (TGF-
β1), basic fibroblast growth factor (bFGF)
and vascular endothelial growth factor
(VEGF). Plasminogen activator inhibitor-1
(PAI-1) is a primary inhibitor to inhibit the
activation of both tPA and uPA.
PAI-1 is a regulator of PA system designated as P1-P1’ residues of the RCL (Figure 2). The
amino acids located N-terminal from this site are referred to
as P-residues P16-P1 and the C-terminal residues are P’-
PAI-1 is an important regulatory protein involved in the
residues P1’-P13’ (Figure 2A). The strength of binding be-
proteolytic [6,7] and fibrinolytic [8,9] pathways of plasmino-
tween the P1 residue and target protease determines the
gen activator. It is the major inhibitor of two types of plasmi-
nogen activator, i.e. tPA and uPA. tPA binds to fibrin and inhibitory activity of the serpin family protein . PAI-1
convert plasminogen to plasmin in the clot and is known as inhibits PA by the formation of a covalent complex, thus
the primary fibrinolytic activator (Figure 1). Distinctly, blocking PA for further interaction with its substrate .
uPA has very low affinity for fibrin, but it is the major PA The inhibitory process involves the formation of a noncova-
form expressed by the migrating cells and its activity is me- lent reversible Michaelis-like complex, followed by an acyl
diated by uPA receptor (uPAR). uPA binds to uPAR on cell intermediate and finally the formation of an ester bond be-
surface to initiate a proteinase activity, which in turn leads to tween the carboxyl group of the P1 residue and the hydroxyl
the activation of plasmin [11-13]. Plasmin is a proteinase group of the serine residue of protease (Figure 2A upper
exhibiting a wide range of substrate spectrum. It is able to panel). Once the initial complex is formed, the P1-P1’ bond
degrade many glycoproteins and proteoglycans of extracel- in the RCL is cleaved, and immediately followed by an inser-
lular matrix (ECM) such as laminin, fibronectin, vitronectin, tion of the P1-PA complex from the initial interaction site into
fibrin, etc. . It also can activate other proteinases such as the opposite side of the PAI-1. This causes a distortion of
matrix metalloproteinase (MMP)-1, -3 and -9 , and activate the protease and inhibits its catalytic activity (Figure 2B). In
or release growth factors from ECM including latent trans- this covalent complex, the PAI-1 is cleaved at the P1-P1’ site
forming growth factor (TGF-β) , basic fibroblast growth implicating that the inhibitory activity of PAI-1 is limited to a
factor (bFGF) , and vascular endothelial growth factor single encounter with its target protease [26-28]. Besides the
(VEGF)  (Figure 1). Cellular releasing of plasmin can active and cleaved forms, there exists a latent form of PAI-1
therefore promote ECM degradation and cell migra- that can be transited from both active and cleaved forms.
tion/invasion . By preventing the activation of plasmin, The active form spontaneously converts to the latent form
the biological function of PAI-1 is not restricted in the vascu- with a half-life of < 1 h . In this structure, the entire amino
lar fibrinolytic system, PAI-1 can play an important role in terminal side of the RCL is inserted as the central strand of β-
many other physiological and pathological processes of sheet A (Figure 2C). This increases the stability of PAI-1 but
tissue remodeling including wound healing, embryogenesis, lacking of its inhibitory activity. The latent form can be reac-
and tumor cell migration and invasion . tivated partially by denaturing agents .
Molecular structure of PAI-1 and its interaction with Interaction of PAI-1 with proteins other than PAs
In addition to the ability to bind and inactivate PAs, PAI-1
PAI-1 is a single-chain glycoprotein belonging to the ser- also has other ligands that might mediate functions of PAI-1.
pin family. All members of this family consist of about 400 These ligands include glucosaminoglycans (eg. heparin),
amino acid residues and have apparent molecular weights ECM components (eg. vitronectin), and scavenger recep-
from 38 to 70 kDa, depending on their degree of glycosyla- tors such as members of the low-density lipoprotein recep-
tion . From the nucleotide sequence analysis, PAI-1 tor (LDLR) superfamily, specifically the lipoprotein receptor-
cDNA encodes a protein containing 402 amino acids with a related protein (LRP)[33-37].
predicted nonglycosylated molecular mass of 45 kDa .
The mature secreted form of PAI-1 consists of 379 amino Vitronectin
acids and contains ~13% carbohydrate, which increases the Vitronectin is a 459-aa glycoprotein. It is divided into a
molecular weight to ~50 kDa. The tertiary structure of the number of specific domains, including the 44-residue soma-
serpin family protein exhibits three β-sheets (A, B and C), tomedin B (SMB) domain at the amino terminus, the connect-
nine α-helices, and an exposed reactive center loop (RCL) ing region that is immediately adjacent to the SMB domain
that contains the reactive site Arg346-Met347 for the target ser- and contains a single RGD sequence for binding with in-
ine protease . The reactive residues Arg346-Met347 are tegrin, and two hemopexin-like repeats. Vitronectin is rela-
26 Print ISSN 1816-0735
PAI-1’s Expression, Biological Function, and Effects on Cancer Development
Figure 2: Molecular structure of PAI-1 and
its interaction with uPA. (A) PAI-1 contains
an exposed reactive center loop (RCL)(red).
The active site Arg346 (red ball)-Met347 (green
ball), designated as P1-P1’ residues, is
responsible for contacting with its target
protease (blue ball). The inhibitory process
involves the formation of a reversible acyl
intermediate, cleavage at the P1-P1’ bond,
and the final formation of an irreversible
ester bond between the carboxyl group of
the P1 residue and the hydroxyl group of the
serine residue of the protease. (B) Once the
initial acyl intermediate is formed, the P1-P1’
bond in the RCL is cleaved, and immediately
followed by a translocation of the P1-PA
complex from the initial interaction site to
the opposite side of the PAI-1. This causes
a distortion of the PA and thus inhibition of
the proteinase activity. (C) Besides the
active and cleaved forms, there exists a
latent form of PAI-1. In this structure, the
entire amino-terminal part of RCL (i.e. P1-
P16) is inserted as the central strand of β-
sheet A. This increases PAI-1’s stability but
decreases its inhibitory activity.
tively unique among adhesive proteins not only because tors such as lipoprotein receptor-related protein (LRP).
PAI-1 binds to it with high affinity but also cells can attach to After PAI-1 is complexed with proteases, the heparin-binding
it through integrins, uPAR, or both [38,39](Figure 3A). Vi- domain of PAI-1, which contains a cryptic receptor-binding
tronectin-bound PAI-1 inhibits the proteolytic degradation of site (Lys69) with the high affinity for LRP, becomes exposed
ECM by inhibiting uPA/tPA, which in turn inhibits cell migra- and accessible for LRP binding. The complex is endocyto-
tion and invasion . The binding site of vitronectin for sed by binding to clathrin-coated pit-localized endocytosis
PAI-1 is localized to the SMB domain . All of the active receptors (Figure 3C and Figure 4). The uPA and PAI-1 then
PAI-1 in plasma circulates in complex with vitronectin , undergo lysosomal degradation, whereas uPAR is recycled
and the binding with vitronectin increases the stability of and reappears on the cell surface  (Figure 4). However,
PAI-1 . In tissue, vitronectin also binds to uPAR  and another report demonstrate that GPI-anchored uPAR is also
the binding site for uPAR is located in the SMB domain as endocytosed by piggybacking on LRP and that direct bind-
well . The binding sites for PAI-1 and uPAR are partially ing of occupied uPAR to LRP is essential for internalization
overlapping but not identical. The affinity of PAI-1 for the of occupied uPAR . Especially, direct binding of domain
SMB domain is much higher than that of uPAR  (Figure 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-
3B). Thus, PAI-1 can competitively inhibit the uPAR- 1-occupied uPAR . PAI-1 also contains a cryptic recep-
dependent attachment of cells to vitronectin tor-binding site that is exposed upon complex formation with
[38,39,45](Figure 3B). Binding of PAI-1 to SMB also inhibits tPA, and plays a role in the clearance of tPA . These
integrin-mediated cell adhesion by sterically blocking the studies suggest that PAI-1, by interacting with uPA/uPAR
adjacent RGD site  (Figure 3B). The stability of PAI-1 in and LRP, can be a potent chemoattractant molecule, which
plasma is increased by 2-fold when binding with vitronectin induces cell migration via regulating morphological attach-
. The crystal structure of the PAI-1–SMB complex sug- ment/dettachment changes (Figure 4). Recently, PAI-1 was
gests a simple mechanism for the stabilization of PAI-1 activ- reported to induce cell migration with corresponding cy-
ity: SMB slows the transition of PAI-1 to the latent form by toskeleton reorganization and phosphotyrosine redistribu-
blocking the associated sliding movement of strands 1A and tion . Using several PAI-1 mutants and specific inhibi-
2A of the main β-sheet into gap between helices E and F  tors, the activity of PAI-1 was shown not to depend on its
(Figure 2B). By binding to vitronectin, the target specificity interactions with uPA, tPA or vitronectin, but rather with LRP.
of PAI-1 is altered, because interaction with vitronectin en- This interaction leads to the activation of the Jak/Stat signal-
ables PAI-1 to inhibit another serine protease, thrombin . ing pathway and the induction of cell migration .
Heparin Biochemical function of glycosylation of PAI-1
It has been demonstrated that the PAI-1-binding site on vi-
tronectin is adjacent to a heparin-binding site . In addi- PAI-1 has 3 potential sites for N-linked glycosylation. A
tion, a heparin binding site can be predicted from the amino heterogeneous glycosylation pattern was noted to occur at
acid sequence of PAI-1, which is conserved in antithrombin the Asn209 and Asn265 sites of PAI-1, while the Asn329 site is
III and heparin cofactor II. Consequently, It have been inves- not utilized for glycosylation yet in human cell lines .
tigated that PAI-1 indeed interacts with heparin. In the pres- The latent transition of non-glycosylated PAI-1 was more
ence of heparin, the reactivity of PAI-1 toward thrombin is easily enhanced by a non-ionic detergent if comparison with
substantially increased. In contrast to vitronectin, heparin glycosylated PAI-1. The glycosylation at Asn265 seems to
does not stabilize the active conformation of PAI-1 [50,51]. determine the conformation of PAI-1. Also, the PAI-1 binding
protein vitronectin may reverse the conformational changes
Lipoprotein receptor-related protein (LRP) induced by the lack of glycosylation at Asn265 .
Once PAI-1 forms the complex with uPA that is specifi-
cally bound to uPAR, it loses its high affinity for vitronectin
but instead increases its affinity toward the clearance recep-
2005 MedUnion Press 27
Lee et al. J. Cancer Mol. 1(1): 25-36, 2005
Figure 3: PAI-1 involved in cell adhesion.
(A) Vitronectin contains the somatomedin B
(SMB) domain at its amino terminus, and
immediately adjacent to the SMB domain is
the single RGD sequence that is a binding
site for integrins. Vitronectin is relatively
unique among adhesive proteins not only
because PAI-1 binds to it but also because
cells can attach to it through integrins,
uPAR, or both. The binding sites of vi-
tronectin for uPAR and PAI-1 are both
located in the SMB domain. (B) The binding
sites of vitronectin for PAI-1 and uPAR are
partially overlapping but not identical. The
affinity of PAI-1 for the SMB domain is much
higher than the affinity of uPAR for this
domain. Thus, PAI-1 can competitively
inhibit the uPAR-dependent attachment of
cells to vitronectin. Binding of PAI-1 to SMB
also inhibits integrin-mediated cell adhesion
via a steric hindrance to the adjacent RGD
site. (C) PAI-1 can bind with uPA/uPAR
complex. This association renders the
cryptic receptor-binding site (CRB) of PAI-1
exposed out and increases PAI-1’s affinity
for the clearance receptor lipoprotein receptor-related protein (LRP). By binding to clathrin-coated pit-localized endocytosis receptors, the complex
of PAI-1/uPA/uPAR can be endocytosed. The PAI-1 and uPA then undergoes lysosomal degradation.
Many factors regulate PAI-1 expression in vitro and in Factors regulate PAI-1 expression
vivo Expression of PAI-1 can be regulated at the transcriptional
level by many factors including: growth factors and cyto-
kines (e.g. TGF-β1, interleukin-1, FGF, and VEGF), hormones
PAI-1 promoter (e.g. glucocorticoids and insulin), inflammatory factors (e.g.
The human PAI-1 gene is located on the long arm of tumor necrosis factor-α and lipopolysaccharide), glucose or
chromosome 7. The genomic DNA of human PAI-1 is ~12.2 lipid metabolites (e.g. glucose, free fatty acid, triglycerol, and
kb long, comprising nine exons and eight introns [21,57]. very-low-density lipoprotein), vascular tone regulating fac-
There are two distinct transcripts (3 and 2 kb, respectively) tors (e.g. angiogentensin II); chemicals (e.g. phorbol ester),
of PAI-1 mRNA with different lengths of polyadenylation in and other environmental or physical factors (e.g. reactive
the 3’ untranslated region (3’UTR)[21,58]. The sequence of oxygen species, hypoxia, stress, wound, adhesion to matrix).
5'-flanking DNA contains the essential cis-acting elements Some examples are described as below:
CCAAT and TATAA for RNA polymerase binding . Sev- TGF-β1: TGF-β1 is one of the earliest cytokines that was
eral known consensus sequences of regulatory elements are described to regulate PAI-1 expression at the transcriptional
located in the region of ~800 bp in front of the PAI-1 tran-
level. TGF-β1 is abundant in the α-granules of platelets.
scription initiation site. These cis-acting elements and the
After vascular injury, it can be released from platelets and
trans-acting regulatory factors involved in the regulation of
stimulate local endothelial PAI-1 expression. This will slow
PAI-1gene expression include: SP-1 (-76 to -71 and -44 to -39)
down the lytic process of thrombin, and result in increase of
[60,61], CTF/NF-1 (-119 to -105, -477 to -445 and -542 to -519),
fibrin deposit and progression of atherosclerosis. The TGF-
AP-1 (-65 to -50), AP-2 and SP-1-like (-82 to -65), and two
β1-responsive CAGA boxes in the PAI-1 gene promoter are
repressor elements (-764 to -628 and -266 to -188). Inter-
the binding sites for SMA/Mothers against decapentaplegic
estingly, wild-type p53 acts as a trans-activator and mutant
(Smad) proteins, which play the key role in TGF-β1-mediated
p53 (His273) acts as a repressor to the binding region at -160
activation of PAI-1 transcription .
to -139 of PAI-1 promoter . There are three CAGA con-
Inflammatory factors: Lipopolysaccharide (LPS) induces
served elements (-280, -580 and -70) that are specific for
PAI-1 levels in the plasma of patients with Gram-negative
TGF-β1-activated Smad 3 and Smad 4 binding in the PAI-1
septicemia and in the cultured human and bovine endothelial
promoter . One hypoxia-responsive element at the hu-
cells . LPS stimulates PAI-1 synthesis but decreases tPA
man PAI-I promoter -194 to -187 is identified, and is neces-
production in endothelial cells, resulting in an antifibrinolytic
sary and sufficient for hypoxia-mediated response
outcome. The stimulatory effect of LPS on PAI-1 expression
is mediated in part by tumor necrosis factor-α (TNF-α). The
5' distal TNF-α-responsive element of PAI-1 gene located 15
PAI-1 mRNA stability kb upstream of the transcription start site that is a conserved
The level of PAI-1 gene expression is determined not only NF-κB-binding site and mediates PAI-1’s response to TNF-α
by the rate of gene transcription but also by the rate at which .
the mRNA is degraded. The AU-rich sequence in the 3’UTR Lipids: Free fatty acids induces PAI-1 expression by acti-
of PAI-1 transcript determines its mRNA stability [66,67]. vating a transcription factor to bind with the sequence 5’-
Moreover, PAI-1 mRNA is especially degraded easily when TG(G/C)1-2CTG-3’ that is repeated four times in the PAI-1
cells have been treated with 8-bromo-cAMP . Using se- promoter (-528 and –599). A very-low-density lipoprotein-
rial deletion and insertion of PAI-1 3’UTR sequence, the re- responsive element that locates at the PAI-1 promoter region
gion from 2926 to 3054 of PAI-1 mRNA is defined as PAI-1 from -672 to -657 is responsible for the effect of plasma
cAMP-responsive element (PAI-1 CRE) . Recently, a PAI- triglycerides on PAI-1 expression . Notably, the 4G/5G
1 RNA binding protein 1 (PAI-RBP1) that specifically inter- polymorphism site locates at -675. Higher level of PAI-1 syn-
acts with the PAI-1 CRE has been identified to involve in thesis is associated with the 4G/4G genotype. The 4G/4G
cAMP-mediated PAI-1 mRNA degradation . allele is preferentially bound with an enhancer protein,
whereas the 5G/5G allele is bound with an enhancer plus a
28 Print ISSN 1816-0735
PAI-1’s Expression, Biological Function, and Effects on Cancer Development
Figure 4: PAI-1 involved in cell migration.
PAI-1 binds uPA/uPAR complex. This leads
to a large shift in PAI-1’s relative affinity
from vitronectin to the clearance receptor
LRP. Subsequently, PAI-1, LRP, uPA/uPAR,
and associated integrins will be endocyto-
sed. Cell will detach not only from the
vitronectin but also the general matrixes. In
the endosome, PAI-1/uPA complex sepa-
rates from the receptors and is targeted to
the lysosome for degradation. The LRP,
uPAR and integrins are then recycled to
another edge of the cell surface for the next
attachment to ECM. This model provides
for the PAI-1-mediated cycled attachment-
detachment-reattachment of integrins that is
necessary for cell migration.
suppressor, resulting in lower levels of PAI-1 transcription. PMA-treated cells. The PMA response elements (i.e. AP-1
This polymorphism site responds to triglyceride. Increased site) in the PAI-1 gene promoter are also identified .
levels of PAI-1 will occur in the individuals with the 4G/4G Hypoxia: Oxygen deprivation, as occurred during tissue
genotype and elevated triglyceride levels in plasma [75,76]. ischemia, usually results in coagulation. When mice are
Despite the plasma levels of PAI-1 seem to be the result of placed in a hypoxic environment, the plasma levels of PAI-1
both environmental and genetic influences, many studies increase in a time-dependent manner accompanied by an
have reported that higher PAI-1 plasma levels occur in indi- increase in plasma PAI-1 activity . Northern blot analysis
viduals with 4G/4G genotype [77-80]. However, the data on of the hypoxic murine lung tissues have demonstrated an
the clinical relevance of these polymorphisms are contro- increase in PAI-1 mRNA compared with normoxic controls
versial . The transcription factor PPARγ may also par- . In contrast, the levels of transcripts for both tPA and
ticipate in PAI-1 regulation, and PPARγ might involve in vas- uPA are decreased under hypoxic conditions. Several puta-
cular diseases . tive hypoxia-responsive elements (HRE-1, at -158 to –151;
Glucose and Insulin: In pig aortic endothelial cells, the HRE-2, at -194 to -187; HRE-3, at -453 to -446; HRE-4, at -566
increase of PAI-1 secretion is significantly associated with to -559; HRE-5, at -681 to -674) were identified in the human
extracellular glucose concentration . It has also been PAI-I gene promoter . Reporter gene assays show that
found that insulin stimulates synthesis of PAI-1 in human the HRE-2 site is necessary and sufficient for the hypoxia-
hepatic cells . Glucose-induced PAI-1 expression could induced PAI-1 expression .
interpret the frequent occurrence of attenuated fibrinolytic Cell adhesion: In anchorage-dependent cells, the level of
activity of plasma and premature cardiovascular disease in PAI-1 mRNA expression is increased when cells begins to
the patients with type II diabetes mellitus. Glucose regulates attach onto the substrate and subsequently turns to be down
PAI-1 gene expression through two adjacent SP-1 sites lo- regulated when cells have attached already . The PAI-1
cated between -85 and -42 of the PAI-1 5'-flanking region . gene expression is induced only in adherent but not in non-
Release of a transcriptional repressor from the SP-1 com- adhered cells. Regulation of PAI-1 gene expression by cell
plexes may explain the induction of PAI-1 gene under high adhesion can be initiated through the interaction between
glucose conditions in vascular smooth muscle cells . vitronectin, fibronectin or collagen and integrins, and is me-
Hyperglycemia stimulates Rho-kinase activity via PKC and diated by PI-3 kinase/Akt and MEK/ERK pathways [92-94].
oxidative stress-dependent pathways, leading to increased
PAI-1 gene transcription .
The role of PAI-1 in regulation of cell adhesion, migra-
p53 and the cell cycle: A p53 binding site in the region -
tion and invasion
160 to -139 of the human PAI-1 gene promoter has been
identified that is responsible for stimulation of the PAI-1
transcription by p53 . Conversely, a p53 mutant (His273) PAI-1 in cell adhesion
inhibits PAI-I promoter activity. This result suggests that an PAI-1 has dual roles in regulation of cell adhesion. uPA
oncogenic p53 form may reduce PAI-1 expression and thus functions as an anti-adhesion molecule by proteolytic deg-
alter the plasminogen/plasmin system during tumor pro- radation of ECM components. As an inhibitor of uPA, PAI-1
gression. In addition, PAI-1 is expressed by a cell cycle- would be expected to promote adhesion. Indeed, PAI-1 has
dependent manner with increasing expression during growth the ability to selectively protect vitronectin from proteolysis
activation (G0-to-G1 transition), and PAI-1 could therefore be by inhibiting local plasminogen activator and thus stabilizes
an indirect cell proliferation indicator [86,87]. the vitronectin-dependent cell adhesion (Figure 3A).
Phorbol Ester: Phorbol 12-myristate 13-acetate (PMA) in- However, PAI-1 also exhibits an anti-adhesive function. Its
duces the PAI-1 levels in human rhabdomyosarcoma and association with vitronectin blocks the binding of both uPAR
hepatocellular carcinoma cell lines [88,89]. There is also an and integrin to vitronectin [39,96-99](Figure 3B). In addition,
increase in the PAI-1 mRNA and gene transcription levels in an anti-adhesive effect of PAI-1 can be through the down-
2005 MedUnion Press 29
Lee et al. J. Cancer Mol. 1(1): 25-36, 2005
Figure 5: The transcription regulatory
sites in the PAI-1 promoter. PAI-1 gene
expression is regulated by many intrinsic
factors (e.g. cytokines, growth factors,
hormones, and lipids) and extrinsic factors
(e.g. physical injury and DNA-damaging
agents). The sequence of 5'-flanking DNA
contains the essential cis-acting elements
CCAAT and TATAA for RNA polymerase
binding. Several known consensus se-
quences of regulatory elements are located
in the region of ~800 bp in front of the PAI-
1 transcription initiation site. These cis-
acting elements and the trans-acting
regulatory factors include: SP-1 (-76 to -71
and -44 to -39), CTF/NF-1 (-119 to -105, -477
to -445 and -542 to -519), AP-1 (-65 to -50),
AP-2 and SP-1-like (-82 to -65), and two
repressor elements (-764 to -628 and -266
to -188). Interestingly, wild-type p53 acts
as a trans-activator and mutant p53 (His273)
acts as a repressor to the binding region
at -160 to -139. There are
three CAGA conserved elements (-280, -580 and -70) that are specific for TGF-β1-activated Smad 3 and Smad 4. One hypoxia-responsive element at
the human PAI-I promoter -194 to -187 is identified, and is necessary and sufficient for hypoxia-mediated response.
regulation of the integrin levels on cell surface by the ability to inhibit the proteinase activity (110-112), but co-
mechanism that PAI-1 binds and induces LRP-mediated en- expression of uPA, uPAR and PAI-1 is required for optimal
docytosis of PAI-1/uPA/uPAR/integrin complex (Figure invasiveness of human lung cancer cells . In addition,
3C). transfection of PAI-1 cDNA into PC-3 prostate carcinoma
cells has been reported not to alter cell invasiveness .
PAI-1 in cell migration
Cell migration is the locomotion of a cell over an ECM
PAI-1 in tumor growth, angiogenesis and metastasis
substratum. uPA has been recognized as to stimulate cell
migration by catalyzing plasminogen activation for proteoly-
sis of substratum and thus releasing cells from the substra- Association of uPA with cancer development has been no-
tum . PAI-1 would be expected to inhibit plasminogen ticed for decades. A simple conception is that ECM and
activation-dependent cell migration. However, it has been basement membrane are degraded through the proteinases
shown that PAI-1 can directly block cell attachment and mi- that are activated by uPA during cancer cell invasion. High
gration by a mechanism independent of its anti-proteolytic levels of uPA in extracts of primary breast carcinoma can be
activity [102,103]. PAI-1 can play a role in cell migration used to predict an early relapse [115,116]. Several studies
owing to its dual roles in regulating the cell adhesion. It can with specific disruptions of the genes for plasminogen and
be either pro-migratory or anti-migratory, depending on uPA also support the hypothesis that uPA activation is a
whether PAI-1 locates at the leading or trailing edge of the rate-limiting step for tumor growth, local invasion and dis-
cell and on the PAI-1 concentration [97,102-105]. Once PAI-1 tant metastasis [117-121]. However, as an inhibitor of uPA
is binding with the uPA/uPAR complex, PAI-1 decreases its and originally regarded as an anti-cancer molecule, PAI-1 is
affinity for vitronectin but increases its affinity for the clear- surprised to be a poor prognostic marker in breast and other
ance receptors LRP and very-low-density lipoprotein recep- types of cancer [101,122-126]. What on earth is the role of
tor [37,51]. Subsequently, cell will detach not only from the PAI-1 during tumor cell progress toward malignancy? It is
vitronectin but also the general matrixes by the clearance an important stimulant for cancer scientists to investigate
receptor-mediated endocytosis mechanism , because the the biological function(s) of PAI-1 in tumor. PAI-1 has been
endocytosis will recruit the PAI-1/uPA/uPAR complex and extensively investigated in tumor growth, invasion, and me-
associated integrins [106-108]. In the endosome, PAI-1/uPA tastasis by experimental animal models (some important
complex separates from the receptors and is targeted to the examples summarized in Table 1). The results obtained are
lysosome for degradation. The clearance receptors, uPAR quite controversial as different experimental systems are
and integrins are then recycled to another edge of the cell used. For example, high levels of PAI-1 expression in hu-
surface. Thus this model provides for the cycled attach- man or murine cancer cells were associated with the retarda-
ment-detachment-reattachment of integrins that is necessary tion of tumor growth, invasion, and metastasis in immunode-
for cell migration . Hence, the cell migration is not only ficient mice [112,114,127]. Application of high level of PAI-1
as simple as through the proteolysis of ECM or blocking the protein to the immunodeficient mice bearing transplanted
binding between vitronectin and integrin, but also through human tumors caused tumor growth inhibition [128,129], but
the induction of endocytosis by PAI-1-conjugated uPA/uPAR low level of PAI-1 enhanced tumor growth . Deficiency
complex (Figure 4). in PAI-1 or uPA expression in host mice prevented murine
T241 fibrosarcoma growth compared with control wild-type
PAI-1 in cell invasion mice . Also, PAI-1 deficiency in the host mice pre-
When cells progress from migration to invasion, not only vented local invasion and tumor vascularization of trans-
locomotion but also penetration through ECM of the cells planted malignant keratinocytes . While this prevention
can be observed. In the cell invasion models, the proteoly- in PAI-1-deficient mice was aborted by the exogenous ade-
sis of ECM by uPA is well associated with the cellular pene- noviral vector expressing PAI-1, the tumor invasion and an-
tration in ECM. However, the observatory results of PAI-1 giogenesis were restored . This PAI-1-recovered tumor
action influencing cell invasion are not consistent. Some vascularization is through its proteinase inhibition activity
reports suggest that PAI-1 inhibits cell invasion through its but not through its interference with vitronectin-integrin
30 Print ISSN 1816-0735
PAI-1’s Expression, Biological Function, and Effects on Cancer Development
Table 1: Some important examples studying the roles of PAI-1 in cancer development by experimental animal models
Model System PAI-1 Effect Reference
Overexpression of PAI-1 in PC-3 human prostate carcinoma Overexpression of PAI-1 reduced the aggressive phenotype Soff et al. 1995 
in athymic mice of PC-3 human prostate carcinoma.
B16 melanomas implanted into PAI-1-overexpression or No difference in tumor growth, metastasis, and survival was Eitzman et al. 1996 
PAI-1 deficient mice observed between control and PAI-1-deficient or -
Intraocular melanomas by adenovirus-mediated gene trans- Metastatic tumor burden was reduced and host survival was Ma et al. 1997 
fer of PAI-1 in an athymic mouse model significantly prolonged.
LNCaP prostate carcinoma in SCID mice treated with mu- PAI-1 inhibited tumor growth through its inhibition of PA Jankun et al. 1997 
tated form of PAI-1 to confirm a correlation between the activity.
inactivation of uPA and tumor size
Malignant keratinocytes implanted into PAI-1-deficient Deficient PAI-1 expression in host mice prevented invasion Bajou et al. 1998 
mice and tumor vascularization of transplanted malignant kerati-
nocytes. But it was restored by intravenous injection of a
adenoviral vector expressing human PAI-1.
T241 fibrosarcomas implanted into uPA or PAI-1 deficient uPA or PAI-1 deficiency disturbed tumor growth. Only PAI- Gutierrez et al. 2000 
mice 1 deficiency prevented angiogenesis.
Chicken chorioallantoic membrane treated with pharmacol- PAI-1 inhibited angiogenesis either through inhibiting pro- Stefansson et al. 2001 
ogical levels of PAI-1 mutants teinase activity or blocking cellular access to vitronectin.
Malignant keratinocytes implanted into PAI-1 deficient PAI-1 restored tumor vascularization through inhibition of Bajou et al. 2001 
mice and treated with adenoviral PAI-1 mutants proteinase activity but not by interacting with vitronectin.
M21 human melanomas in nude mice treated with different PAI-1 stimulated tumor growth and angiogenesis at low McMahon et al. 2001 
concentrations of PAI-1: matrigel implants in wild type amounts while it inhibited tumor growth at higher amounts.
mice treated with increasing concentrations of PAI-1
Overexpression of PAI-1, PAI-1 (VN-), PAI-1 (INH-) PAI- Only overexpression of wild-type PAI-1 reduced the burden Praus et al. 2002 
1 (VN-, INH-) and tumor cell migration in vitro and metas- of metastasis by 68% compared with the control.
tasis in mice in vivo
Aortic ring explant in PAI-1 deficient mice treated with PAI-1 stimulated angiogenesis at low doses while it inhib- Devy et al. 2002 
increasing concentration of PAI-1 or adenovirus expression ited angiogenesis at higher doses.
of PAI-1 mutants.
T241 fibrosarcomas implanted into PAI-1 deficient mice PAI-1 deficiency had no effect on tumor growth and angio- Curino et al. 2002 
MMTVPym-T-induced breast tumor that mimic human No difference of breast tumor development and vascular Almholt et al. 2003 
mammary adenocarcinoma in PAI-1 deficient or normal density was observed between PAI-1 deficient and normal
Doses of recombinant PAI-1 protein intraperitonealy in- PAI-1 exhibited both pro- and anti-angiogenic effects de- Lambert et al. 2003 
jected in PAI-1 deficient animals to see the angiogenic pending on the dose and merely through its anti-proteolytic
potential with a model of laser-induced choroidal neovascu- activity.
PAI-1-overexpressed PVDA keratinocytes transplanted into Overexpression of PAI-1 in host or in tumor cells reduced Bajou et al. 2004 
PAI-1-overexpressed, -deficient or wild type mice tumor development in vivo. High level of PAI-1 in cancer
cells did not overcome the PAI-1 deficiency in the host for
Low grade (HaCaT II-4) and high grade (HaCaT A5-RT3) Tumor incidence was reduced for HaCaT II-4 (low grade) Maillard et al. 2005 
of malignant skin keratinocytes subcutaneously injected in cells in PAI-1 deficient mice, but not reduced for HaCaT
PAI-1-deficient and wild type mice A5-RT3 (high grade) cells.
binding . Many studies have suggested that PAI-1 at cells were transplanted into the PAI-1-deficient mice, the
high level prevents tumor growth through inhibiting angio- PAI-1-overexpressed cells did not compensate the PAI-1
genesis, while enhances tumor growth by promoting angio- deficiency in the host to recover the angiogenesis and tu-
genesis at low level [129,133,134]. Recently, many investiga- morigenesis . However, some reports exhibit no sig-
tions more support that null expression of PAI-1 in the host nificant difference in the tumor growth and angiogenesis
and supraphysiologic level of PAI-1 expression either by between PAI-1-deficient and wild-type mice. For example,
host or tumor cells exhibit preventive effect on tumor growth when intravenous inoculation of murine melanoma cells into
and angiogenesis, but the physiological concentration of PAI-1-overexpressed or PAI-1-deficient mice in comparison
PAI-1 promotes in vivo tumor invasion and angiogenesis with wild-type control, no differences were observed in the
[135,136]. Interstingly, when the PAI-1-overexpressed tumor extent of pulmonary metastasis, tumor size and overall sur-
2005 MedUnion Press 31
Lee et al. J. Cancer Mol. 1(1): 25-36, 2005
vival in these mice . PAI-1 deficiency has also no effect Is PAI-1 a target or drug for cancer therapy
on the tumor growth and angiogenesis of the transplantation
of T241 fibrosarcoma cells . One report illustrates that
Since the early observations of elevated PAI-1 expression
using MMTVPym-T-induced breast cancer to mimic sponta-
in several tumor types, PAI-1 had been expected to be a po-
neously human mammary adenocarcinoma , there are
tential target for cancer therapy. But actually, it still remains
no differences in the primary tumor growth and vascular
a controversy of the role of PAI-1 in tumorgenesis. From
density between PAI-1-deficient and wild-type mice . In
many experimental results, low level (physiological level) of
addition, PAI-1 deficiency is not sufficient to prevent neo-
PAI-1 in the host may facilitate angiogenesis and tumor
plastic growth of the aggressive tumors in human skin, it
growth [129,131,133-136]. In PAI-1-deficient background,
still exerts its tumor-promoting effect in a tumor stage–
angiogenesis and tumor will not progress [130,131]. Con-
dependent manner .
versely, many other studies indicate that administration of
The proangiogenic effect of PAI-1 at suitable concentra-
high level (pharmacological level) of PAI-1 seems to prevent
tions may be through the inhibitory activity of PAI-1 to limit
the angiogenesis, tumor growth and metastasis
uPA-dependent plasminogen activation, and thus stabilize
[112,114,127-129]. It is notable that all these results are de-
the basement membranes around newly formed vessels and
rived from the model of tumor transplanted, and many ex-
also stabilize the matrix scaffold required for endothelial cell
periments with different cell types or different protocols
migration and the assembly of endothelial cells into capillar-
exhibit the different conclusions. The only one study that
ies. A critical balance between proteases and their inhibitors
used spontaneously induced tumors in the PAI-1-deficient
is thought to be essential for optimal tumor cell invasion and
host also shows no correlation of PAI-1 with tumorigenesis
angiogenesis [113,142]. Elevated expression of PAI-1 in
tumor cells tends to inhibit tumor growth, invasion, and me-
However, administration of pharmacological levels of PAI-
tastasis. This result is opposite to the clinical observation
1 to inhibit tumor growth and angiogenesis has been dem-
that PAI-1 overexpression correlates with the malignancy in
onstrated by many reports [112,114,127-129,133,134,136].
breast or colon cancer. By immunohistochemistry assay,
Because PAI-1 is multifunctional and exhibits short half-life
the PAI-1 is expressed in the leading edge of myofibroblasts
in vivo, more studies needed to be performed to render PAI-1
in breast cancer tissues, which is responsible for the corre-
really usable in the treatment of cancer. It should be con-
lation between the PAI-1 level in breast tumor extracts and
cerned that if treatment of high level of the PAI-1, the inhibi-
poor prognosis . Similar observation is also obtained in
tory activity of PAI-1 may increase the rate of the process of
colon cancer . These results are consistent with other
atherosclerosis and thrombosis. The strategy of using the
results that PAI-1 is mostly expressed by stromal cells but
specific mutant is intriguing to scientists. For example, the
not by tumor cells [131,140]. Moreover, whatever the
mutant of PAI-1 that interferes with the binding of integrin
amounts of PAI-1 are produced by tumor cells, blood vessels
with vitronectin but without inhibiting PA activity is found to
are limited under the collagen gel and tumor cells fail to in-
inhibit angiogenesis in vivo, but not found to promote
vade in the PAI-1-deficient host tissue . These findings
thrombosis because it does not inhibit fibrinonlysis .
suggest that the site of PAI-1 production (stromal cells
Up to date, more than 600 PAI-1 mutants have been prepared
rather than tumor cells) is a critical factor in tumor angio-
and all these mutants have been utilized for studying the
genesis rather than the total amount of PAI-1 in tumor.
structure-and-function of PAI-1 . The availability of
However, some reports exhibit no differences in the tumor
these mutants and the information of the PAI-1’s function
growth and angiogenesis between PAI-1-overexpressed or -
may provide us some directions to develop useful agents to
deficient and wild type mice. These controversial results
inhibit tumor angiogenesis and growth. As demonstrated by
may implicate that the influence of diverse factors exist on
many studies, PAI-1 has been administrated in its protein
animal tumor models, such as cell numbers implant, cell
form. Delivery of PAI-1 gene into the target tissue can be
types (stages of malignancy), expression of some other
another considerable way. Adenovirus-mediated PAI-1 gene
types of PAI (PAI-2 or PAI-3), and the activation of PA system.
expression is found to reduce the tumor growth, migration
Nevertheless, in despite of many reasons for the incompati-
and metastasis in vivo [127,149,150]. Some factors, such as
ble results, most of these studies result from the transplan-
the efficiency of the gene delivery system (adenovirus, ret-
tation of tumor models that may not be reflected onto the
rovirus or liposome), the design for PAI-1 expression con-
real pathogenesis of cancer. Till now, only one report illus-
struction (driven by which promoter and what the mutant
trates the spontaneous development of primary breast tu-
design), and the possibility of accompanying side effects
mors, but shows no significant difference in the rates of
during the delivery process, are all needed to concern. An-
tumor formation, growth, angiogenesis, and metastasis.
other intriguing strategy is to develop antibodies that par-
From these results, how does PAI-1 involve in the real proc-
tially modulate PAI-1’s function. It has been demonstrated
ess of tumorigenesis is still not certified. It still needs to
that antibodies to PAI-1 can suppress the metastasis of hu-
accumulate more investigations using such spontaneous
man tumor cells in mouse xenograft models [151-153]. In
addition, the scientists have screened some small molecules
It has been suggested recently that the prognostic impact
bearing the specific inhibitory activity on PAI-1 [154-156],
of PAI-1 is not based on its involvement in angiogenesis
although the detailed mechanisms of these small molecules
alone . Another important efferent is that high levels of
are unclear yet.
PAI-1 contribute to tumor growth by inhibiting apoptosis of
tumor cells. Addition of PAI-1 to culture medium can inhibit
tumor cell apoptosis . The spontaneous fibrosarcoma Conclusion
with wild-type PAI-1 shows less sensitivity to chemotherapy-
elicited apoptosis . This phenomenon was not noted in Further studies should be done to fully realize PAI-1’s bio-
PAI-1-deficient fibrosarcoma. However either in wild type or logical functions in vivo. For understanding of PAI-1’s role
PAI-1 deficiency, the mice display similar sensitivity to the in cancer development, accumulation of more data from
treatment of etoposide . Hence, PAI-1 might regulate consistent animal models and protocols is necessary.
apoptosis of transformed cells but not normal cells, suggest- Moreover, spontaneous tumorigenesis models should be
ing a differential effect of PAI-1 between cancer cells and adopted to investigate the actual role of PAI-1 in tumori-
normal cells. genesis. The developments of several PAI-1 mutants, gene
delivery systems, and some antibodies or small molecules
for antagonizing the partial or complete PAI-1’s function may
32 Print ISSN 1816-0735
PAI-1’s Expression, Biological Function, and Effects on Cancer Development
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