Keywords gene regulation; plasminogen activator inhibitor type 2; protease inhibitor; serpin Correspondence R.. This was first revealed over a decade ago, when it was shown that the incre
Trang 1The undecided serpin
The ins and outs of plasminogen activator inhibitor type 2
Robert L Medcalf and Stan J Stasinopoulos
Australian Centre for Blood Diseases, Monash University, Prahran, Victoria, Australia
Introduction
The plasminogen activating cascade became a much
investigated enzyme system during the early 1980s,
mainly for its role in maintaining vascular patency and
for its effect on the extracellular matrix in the context
of wound healing and cell migration The controlled
generation of the powerful protease, plasmin, from
its precursor plasminogen seemed to be a relatively
straightforward process at the outset: two serine
pro-teases had been identified that could specifically cleave
plasminogen and produce active plasmin These
pro-teases (tissue-type- and urokinase-type plasminogen
activator; tPA, uPA) were in turn specifically inhibited
by plasminogen activator inhibitors (PAIs)-types 1 and
2, both of which belong to the serine protease inhibitor
(serpin) superfamily Other cofactors, such as the
ser-pin alpha2 antiplasmin, the urokinase receptor (uPAR)
and fibrin, were also shown to play important roles in
regulating plasmin formation and activity [1] This may have been the general consensus in the late 1980s, but nowadays it has become clear that many of the individual components of the fibrinolytic⁄ plasminogen activating system perform other roles that could not have been foreseen tPA, for example, is not just a
‘plasminogen activator’; it is now widely appreciated for its role in the central nervous system [2,3] Although it can act on its classical substrate, plasmino-gen, in this compartment, it also associates with other targets, and in some cases can even act like a cytokine
to activate microglial cells without engaging its cata-lytic properties [4] Similarly, the two plasminogen acti-vator inhibitors are now known to perform additional functions PAI-1 can act as an accessory protein that modulates the association of the uPA receptor with in-tegrins This association, in turn, influences cell migra-tion independently of the PAI-1 protease inhibitory activity [5,6]
Keywords
gene regulation; plasminogen activator
inhibitor type 2; protease inhibitor; serpin
Correspondence
R L Medcalf, Australian Centre for Blood
Diseases, Monash University, 6th Floor
Burnet Building, 89 Commercial Road,
Prahran, 3181 Victoria, Australia
Fax: +61 39903 0228
Tel: +61 39903 0133
E-mail: Robert.medcalf@med.monash.edu.au
(Received 31 March 2005, accepted 13 July
2005)
doi:10.1111/j.1742-4658.2005.04879.x
Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine protease inhibitor (serpin) with unique and tantalizing properties that is generally considered to be an authentic and physiological inhibitor of uro-kinase However, the fact that only a small percentage of PAI-2 is secreted has been a long-standing argument for alternative roles for this serpin Indeed, PAI-2 has been shown to have a number of intracellular roles: it can alter gene expression, influence the rate of cell proliferation and differ-entiation, and inhibit apoptosis in a manner independent of urokinase inhi-bition Despite these recent advances in defining the intracellular function
of PAI-2, it still remains one of the most mysterious and enigmatic mem-bers of the serpin superfamily
Abbreviations
ARE, AU-rich element; IL, interleukin; K5, keratin 5; LPS, lipopolysaccharide; ov, ovalbumin; PAI, plasminogen activator inhibitor; PAUSE-1, PAI-2-upstream silencer element-1; Rb, retinoblastoma; serpin, serine protease inhibitor; TNF, tumour necrosis factor; tPA, tissue-type plasminogen activator; TTP, tristetraprolin; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor.
Trang 2For PAI-2, there was a strong suspicion soon after
its discovery that the real function of this inhibitor
had been overlooked From a teleological viewpoint,
a non-uPA inhibitory role was expected, as the
majority of PAI-2 was found in a location where its
intended or perhaps presumed natural target (i.e
uPA) did not even exist, that being the cell cytosol
[7] This minireview will focus on the cellular and
molecular biology of PAI-2 and highlight some of
the most recent findings on the role and impressive
pattern of regulation of this enigmatic protease
inhi-bitor Although new data is emerging, PAI-2 is still
one of the most cryptic protease inhibitors known
and its role in biology and pathophysiology is still
being unravelled
General biology of PAI-2
PAI-2 was defined as a placental tissue-derived uPA
inhibitor over three decades ago [8], which was
subse-quently verified by others [9,10] Human PAI-2
con-sists of a single chain protein of 415 amino acids
encoded by a 1900 bp PAI-2 transcript and is highly
homologous with mouse and rat PAI-2 PAI-2 exists
predominantly as a 47 kDa nonglycosylated
intracellu-lar form [7], however a small percentage of PAI-2
is able to enter the secretory pathway by a process
referred to as facultative translocation [11] and
secre-ted as a 60 kDa glycosylasecre-ted protein The basis for this
bi-topological distribution is due to the lack of a
con-ventional hydrophobic amino-terminal signal sequence
Instead, PAI-2 possesses an inefficient internal signal
sequence [12] This bi-topological (intracellular⁄
extra-cellular) expression pattern of PAI-2 has also been
shown for a related serpin known as maspin [13] and
this feature remains one of the most intriguing aspects
of PAI-2 (and maspin) biology Because the uPA
inhibitory capacity of both forms of PAI-2 seems to be
similar, it was considered early on that the release of
high local concentrations of nonglycosylated PAI-2
from dead or dying cells at sites of inflammation may
provide an immediate source of enriched uPA
inhibi-tory activity [14] While anecdotal evidence would
cer-tainly support this, the growing consensus of opinion,
however, is that PAI-2 possesses an as yet ill-defined
intracellular role
Structural considerations
Based on a number of criteria, PAI-2 has been
classed as a member of the ovalbumin subfamily of
serine protease inhibitors known as the ovalbumin
(ov)-serpins, with ovalbumin being the prototypical
member of this family [15] Ovalbumin-serpins share
a common genomic structure and all lack conven-tional signal sequences and are, for the most part, located intracellularly Closer examination of the genomic structure of PAI-2 revealed another distinctive feature, that being an extension of exon 3 that encoded
a unique domain bridging helices C and D of the pro-tein This so-called C-D interhelical domain [16], other-wise known as the C-D loop, has since been implicated
in the function of PAI-2 Glutamine residues in the C-D loop can be crosslinked by tissue transglutaminase and factor XIII to structures in trophoblasts and to fibrin [16–18] Moreover, the C-D loop has been shown to bind noncovalently to annexins, retinoblastoma protein and a number of unidentified proteins [19,20] Using the expressed C-D interhelical loop as bait, Fan
et al identified the b1 subunit of the proteosome as
a binding partner [21] The physiological relevance
of these findings remains to be clarified, but none-theless points to diverse roles of the C-D loop in PAI-2 function
Polymerization of PAI-2 Many serpins have been shown to undergo loop sheet polymerization Generally, polymerization occurs due
to a genetic aberration, which results in serious patho-logical consequences due to conformational changes of these proteins [22] PAI-2 is also able to polymerize, but in contrast to the other polymerizing serpins this is not a consequence of a mutation in the PAI-2 gene, nor is it associated with any known pathologies Indeed, PAI-2 displays conformational plasticity and is the only known wild type serpin to form polymers spontaneously and reversibly under physiological con-ditions [23] Furthermore, this is influenced by the redox status of the cell: PAI-2 can exist in either a sta-ble monomeric or a polymerogenic configuration, the latter stabilized by disulfide bonds that connect a cys-teine residue within the C-D loop to another cyscys-teine residue at the bottom of the molecule [24] The mono-meric form is also stabilized by binding to vitronectin while retaining its inhibitory activity Under conditions
of oxidative stress, the polymerized inactive configur-ation of PAI-2 can form but whether this has any other impact on cell function is unknown More recently it was shown that the C-D loop within the sta-ble monomeric form of PAI-2 is mobile and that the monomeric and polymerogenic forms of PAI-2 were interchangeable [25] Hence, not only does PAI-2 exist
as a bi-topological protein, it can also exist in different conformational forms within the intracellular compart-ment
Trang 3Expression pattern of PAI-2 and its role
in pregnancy
Under normal conditions, PAI-2 has a restricted tissue
distribution pattern with expression detected at high
levels in keratinocytes, activated monocytes and the
placenta [26] Lower constitutive levels of PAI-2 are
also found in other cells, including cells of neuronal
origin [27] Plasma levels of PAI-2 are usually low or
undetectable; however, they rise significantly in some
forms of monocytic leukaemia [28] One of the most
physiologically striking observations for PAI-2
con-cerns its association with pregnancy Plasma levels of
PAI-2 increase impressively during the third trimester
of pregnancy (up to 250 ngÆmL)1) and are maintained
at these levels for up to 1 week postpartum and then
rapidly decline [10] The tissue source of plasma PAI-2
is the placenta itself Indeed, PAI-2 is highly expressed
in trophoblasts [29,30] and it was conjectured that
PAI-2 acted to protect the placenta from proteolytic
degradation towards the end of the gestational period
and to regulate postpartum haemostasis However, a
placental associated PAI-2 sensitive protease is yet to
be described Perhaps the role of PAI-2 in the placenta
is unrelated to protease inhibition In this regard, it is
interesting to point out that PAI-2 forms complexes
with other placental proteins, including vitronectin
[9,31], but the functional significance of this in terms
of placental biology is unknown
The association of PAI-2 with pregnancy and its
placenta-specific expression suggested that PAI-2
might perform a critical function during foetal
devel-opment If this were indeed the case, one would have
predicted a developmental abnormality in PAI-2–⁄ –
mice Mice with a targeted deletion in the PAI-2 gene
have been described but these mice have not as yet
displayed any noticeable phenotype [32] at least under
normal, nonchallenging conditions To exclude the
possibility that the lack of effect was due to
redund-ancy with PAI-1, a double knock-out mouse was
pro-duced that harboured a disruption at both the PAI-1
and PAI-2 loci Still, no obvious phenotype was seen
Given the high degree of PAI-2 expression in the
human placenta, it was surprising at first glance that
foetal development and reproduction was undisturbed
in PAI-2–⁄ – mice However, no firm conclusions can
be drawn from this as, unlike the human situation,
PAI-2 is not found in the mouse placenta It is
indeed a strange curiosity that the presence and
regu-lation of placental PAI-2 is not conserved in the
mouse However, this important data has only been
presented as a statement within a review article [33]
and additional supportive information would be
welcomed on the presence or absence of PAI-2 in the mouse placenta
The role of PAI-2 in the skin PAI-2 expression within the skin is restricted to the upper layers of the dermis PAI-2 has also been repor-ted to inhibit keratinocyte proliferation [34] and to play a role in keratinocyte differentiation [34] A cleaved form of PAI-2 has been found in keratinocytes [35] implying that PAI-2 itself is a substrate for a pro-tease in these cells
To determine the consequences of dysregulation of PAI-2 on epidermal differentiation, Zhou et al [36] produced transgenic mice that overexpressed PAI-2 in the proliferating layers of mouse epidermis and hair follicle cells by placing the PAI-2 transgene under the control of the keratin 5 (K5) promoter Although the presence of PAI-2 had no effect on skin morphology
or proliferation under normal conditions, the PAI-2 overexpressing mice were found to be highly suscept-ible to chemically induced papilloma formation The means by which PAI-2 promoted papilloma formation
is unknown, but may have been related to its reported antiapoptotic effect (see below) since cessation of tumour promoting treatment in control mice resulted
in extensive apoptosis of the papilloma but not in the K5-PAI-2 transgenic mouse
Leukocyte biology Monocytes and macrophages express PAI-2 and levels are impressively increased in these cells following sti-mulation with tumour necrosis factor (TNF) [14] and lipopolysaccharide (LPS) [37,38] Induction of PAI-2 gene expression has been associated with monocyte dif-ferentiation, at least in the U-937 monocyte-like cell system [39], suggesting a role for PAI-2 in this process
In the mouse system PAI-2 does not appear to be indispensable for leukocyte development as PAI-2–⁄ – mice exhibit normal leukocyte recruitment and appear
to differentiate normally [32]
Novel insights into the role of PAI-2 in monocytes came from studies using THP-1 cells Unlike all other widely used monocyte-like cell lines (e.g U-937, K562, HL60) that express endogenous PAI-2, the THP-1 monocytic cells provided a notable exception to this rule THP-1 cells bear many features common to regu-lar mononuclear phagocytes, but are closer in pheno-type to a mature monocyte than other monocytic cell lines (i.e U-937, K562) Although the expression pat-tern of THP-1-derived uPA and its receptor (uPAR) is similar to that observed in other monocytic cell lines
Trang 4[40,41], THP-1 cells do not express a functional PAI-2
protein [40] These authors demonstrated that
THP-1-derived PAI-2 was functionally inactive while the PAI-2
transcript in these cells was truncated
The molecular basis for the aberrant production of
PAI-2 in THP-1 cells is due to a translocation anomaly
[42] The complete absence of a functional PAI-2 in
these cells defined THP-1 cells effectively as a human
monocytic PAI-2–⁄ –cell line To take advantage of this
PAI-2–⁄ –cell line, Yu et al [43] produced stable THP-1
cell lines that expressed either wild type PAI-2 or a
PAI-2 mutant containing an alanine substitution at the
P1 position (Arg380) The presence of wild type PAI-2
caused a significant decrease in THP-1 cell
prolifer-ation, reduction in DNA synthesis and a phenotypic
change following phorbol ester-induced differentiation
The ability of PAI-2 to alter the differentiation process
was dependent on its active form as cells expressing
PAI-2Ala380 did not display these changes This study
demonstrated for the first time an intracellular role for
active PAI-2 in monocytes These results were
con-sistent with the possibility that PAI-2 disrupted an
intracellular protease(s) that was involved in cell
prolif-eration and⁄ or differentiation although no such target
protease has been detected thus far
PAI-2 is also present at very high levels in
eosino-philic leukocytes Indeed the level of PAI-2 in these
cells was shown to be the highest among all other
leu-kocyte subtypes [44] Furthermore, PAI-2 was localized
to eosinophil-specific granules and shown to be still
capable of inhibiting urokinase It was suggested that
PAI-2 might play a role in eosinophil mediated
inflam-mation and tissue remodelling
Role of intranuclear PAI-2
A number of Ov-serpins have been detected within the
nuclear compartment, including bomapin, PI-9, and
maspin [45–47] PAI-2 has also been shown to have a
nuclear presence [20,45,46] yet the physiological role of
PAI-2 in this compartment is unknown However, in
a study by Darnell et al nuclear-located PAI-2 was
shown to bind to retinoblastoma protein (Rb) via its
CD-loop [20] Rb is a prototypical tumour suppressor
gene and critical cell cycle regulator that targets the
E2F family of transcription factors [48] PAI-2
colocal-ized with Rb and, interestingly, inhibited Rb turnover
by protecting Rb from proteolysis [20] This in turn
led to an increase in Rb protein levels and
Rb-medi-ated activities including the transcriptional repression
of oncogenes This is a curious finding because PAI-2–⁄ –
mice do not appear to have any change in cell number,
and it would be predicted that Rb turnover would be
accelerated in PAI-2–⁄ – mice freeing E2Fs to mediate proliferation Although additional evidence is required
to explore the consequences of PAI-2 and Rb inter-action, these data underscore a novel and previously unsuspected intranuclear role for PAI-2
The role of PAI-2 in metastatic cancer, apoptosis and infection
Cancer
A number of in vivo studies have assessed the prognos-tic relevance of tumour- and stromal-derived PAI-2 in the metastatic spread of cancer of the neck, lung and breast [49–53] The only established protease target for PAI-2, namely uPA, is strongly implicated in facilita-ting cell dissemination in the context of tumour meta-stasis and it is likely that the beneficial effect of PAI-2 seen in these studies is simply via uPA inhibition Overexpression of PAI-2 in melanoma cells prevented spontaneous metastasis of transplanted cells in scid mice [54], while overexpression of PAI-2 in HT-1080 cells has also been shown to reduce uPA-dependent cell movement in vitro and metastatic development
in vivo [55] The ability of PAI-2 to selectively bind to cell surface bound uPA (via uPAR) and subsequently
be internalized [56] has prompted studies to assess the effectiveness of PAI-2 as a delivery vehicle for isotopes (213Bi) and toxins that can be targeted to uPA-bearing cancer cells This approach has provided positive out-comes at least in some preclinical studies [57–59]
Apoptosis Circumstantial evidence that first implicated PAI-2 as
an inhibitor of apoptosis came from genetic associ-ation studies with BCL-2 [60] The BCL-2 proto-oncogene was discovered over 15 years ago as the archetype inhibitor of apoptosis Evidence that BCL-2 was playing such a role in humans came from studies
in patients with follicular lymphoma In these patients,
a translocation event occurs between chromosomes 14 and 18 t(14; 18) that brings the BCL-2 gene into juxta-position with the locus of the immunoglobulin heavy chain, resulting in overexpression of BCL-2 [61] This
in turn inhibits the apoptotic process of the lym-phoma The relevance of this to PAI-2 stemmed from the fact that the PAI-2 gene is located less than
300 mbp from the BCL-2 gene and is translocated along with BCL-2 in patients with follicular lym-phoma PAI-2 and BCL-2 also share structural similar-ities and it was proposed that the function of PAI-2 may overlap with BCL-2 So with this background, a
Trang 5number of publications in the mid-1990s provided
in vitroevidence that PAI-2 could inhibit TNF-induced
apoptosis in HT-1080 fibrosarcoma cells [62] and HeLa
cells [63] A cleaved form of intracellular PAI-2 has
been found in ND4 monocytes undergoing apoptosis
[64] In no case has an intracellular PAI-2-sensitive
proteinase been identified Other reports, however,
have provided contradictory data [18] One argument
in the interpretation of the significance of PAI-2
dur-ing apoptosis concerns the level of enforced expression
of PAI-2 in the model systems used In most of these
in vitro studies, PAI-2 was overexpressed in cells that
either did not make PAI-2 at all or were expressed to
levels that well exceeded endogenous expression levels
Under these conditions, PAI-2 may indeed inactivate
one or more intracellular proteases, but whether this
genuinely reflects the in vivo role of PAI-2 can be
rea-sonably debated
Viral infection
Evidence to suggest that PAI-2 participates in the host
response to alphaviral infection is based on
over-expression studies in HeLa cells The protective effect
of PAI-2 was indirect, as PAI-2 appeared increase
interferon levels which then triggered an increase in
the expression of a battery of antiviral genes [65]
Shafren et al [66] also demonstrated the same PAI-2
over-expressing HeLa cells were protected from lytic
infection by human picornaviruses In this case, PAI-2
promoted the transcriptional down-regulation of
sur-face expression of picornavirus receptors (decay
accel-erating factor, intercellular adhesion molecule-1 and
coxsachievirus-adenovirus receptor; DAF, ICAM-1
and CAR, respectively) These observations further
support the growing body of evidence [42,43] that
intracellular expression of PAI-2 is linked to a
signal-ling pathway(s) that can reprogram gene expression
One may even speculate that PAI-2 could play a role
in the innate immune response since its expression is
commonly associated with inflammation and the host
response to infection
PAI-2 gene expression and regulation
Based on data accumulated over the past 17 years, it is
evident that the PAI-2 gene expression can be induced
by a wide range of agonists Moreover the level of
PAI-2 gene induction in some examples is quite
extra-ordinary Agonists of PAI-2 induction include growth
factors (transforming growth factor-b, epidermal
growth factor and monocyte-colony stimulating
fac-tor; TGFb, EGF and M-CSF, respectively), hormones
(retinoic acid, dexamethasone and vitamin D3), cyto-kines [TNFa, interleukin (IL)-1 and IL-2)], vasoactive peptides (angiotensin II), toxins (dioxin and endotoxin) and tumour promoters (phorbol esters and okadaic acid) [26,67] PAI-2 mRNA expression is also strongly increased by the excitotoxic glutamate analogue, kainic acid in neuronal cells in vivo [27]
PAI-2 was cloned by groups that had an intent focus on the cell and molecular biology of PAI-2 [39,68,69], and by others inadvertently through differ-ential gene expression studies For the latter, PAI-2 was identified as a TNF responsive gene in monocytes and fibroblasts [70,71] and as a dioxin responsive gene
in keratinocytes [72] Microarray studies identified PAI-2 as an inducible gene in response to IL-5 [73], factor 7⁄ tissue factor [74], and again by TNF [75] Dif-ferential gene expression profiling (SAGE) of LPS-trea-ted primary human monocytes identified PAI-2 as the third most inducible gene being induced 105-fold by this agent [37] In a microarray study to identify Lp(a) inducible genes in human monocytes, PAI-2 mRNA was found to be the most induced transcript from a screen of 8000 cDNAs [76] These latter studies pro-vide further epro-vidence of the diverse repertoire of agents that strongly regulate PAI-2 expression and by associ-ation, PAI-2 is likely to play a role in the biological consequences initiated by these agents
The impressive magnitude of induction by such a variety of biological agents prompted many laborator-ies to explore the transcriptional and post-transcrip-tional processes underlying PAI-2 expression
Transcriptional regulation of PAI-2 expression Run-on transcription assays provided direct evidence that the induction of PAI-2 expression in U-937 cells following phorbol ester treatment involved dramatic increases in the rate of PAI-2 transcription [39] Similar studies in HT-1080 fibrosarcoma cells demonstrated a transcriptional component following TNF-mediated induction of PAI-2 expression [14] These studies led to
an analysis of the PAI-2 promoter [77,78] DNase-1 protection experiments indicated that the proximal region of the PAI-2 promoter possessed a congested arrangement of cis-acting elements Of these, only the AP1-like elements, AP1a (TGAATCA, )103 to )97) and AP1b (TGAGTAA, )114 to )108), and a cAMP responsive element (CRE)-like element (TGACCTCA, )187 to )182) [77,79] were shown to have functional activity during transcriptional regulation Curiously, a repressor element located between)219 and )1100 was suggested to play a role during TNF induction [80] The identification of the exact sequence within this
Trang 6region and trans-acting factors responsible for this
activity have not been reported Antalis et al [81]
characterized 5.1 kb of 5¢ flanking region in U937 cells
by deletion analysis and found a silencer between
)1977 and )1675 that acts in an orientation- and
position-independent but not cell-specific manner The
silencer activity was localized to a 28 bp sequence
containing a 12 bp palindrome at position )1832,
CTCTCTAGAGAG, which was termed
PAI-2-upstream silencer element-1 (PAUSE-1) Later analysis
defined the minimal functional PAUSE-1 element as
TCTNxAGAN3T4, where x¼ 0, 2 or 4 [82]
UV-cross-linking analyses determined that the PAUSE-1 binding
protein was 67 kDa, but its identity remains
unknown In their study, PAUSE-1 was not
character-ized in the context of TNFa induction and it would
be worthwhile to explore the relationship between
PAUSE-1 and the element that selectively represses
TNFa inducibility in HT-1080 cells [80]
Post-transcriptional regulation of PAI-2 expression
As mentioned earlier, PAI-2 is one of the most highly
regulated genes known, at least in terms of the
magni-tude by which it is induced by growth factors,
hor-mones, cytokines [73,83] and tumour promoters
[39,84] Although PAI-2 induction involves substantial
changes at the level of transcription,
post-transcrip-tional events are also important in modulating its
expression This was first revealed over a decade ago,
when it was shown that the increase in PAI-2 mRNA
after synergetic stimulation by phorbol myristate
ace-tate and TNFa (1000- to 1500-fold) could not be
accounted for by an increase in PAI-2 transcription
rate alone (50-fold), suggesting that
post-transcrip-tional processes influence PAI-2 gene expression [84]
The PAI-2 transcript has since proven to be a
valu-able model to study post-transcriptional regulation,
most notably at the level of mRNA instability
PAI-2 mRNA contains a functional nonameric (UUAUUUAUU) AU-rich element (ARE) in its 3¢-un-translated region [85] Mutagenesis of this element par-tially stabilized the normally unstable PAI-2 mRNA, hence revealing a functional role for this motif [85,86] This element also provides binding sites for several ARE binding proteins, including the stabilizing protein HuR [86] and the mRNA destabilizing protein tristetr-aprolin (TTP) [87] HuR is a member of the Hu family
of mRNA binding proteins and has been associated with promotion of mRNA stability [88] TTP, on the other hand, is a potent mRNA destabilizing protein that associates with ARE elements in cytokine tran-scripts, including TNFa [89] and IL-3 [90] Overexpres-sion of TTP in HEK 293 cells transfected with a constitutively active PAI-2 expression vector resulted
in loss of PAI-2 mRNA, suggesting that TTP can indeed regulate PAI-2 expression [86] Other cytoplas-mic and nuclear proteins also bind to the ARE with the PAI-2 3¢-UTR [85,86] but these are yet to be iden-tified The PAI-2 transcript also possesses another instability determinant located within exon 4 of the PAI-2 coding region [91] UV-crosslinking studies have identified two RNA-binding proteins (approximately 50–52 kDa) that specifically interact with this sequence Taken together, the data published to date suggest that PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3¢-UTR It remains to be determined whether these instability elements in the coding region and the 3¢-UTR act in a coordinated fashion to control PAI-2 mRNA stability (Fig 1)
Conclusion PAI-2 has been implicated in many facets of biology some of which are unrelated to its ability to inhibit extracellular uPA However, the ability of PAI-2 to reduce the metastatic potential of a number of cancers,
Fig 1 Schematic representation of
regula-tory domains within the PAI-2 transcript that
influence PAI-2 expression at the
post-tran-scriptional level At least two domains exist:
one within exon 4 (E4) of the coding region
and the other within the 3¢-UTR Proteins
that have been shown to bind to these
regions in vitro are shown See text for
details E, exon.
Trang 7presumably via inhibition of extracellular or
cell-sur-face bound uPA, is arguably the most consistent and
physiologically relevant finding to date Nonetheless,
the response of the PAI-2 gene to such a diverse
reper-toire of agonists and the impressive magnitude of
induction in leukocytes in response to toxins and
cytokines invokes PAI-2, albeit circumstantially, with
inflammation, tissue repair and possibly the innate
immune response Similarly, the evidence linking PAI-2
with apoptotic processes, Rb turnover, cell
prolife-ration and differentiation is substantial and gaining
momentum but more direct and physiologically
focused experiments are needed in order to define its
undisputed intracellular function It is anticipated that
this information will be forthcoming through a more
extensive analysis of the PAI-2–⁄ – mice Results from
these experiments are eagerly awaited
Acknowledgements
This study was supported by grants obtained by RLM
from the National Health and Medical Research
Council of Australia
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