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Leavesley, David, Kashyap, Abhishek, Croll, Tristan, Sivaramakrishnan,Manas, Shokoohmand, Ali, Hollier, Brett, & Upton, Zee(2013)Vitronectin - Master controller or micromanager?IUBMB Life, 65(10), pp. 807-818.

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https://doi.org/10.1002/iub.1203

 

Vitronectin  –  Master  Controller  or  Micromanager?   1  

 

Invited Review – IUBMB Life

Vitronectin – Master Controller or Micromanager? Authors. David Leavesley*, Abhishek Kashyap, Tristan Croll, Manaswini Sivaramakrishnan, Ali Shokoohmand, Brett Hollier & Zee Upton. Address. Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia.

*Corresponding Author: d.leavesley@qut.edu.au

Keywords. Vitronectin, Extracellular Matrix, Growth Factor, Multi-protein Complex, Thrombogenesis, Coagulation,

 

2   Vitronectin  –  Master  Controller  or  Micromanager?  

 

Summary

The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key

controller of mammalian tissue repair and remodelling activity is emerging from nearly 50

years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out

mouse was found to be viable and to have largely normal phenotype. However, diligent

observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound

healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of

thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus

grows in an organised structure. In addition to closing the wound, the thrombus also serves to

protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair

mediators and provides a provisional scaffold for the repairing tissue. In the absence of

vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins.  

Our data demonstrates that a wide variety of biologically active species associate with VN.

While initial studies were focused on mitogens, other classes of bioactives (e.g.

glycosaminoglycans, metalloproteinases) are now also known to specifically interact with

VN. Many of these interactions are long-lived, often resulting in multi-protein complexes,

while others are stable for prolonged periods. Multiprotein complexes provide several

advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-

stimulation of cell surface receptors and thereby enhancing cellular / biological responses.

We contend that these, or equivalent, multi-protein complexes mediate vitronectin

functionality in vivo. It is also likely that many of the species demonstrated to associate with

vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes.

Thus the predominant biological function of vitronectin is that of a master controller of the

extracellular environment; informing, and possibly instructing cells ‘where’ to behave,

‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

 

Vitronectin  –  Master  Controller  or  Micromanager?   3  

 

Introduction – What is VN?

An ability to repair damaged tissue(s) is essential to sustain life in all multicellular

organisms. We remain in awe of the ability of some amphibians (e.g. salamander) and lizards

to completely regenerate lost appendages with fully restored functionality. The biological,

physiological and molecular events that underlie tissue repair and regeneration has been a

focus of human intrigue and experimentation for centuries. However, only recently are we

beginning to understand how complex this challenge is in higher order organisms, especially

those with multiple and complex tissue types. It requires a cascade of temporal and spatial

molecular and cellular events, of which absent or defective function results in reduced

individual fitness and life expectancy. In this brief review we introduce and discuss the

emerging concept of the cellular glycoprotein vitronectin as a biological ‘superglue’ and key

controller of mammalian tissue repair and remodelling.

Vitronectin (VN) was first described as an adhesive protein that supported the

attachment and spreading on glass (Latin: vitreous, ‘of glass’) of ‘unadapted’ (primary) cells

(1). It is the property of serum that enables mammalian cells to adhere to culture vessels,

supporting cell survival and propagation. Therein lies the first clue to its biological function.

Since this initial report, VN has been independently “rediscovered” by several investigators

and was reported variously as ‘serum spreading factor’ (2), ‘S protein’ (of complement,

equivalent to ‘Protein X’, and not to be confused with ‘Protein S’) (3), Plasminogen activator

inhibitor-1 binding protein (4), somatomedin B (SMB, 5) and ‘epibolin’ (6). It shares

structural similarities with the membrane-attack-complex inhibitor (of complement) (7),

plasminogen activator inhibitor-1 (PAI-1) binding protein (PAIBP) (4) and with the matrix

metalloproteinase (MMP)-21 (8). Indeed we have used this similarity to MMP and the well-

described hemopexin-like repeats with the solution for the SMB domain to create a new

model of VN (Figure 1).

Physico-chemical analysis of VN purified from human tissues indicates that it

possesses a high degree of conformational flexibility (Fig 1). It is present in vivo as

monomeric (native) and multimeric (termed ‘denatured’) forms, depending upon its

association with other molecular species and the activity of proteolytic enzymes, and includes

conserved structural motifs that confer VN with specialised functions (9; 10). The 44-residue

N-terminal SMB domain is frequently found as a biologically active cleavage product,

reported to homodimerise  and  bind  with  the  urokinase  receptor  (uPAR)  and  PAI-­‐1  (11).

 

4   Vitronectin  –  Master  Controller  or  Micromanager?  

 

In spite of these reports, VN is commonly considered to be an incidental component of the

extracellular matrix (ECM), primarily associated with endothelial and perivascular tissue. In

contrast to ‘classical’ ECM glycoproteins such as collagen (COL), fibronectin (FN) and

laminin, which have structural functions, VN is a ‘matricellular’ protein. Matricellular

proteins are “modular, extracellular proteins whose functions are achieved by binding to

matrix proteins as well as to cell surface receptors, or to other molecules such as cytokines

and proteases that, in turn, interact with the cell surface” (12). Thus while not possessing

bona fide structural credentials, nonetheless VN is a critical component of the extracellular

space.

VN can be detected in most tissues, particularly after exposure to trauma or stress (13).

Vitronectin is present at high concentrations in the peripheral vasculature (200-400 µg/mL)

(14), and is synthesised predominantly by hepatocytes in the liver. Given the abundance of

VN in blood plasma (c.f. FN 300-500 µg/mL) the first decades of VN research were largely

focused on how VN contributed to homeostasis and innate immunity (15; 16). However,

recent evidence indicates VN has a wider role consistent with its matricellular activity:

modulating biological functions critical for the survival of the organism. In this review we

discuss the functional dependency many endogenous regulatory species have for VN,

providing evidence that VN should be viewed as a molecular organiser or, micromanager.

The synthesis of VN by somatic tissues is minimal in comparison to hepatocyte

production (17). VN is synthesised as a precursor species with limited activity and is

transported into the interstitial space via receptor-mediated exo- and transcytosis (13, 18).

Messenger RNA encoding VN (gene VTN, on 17q11.2 [HGNC:12724]) is detectable in most

cell and tissue types, and its expression is frequently upregulated in tissues experiencing

stress (e.g. proinflammatory response) (19). When tissue is subjected to stress,

immunoreactive VN accumulates in the extravascular space. It also accumulates in the

interstitial space around tumour islands (carcinomas and sarcomas). Deposits of VN are

notably enriched in immune complexes associated with chronic inflammatory diseases (e.g

rheumatoid arthritis) and chronic fibrotic diseases (lung, liver and kidney fibrosis) 19). In

response to acute tissue injury, VN interacts directly with elements of the complement (20)

and coagulation cascades (21), through which it impacts upon thrombus formation, stability

of vessel occlusion and subsequent inflammation and immune responses. This activity is

largely a consequence of circulating in complex with PAI-1 (22). Thus, VN participates in

 

Vitronectin  –  Master  Controller  or  Micromanager?   5  

 

key physiological events that take place during the re-establishment of vascular homeostasis,

wound repair and tissue regeneration. Confocal microscopy has localised VN to unidentified

cytoplasmic structures within necrotic cells but it is not found associated with early apoptotic

cells (i.e. cells with compromised membrane integrity) (23). VN accumulation in sites of

injury, has been interpreted as leakage from local vessels. It is conjecture whether VN acts as

an alarm signal, phagocytic signal (opsonin), immunosuppressant, or has some

protective/sequestration function in this situation.

Vitronectin is also associated with a number of ‘solid’ human tumours, in particular

aggressive and metastatic tumours (24, 25, 26). As evident in traumatised tissue (above), VN

is frequently enriched in the pericellular stroma, the interstitial boundary evident between

tumour cell islands/clusters and adjacent ‘normal’ connective tissue (27, 28). Less surprising,

VN is also found in the walls of tumour-associated blood vessels (29). These observations

suggest that VN fulfils a key function, as yet uncharacterised, at structure-function

boundaries. In contrast, ‘normal’ or homeostatic tissues are at best ‘weakly reactive’ for VN.

In uninjured tissues immunoreactive VN is usually limited to ducts and small blood vessels as

a homogeneous, weak staining reaction (26).

Structure and Function (monomeric/polymeric)

The mature VN polypeptide is stabilised by multiple disulphide bonds located at the

carboxyl polycationic domain and amino SMB terminal domain. It is extensively decorated

with: (a) saccharides at three N-glycosylation motifs (Fig 1); (b) at least two sulphates on

tyrosine residues (27); and (c) multiple phosphate groups on threonine residues (28).

Vitronectin exhibits a high degree of conformational flexibility (9) which presumably is

determined by the local aqueous environment. Small angle X-ray scattering experiments

demonstrate that in aqueous phase VN monomers assume a ‘peanut-shaped’ globular

conformation ~11 nm long and ~5 nm wide (29). In this configuration VN is largely

unreactive and has little binding affinity towards heparin, presumably because the cell

recognition RGD and polycationic (heparin-binding) motifs are structurally hindered (Fig 1).

In non-aqueous environments (e.g. associated with ‘insoluble’ ECM fibrils), functional

studies suggest VN ‘denatures’ and self-assembles into multimeric meshworks (9). In this

configuration, previously cryptic functional motifs become available and confer new

functional attributes to the VN molecule.

 

6   Vitronectin  –  Master  Controller  or  Micromanager?  

 

The human VN monomer is synthesised as a precursor polypeptide of 478 amino acids

(75 kDa) including a 19-amino acid signal peptide (16) (Fig 1). Polysaccharides account for

~30% of the mature mass. Additionally, VN is a substrate for the transglutaminases including

factor XIIIa, allowing its covalent incorporation into fibrin clots and/or ECM (30). In some

tissues the 44 aa N-terminal fragment is cleaved, releasing the cysteine-rich SMB peptide

(31). Further processing cleaves the 75 kDa mature protein at amino acid (aa) 379 into

daughter polypeptides of 65 kDa and 10 kDa, linked by a disulphide bridge. With a core

polypeptide comprising multiple structural functions residing in four distinct domains (Fig 1),

interaction with partner molecules, especially glycosaminoglycans (GAGs), confer mature

VN with dynamic structural flexibility. Unsurprising each conformation supports distinct

biological activity. The function of VN is determined by, and dependent upon, interactions

with other species (e.g. divalent cations, GAGs) which induce conformational change and

self-assembly into heterogeneous multimolecular complexes (9). Protease activity and/or

physical interactions with ‘target’ species (usually called ligands) cause VN to ‘denature’ and

refold into a conformation predisposed to increasing the affinity of such interactions (Fig 1),

and coincidently self-assemble into fibrils forming a heterogeneous meshwork (extracellular

matrix) with other extracellular glycoproteins (16). The multimeric state further reveals

previously cryptic sites, dramatically enhancing VN activity. In addition to the

aforementioned SMB domain, VN includes the conserved consensus cell-recognition motif

Arg-Gly-Asp (RGD), a flexible connecting region, and two hemopexin-like domains of two

similar halves, approximately two hundred amino acid residues large formed from a basic

repetitive unit of 35 ≥ 45 residues connected by a histidine-rich hinge region (32). The

hemopexin domains are speculated to prevent haem-mediated oxidative stress through

sequestration (33). A strongly basic region adjacent to the C-terminus (aa 348-379) assembles

as a groove supporting interactions with molecular species containing polyanionic domains

(e.g. GAGs, proteoglycans, metabolites) (reviewed in 16). The structural domains present in

VN are largely responsible for the polyfunctional biological activity evident in experimental

analyses of this multi-talented extracellular organiser.

In the ‘denatured’ conformation, VN interacts with diverse molecular species present

in the extracellular and interstitial milieu. Complement intermediates C5b-C6, C5b-C7, and

complement factors C8 and C9 interact directly with the heparin-binding domain of VN

(Table 1) (7, 34). PAI-1 and fibrinogen (gamma chain) directly associate with unreactive

circulating plasma VN (21, 35). Perforins produced by cytolytic T-cells interact with the

 

Vitronectin  –  Master  Controller  or  Micromanager?   7  

 

polycationic region. Thus these antimicrobial polypeptides are effectively captured and stored

within the provisional ECM and coincidently inhibit lytic activity. Proteases critical to the

coagulation cascade, PAI-I (36), thrombin, TAT III complex (37), fibrinogen (and fibrin),

collagen I and III, urokinase and proteolytic fragments derived from their activity (e.g.

angiostatin/plasminogen, anastellin) (reviewed in 13,16) have all been identified to associate

with multimeric, or denatured VN. Interestingly, and of potential significance, many of these

interaction are mediated via the glycosaminoglycan intermediates which bind to VN through

the basic consensus heparin binding groove (residues 348 - 379) in the carboxyl-terminus

(38).

Vitronectin participates in the earliest response to tissue trauma. Measured at micromolar

concentrations in plasma, VN accumulates as complexes with fibrinogen and with PAI-1.

These interactions stabilise fibrinogen and PAI-1, preventing unintended coagulation and

fibrinolysis, respectively (39, 40). Upon activation of the clotting and coagulation cascades

(triggered by exposure to air, debris from damaged cells, or endogenous alarm signals),

plasma VN undergoes rapid conformational changes, self-assembling into fibrils and

exposing cell-binding and glycosaminoglycan binding motifs. Multimerisation reveals

previously cryptic domains/sites dramatically increasing local concentrations of available

binding sites and precipitating interactions that support the assembly of the heterogeneous

multiprotein thrombus complex: a fibrillar meshwork of fibrin, fibronectin, von Willebrand

factor (vWF), complement pathway (e.g. factor XIII), coagulation intermediates (e.g. factor

VIII) and cell debris (e.g. necrotic cell fragments). While the clot seals the wound (protecting

underlying tissue from exposure to air) and re-establishes haemostasis, it coincidently

supports cell-specific survival, cell infiltration and tissue repair as a provisional ECM. Thus

the clot is both a reservoir of biologically active species and metabolites (41) and a

provisional ECM. Molecular crowding ensures bioactive species remain at high local

concentrations, supporting beneficial and/or deleterious physiological processes. Key

regulators of clot integrity and stability, tissue plasminogen activator (tPA) and urokinase

plasminogen activator (uPA), attenuate further activation of plasmin from plasminogen

precursors sustaining clot integrity. Fibrin break-down (fibrinolysis) releases fibrin

degradation products, some known to act upon thrombin and inhibit further clot formation,

while others act upon blood vessel growth and permeability (42).

 

8   Vitronectin  –  Master  Controller  or  Micromanager?  

 

VN interacts directly, and indirectly, with a repertoire of extracellular species which

support diverse biological and physiological functions (summarised in Table 1). While many

such interactions are predicted from the presence of consensus sequences within the primary

sequence of VN, a large number of interactions are complex in nature, dependent upon post-

translational modifications (e.g. glycosylation, conformational rearrangements) and less well-

characterised interactions with intermediate species (e.g. glycosaminoglycans, polysulphated

proteoglycans, argonaute, part of the RNA-induced silencing complex (RISC)(38).

GAGs are classically thought to function in the main as inert ‘space-filling’

molecules. With a net negative charge, GAGs attract cations (e.g. Na+, Ca2+), positively

charged metabolites and glycoproteins. Their high net charge also attracts and organises the

surrounding water, resulting in a semi-aqueous ‘hydrogel’ state. GAGs can be viewed as a

hydrostatic ‘straight-jacket’, which restricts molecular flexibility and maintains order (43,

44). They also function as intermediates linking unrelated glycoproteins to multimeric VN

via the polycation groove (45). Thus the pericellular microenvironment, also called

glycocalyx, is a rich heterogeneous pool of well-ordered proteins, saccharides, lipids and

metabolites. Specific cell surface receptor systems translate the composition of this external

ECM via outside-in ‘feedback’ signalling pathways. VN, for example, is recognised by the

VN-binding integrins αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αIIbβ3 (46).

Why so many ‘VN receptors’? At the very least these distinct receptors suggest

specialisation and/or divergent functions. VN ligation of integrins and subsequent

phosphorylation of intracellular targets causes integrins to become clustered in the plane of

the cell membrane. This aggregation is thought to precipitate the assembly of protein rafts

comprising integrins, cytoskeletal proteins and signalling molecules, visible as punctate

patterns under differential interference contrast microscopy and known as focal adhesion

contacts (47). Ninety percent of cellular phosphoproteins are located within such multiprotein

rafts at the cell membrane (48). Focal adhesion contacts are interpreted to represent intimate

cell linkages between elements of the ECM and transmembrane outside-in signalling

cascades. These signalling cascades ensure the cell response is appropriate to the

environment. In the context of tissue injury, VN provides a foundation scaffold for both the

provisional ECM and coincidently facilitates inflammatory and immune cell infiltration and

coincidently healing somatic tissue.

 

Vitronectin  –  Master  Controller  or  Micromanager?   9  

 

Given the body of experimental evidence demonstrating the earliest responses to

tissue injury are orchestrated by VN, it came as a surprise to discover mice in which VN

expression had been ‘knocked out’ developed normally and had normal blood chemistry (45).

In a series of careful studies Jang et al. (46) subsequently revealed the VN-KO animals suffer

from delayed healing. Thrombogenesis is decreased and fibrinolysis is increased in the VN-

KO animals, thereby delaying new blood vessel growth and thus wound healing (46). New

blood vessels that do form are ‘leaky’ due to the loss of vascular endothelial (VE)-cadherin, a

molecule that mediates strong cell-cell attachment and forms the ‘water-proof’ seal between

adjacent endothelial cells (47). Macrophage infiltration is also notably reduced, while the

levels of plasma tPA and uPA remained elevated in VN-KO mice. Thus the resolution of

inflammation is also indicated to be defective. The molecular basis of this pathology remains

to be elucidated.

VN in cancer progression

Upon interacting with cell surface receptors, VN triggers intracellular signalling cascades,

which affect critical cellular events, including cell attachment, spreading, migration and

survival (45). Hence, VN occupies a prominent role in tissue repair and homeostatic

processes. Considering the wealth and diversity of published experimental data, VN might be

interpreted as the master controller, the organiser who intimately informs the cell what’s

happening in its immediate microenvironment. Evidence supporting such a key role is also

implicated by the contribution of VN to the development and progression of cancer. These

similarities were sufficient to prompt Dvorak (48) to describe cancer as “wounds that do not

heal”. Mammalian development, wound repair and cancer share extensive common features

of phenotype, physiology and biochemical systems.

Through similar interactions with integrins and uPAR, VN is reported to modulate

cancer metastasis, possibly functioning as a scaffold, or ECM support for tumour cell

migration during invasion, and/or endothelial cell migration during angiogenesis (24, 26, 49).

Evidence supporting this concept is the migration of the highly metastatic MDA-MB-231 and

MDA-MB-435 breast cancer cell lines when assayed in vitro is inhibited when αvβ3 and

αvβ5 VN-receptors (VN-binding integrins) are blocked with antagonists (50). The αvβ3 and

αvβ5 integrins are also critical to angiogenesis; required to sustain tumour growth greater

than 1≤ 2 mm, and subsequent tumour cell dissemination (47, 51). Collectively the weight of

 

10   Vitronectin  –  Master  Controller  or  Micromanager?  

 

in vivo and in vitro evidence overwhelmingly supports a critical role for VN as a foundation

ECM, or master organiser, supporting somatic tissue repair and cancer progression through

direct interactions with binding partners which include PAI-I, uPAR and integrins.

Subsequently, VN supports tumour cell survival and growth indirectly by supporting

angiogenesis.

VN supports angiogenesis

In order to survive and thrive all eukaryotic tissues require a blood supply. This is as true for

tumour cells as it is for somatic tissues. Indeed without vascularisation tumours cannot grow

beyond 1–2 mm3, due to a lack of oxygen and other essential nutrients (52, 53). Fundamental

to new blood vessel growth and survival is the endothelial basement membrane. The

basement membrane provides anchorage through which cell adhesive receptors, including

integrins, interact and provide the mechanical tension required for blood vessel

morphogenesis (54). Dynamic remodelling of the endothelial ECM, particularly by

membrane-type matrix metalloproteinases (MT-MMPs), co-ordinates de novo formation of

new vessel tubes. Sprouting endothelium aligns and assembles into new tubes with a new

vascular basement membrane, a specialised ECM that provides anchorage as well as a

reservoir for cytokines and growth factors that convey specific and specialised signalling

functions to the developing vessels. Interestingly the VN receptor, αVβ3, is expressed at high

levels in endothelial cells during angiogenesis (55).  

Although angiogenesis was not found to be impaired in VN-KO mice, persuasive evidence

indicates that VN is pivotal to this process. Upon addition of VN receptor antagonists new

blood vessel growth is effectively stopped during both physiological and tumour

angiogenesis (55, 56). Conversely VN and fibronectin also provide essential sources of

endogenous angiogenic peptides (e.g. anastellin) and partner molecules for functional

multiprotein complexes (e.g. anti-thrombin III) (57).  

In addition to key angiogenic functions, VN is required to facilitate tumour cell invasion. As

well as providing a provisional ECM for invading tumour cells, VN localises functional

MMPs to the tumour-ECM boundary (58). VN-immobilised MMPs cleave adjacent ECM

species (including VN), allowing tumour cells to penetrate constraining interstitial tissue

ECM. Further, it facilitates integrin-mediated tumour cell motility (59). Recent evidence for

the integrin-dependence of ‘cancer stem cells” (CSC) and metastatic dissemination and/or

 

Vitronectin  –  Master  Controller  or  Micromanager?   11  

 

residual disease has been described (60). Notwithstanding, CSC survival and subsequent

progression remains dependant upon interactions between CSCs and the local

microenvironment (61).  

VN confounds serum-free tissue culture data

It is time to re-evaluate and reinterpret our past in vitro studies in terms of our current

understanding of the informative in vivo pericellular environment, especially when it comes

to cellular responses to growth factors. Extensive evidence indicates that VN present within

serum is the primary and the most effective species that supports cell attachment and

spreading on tissue culture (TC) plastic (62, 63, 64). This is attributed to the fact that at serum

concentrations of 2%, or greater, VN is the most prominent ECM protein to adsorb onto TC

plastic, when compared to other ECM proteins such as FN and laminin (65). Interpretation of

data gained from such in vitro assays invariably fail to account for the competative activity of

albumin, IgG, alpha-1-antitrypin and other non-adhesive factors present in the serum (66, 67).

On the contrary, VN outcompetes all of these plasma proteins to adsorb readily to TC plastic

(68, 69) in the order of 42-55 ng/cm2 (65).

Until recently, in vitro studies investigating the effects of growth factors on cell

functions were performed with (a) serum-starved cells seeded in media containing the growth

factor/s of interest together with at least 0.5 to 2% serum, or (b) on pre-plated cells starved

from serum for 4-24 hours. In either case, VN adsorbed from serum-containing media (63)

remains present for the duration of the assay. Thus VN can localise endogenous (serum)

growth factors more effectively and induce synergistic and/or confounding effects on the

cells. Interpreting data from such experiments is ambiguous, since it is unclear if the observed

response(s) is a response to the growth factor (GF), or a response to residual

GF:VN/GF:ECM associations. Given what we now know, care should be taken when

interpreting data investigating the effects of growth factors on cell behaviour, especially

when cells are exposed to even the smallest presence of serum. Hence, the contribution of

VN and VN-binding integrins should be acknowledged and studied alongside the classical

growth factor:receptor-mediated effects.

With this in mind we re-evaluated our in vitro experimental design several years ago.

We assembled (pre-bound) complexes of mitogen/growth factors and ECM species for our in

vitro experiments (70, 71, 72, 73, 74). This approach acknowledged the evidence that cells

 

12   Vitronectin  –  Master  Controller  or  Micromanager?  

 

and cell surface ‘receptors’ do not encounter extracellular species in isolation, nor in fluid

phase. In vivo the pericellular environment is a hydrogel of aqueous water organised by the

glycocalyx of extracellular GAGs and metabolite polyions (75). In such a constrained

environment molecular crowding ensures frequent and prolonged interactions are statistically

favoured, can be sustained, and effectively ensure ‘high’ local concentrations of the

interacting partners (76).

Applying this strategy revealed that VN is able to assemble multiprotein complexes

with a variety of biologically active species. Biochemical characterisation studies of insulin-

like growth factor (IGF)-II were found to be reproducibly ‘contaminated’ with an

unidentified 70 kDa serum species; later identified as VN (76). At the time, why IGF-II was

‘contaminated’ with VN when the closely related mitogen, IGF-I was not, could not be

explained. Subsequent work discovered that IGF-I can also associate with VN, however, only

via intermediates, select insulin-like growth factor binding proteins (IGFBP) (77). The most

surprising aspect of these studies was the observation that in the presence of the VN

‘contamination’ cellular responses to IGF-II where greater than uncontaminated IGF-II! The

contaminating VN clearly contributed functional properties that were not induced when it

was absent. We now recognise that VN acts as a scaffold for IGFs, and several other

mitogens, and presents bioactive species to cognate cell surface receptors in the context of

simultaneous interactions with the VN receptor, and potentially other species in vivo (e.g.

GAGs). This makes sense of observations of molecular rafts within the cell membrane (78)

that effectively concentrate receptor and signalling pathway components in close proximity,

dramatically increasing effective signal transduction and cell response. Coactivation of the

VN receptor and IGF receptor is necessary for the enhanced response.

 

VN the master organiser

To date, most in vitro studies examining VN have employed the ‘solution in a test-tube’

approach. This strategy, while simple and convenient, is completely unlike the in situ

environment in which the experiment occurs. This approach denies processes that take place

at the molecular scale, that do occur in vivo. For example, the constraints imposed by even

transient interactions with large ‘insoluble’ structural polymers common in the ECM (e.g.

collagens) change the dynamics of transit binding events and possibly also induce

conformational reorganisation. Introduced above, VN responds to interactions with itself

 

Vitronectin  –  Master  Controller  or  Micromanager?   13  

 

and/or unrelated molecular species, including other ubiquitous ECM polymers (fibronectin,

collagens, fibrin, poly-sulphated proteoglycans, etc.) and charged small metabolites (cations,

glycopeptides, etc.) with conformational reorganisation. Dynamic responsiveness imparts VN

with novel functional properties appropriate to the local needs. This property could explain

the sometimes contradictory and confounding experimental observations present in the VN

literature.

The evidence indicating VN primarily functions as a micromanager includes the

original description of ‘alpha-one protein’ by Holmes (1). Holmes used soda glass beads to

both isolate VN from human serum and later glass and polystyrene treated with VN to culture

‘unadapted’ HeLa, human conjunctiva and human heart cell lines for several years (1). Today

we have replaced glass vessels with plasma-treated polystyrene but still require serum,

containing substantial concentrations of VN, to support cell attachment and proliferation. As

mentioned earlier, serum from which VN has been depleted fails to support mammalian cell

attachment and survival (63, 64, 79). One explanation for this observation is that in the

absence of VN, the well-described structural components of coagulation (fibronectin,

fibrinogen, collagens and serine protease zymogens) are insufficient to self-assemble an

appropriately organised provisional ECM capable of engaging cell attachment systems that,

in turn, trigger classical ‘outside-in’ signalling cascades (Fig 3) (80, 81, 82). Intriguingly, the

cell recognition systems that interact with the ECM microenvironment also have the capacity

to propagate reverse ‘inside-out’ signalling events (82) and cross-talk with diverse unrelated

transmembrane receptor systems (83, 84, 85). It remains to be established if VN participates

directly in the molecular events that take place in the ECM, or is it merely a passive scaffold

structure that organises and micromanages the local hydrogel milieu into appropriate

functional structures recognised and bound by cell surface receptor systems.

Throughout this brief review of the ubiquitous extracellular glycoprotein vitronectin,

we present and discuss experimental evidence suggesting that VN is more than a structural

molecule; it is a fundamental organiser, or micromanager of the extracellular

microenvironment. Assembling a functionally integrated, informative and responsive

structure is essential to mammalian cell and tissue survival. We contend that VN is a

principle conductor orchestrating many of the essential processes that contribute to cell

function. We have also highlighted the importance of confounding effects of ECM proteins

 

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present in conventional serum-free tissue culture studies and draw attention to the importance

of re-evaluating these findings in light of the evidence outlined in this brief review.

 

 

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Table 1: Molecular species reported to interact with Vitronectin

Name of Interactor Experimental Evidence Interaction Reference

Epidermal growth factor (EGF) In Vitro Direct 86 Fibroblast growth factor; basic (bFGF) In Vitro Direct 86 IGF binding protein 2 (IGFBP-2) In Vitro Direct 77 IGF binding protein 3 (IGFBP-3) In Vitro Direct 77 IGF binding protein 4 (IGFBP-4) In Vitro Direct 77 IGF binding protein 5 (IGFBP-5) In Vitro Direct 87 Insulin-like growth factor II (IGF-II) In Vivo Direct 76 Tumour necrosis factor receptor superfamily, 11B (TNF-R IIB) In Vivo Direct 88 Hepatocyte growth factor (HGF) In Vitro Direct 89 Proopiomelanocortin (POMC, beta endorphin) In Vitro Direct 90 Transforming growth factor (TGF) beta 1 (TGF β1) In vivo; In vitro Direct 91 TGF beta 2 (TGF β2) In Vitro Direct 86 Vascular endothelial growth factor A (VEGF A) In Vitro Direct 86 Sonic Hedgehog (Shh) In Vitro Direct 92 Fibrinogen, gamma chain In vivo; In vitro Direct 21 Plasminogen activator inhibitor 1 (PAI-1, Serpine 1) In vivo; In vitro Direct 93 Plasminogen activator receptor, urokinase type (uPAR, CD87) In vivo; In vitro Direct 41 Gelatinase A/Type IV collagenase (MMP-2, EC 3.4.24.24) In Vitro Direct 94 Matrilysin (MMP-7, EC 3.4.24.23) In Vitro Direct 95 Matrix metalloproteinase-26 (MMP-26, EC 3.4.24.1) In vitro Direct 96 Neurosin (KLK6, EC 3.4.21.B10) In vivo; In vitro Direct 97 Casein kinase II (EC 2.7.11.1) In vivo; In vitro Direct 28 Protein kinase C, alpha (EC 2.7.11.13) In Vitro Direct 98 Protein kinase C, beta 1 (EC 2.7.11.13) In Vitro Direct 98 Protein kinase C, gamma (EC 2.7.11.13) In Vitro Direct 98 Integrin beta 1 (CD29) In Vitro Direct 99 Integrin beta 3 (CD61) In Vitro Direct 99 Integrin, beta 6 In Vitro Direct 100 Integrin beta 8 In Vitro Direct 101 Integrin alpha 8 In Vitro Direct 102 Plasminogen In Vitro Direct 103 Kininogen (Fitzgerald factor) In vivo; In vitro Direct 104 Amphiphysin1 In Vitro Direct 105 Angiostatin (plasminogen fragment) In Vitro Direct 103 Angiopoietin-1 In Vitro Direct 104 Angiopoietin-2 In Vitro Direct 104 Anti-thrombin III (AT III, Serpine C1) In vivo; In vitro Direct 105 Protein kinase A, cAMP-dependent (EC 2.7.11.11) In Vitro Direct 106 Nectin-3, Poliovirus receptor-related protein (CD113) In Vitro Direct 107 Nectin-5, Poliovirus receptor (PVR) (CD155) In Vitro Direct 108 Lacritin In Vitro Direct 109 Collagens, α1A, α2A, In Vitro Direct 110 SPARC (osteonectin, BM40) In vivo; In vitro Direct 111 β-Endorphin In Vitro Direct 90 Syndecan-1 (CD138) In Vitro Direct 112 Syndecan-2 (HSPG-1, fibroglycan) (CD362) In Vitro Direct 112 Syndecan-4 (Amphiglycan) In Vitro Direct 112 Heparin cofactor II, Prothrombin In vivo; In vitro Complex 113 Betaglycan (TGFβ-RIII) In Vitro Complex Mol. Endo.,

 

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2009 (114) A disintegrin-like and metalloproteinase with thrombospondin type motif (ADMATS2; ADAMTS3) - heparan sulphate proteoglycan (HSPG) In Vitro Complex 115

IGF-I - IGFBP-2, 3, -4, -5; (IGF-I:IGFBP-2/-3/-4/-5) In Vitro Complex 77 Fibroblast growth factor 1 - HSPG In Vitro Complex 56 Interleukins (2, 3, 4, 5, 6, 7, 12) - HSPG In Vitro Complex 116 Chemokines (C-C motif) - HSPG In Vitro Complex 115 Granulocyte-Macrophage Colony Stimulating factor (GM-CSF) - HSPG In Vitro Complex 117 Interferon-γ - HSPG In Vitro Complex 118 Tumour Necrosis Factor alpha (TNF-α) - HSPG) In Vitro Complex 118 Integrin alpha V - Integrin, beta 3 (αvβ3) In vivo; In vitro Complex 119 Integrin alpha V - Integrin, beta 5 (αvβ5) In vivo; In vitro Complex 120 Integrin alpha V - Integrin, beta 6 (αvβ6) In vivo; In Vitro Complex 100 Integrin alpha 8 - Integrin, beta 1 (α8β1) In Vitro Complex 102 Thrombin - Antithrombin III - HSPG In vivo; In Vitro Complex 121 Plasminogen activator inhibitor 1 - Thrombomodulin In Vitro Complex 122 Galectin 1 - Thrombospondin I - Fibronectin 1 – Secreted phosphoprotein 1 In Vitro Complex 123

 Legend Table 1: Molecular species reported to interact with Vitronectin Molecular species (human only) reported to interact with vitronectin in vivo and/or in vitro. Species demonstrated to interact directly with vitronectin are listed, and species that interact indirectly, via complex formation, are listed and shaded.

Abbrev. EGF: Epidermal growth factor; bFGF: Fibroblast growth factor, basic; IGFBP: IGF binding protein-2; -3; -4; -5; IGF-I: Insulin-like growth factor I; IGF-II: Insulin-like growth factor II; TNF-R IIB: Tumour necrosis factor receptor superfamily, 11B; HGF: Hepatocyte growth factor; POMC: Proopiomelanocortin (beta endorphin); TGF: Transforming growth factor-beta 1; -beta 2: VEGF: Vascular endothelial growth factor; Shh: Sonic Hedgehog; PAI-1: Plasminogen activator inhibitor 1 (Serpine 1); uPAR: Plasminogen activator receptor, urokinase type (CD87); MMP-2: Gelatinase A/Type IV collagenase; MMP-7; Matrilysin; MMP-26: Matrix metalloproteinase-26; KLK6: kallikrein-6, Neurosin; AT III: Anti-thrombin III (Serpine C1); PVR: Poliovirus receptor (Nectin-5, CD155); SPARC: SPARC: secreted protein acidic and rich in cysteine, osteonectin (BM40); HSPG: Heparan Sulphate Proteoglycan; ADMATS: A disintegrin-like and metalloproteinase with thrombospondin type motif; TNF: Tumour Necrosis factor.

 

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Figure 1: Structural domains of vitronectin (predicted tertiary structure).  The NMR structure of the somatomedin B (SMB) domain (PDB accession ID 2jq8) was fused to a homology model of the remainder of the protein generated by the SWISS-MODEL repository by overlaying three contiguous residues found in both structures. This model is based upon the crystal structure of pro-MMP1 (PDB accession ID 1su3), with which vitronectin shares 12% sequence identity. After extensive energy minimisation to resolve steric clashes, the structure was relaxed in a short 1 ns simulation in explicit water using NAMD. To generate the open conformation, an interactive in vacuo simulation was carried out in which only residues 1 to 60 (comprising the SMB domain and the flexible peptide linking it to the polyanionic region) were free to move. The SMB domain was pulled out of place using steering forces averaged over residues 1-40, with each atom experiencing a force scaled by its atomic mass. Both structures were then equilibrated in explicit water at 300K for 10 ns.

This model supports the contention that interactions with the SMB domain act to maintain the N- and C-terminal domains in a structured state, and that its disruption or removal triggers an order-to-disorder transition exposing previously cryptic RGD peptide and HBD recognition motifs.

HBD: Heparin binding domain; TG: Transglutaminase motif; SMB: Somatomedin B domain; RGD: Arginine-Glycine-Aspartic acid (cell adhesion motif); G: consensus glycosylation motif; P/S: consensus phosphorylation / sulphation motif.

 

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Statement – Conflict of Interest The Authors have purchased shares in Tissue Therapies Ltd., an enterprise spun-out from the Queensland University of Technology, Brisbane, to commercialize some of the technology described in this manuscript.