Características del territorio peruano

Integrins are not only adhesion molecules, b u t they also play a role in signal transduction into and out of cells (Hynes, 1992; Juliano and H askill, 1993; Adam s and W att, 1993). The initial step in integrin m ediated signalling is the activation of the receptor, via coupling of the receptor to soluble m ediators (e.g. horm ones or cytokines) a n d /o r solid phase reactants (ECM proteins or other cells). Thus, a signalling cascade, such as the release of lip id second m essengers, activation of protein kinases (Malik and Parsons, 1996; Parsons 1996), and changes in intracellular pH and calcium (LaFlamme and Auer, 1996). Thus in te g rin signalling regulates cell proliferation, d ifferen tiatio n and survival, as well as adhesion (Meredith et al, 1996) (Summarised in Figure 1.4).

One of the first observations in fibroblasts adherent to fibronectin w as the activation of the N a + /H + antiporter th at leads to cytoplasm ic alkalisation (Hynes, 1992; Schwartz, 1994). The increase in intracellular p H leads to the activation of the cell from a quiescent state, due to the integrin activation of lipid m essengers. Integrin-ligand binding triggers the phosphatidyl inositol pathw ay by activating the tyrosine kinase dependent phospholipase Cy. PLC h y d ro ly s e phosphatidylinositol bisphosphate (PIP2) to inositol phosphate and diacylglycerol, both involved in the regulation of intracellular calcium and the

Ligand Integrin Interaction

(s^

( Grb2 ) Sos-1

\

PI3-K PIP2 DAG PIP3 Actin-cytoskeleton Rearrangem ent PKC Ca^+Z Calmodulin- Dependent Enzymes Gene Expression

Change in cell shape e.g. adhesion/m otility

p H Increase ATPase Ca^+ p um p Activation ppéO^^c MAPK Cytoskeletal Changes

I

I

Figure 1.4 In teg rin d e p e n d e n t sig n allin g pathw ays. Details are described w ith in the text. Figure m odified from Marshall, 1995.

Chapter 1: Introduction 75

activation of protein kinase C (PKC), w hich in tu rn activates the N a+ /H + antiporter (Meredith et al, 1996).

O ther m ain signalling events include the phosp h o ry latio n of tyrosines on integrin-associated proteins, for example, tyrosine kinases of the src family and focal adhesion kinase (FAK) are involved in the focal adhesion complex. These complexes are thought to be signal transduction "organelles' (Richardson and Parsons, 1995). The initial phosphorylation of FAK by the focal adhesion associated kinase ppl25^^^, leads to induction of the signalling cascade, the p h o sp h o ry la tio n of FAK co n tro llin g the cascade. A m ajor site of phosphorylation is tyrosine 397, an autophosphorylation site, w hich in tu rn leads to the generation of a src binding site called the src homology-2 (SH2) dom ain. A dditional tyrosine phosphorylation of FAK in its catalytic dom ain is also im p o rta n t for function (M alik an d Parsons, 1996). O nce FAK is p h o sp h o ry lated and a src is associated there is in increase in the tyrosine phosphorylation of the cytoskeletal proteins paxillin, tensin and p l3 0 (Fetch et al, 1995). Subsequent to the protein complex phosphorylation, the activated FAK also binds to the SH2 dom ain of the carboxyl-term inal src kinase, csk, w hich negatively regulates src activity (Sabe et ah, 1994).

Therefore, FAK acts as a central co-ordinating point for integrin m ediated signalling, resulting in a change in cellular physiology, such as m igration and m etastasis, w here the FAK protein is upregulated due to an over expression of its mRNA (Weiner et ah, 1993).

A further functional role for integrin m ediated signalling is the regulation of cell proliferation. The cascade originates w ith the phosphorylation of FAK and the association of the adapter protein, GRB2, by its SH2 dom ain. This provides a linking route to the mitogen-activated (MAP) kinase pathw ay via ras. SOS, a

rasG D P /G T P exchange p ro tein , b in d s to the G R B 2 /F A K /src com plex (Schlaepler et al., 1994). This protein complex leads to the activation of MAP kinases, w hich in tu rn activates a num ber of transcription factors, including c-jun, c-fos, c-myc, which are involved in regulating grow th and differentiation (Davis, 1993).

Finally, integrins have a role in cell survival. Adhesion to the ECM is required for grow th and survival (LaFlamme and Auer, 1996). Evidence has show n that integrin engagem ent triggers signals that m ay inhibit apoptosis (program m ed cell death) (Re et ah, 1994; Brooks et ah, 1994a). The signals involved have not been identified, however, ligand occupancy alone is not sufficient and changes in cell shape and spreading m ay be involved (M eredith et ah, 1993). It is likely th at tyrosine p h o sp h o ry latio n is involved, since treatm en t of cells w ith van ad ate, a phosphotyrosine inhibitor, can overcom e the req u irem en t of adhesion for apoptosis (Re et ah, 1994). Specific integrin heterodim ers have been show n to play significant roles in anti-apoptotic activity. If the aVp3 receptor is blocked by antibodies during angiogenesis in chick em bryos, both cellular proliferation ceases and apoptosis is induced (Brooks et ah, 1994a; Brooks et ah, 1994b). Similar results have been show n by preventing m am m ary epithelium interactions by inhibiting the p i integrins (B oudreau et ah, 1995; H ow lett et ah, 1995).

This brief description of integrin m ediated signalling show s th at on integrin activation, via ligand or exogenously induced, cellular b eh av io u r can be regulated. The signalling cascades are generated from a num ber of stim uli, be they h o rm o n e or g ro w th factor, or th ro u g h EC M -integrin b in d in g , are integrated in order for the cell to respond during w hich there is a great deal of cross-talk betw een the pathways.

Chapter 1: Introduction 77

1.19 A dhesion m olecules in bone

Several adhesion molecules have been found in bone, w ith recent literature sh o w in g th eir expression in stro m al cells, osteoblasts, osteocytes an d ch o ndrocytes; the ad h esio n m olecules id en tified in clu d e the in te g rin s discussed. There are, however, discrepancies in the expression phenotype, and thus confusion in their function.

Of the cells in bone osteoclasts have been studied in the greatest detail and their molecular phenotype and functions are better understood. Osteoclast express integrin receptors, lim ited to three receptors in the m ature cell: aV p3 the classical' vitronectin receptor, a 2 p l a collagen/ lam inin receptor and, a V p l, a further vitronectin receptor' (Nesbitt et a l, 1993). There is evidence th at integrins act du rin g the developm ent and differentiation of osteoclasts. The num ber of other adhesion molecules expressed by osteoclasts is lim ited to CD44 (N akum ura et al., 1995; N akam ura and Ozawa, 1996) and some m em bers of the cadherin family (Mbalaviele et al, 1995). Functionally, there is a strong relationship by w hich inhibition of osteoclast integrins leads to a d o w n regulation of osteoclastic bone resorption (Shankar and H orton, in press).

1.19.1 T he role of integrins in osteoclastic bone resorption

H orton et a l, (1985a) originally suggested th at ad h esio n receptors h a d a significant functional role in osteoclast biology - especially in m o d eratin g osteoclastic resorption. The antibody 13C2 w as found to inhibit in vitro resorption of osteoclasts obtained from osteoclastoma (Chambers et a l, 1986). Subsequent w ork established th at the inhibitory effect w as m ediated via the aVp3 vitronectin receptor (Davies et al, 1989).

O ther detailed biochemical analysis (Nesbitt et ah, 1993; review ed in H orton an d R odan, 1995) dem onstrated th at m am m alian osteoclasts express three integrin dim ers (data sum m arised in H orton and Rodan, 1995). Findings betw een different studies have been consistent and integrin expression has been verified in different species. However, there is evidence to suggest that osteoclasts express other integrin subunits. H ughes et al. (1993) and Grano and colleagues (1994a) reported that a5 and a3 are expressed, although this has not been a consistent finding. H ow ever, these m ay be expressed by im m ature osteoclasts, as osteoclasts prepared from anim al m arrow are obviously present at all stages of developm ent. There are some species differences in osteoclast integrin expression; for example, avian osteoclast express a 5 p l and aV p5 in addition to aV p3 (Duong et al, 1992; Ross et ah, 1993), and possibly p2 integrins (A thanasou et ah, 1992); data reviewed in H orton et ah, 1996c).

In vitro assays using cells from several species have show n th at osteoclast adhesion to bone involves the interaction betw een osteoclast integrins and the ECM p ro tein s (Flores et ah, 1992; H elfrich et ah, 1992; Ross et ah, 1993; sum m arised in H orton and Rodan, 1995). The m ain adhesive interaction ap p ears to be th at of the vitronectin receptor, w hich m ediates RG D -peptide d e p e n d e n t a d h esio n to a large n u m b er of R G D -containing p ro tein s. M am m alian, b u t not avian osteoclasts, adhere to Type I collagen using the a 2 p l receptor complex. This osteoclast integrin-collagen adhesion is RGD peptide sensitive, unlike collagen binding by integrins of other cells (Helfrich et ah, 1996).

1.19.2 Role of the aVp3 receptor in osteoclast adhesion

Inhibition of osteoclast adhesion and resorption u sing function blocking antibodies, targeting the vitronectin receptor, and RGD-peptides affecting the aV p3 and a 2 p l integrins of osteoclasts can control bone resorption. Data to

Chapter 1: Introduction 79

su p p o rt this w as provided by Sato and colleagues (1990) w ho used the RGD- sequence containing snake venom protein, echistatin, w hich also blocked bone resorption. Furtherm ore, studies using RGD peptides an d peptidom im etic agents (Nickols et al., 1995), other snake venom s including kistrin, aV and p3 antibodies, and antisense oligonucleotides (one of the topics of this thesis) have all show n th at targeting the integrin receptors of osteoclasts has dram atic, inhibitory effects on bone resorption.

Thus, the vitronectin receptor (VnR) is an im portant stru ctu re involved in osteoclast-m atrix interactions. Originally, Lakkakorpi and co-w orkers (1991; 1993) localised the VnR expression to specific areas of the osteoclast m em brane; in rat osteoclasts both the ruffled border and the basolateral m em branes were positive, w here areas that correspond to the sealing zone rem ained unstained.

H ow ever, other w ork appears to indicate the preferential localisation of aVp3 in the sealing zone membrane, the podosom e (Reinholt et a l, 1990). Podosom es are th o u g h t to associate w ith osteoclast integrins, especially th e aV p3 tran sm em b ran e receptor. Since the podosom e ring is the actual p o in t of attachm ent, th en integrins m ay be expressed in the sealing zone directly. H ow ever, the tight apposition of the sealing m em brane w ith the bone surface is approxim ately 0.2 to 0.5 nM, which is too narrow for an integrin molecule, of approxim ately 20 nM in length, to be the actual osteoclast adhesion molecule. O ther data from H ultenby et al. (1993) and Neff et al. (1996) also suggest the p resen ce of in teg rin s in the sealing z o n e/p o d o so m e ring. C onversely, L a k k a k o rp i et a l, (1993) have used a w ide variety of integrin antibodies, targ etin g b o th extra- and intracellular epitopes, and have not detected the expression of integrins w ithin the sealing zone. One explanation for these conflicting data is that the integrin positivity show n by Neff et al. (1996) was in

fact on the basolateral membrane, aw ay from the tight seal or on non-polarised, non resorbing osteoclasts.

The vitronectin receptor m ay have a role in other stages of the resorption cycle. A p3 integrin colocalises w ith talin and vinculin in the osteoclast podosom e, w hich is very likely to be aVpS (Zambonin-Zallone gf a l, 1989). The osteoclast is also a m igratory cell and Lakkakorpi et a l, (1993) subsequently found that aV p3 is expressed at the leading edge of osteoclasts m igrating on bone, a region rich in podosom e structures (Lakkakorpi and V aananen, 1991; N esbitt and H orton, u n p u b lish ed data). A nti-aV antibodies colocalise w ith the b ro ad vinculin ring formed prior to the sealing zone, and also localise w ith the double vinculin ring found in polarised, resorbing osteoclasts; how ever, no staining w as p resent in the tight seal zone. How ever, there w as a lack of aV and p3 colocalisation suggesting other, possibly a V p l, integrins were expressed at this site (Nesbitt et al, 1993).

Therefore, the aV p3 integrin is not only involved in osteoclast adhesion and resorption, b u t also in osteoclast m igration to sites of "new' resorption and m ay be involved in phases of the resorption cycle, prior to tight seal attachm ent. The m olecular m echanism of the attachm ent process in the established clear zone of a resorbing, non-m igratory osteoclast still rem ains to be established (Vaananen and H orton, 1995).

1.19.3 The function of other osteoclast integrins

O steoclasts also express the a 2 p l and a V p l receptors. H elfrich et a l, (1996) have show n that p i, bu t not p3 integrins m ediate osteoclast adhesion to native collagens, m ainly via a 2 p i. Furtherm ore, blockade of p i in teg rin s w ith antibodies also inhibits bone resorption in vitro in isolated osteoclast assays (Horton et a l, 1995; H elfrich et a l, 1996), although the exact m echanism of

Chapter 1: Introduction 81

action has not yet been defined. Recent data has also show n the relevance of th e p i su b u n it in osteoclast function, by tem porarily knocking o u t p ro tein expression (and function) using antisense oligodeoxynucleotides (Townsend and H orton, 1996a; 1996b and herein).

1.19.4 In vivo roles of osteoclast integrins

A red u ctio n in blood calcium concentrations (hypocalcaem ia) h as been dem onstrated w hen using the snake venom peptides, echistatin and kistrin, in rats in vivo (Fisher et al, 1993; King et a l, 1994). Echistatin w as u sed in the PT H -infused thyroparathyroidectom y (TxPx) m odel an d kistrin in PTHrP- in d u ce d hypercalcaem ia. Cyclic RGD -containing p ep tid es an d p ep tid o - mimetics also induce hypocalcaemia in the PTH-infused TxPx m odel, although these are less potent than snake venom peptides (Nickols et al, 1995).

In vivo studies using RGD-containing and absent (m utant) echistatin (Fisher et a l, 1993; Sato et a l, 1994) su g g est th a t in te g rin s are m e d ia tin g the hypocalcaem ic effect by inhibiting osteoclastic bone reso rp tio n an d th at sufficient, active levels can be attained in bone. These data do n o t exclude b in d in g to integrins elsew here in the body w hich m ay also be involved in calcium homeostasis.

The involvem ent of osteoclast integrins in vivo w as firstly show n u sing the antibody, F l l , to the rat p3 integrin which resulted in hypocalcaemia in the rat TxPx m odel (Grippes et al, 1996). A direct inhibition of osteoclasts in bone via a p3 integrin receptor (aVp3) is supported by the localisation of F l l antibody to osteoclasts in situ after in vivo adm inistration. O ther research using echistatin or mimetics infused into the animal lead to a complete block in the acute loss of trabecular bone seen in secondary hyperparathyroidism (Yamamoto et a l, 1993) and following ovariectom y in the m ouse (Yamamoto et a l, 1994; Nickols et al.

1995). This data strongly suggests that the inhibitory effect of RGD occurs via a direct action on bone, via the aVpS integrin on osteoclasts.

1.19.5 N on integrin receptors in osteoclasts

M any studies have been carried out to determ ine the expression of non-integrin adhesion receptors in osteoclasts. H orton and Davies, (1989) failed to identify o th er ad h esio n receptors. H ow ever, m ore recent, p o w erfu l m olecular approaches have provided prelim inary data indicating that osteoclasts express E-cadherin (Mbalaviele et al., 1995), the 67 kDa lam in in recep to r Mac-2 (Takahashi et al, 1993) and CD44 (Athanasou and Quinn, 1990; N akam ura et ah, 1995; N akum ura and Ozawa, 1996). It is likely that some of these proteins are not d o m in an t in m ature osteoclasts or only present on a su b p o p u latio n of 'im m ature' osteoclasts. It is possible th at they are involved in osteoclast developm ent, fusion and functional m aturation from haem opoietic stem cells. Prelim inary imm unoelectron microscopic data suggest that CD44 m ay m ediate interactions of osteoclasts w ith osteoblasts, b u t not bone m atrix (N akam ura et ah, 1995). The question of w hether CD44 is involved in osteoclast developm ent has not been answered, yet recent confocal microscopy data suggests th at CD44 links w ith m oesin and the actin filament system and is th u s involved in the cytoskeletal organisation of the cytoskeleton in osteoclasts (N akam ura and Ozawa, 1996).

1.19.6 A dhesion molecules and osteoclast development

To d e te rm in e w h ich ad h esio n m olecules are im p o rta n t in o steo clast d ev elo p m en t from stem cells to com m itted, m ononuclear, post-m ito tic precursors (functional mononuclear osteoclasts) has been difficult. Identifying cells at different stages in development, has complicated the search for the role of adhesion molecules. How ever, antigenic m arkers of osteoclasts have now allowed the identification of some of the differentiation stages. Evidence has

Chapter 1: Introduction 83

been indirect and gained by using antibody or peptide inhibition in short term bone m arrow cultures treated w ith la,25-dihydroxyvitam in D3. For example, rodent osteoclast developm ent can be inhibited by the RGD-containing snake venom proteins, echistatin (Tanaka et al., 1991) and kistrin (Sum itani et ah, 1991), stro n g ly im p ly in g a role for v itro n e ctin recep to r. H o w ev er,

In document Factores socioeconómicos, ambientales y situación del abastecimiento de agua para consumo humano en el Centro Poblado de Accaso del distrito de Pilcuyo Puno (página 39-43)