FIRST INTERNATIONAL SYMPOSIUM ON THE SYNOVIUM-PART 3

Tyrosine Kinase Signal Transduction

in Rheumatoid

Synovitis

William V. Williams, Joan M. VonFeldt, Thaila Ramanujam, and David 6. Weiner Explants of synovial cells in rheumatoid arthritis display a transformed phenotype with focus formation and anchorage-independent growth. Many of the cytokines that activate these fibroblasts mediate their action through tyrosine kinase growth factor receptors. Mechanisms of signal transduction via such tyrosine kinases are therefore relevant to the pathogenesis of rheumatoid lesions. Data are presented using the neu oncogene product ~185”” as a model system to explore signal transduction by receptor tyrosine kinases. Evidence is shown that increased tyrosine kinase activity in the oncogenic form of this protein may result from dimerization of the tyrosine kinase receptor. In the normal cellular counterpart of ~185”“. dimerization appears to be mediated by the action of an as yet unidentified ligand. Dimerization also ap-

T

HE SYNOVIUM in rheumatoid arthritis (RA) is proliferative and destructive, sharing many properties of a cancerous growth. The synovial lining cells proliferate and invade neighboring cartilage, bone, and tendons. Yet while

Abbreviations: B104 cell, rat neuroblastoma that expresses the oncogenic form of neu; B104-1-1 cell, NIH3T3 cell that expresses the oncogenic form of neu; BT474 cell, human breast adenocarcinoma cell; CSF-1 or MCSF, colonystimulating factor 1 or macrophage colony-stimulating factor; c-fms/CSF-l-R, the CSF-1 receptor; DBW-2, antisera that binds specifically to the rat and human forms of p185”‘“; DHFR/GB cell, murine fibroblast (NIH3T3 cell) that expresses the normal form of the rat neu protooncogene; DSP, dithiobissuccinimidyl proprionate; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EGFR, EGF receptor; FCS, fetal calf serum; BFGFR, basic fibroblast growth factor receptor; G protein, guanosine triphosphate-binding protein; GAP, the ras guanosine triphosphatase-activating protein; IGF, insulinlike growth factor; IGFR-I, IGF receptor; mAb, monoclonal antibody; neu, HER2, or c-erbB-2, a receptorioncogene in the EGFR family; OA, osteoarthritis; p185”“, the protein product of the neu, ic-erbB-2/HER2 oncogeneslprotooncogenes; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PLCy, phosphohpase C-y; RA, rheumatoid arthritis; RER, rough endoplasmic reticulum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TGFo, transforming growth factor a; TGFB, transforming growth factor p.

Seminarsin

pears to be important in signal transduction mediated by epidermal growth factor, plateletderived growth factor, and colony-stimulating factor 1. These cytokines also alter the phenotype of rheumatoid synovial fibroblasts to resemble transformed fibroblasts. Additionally, preliminary data that suggest increased tyrosine kinase activity in rheumatoid arthritis synovia compared with osteoarthritis synovia are presented. Molecular characterization of tyrosine kinase receptors will be an important direction for future studies of the pathogenesis of rheumatoid disease. Copyright 0 1992 by W.B. Saunders Company INDEX WORDS: Tyrosine kinase; rheumatoid arthritis; growth factors; signal transduction; oncogenes.

From the Rheumatology Section, Department of Medicine, University of Pennsylvania School of Medicine; the Pediatric Rheumatology Section, Childrens Hospital of Philadelphia; and the Biotechnology Center, Wiitar Institute ofAnatomy and Biology, Philadelphia, PA. W I/: Williams was supported by an NIH FIRST award and grants from the Lupus Foundations of Philadelphia and Pennsylvania, the American Federation for Aging Research, the Sclerodenna Federation and Research Foundation, and the Arthritis Foundation, Eastern Pennsylvania Chapter. D.B. Weiner was supported by grants from the American Foundation for AIDS Research, the Council for Tobacco Research, and an NIH FIRST award. Presented at the First International Symposium on the Synovium, September 1990. William V. Williams, MD: Assistant Professor, Rheumatologv Section, Department of Medicine, University of Pennsylvania School of Medicine, and Pediatric Rheumatology Section, Children’s Hospital of Philadelphia; Joan M. VonFeldt, MD: Fellow, Rheumatology Section, Department of Medicine, L’niversity of Pennsylvania School of Medicine; Thaila Ramanujam, MD: Fellow, Rheumatology Section, Department of Medicine, University of Pennsylvania School of Medicine; David B. Weiner, PhD: Assistant Professor, Rheumatology Section, Department of Medicine, University of Pennsylvania School of Medicine, and Director, Biotechnology Center, Wistar Institute ofAnatomy and Biology. Address reprint requests to William V. Williams, MD, Rheumatology Section, 570 Mahoney Bldg, Hospital of University of Pennsylvania, 3600 Spruce St, Philadelphia, PA I91044283. Copyright 0 I992 by W B. Saunders Company 0049-0172192/2105-0005$5.00/O

Arthritis andRheumatism, Vol21, No 5 (April), 1992: pp 317-329

317

318

mimicking the unbridled growth of a malignancy, these cells do not display the metastatic phenotype. Explants of these synovial cells display a transformed phenotype, with prominent nuclei, foci formation, and anchorage-independent growth. In long-term culture, these cells lose these tumorlike properties but can acquire them anew under certain conditions,’ including the addition of specific growth factors to culture media. These observations emphasize the importance of cytokines in the perpetuation of this transformed phenotype in RA. Many cytokines that activate these fibroblasts mediate their action through tyrosine kinase receptors. The most well characterized of these is plateletderived growth factor (PDGF), which has been shown in synovial tissue and induces synovial fibroblast proliferation, anchorage-independent growth, and focus formation.‘,2 Both epiderma1 growth factor (EGF) and transforming growth factor a (TGFcx)~ stimulate EGF receptor (EGFR) and induce cell growth. Basic fibroblast growth factor has also been shown to induce growth of synovial fibroblastlike cells.“-’ It is probable that some of these growth factors and their receptors are the normal cellular counterparts of oncogenes, termed protooncogenes. Activation of these growth factor receptors may mediate many of the unregulated phenotypic characteristics shown by RA synovium. These growth factor receptors, part of the tyrosine kinase family of receptors, are an area of intense research. Dysregulation of cell growth is a central feature in the development of several pathological states. Studies have detailed the ability of single gene products to transform normal cells (such as fibroblasts) from a normal, contactinhibited phenotype to an unregulated phenotype typical of cancer cells. Oncogenes have been described in a variety of cancerous tissues and are believed to be ultimately involved in the pathogenesis of many human tumors.‘.’ The normal cellular counterparts of oncogenes, called protooncogenes, are expressed in many normal tissues, where they have demonstrated roles in the regulation of cell growth and/or fetal development. Several families of oncogenes participate in various aspects of signal transduction and/or cellular proliferation responses. As such, oncogenes and protoonco-

WILLIAMS

ET AL

genes can be subdivided “anatomically” as residing or acting at the cell membrane, as participating in signal transduction from the cell membrane to the nucleus, and as nuclear/DNAbinding proteins that directly affect gene transcription. Examples of membrane-associated oncogenes are sis, a PDGF homolog that transforms cells by constituitively activating the PDGF receptor (PDGFR),’ and neu or c-erbB-2, a growth factor receptor with intrinsic tyrosine kinase activity.‘” Intracellular/signal transducing oncogenes are typified by the ras oncogene, which is a gunosine triphosphate-binding (G) protein analog.‘.‘~” G proteins are involved in transduction of signals from membrane receptors (ie, P-adrenergic receptors) to intracellular enzymes (ie, adenylate cyclase) with subsequent generation of second messengers. Nuclear/ DNA-binding oncogenes include myc, myb, fos, and jun. These proteins bind DNA at the promoter regions of genes and regulate transcription of the linked gene.7.x~‘2.” Although all classes of oncogenes and protooncogenes play critical roles in regulatory cellular growth, the membrane-associated class is of particular interest because of its location on the cell surface. These oncogenes and protooncogenes are accessible to various agents without the requirement that such agents transverse the cell membrane. Therefore, therapeutic agents may be developed that regulate the behavior of membrane-associated oncogenes and protooncogenes that are incapable of penetrating into cells yet still have a profound effect on cell physiology. The vast majority of membrane receptorlike oncogenes characterized to date are tyrosine kinases. This points out the broad tissue distribution of these receptors and their impact on cellular processes. The role of the corresponding protooncogenes in regulating cellular growth and differentiation in a wide range of tissues has also become apparent. The general organization of proteinityrosine kinases is shown in Fig 1. Important members of this gene family include PDGFR-A and -B,14.15 EGFR,‘“” insulinlike growth factor receptor (IGFR-I),2” the insulin receptor,2’-‘3 the basic fibroblast growth factor receptor (BFGFR),” the colony-stimulating factor 1 (CSF-I or MCSF) receptor (c-fms),” and the human counterpart of the rte~ gene c-erbB-2

TYROSINE

KINASES IN RHEUMATOID

Insulin-R EGFR HERZheu IGF-1-R HERJ/c-erbB-3 IRR

Fig 1: growth

319

SYNOVITIS

PDGF-R-A P;;f;R;(” _ _

phosphorylate one another and regulate each other’s expression.29,30 Understanding the tyrosine kinase growth factor receptors expressed in a particular tissue reveals a great deal about the growth regulation of that tissue. We present a brief review of the literature on the tyrosine kinase receptors and some of our own data on tyrosine kinase activity involving the neu oncogene. These results show that increased tyrosine kinase activity may result from oligomerization of these receptors. A single amino acid substitution in the transmembrane region of neu greatly enhances tyrosine kinase activity and subsequent cell growth. This suggests methods of exploiting our knowledge of the tyrosine kinase receptors in designing therapeutics.

FGF-R J&

MATERIALS

Xmrk

c-kit

General organization

of tyrosine kinase

factor

receptors.

The

receptors

are

grouped into four families based on their overall structural organization.

Groups I and II are char-

acterized by cysteine-rich external domains (cys) and an internal protein tyrosine kinase domain (PTK). Groups Ill and IV are characterized by external immunoglobulinlike domains (lg) and a kinase insert (KI) within the PTK domain. Examples of each family include I, EGFR, c-erbb-2/ HERSlneu;

II,

insulin

receptor,

PDGFR-A and -B, c-fmslCSF-IR;

IGFR-I;

III,

IV, FGFR. (Mod-

ified from Ullrich and Schlessinger.“‘)

AND METHODS

Cell Lines

DHFR/G8 cells are murine fibroblasts (NIH3T3 cells) that express the normal form of the rat neu protooncogene,31B32 B104 is a rat neuroblastoma that expresses the oncogenic form of neu, 31,32and B104-l-l cells are NIH3T3 cells that express the oncogenic form of neu. These cells were grown and maintained as previously described.31,32 BT474 cells, human breast adenocarcinoma cells, were grown and maintained as previously described.*’ Human foreskin fibroblasts, obtained from American Type Culture Collection, were grown and maintained as described.33 Cell Labeling and Immunoprecipitation

or HER2.26 The role of these specific receptors in regulation of tissue growth is a function of their tissue distribution. Thus, the EGFR is located primarily in epidermal tissues, PDGF-R in endothelial cells and other endodermderived tissues, BFGFR in fibroblasts and mesenchymal tissues, c-fms in monocytes and macrophages, and c-erbB-2 in epithelial tissues.27 In addition, recent studies have shown the phenomenon of receptor “cross-talk.” For example, the presence of the cellular neu protein or EGFR singly, even at high levels on the same cells, does not produce oncogenic transformation. However, overexpression of neu and the EGFR in the same cell results in cellular transformation.28 Interestingly, these receptors can also

Cell labeling and immunoprecipitation have been described in detai1.31.32 Briefly, 1 x lo6 cells were plated and cultured for 24 hours, and [32P]adenosine triphosphate (ATP) (Amersham, Arlington Heights, IL) was added at 0.5 mCi/mL in 5% fetal calf serum (FCS)/phosphate-free medium for 6 hours. The cells were then washed in cold phosphate-buffered saline (PBS) with 400 umol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, and 400 kmol/L sodium orthovanadate and lysed in lysis buffer (1% NP40; 0.1% deoxycholate; 0.1% sodium dodecyl sulfate; 0.15 mol/L NaCl; 0.01 mol/L sodium phosphate, pH 7.4; 1% Trasylol; 1 rnmol/L phenylmethylsulfonylfluoride (PMSF);

320

2 mmol/L EDTA; 10 mmol/L NaF; 10 mmol/L sodium pyrophosphate, 400 bmol/L sodium orthovanadate; 10 mmol/L iodoacetamide; and 1 mmol/L ATP; all from Sigma, St Louis, MO) for 30 minutes. Precleared supernatants were subjected to immunoprecipitation with the following antibodies: anti-rat neu mAb 7.16.434”6; rabbit anti-rat/human neu antisera DBW227,29X37; anti-PDGFR antisera33.“8; anti-EGFR monoclonal antibody (mAb)39; anti-phosphotyrosine mAb.32 Immunoprecipitates were boiled in Laemmli’s sample buffer and analyzed in 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Dried gels were exposed to prefogged film (Kodak, Rochester, NY) at - 70°C. For reducing gradients, immunoprecipitates were run in lanes with added 2-mercaptoethanol, and the gels were run overnight at low voltage to allow diffusion of the reducing agent across the gel. Dried gel was autoradiographed as noted above. Chemical Cross-Linking The homobifunctional cross-linking reagent dithiobissuccinimidyl proprionate (DSP; Pierce Chemical Co, Rockford, IL) was used to crosslink spatially related polypeptides on the cell surface as described.31 After cell labeling with [32P]ATP, cells were incubated with DSP for 30 minutes, lysed, immunoprecipitated, and analyzed as described above.

WILLIAMS ET AL

RESULTS

Synovial Tyrosine Kinase Activity Tyrosine kinases catalyze the phosphorylation of tyrosine residues. This can be used to quantitate tyrosine kinase activity in synovial membrane preparations. We initiated these studies by comparing the intrinsic tyrosine kinase activity in RA and osteoarthritis (OA) synovium. This study used extracts of whole synovial tissue (three RA and three OA synovia showing typical pathological appearances histologically). The rest&s are shown in Fig 2. Although still preliminary, these data indicate potentially higher intrinsic tyrosine kinase activity in RA synovial tissue consistent with increased propensity for cell growth. Efects of Tyrosine Kinase Ligands on Synoviocyte Growth Most of the information regarding synovial tyrosine kinases is derived from functional studies of synovial cells grown in vitro. These studies indicate a propensity for these fibroblastlike 5000 U

RA Synovium

--b

OA Synovium

4000 -

3000 -

Synoviocyte Culture With Growth Factor Stimulation Synovial cells were harvested from rheumatoid knee synovial tissue at the time of joint replacement. The synovial tissue was examined pathologically and showed the characteristic changes of rheumatoid synovium with proliferation of the lining cells and infiltration with mononuclear cells. The adherent cells were isolated by growth on tissue culture grade T-75 flasks (Corning, Wexford, PA) as previously described.2 The cells were exposed to the various growth factors 48 hours after harvest. Growth factor concentrations were PDGF, 50 ng/mL; EGF, 25 ng/mL; BFGF, 50 ng/mL (all from Amgen Biologicals, Thousand Oaks, CA). Cells were photographed more than 1 week later.

,001

.Ol

.l

MEMBRANE

Fig 2:

Tyrosine

1

10

CONCENTRATION

kinase activity

100

(kg/ml)

in RA and OA

synovium. Tyrosine kinase activity was determined as described.3z The mean tyrosine-specific counts per minute incorporated for three samples each are compared with increasing concentrations of membrane proteins.

TYROSINE

KINASES IN RHEUMATOID

321

SYNOVITIS

to proliferate in response to several growth factors that activate tyrosine kinase receptors. Figure 3 shows synoviocytes in tissue culture under the influence of some of these growth factors. Note that synoviocytes grown in the presence of the tyrosine kinase-stimulating ligands PDGF (Fig 3B) and EGF (Fig 3C) grow in clusters, similar to the foci described for the growth of transformed (cancer) cells. Synoviocytes grown in the absence of exogenous growth factors typically lose this phenotype with passage in culture (Fig 3A), and PFGF (another tyrosine kinase-stimulating ligand) does not induce this phenotype (Fig 3D). This suggests a

cells

role for EGF and PDGF in inducing the transformed characteristics of rheumatoid synovium. Dimerization and Activity of Receptor Tyrosine Kinases

To explore the question of signal transduction by receptor tyrosine kinases, we used the neu oncogene product ~185”” as a model system. Oncogenic and protooncogenic forms of ~185”‘”differ by a single amino acid substitution in the transmembrane region (Val + Glu 664). These proteins differ markedly by their degree of tyrosine kinase activity. This difference can be best examined in the cell lines DHFR/G8

Fig 3: Synovial morphology after culture with growth factors representing tyrosine kinase receptors. Synovial fibroblastlike cells were grown as described in Materials and Methods in the presence or absence of various growth factors. (A) No growth factor; (B) PDGF, 50 ng/mL; (Cl EGF, 25 ng/mL; (D) BFGF, 50 ng/mL. Note the cellular aggregates reminiscent of focus formation seen in transformed fibroblasts for the PDGF and EGF groups.

322

and B104-1-1. These cell lines were derived from NIH3T3 cells (murine fibroblast ceil line) subsequently transfected with the normal protooncogenic form of neu (DHFR/G8 cells) or the oncogenic form of neu (B104-l-l). B104-l-l cells exhibit a typical transformed phenotype characterized by focus formation, colony formation in soft agar, and ability to form tumors in nude mice,““3h while DHFR/G8 cells do not have these characteristics. To visualize the intrinsic tyrosine kinase activity, these cells were labeled with [3zP]ATP and lysed, and phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibodies. When separated by SDS-PAGE, the labeled phosphotyrosine-containing proteins can be visualized by autoradiography (Fig 4). A much greater amount of phosphotyrosine-containing proteins is present in B104-l-l cells (lanes C and D) than in DHFR/G8 cells (lanes A and B). This suggests higher intrinsic tyrosine kinase activity in the oncogenic neu-expressing cells, which is supported by direct assays of tyrosine kinase activity in these two cell lines.32 The point mutation that causes oncogenic transformation by lzeu must affect signal transduction in some way that leads to increased tyrosine kinase activity. To investigate potential structural changes induced by this mutation, biochemical analysis of ~185”” was carried out. Initial studies indicated that the overall molecular mass and isoelectric point of the oncogenic and protooncogenic forms was similar.“‘-35We next evaluated the state of aggregation of ~185”‘” in the cell membrane. These studies used chemical cross-linking reagents to covalently stabilize any intermolecular interactions present. Figure 5 shows the effect of the ~185”‘” protein in the different cell lines in the presence of DSP, a homobifunctional cross-linking agent. The DHFR/G8 cell line shows a predominance of monomeric ~185”‘” proteins (band in the lower part of the gel). In addition, trace amounts of a high-molecular-weight protein (band at the top of the gel) are detected. This high-molecularweight form markedly increases in the presence of DSP, suggesting close proximity of the ~185”’ proteins to allow for dimerization with the cross-linking agent. The B104 cell line, which expresses low levels of the oncogenically transformed ~185”“”protein (see lower band), shows

WILLIAMS

DHFR/G8

ET AL

8104-1-l

AB

C

D

200

97

68 43

23

Fig 4:

Tyrosine

kinase activity

formed phenotypic D) compared

8104-1-l

of the trans-

ceils (lanes C and

with the normal

ceils DHFFVG8

(lanes A and B). The tyrosine kinase activity is visualized by labeling cells with [32P]ATP, lysing cells, immunoprecipitating

the phosphotyrosine-

containing

antiphosphotyrosine

proteins

with

antibodies, and analyzing the immunoprecipitate by SDS-PAGE and autoradiography. Note the multiple distinct bands in A and B corresponding to phosphotyrosine-containing cellular proteins. In contrast, the greater amount of phosphotyrosine-containing proteins in B104l-l cells results in a more intense signal, blurring the bands. This suggests increased tyrosine kinase activity in the 8104-1-l cells compared with DHFWG8 cells.

TYROSINE KINASES IN RHEUMATOID

SYNOVITIS

323

200

DHFWG8 Fig 5:

6104

Cross-linking

analysis of ~185”‘” onco-

genie and protooncogenic homobifunctional

6104-l-l phosphoproteins.

The

cross-linking reagent DSP was

used to cross-link molecules within a 12 A proximity. The cells were incubated for 30 minutes in DSP and subsequently tated,

and separated

lysed, immunoprecipiby SDS-PAGE

sence of reducing agents. (-), linking reagent;

(+),

in the ab-

no use of cross-

preincubation

with

DSP.

the presence of a higher proportion of dimers in the absence of DSP and a marked increase in these high-molecular-weight aggregates with the cross-linking agent. This is more apparent for the B104-l-l cell lines, which express more ~185”” protein in both the monomeric and dimeric form. Note the strong band at the top of the gel for B104-l-l even in the absence of DSP, suggesting a high level of dimeric ~185”‘” proteins even without cross-linking. Thus, while the protooncogenic ~185”‘”protein (cell line DFRI G8) shows a modest increase in cross-linkable complex, the oncogenic p185”‘” proteins (expressed in the cell lines B104 and B104-l-l) showed greater than 70% aggregation in the presence of the cross-linking agent.31 This suggests dimerization of this oncoprotein and would explain how the increased tyrosine kinase activity would be effected. The importance of disulfide linkages in the high-molecular-weight forms of the ~185”‘”protein has been demonstrated previously.3’ Our results showed the loss of dimers when the oncogenic protein ~185”‘” was reduced with a 2-mercaptoethanol gradient. The protoonco-

genie ~185”‘” does not show the presence of large numbers of dimers. The absence of these dimeric forms in the normal neu receptor of rats and humans, as well as the PDGFR and EGFR (present in synovial cells), is shown in Fig 6. An SDS-PAGE incorporating a 2-mercaptoethanol gradient shows the absence of a high-molecular weight dimer. The proteins show an increase in size with reduction, typically seen as the molecules unfold from their intact, more compact shape. However, unlike the ~185”‘” oncogenic protein, in which the high-molecular-weight species disappear dramatically during reduction, there is no evidence of a high-molecularweight band disappearing in the normal growth receptors. This is consistent with the presumption that these growth factors are protooncogenelike in their unaltered state. DISCUSSION

Molecular Mechanisms of Tyrosine Kinase Activity and Regulation

To understand how tyrosine kinases regulate cellular growth, one must appreciate their molecular structure and how it translates into function. The tyrosine kinase growth factor receptors all have similar molecular organizations. There is an external ligand-binding domain, which is the basis for grouping the various tyrosine kinases into subtypes (Fig 1).4@43 Subtype I includes c-erbB-2 and the EGFR, and these receptors have two cysteine-rich external domains. Subtype II includes the insulin receptor and the CSF-1 receptor, which exist as disulfide-linked homodimers with a single cysteine-rich domain on each subunit. Subtype III includes PDGFR-A and PDGFR-B, which possess five external immunoglobulinlike domains. Subtype IV includes the PFGFR and is characterized by three external immunoglobulinlike domains. All of the tyrosine kinases have one transmembrane region, generally devoid of charged amino acid residues.‘” The kinase domain resides intracellularly and possesses several distinct sites. Critical for catalytic activity are the ATP-binding site and the substratebinding domains.40-42These are separated in the primary sequences of the proteins. The r-phosphate group of the ATP bound to the ATPbinding site is donated to the target tyrosine of the substrate.43-48Recent studies, including our

324

WILLIAMS

ET AL

tyrosine kinase. In fact, some tyrosine kinases exist as membrane-associated multiprotein complexes, whereas the ATP-binding site resides on a different molecule than the substrate-binding kinase domain.5h This implies that oligomerization of these receptors is required for high levels of enzymatic activity (Fig 7). Our studies of the transforming lzeu oncoprotein and the protooncogene support this hypothesis. We showed that the single amino acid change in the transmembrane region of neu (val + glu at position 664) altered the associative properties of p185”” such that the transforming oncoprotein exists primarily as a homodimer”’ (see Fig 2 for schematic). This is associated with markedly elevated tyrosine kinase activity in the transforming oncoprotein compared with its normal cellular analog.‘”

PDGF-R

Ligand or Mutation-induced Receptor Aggregation

EGF-R

Fig 6:

An SDS-PAGE incorpo-

rating a 2-mercaptoethanol

gra-

dient of the normal neu receptor of rats and humans, as well as

the

PDGFR

(present

and

in synovial

These

results

sence

of

weight

show

EGFR cells). the

ab-

a high-molecular-

dimer.

The

proteins

show an increase in size with 2-mercaptoethanol,

l-l-NewR

pact shape; however, unlike the ~185”‘” oncogenic protein, in which the high-molecular-

Fig 7:

weight

strates, there must be interaction

species disappear

matically

NR -

typically

seen as the molecules unfold from their intact, more com-

R

during

dra-

reduction,

Signal transduction

via tyrosine kinases.

For tyrosine kinases to phosphorylate catalytic

domains.

their sub-

between

In this scheme,

two

the y-PO,

there is no evidence of a high-

group of ATP bound to the ATP-binding

molecular-weight

one catalytic domain is donated to the substrate bound to the substrate-binding site of a second

band disap-

pearing in the normal growth receptors.

own, indicate that this reaction does not occur intramolecularly.3’~49~55 Instead, for phosphorylation of the substrate to occur it must be brought in proximity to an ATP bound to a second

site of

catalytic domain. For some tyrosine kinases, this involves ligand- or receptor-induced dimerization, while for others it is the result of a conformational change bringing two linked catalytic domains in proximity to one another.53’55.9”” R, regulatory site (phosphorylation site); ATP, ATPbinding site; S, substrate-binding site.

TYROSINE KINASES IN RHEUMATOID

SYNOVITIS

These studies, which were key in identifying signal-transduction pathways used by tyrosine kinases, were among the first to suggest a role for the transmembrane region of proteins aside from simply anchoring proteins in the cell membrane. In addition to the kinase domain, the intracellular segment of these receptors typically possesses several regulatory domains. This has been best characterized for the EGFR, in which a threonine residue (Thr654) acts as a protein kinase C-phosphorylation site.43X57 This site is located in the juxtramembrane domain, just inside the membrane between it and the ATPbinding site. Phosphorylation of this residue decreases tyrosine kinase activity and downmodulates the receptor.57-60The insulin receptor seems to have similar regulatory properties61 There are also several “autophosphorylation” sites, located near the carboxy terminus of these receptors.62@ Phosphorylation of these tyrosine residues may either enhance or inhibit tyrosine kinase activity, depending on the specific residues invo1ved.65-67 Just as dimerization of membrane tyrosine kinase activates kinase activity, it may also generate signals leading to eventual downmodulation of receptor expression. This is typified by the ability of anti-pl85”‘” antibodies to down-modulate oncoprotein expression upon receptor cross-linking,34.3s perhaps because of the eventual effects of signal transduction with feedback regulation of receptor expression. Alternatively, it is likely that antibody-mediated receptor aggregation does not maintain the integrity of the receptors, and therefore aggregation and signal transduction may not be linked in this specific example. Although the details of this regulation remain to be determined, it is likely caused by the effects of tyrosine phosphorylation of specific substrates, which activates other signaling pathways. Many signaling pathways appear to be affected by these receptor tyrosine kinases. Phospholipase C-7 (PLCy) has been shown to be a likely substrate for both the EGFR and the PDGFR,@-‘* and this may enhance phospholipase activity after phosphorylation.69,71,73 The rus GTPase-activating protein GAP is a substrate,74 implicating tyrosine kinases as regulators of G-protein-linked signaling pathways. The c-rafprotooncogene may be a substrate of the PDGFR,75 and our recent

325

studies indicate that lipocortin is a potential substrate.76 The many substrates for tyrosine kinases critical in many intracellular signaltransduction pathways indicates the central role of these receptor/enzymes in regulating cell physiology. Tyrosine Kinase Receptors as Therapeutic Targets

One of the most dramatic examples of a therapeutic agent directed at a membraneassociated oncogene comes from work on the c-erbB-2 or neu oncogene.34”6 “Neu” was first described by Weinberg’s laboratory in studies of ethylnitrosourea-induced rat neuroblastomas.“-@’ These neuroblastomas were developed by prenatal exposure of rats on gestational day 14 to 16 to ethylnitrosourea. Cell lines were developed from these tumors and found to possess a typical transformed phenotype including focus formation in vitro, growth on soft agar, and tumor development when injected into nude mice. High-molecular-weight DNA was extracted from these neoplastic cells and transfected into NIH 3T3 cells. Some of the transfected cells became similarly transformed. The neu oncogene was subsequently cloned from these cells and found to possess an overall organization typical of membrane-associated tyrosine kinases.77-80This includes two cysteinerich external domains, presumably involved in ligand binding, a single transmembrane domain, and an intracellular domain including an ATPbinding site, a kinase domain, autophosphorylation sites, and regulatory sites (eg, protein kinase C-phosphorylation sites). The normal cellular counterpart of neu was subsequently identified and found to differ from the oncogene form by a point mutation in the transmembrane domain.81 Valine at position 664 in the protooncogene product is replaced by glutamic acid in the oncogenic form of the protein. This single amino acid substitution was found to be responsible for the transforming properties of the oncogenic form of neu. Monoclonal antibodies were developed against the extracellular domain of the transforming neu oncoprotein ~185”‘” through the immunization of mice with neu-transfected cells.34The resulting antibodies were capable of cross-linking the oncoproteins on the cell surface, resulting in their down-modulation.‘4”6 This completely reversed the transformed phe-

326

notype of the cells in vitro. In addition, injection of the monoclonal antibodies to nude mice and rats bearing neu-induced tumors resulted in tumor regression.35x3bSimilar studies have been performed with antibodies to the EGFR, using a monoclonal antibody (425) that inhibits growth of EGFR-bearing cells.82 These findings indicate that removal of tyrosine kinase activity from the cell membrane disconnects signal transduction. Antibodies that target such membraneassociated tyrosine kinases may profoundly alter the physiology of cells bearing these receptors. Thus, in a wide range of circumstances, antibody molecules that target tyrosine kinase growth factor receptors may have clinical utility. Synovial Tyrosine Kinases

The most often implicated tyrosine kinase ligand in rheumatoid synovium is PDGF, which has been shown in synovial tissue and induces synovial fibroblast proliferation, anchorageindependent growth, and focus formation.4.5.R3-85 The PDGFp receptor has also been detected on synovial endothelial cells” and fibroblastlike cells.” immunohistochemically. Additionally, Remmers et al described increased tyrosine phosphorylation and expression of the protooncogene products fos and myc in rheumatoid synovium compared with osteoarthritis synovium.87 The PDGF receptors, members of the tyrosine kinase family of growth factor receptors, have been further characterized. The alpha receptor, in the dimeric form, binds with high affinity to all three isoforms of PDGF, whereas the beta receptor binds only to the BB isoform of PDGF. Variation in affinity binding of these receptors and how this binding affects the triggered cellular events remains to be elucidated. It is possible that in rheumatoid synovium, PDGF binding somehow induces dimerization of the receptor, resulting in increased tyrosine kinase activity. EGF has also been observed in synovial tissue’ and found to induce growth of synovial fibroblasts,*’ as does TGFa,*” another ligand for the EGFR. EGF has been associated with the presence of neovascularization in synovial tissue and implicated as an angiogenic factor that promotes the growth of pannus in RA.3 EGF is localized to the rough endoplasmic reticulum (RER) of the type B (fibroblastlike) cells and

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found on the surface of the type A (monocytelike) cells, suggesting activation of the type A cells by the fibroblast cells.” BFGF has also been implicated as a mitogen in the pathogenesis of RA. It has been detected in the culture supernatants of patients with RA and shown to induce growth of these cells.‘-h Similarly to EGF, BFGF has been identified as an angiogenic factor. Melnyk et alb detected expression of the BFGF gene and the presence of high- and low-affinity receptors in cultured synoviocytes. This group therefore postulates that BFGF derived from synovial lining cells stimulates the proliferation of these cells in an autocrine manner. Insulinlike growth factors (IGFs), another group of tyrosine kinase-dependent growth factors, stimulate chondrocyte proteoglycan synthesis. In arthritis there is inhibition of chondrocyte proteoglycan synthesis and degradation of cartilage matrix, and it may be that there is dysregulation of IGF in RA. Although this study indicates a likely role for the corresponding growth factor receptors in supporting synovial proliferation, it does not indicate which receptors predominate in vivo or show whether additional tyrosine kinases might be present. In addition, growth factors whose receptors have not been characterized (such as TGFB) also have marked effects on synovial physiology.x’~x’~“’ Through examination of synovial tissue and cells for expression of tyrosine kinases in a general sense, insight will be gained into the expression levels of the receptors for the known tyrosine kinase-related growth factors, and potential novel tyrosine kinases may be detected that are involved in pathogenesis. SUMMARY

In spite of significant advances in understanding the pathogenesis of RA, current therapy is still difficult, toxic, and often ineffective. A major focus of investigation is the tumorlike character of synovial tissue in RA. The presence of high levels of tyrosine kinase activity, as well as the identification of cytokines that serve as Iigands for tyrosine kinase growth factor receptors in rheumatoid synovium, suggests a role for synovial tyrosine kinases in mediating the transformed phenotype and invasive characteristics of synovium. Advances in molecular biology and rational drug design have shown methodologies

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that can be applied to therapeutic development once target receptors are identified. By characterizing cell-surface receptors critically involved in the pathogenesis of synovial proliferation, specific targets for development of future therapies will be identified. Protein tyrosine kinases, because of their intimate relationship to control of cellular growth, are attractive potential targets for designing such novel therapeutics. Molecular studies should serve to establish the

identity of the major tyrosine kinases involved in synovial hyperplasia. This will have an impact on our understanding of synovial pathogenesis at multiple levels and allow future studies to progress into the development of novel therapeutics. ACKNOWLEDGMENT The authors thank Michael Merva for technical assistame and H.R. Schumacher for critical review of the manuscript.

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Tyrosine kinase signal transduction in rheumatoid synovitis.

Explants of synovial cells in rheumatoid arthritis display a transformed phenotype with focus formation and anchorage-independent growth. Many of the ...
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