Tenascin-C deposition requires β3 integrin and Src

www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 322 (2004) 935–942

BBRC

Tenascin-C deposition requires b3 integrin and Srcq

Yongjian Yang, Dongmin Dang, Seiki Mogi, Daniel M. Ramos*

Department of Stomatology, University of California at San Francisco, San Francisco, CA, USA

Received 30 July 2004

Abstract

In this study we now show that deposition of the mesenchymal matrix marker, tenascin-C (TN-C), is mediated through b3expression and activation of Src. There was a striking upregulation of TN-C matrix organization in cell lines expressing b3 and acti-

vated Src when compared to cell lines with neither of these attributes. When b3 function was suppressed so was the deposition of

TN-C. The same was true for function and activation of Src. When Src was inactive, the deposition of TN-C was low. We also deter-

mined that one of the downstream effectors of Src, MAPK, was also required to promote TN-C deposition. When MAPK activation

was inhibited, TN-C deposition was also decreased. MMP activation is also implicated in TN-C deposition. The broad spectrum

MMP inhibitor, GM6001, suppressed TN-C organization. These results indicate that b3 integrin ligand binding and the activation

of the Src/MAPK/MMP pathway modulate deposition of TN-C.

� 2004 Elsevier Inc. All rights reserved.

Keywords: b3 integrins; ECM; Tenascin-C; Src

Melanoma is caused by malignant transformation of

normal melanocytes. Metastatic spread is a major cause

of death among cancer patients. However, the mecha-

nism underlying tumor cell invasion and metastasis re-

mains poorly understood. The metastasis of tumor

cells is a complex series of interrelated steps and tumor

cell adhesion to the individual components of the extra-cellular matrix (ECM) constitutes a critical step in this

model [1,2]. Adhesion to the ECM is mediated through

the integrin family of cell surface proteins. The integrins

are a family of heterodimeric, integral membrane glyco-

proteins consisting of non-covalently bound a and bsubunits. The multiple heterdomeric ab combinations

mediate specific adhesion to ECM components such as

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.08.009

q Abbreviations: ECM, extracellular matrix; FN, fibronectin; VN,

vitronectin; TN-C, tenascin-C; MMPs, matrix metalloproteinases;

MT1-MMP, membrane type 1-MMP; CASrc, constitutively active Src;

KDSrc, kinase-dead Src; MEK, mitogen-activated protein kinase/

extracellular signal-regulated kinase; MAPK, mitogen-activated pro-

tein kinase; DMEM, Dulbecco�s modified Eagle�s medium.* Corresponding author. Fax: +1 800 7836653.

E-mail address: [email protected] (D.M. Ramos).

fibronectin (FN), vitronectin (VN), and tenascin-C

(TN-C) [3]. Adhesive interactions between integrins

and ECM components are reported to play important

roles in tumorigenesis, invasion, and metastasis [4,5].

Recent work has shown that integrin receptors can

initiate a signal transduction cascade that can affect

many aspects of cell growth [6,7]. av integrins have beenimplicated in mediating cell attachment and spreading

[8], in cell locomotion [9], in management of the extra-

cellular protease cascade [10], in angiogenesis [11], in

apoptosis [12], and in tumor cell invasion and metastasis

[11]. avb3 expression has been shown to be consistently

upregulated with MMP2 [13].

The ECM is a dynamic structure which continually

undergoes modification both by neo-deposition of mol-ecules or by modification of existing ones. Specific ma-

trix components, such as FN, promote stable cell

attachment and encourage cell proliferation. Other

ECM molecules, such as TN-C, are less adhesive and

do not stimulate proliferation. TN-C is associated with

epithelial–mesenchymal interactions during embryogen-

esis [14]. Expression of TN-C is highly restricted in

mailto:[email protected]

936 Y. Yang et al. / Biochemical and Biophysical Research Communications 322 (2004) 935–942

normal adult tissues but is increased during wound heal-

ing and under many pathological conditions [15,16].

Therefore, the aberrant expression of TN-C may be an

important indicator of advanced or aggressive tumor

cell behavior.

The discrete composition of the ECM modulates cellbehavior through its ability to transduce specific signals

initiated through integrin receptors.

Acquisition of specific matrix metalloproteinases

(MMPs) is another characteristic of the progression of

neoplastic disease [10,11].

The purpose of this study was to further investigate

the deposition of TN-C as a direct consequence of the

b3 ligand binding and Src activation.

Materials and methods

Reagents. Hamster anti-mouse to b3 (CD 61) was purchased from

Pharmingen (San Diego, CA). Rat monoclonal antibodies to TN-C

were purchased from Sigma Scientific (St. Louis, MO). Fluorescein

isothiocyanate-conjugated streptavidin was purchased from Amer-

sham (Arlington Heights, IL). Antibodies to MT1-MMP were pur-

chased from Chemicon International (AB815). GM6001 (Calbiochem),

a general inhibitor of all MMPs, was used at a concentration of 10 lM.

Anti-TIMP2 antibodies were purchased from Sigma Scientific. MEK

inhibitor U0126 and the Src inhibitor PP-1 were purchased from

Calbiochem. Antibodies to total ERK (#9102) and to phospho-ERK

(#9101) were purchased from Cell Signaling (Beverly, MA).

Cell culture. The highly metastatic K1735M2 and the poorly met-

astatic K1735C23 murine melanoma cell lines were a generous gift from

Dr. I.J. Fidler (University of Texas M.D. Anderson Hospital and Tu-

mor Institute, Houston, TX) [17]. The highly metastatic K1735M2 cells

express abundant avb3 whereas the poorly invasive K1735C23

cells express little or almost no levels of b3 [18]. Additional cell

lines K1735C23mb3 (b3 sense), K1735M2Tb3 (b3 anti-sense),

K1735C23CASrc (constitutively active Src), and the K1735M2 KDSrc

(kinase dead Src) cells were generated in our laboratory as described

[19]. K1735C23CASrc cell line was produced by the expression of a

constitutively active Src into the K1735C23 cell line. The

K1735M2KDSrc cells were generated by expressing a kinase dead Src

into the K1735M2 cell line. All cell lines were cultured in 10% fetal

bovine serum (FBS) in Dulbecco�s minimal essential medium (DMEM).

Tumor growth and its matrix assembly. C3H/HeN syngeneic mice

were injected with 2 · 105 tumor cells (K1735M2, M2Tb3, M2KDSrc,

C23, C23mb3, and C23CASrc) in 50 ll DMEM transorally into the

floor of the mouth as described previously [20]. After 4 weeks the ani-

mals were sacrificed by cervical dislocation and the tumors were snap-

frozen and prepared for immunofluorescence microscopy as described

[20]. Sections of the frozen specimens were air-dried and immersed in

ice-cold acetone for 5 min. Slides were blocked for nonspecific staining

with 5% fetal calf serum in PBS for 5 min at 37 �C. The blocking

solution was replaced with primary antibody (anti-TN-C or FN 1:200)

for 60 min at room temperature. Sections were then washed three times

for 5 min in PBS and incubated for 30 min at room temperature with a

1:100 dilution of the secondary antibody, which is FITC conjugated

anti-rat or -rabbit IgG (Amersham), washed with PBS, and mounted

with Vectashield (Vector Laboratories, Burlingame, CA).

Conditioned media. To detect levels of TIMP2 the K1735M2,

M2Tb3, M2KDSrc, C23, C23CASrc, and C23mb3 cells were grown

under serum-free conditions for 12 h. The media were collected and

concentrated 20-fold using Microcon YM-10 Centrifugal filters (Mil-

lipore, Bedford, MA).

Detection of TN-C matrices in culture. To detect TN-C organiza-

tion, K1735 cells were plated directly onto uncoated glass coverslips

for 12 h and then fixed with 2% paraformaldehyde for 10 min. Non-

specific binding was blocked with 3% bovine serum albumin (BSA).

The cultures were incubated with polyclonal antibodies to TN-C for

1 h at room temperature, rinsed with PBS, and incubated with biotin-

conjugated goat anti-rat IgG (1:50) for 30 min at room temperature,

followed by rinsing with PBS. The cultures were then incubated with

FITC-conjugated streptavidin (1:100) (Amersham) for 30 min at room

temperature, washed with PBS, and mounted with Vectashield (Vector

Laboratories, Burlingame, CA). The cultures were then examined

using immunofluorescence microscopy for the expression of TN-C.

Matrix isolation. K1735 melanoma cells were seeded at 70% con-

fluence for 12 h. In some experiments the cells were incubated in the

presence of function-blocking antibodies to b3 (20 lg/ml), or the MEK

inhibitor U0126 (0.1 lM), the Src inhibitor PP-1 (1 lM), or the broad-

spectrum MMP inhibitor GM6001 (10 lM). Twenty-five millimolar of

NH4OH was added at 37 �C for 5 min to lyse the cells. The plates were

rinsed with PBS to remove cellular debris. The remaining matrix was

collected and solubilized in 10 mM Tris–HCl, pH 6.8, 8 M urea, 1%

SDS, and 15% b-mercaptoethanol. The samples were then boiled and

separated by SDS–PAGE followed by Western blotting using anti-TN-

C antibodies. The immunoblots were visualized by the ECL system

and Hyperfilm X-ray film (Amersham, Piscataway, NJ).

Western blotting. To harvest the cells the monolayer was scraped

and lysed in 150 mM NaCl, 1 mM EDTA, 20 mM Tris–HCl, 50 mM

NaF, 1 mM Na3VO4, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl

fluoride, 10 lg/ml leupeptin, and 0.05% aprotinin. The lysate was then

centrifuged at 15,000g for 15 min and precleared for 1 h at 4 �C with

protein A–agarose. Protein concentrations were determined by BCA

protein assay kit (Pierce, Rockford, IL). The samples were then sep-

arated by SDS–polyacrylamide gel electrophoresis and transferred to a

nitrocellulose membrane (Micron Separation, Westborough, MA).

Nonspecific staining was blocked with 5% nonfat dried milk overnight

at 4 �C. Next, the membranes were incubated with antibodies to TN-C,

MT1-MMP or TIMP2 at a 1:200 dilution for 2 h, washed and incu-

bated with horseradish peroxidase-conjugated sheep anti-rabbit IgG

(Amersham) for 1 h. The membranes were then analyzed by ECL

chemiluminescence kit (Amersham). Western blotting was quantified

using NIH Image 1.61. Values were expressed as relative value units

(RVU).

Results

Expression of b3 promotes TN-C matrix deposition

We previously demonstrated that expression of avb3suppressed FN assembly in K1735 melanoma [18]. In

this study we focused on the potential modulation of

the ECM component TN-C by the K1735 cells (Table 1).Initial analysis began by culturing the K1735M2,

K1735C23, and K1735C23mb3 cells for 12 h under ser-

um-free conditions. The cultures were then processed for

immunofluorescence microscopy and incubated with

anti-TN-C antibodies [18,21]. The b3-positiveK1735M2 and C23mb3 cells organized a rich TN-C ma-

trix, whereas the K1735C23 cells did not (Fig. 1a, panels

A and E, respectively).TN-C matrix assembly by the M2 and the C23mb3

cells was easily suppressed by expression of a b3 anti-

sense construct (K1735M2Tb3) or the addition of b3function blocking antibodies (Fig. 1a). These results

Fig. 1. Expression of avb3 promotes TN-C deposition. (a) Evaluation

of matrix deposition by immunofluorescence microscopy. K1735M2,

-M2Tb3, -C23, and -C23mb3 (A, B, D, and E, respectively) cells were

grown on glass coverslips for 12 h, fixed and processed for immuno-

fluorescence microscopy using anti-TN-C antibodies. The K1735M2

and the K1735C23mb3 cells were also grown in the presence of

function blocking antibodies to b3 (C and F, respectively). Note that

anti-b3 suppressed TN-C organization. Scale bar = 50 lm. (b) Western

blot analysis of TN-C deposition. The ECM was isolated from the

K1735M2, -M2Tb3, -M2 (plus anti-b3 antibodies, +) C23, C23mb3,and the C23mb3 (plus anti-b3 antibodies, +) cell lines and analyzed by

Western blot with anti-TN-C antibodies. RVU, relative value units.

Table 1

Characteristics of K1735 cell lines

Cell lines b3 Src activity

K1735M2 +++ +++

K1735M2Tb3 +/� +

K1735C23 +/� +/�K1735C23mb3 ++++ +++

K1735M2KDSrc +++ +/�K1735C23CASrc +/� ++++

Y. Yang et al. / Biochemical and Biophysical Research Communications 322 (2004) 935–942 937

indicate that both natural expression as well as forced

expression of the avb3 integrin facilitated a TN-C de-

fault pathway.

Deposition of TN-C was further analyzed by Western

blotting. The extracellular matrix was extracted as de-

scribed in ‘‘Material and methods’’ using a 25 mM solu-

tion of NH4OH/urea. This extract was then separated

by SDS–PAGE. The gel was transferred to a nitrocellu-lose membrane and probed with anti-TN-C antibodies

(Fig. 1b).

Western blotting confirmed the results obtained by

immunofluorescence. Expression of TN-C in the

K1735M2 cells was threefold higher when compared with

the M2Tb3 cells (antisense) or the parental K1735C23

cell line. Function-blocking antibodies to b3 (+), sup-

pressed M2 secretion of TN-C by over 66% (Fig. 1b).When b3 was forcefully expressed in the K1735C23

cells (K1735C23mb3), TN-C secretion was increased

fourfold. In the presence of anti-b3 antibodies TN-C

secretion was suppressed by 75%.

Together, these results demonstrate that both the

expression (both forced and naturally occurring) and

function of avb3 promotes TN-C matrix organization

and establishes a default TN-C pathway.

The role of Src in ECM deposition

Our previous work determined that b3 ligand binding

resulted in the activation of Src [20]. Activation of Src

stimulated cell proliferation and negatively affected FN

assembly [22].

We now wished to determine if Src activation modu-lated TN-C assembly. To evaluate the effects of Src acti-

vation on matrix assembly, we used the K1735C23

CASrc and the K1735M2KDSrc cell lines, which have

been described previously [18]. Briefly, the CASrc and

the KDSrc cell lines were established by retroviral trans-

duction with constitutively active Src or kinase dead Src,

respectively [20]. These cells were cultured for 12 h and

evaluated by immunofluorescence microscopy (Fig.2a). The expression of a KDSrc significantly suppressed

the organization of TN-C, as did incubation of the M2

cells with the Src inhibitor PP-1 (Fig. 2a). In contrast,

the expression of a constitutively active Src (CASrc) en-

hanced TN-C deposition (Fig. 2a). Enhanced TN-C

could be suppressed by incubation with PP1. This indi-

cated that the increased TN-C deposition was specifi-

cally a result of Src activation.Western blotting was done to quantify the immuno-

fluorescence (Fig. 2a, lower panel). The expression of a

KDSrc by the M2 cells or incubation with the Src inhib-

itor PP1 reduced TN-C deposition by 66% (Fig. 2a, low-

er panel). In a similar fashion, the expression of a CASrc

resulted in an 80% increase in TN-C deposition by the

C23 cells (Fig. 2a, lower panel). As demonstrated by

immunofluorescence, incubation with PP1 returned thedeposition of TN-C to baseline levels.

We next wished to evaluate the effect of pharmaco-

logical inhibitor of Src on the organization of TN-C

by the K1735C23mb3 cells which were transduced with

the b3 integrin subunit. The incubation with the Src

inhibitor PP1 totally suppressed TN-C matrix deposi-

tion back to baseline expression levels (Fig. 2a, lower

panel).

In vivo analysis of the ECM

We next investigated TN-C deposition using an in

vivo assay. K1735 tumors were previously generated

through the transoral placement of the different K1735

cell lines into syngeneic C3H/HeN mice [20,22]. The tu-

mors were then sectioned and evaluated by immunoflu-orescence microscopy (Figs. 2b and c). TN-C was

extensively deposited throughout the lesions produced

Fig. 2. Src modulates organization of the ECM. (a) TN-C organization requires active Src. K1735M2, M2KDSrc, K1735C23, and K1735C23CASrc

cells (A, B, D, and E, respectively) were grown on glass coverslips for 12 h and evaluated for TN-C organization. In some experiments the M2 and the

C23CASrc cells were also grown in the presence of the Src inhibitor PP-1 (C and F, respectively). Scale bar = 50 lm. Western blot. The ECM was

extracted from cultures of the M2, M2KDSrc, M2+PP1 (+), C23, C23CASrc, C23CASrc+PP1 (+), C23, C23mb3, and C23mb3+PP1 (+) cells

analyzed by Western blotting with anti-TN-C antibodies. Note that addition of PP-1 to the cultures dramatically reduced deposition of TN-C matrix

formation in all cell lines tested including the K1735C23mb3 cells which forcefully express b3. RVU, relative value units. (b) In vivo determination of

TN-C expression. Tumors derived from the K1735M2 (A) -M2KDSrc (B) -C23 (C, E), -C23mb3 (D), and -CASrc (F) cells were evaluated for the

expression of TN-C by immunofluorescence microscopy. As found in vitro, the expression of b3 or the activation of Src enhanced TN-C deposition.

Scale bar = 50 lm. (c) In vivo determination of FN expression. Tumors derived from the K1735M2 (A), -M2KDSrc (B), -C23 (C,E) -C23mb3 (D),

and -CASrc (F) cells were examined for FN deposition. The expression of b3 or CASrc significantly suppressed the organization of FN in vivo. Scale

bar = 50 lm.


by both the K1735M2 and C23mb3 tumors (Fig. 2b).

These results paralleled those obtained through our in

vitro cell culture system.

TN-C deposition was next evaluated in tumors gener-

ated by the CASrc and the KDSrc cell lines (Fig. 2b). As

found in the K1735 cell cultures, TN-C was highly orga-

nized in the tumors derived from the K1735C23CASrc

cells but not from the KDSrc cells (Fig. 2b).Using the same in vivo system as described above, we

examined the deposition of FN. The expression of FN

was opposite to that described for TN-C in vivo. FN

was highly expressed in both the K1735C23 and the

M2KDSrc tumors. Significantly less FN was detected

in the K1735M2, K1735C23mb3, and the CASrc de-

rived tumors (Fig. 2c). These results suggest that the

expression of b3 and activation of Src suppress the FN

pathway and promote the deposition of TN-C.

Tenascin-C organization is modulated through MEK

Sustained MAPK activation accounts for many fea-

tures of transformation as well as regulating a variety offactors related to cell differentiation [23]. We wished to

determine if deposition of TN-C matrices was dependent

uponMAPK.MAPK is activated via MEK.We used the

MEK inhibitor U0126 to suppressMAPK activation and

analyze the effect this pathway may play in TN-C organi-

zation. The K1735M2 and the K1735C23mb3 cells were


cultured both in the absence and presence of U0126. TN-

C deposition was then analyzed by immunofluorescence

microscopy and Western blotting (Fig. 3a). The addition

of U0126 dramatically suppressed deposition of TN-C

when imaged by immunofluorescence microscopy. When

visualized by Western blotting, we determined U0126suppression to be approximately 80% for both the

K1735M2 and the K1735C23mb3 cell lines (Fig. 3a).

These results indicate that TN-C organization requires

activation of MAPK.

We next wished to demonstrate the effect of U0126 on

the phosphorylation of MAPK. We first evaluated total

ERK1/2 and pERK1/2 levels in the K1735M2 (wt) and

the K1735C23mb3 (b3 overexpressors) at seveal differenttime points (0, 2, 6, and 12 h).We see a gradual increase in

phosphorylation of ERK1/2 over time in both the

K1735M2 and the K1735C23mb3 cells. There was a

slightly higher phosphorylation of p42 (ERK2) when

compared with p44 (ERK1) in both the K1735M2 and

theK1735C23mb3 cells, indicating that the forced expres-sion of b3 behaves in a similar fashion to wild type b3.

Fig. 3. TN-C assembly requires MAPK and MMP activation. (a) Suppressi

cells (C,D) were grown for 12 h on glass coverslips in the absence or presenc

deposition by Western blotting. The ECM was isolated from the K1735M2

inhibitor U0126. Western blotting was performed on the extracted ECM us

cultured in the presence of U0126. A time course was performed using the M2

12 h). Both cell lines phosphorylated ERK2 slightly greater than ERK1 over

12 h time pt in both the M2 and the C23mb3 cells. Note 75–80% inhibition

organization is mediated through MMPs. The K1735M2 and the K1735C23m

broad spectrum protease inhibitor GM6001 (B and D, respectively). Whe

organization of TN-C. Scale bar = 50 lm. Western blotting. K1735M2 and th

GM6001. The ECM was isolated and analyzed by Western blot using anti-T

relative value units.

We next wished to determine the specific effects of

U0126 on ERK1/2 in both the wild type K1735M2

and the K1735C23mb3 cells, which have forced expres-

sion of b3. There was a 75% suppression of p44 (ERK1)

phosphorylation in the presence of U0126 in both the

K1735M2 and the K1735C23mb3 cell lines (Fig. 3a,lower panels). Additionally there was an 80% suppres-

sion of phosphorylation of p42 (ERK2) in both cell lines

(Fig. 3a, lower panels). These results demonstrate the

potent effect of U0126 on the activation of ERK1/2.

This directly suggests that ERK1/2 is involved in the

organization of TN-C matrices.

Suppression of MMPs modulates the organization of

Tenascin-C matrices

Modulation of the ECM occurs both by deposition of

new ECM molecules and degradation of existing mole-

cules. The degradation of the basement membrane and

invasion into the underlying tissue have long been the

histologic criteria for diagnosis of neoplastic disease

on of MAPK inhibits TN-C assembly. K1735M2 (A,B) and -C23mb3e of the MEK inhibitor, U0126. Scale bar = 50 lm. Analysis of TN-C

, -C23b3 cultures both in the presence (+) and absence of the MEK

ing antibodies to TN-C. TN-C deposition was reduced by 75% when

and the C23 cells to evaluate phosphorylation of ERK1/2 (0, 2, 6, and

all time pt. 0.1 lM U0126 was used to suppress ERK1/2 activity at the

of phosphorylation of ERK1/2 in the presence of U0126. (b) TN-C

b3 cells were cultured for 12 h in the absence (A,C) or presence of the

n grown in the presence of GM6001, both cell types decreased the

e -C23mb3 cells were grown for 12 h in the absence or presence (+) of

N-C antibodies. GM6001 decreased TN-C deposition by 80%. RVU,


[24]. The differential organization of TN-C by activation

of Src and MAPK led us to investigate whether MMP

activation was also a participant in this system. To ad-

dress this concern, we grew the M2 and the C23mb3 cul-

tures in the presence and the absence of the general

MMP inhibitor GM6001. When the M2 and theC23mb3 cells were grown in the presence of GM6001,

TN-C matrix assembly was decreased dramatically when

examined by immunofluorescence (Fig. 3b). When visu-

alized by Western blotting we determined this decrease

to be approximately 80% (Fig. 3b).

These results indicate that TN-C deposition into a

three-dimensional matrix requires MMP modification.

MT1-MMP and TIMP2 are differentially expressed in

K1735 cells

Matrix expansion is caused by the overproduction

and/or the impaired proteolytic degradation of the

extracellular matrix. The production of the matrix is a

tightly regulated process. Regulated MMP suppression

and timely activation are important factors modulatingtumor cell behavior. We have previously shown there

is a strict correlation between b3 expression and

MMP2 activation in K1735 cells. It has been indicated

by others that in melanoma cells TIMP2 expression lev-

els might regulate MT1-MMP mediated activation of

pro-MMP2 [25]. We wished to evaluate the relative lev-

els of MT1-MMP and TIMP2 in this system.

By Western blotting, we determined that MT1-MMPwas expressed 50% greater in the K1735M2, C23mb3,and the C23CASrc cells when compared with the

K1735M2Tb3, M2KDSrc, and C23 cell lines (Fig. 4).

The expression of TIMP2 was opposite to that of

MT1-MMP (Fig. 4). TIMP2 expression was fourfold

greater in the conditioned media (CM) from the

M2Tb3, M2KDSrc, and C23 cells compared with CM

Fig. 4. b3 and Src modulate MT1-MMP and TIMP2. Western blot

analysis was performed using anti-MT1-MMP and -TIMP2 antibodies

on whole cell lysate (WCL) and conditioned media (CM) of the

K1735M2, -M2T b3, -M2KDSrc, -C23, -C23mb3, and -C23CASrc cell

lines. Expression of MT1-MMP was 50% greater in the WCL from the

K1735M2, C23mb3, and the -C23CASrc cell lines when compared

with the K1735M2Tb3, -M2KDSrc, and -C23 cell lines. M2Tb3,M2KDSrc, and C23 cells of CM and WCL contained fourfold and

twofold greater levels of TIMP2, respectively, when compared with the

-M2, -C23m b3, and CASrc cell lines. RVU, relative value units.

from the M2, C23mb3, and the C23CASrc cell lines

(Fig. 4). Expression of membrane-bound TIMP2 was

twofold greater in the noninvasive M2Tb3, KDSrc,

and the C23 cell lines compared with their invasive

counterparts (Fig. 4). The relative expression levels of

MT1-MMP and TIMP2 suggest that they are importantmodulators of K1735 tumor cell invasion.

Discussion

In this study we further investigated the post-ligand

binding events displayed by the highly characterized

K1735 murine melanoma cell lines. We wished to fur-ther explore the specific modulators of ECM organiza-

tion, which is an important aspect of tumor cell

biology (invasion and metastasis).

In the current study, we demonstrate that TN-C

organization is regulated through a mechanism distinct

from that previously shown by our group for FN assem-

bly [26]. Expression of avb3 enriches the organization of

TN-C, whereas suppression of its expression or functioninhibits TN-C assembly.

In the current study we demonstrate that the activa-

tion of Src, via avb3 ligand-binding, promotes deposi-

tion of a TN-C matrix assembly. These findings are

opposite to what we previously documented for FN

assembly by the K1735 cells [18]. The deposition of

TN-C indirectly suggests that b3 ligand binding may

promote a less differentiated phenotype [26].Elevated Src expression and kinase activity are com-

mon features of many malignancies [27]. Src family ki-

nases are known to play important roles in integrin

signaling [28]. Therefore, we examined the effect of Src

mutations on TN-C organization. TN-C is typically ex-

pressed in the tumor stroma and by the tumor cells [29]

as well as the vasculature at sites of angiogenesis [30].

Our work indicates that expression of the avb3 inte-grin and activation of c-Src promotes TN-C matrix

assembly. The results of the current study compiled with

our previous work suggest that deposition of TN-C and

FN is differentially regulated via activation or suppres-

sion of Src [18].

Our previous studies showed that Src activation is

downstream of b3 ligand binding in vitro [18,20]. Our

current results indicate that this cross talk modulatesTN-C deposition in vitro. We also evaluated these find-

ings in vivo. K1735 tumors were established by transoral

injection described previously [20]. Tumors derived from

the K1735M2, C23mb3, C23CASrc, and M2KDSrc cell

lines were evaluated for TN-C and FN deposition. The

K1735M2Tb3 cell line did not form tumors. TN-C

was abundant in the b3 positive tumors (K1735M2

and the K1735C23mb3) and in those expressing a con-stitutively active Src (K1735C23CASrc). FN was scar-

cely discernable in the extracellular matrix of the M2,


C23mb3, and the CASrc tumors. This demonstrates that

the cells retain their phenotype and matrix organizing

ability when grown back in their syngeneic host.

The expression of b3, through its activation of Src,

modulates the deposition of the ECM. We hypothesize

that cross talk exists between the b3 integrin and Src.This cross communication may result in a modification

of the ECM with an emphasis placed on TN-C

expression.

The work by our group previously demonstrated that

Src activation resulted in a downstream activation of

MAPK. In our current work we show that the suppres-

sion of MEK inhibited TN-C assembly. Phosphoryla-

tion of ERK1/2 in cells forced to express b3 is similarto that in cells with naturally high level expression of

the integrin. Additionally, TN-C matrix organization

is modulated by the activation of ERK1/2.

Local degradation of connective tissue is essential for

tumor cell invasion and metastasis [25]. Degradation of

the ECM is accomplished through activity of different

MMPs [31]. This hypothesis was verified by inhibiting

the activation of MMPs by incubating the cells in thepresence of GM6001. The addition of GM6001 de-

creased the organization of the TN-C. This indicates

that the activation of MMPs is essential for TN-C depo-

sition by the K1735 cells.

MMP activity is tightly regulated and is subjected to

several layers of control including gene transcription

and inhibition by soluble inhibitors [32]. We evaluated

the expression of MT1-MMP and its inhibitor TIMP2.MT1-MMP is increased in multiple tumors and acti-

vates MMP2. MT1-MMP expression is significantly

higher in the b3 expressing cells and the CASrc cells.

MT1-MMP also activates proMMP2 which is highly ex-

pressed in the b3-positive K1735 cell lines [20]. The pro-

file of TIMP is opposite to that of MT1-MMP.

The expression of TIMP2 in the poorly invasive

K1735 cells imparts this system with an additional levelof regulation.

The results of the current work indicate that the

deposition and architectural outline of the ECM in mel-

anoma is highly dependent on integrin ligand binding.

The adhesion of the avb3 integrin complex to VN results

in Src activation, subsequent activation of MAPK. The

MMPs modulate the eventual outcome of the incoming

ECM signals with a dramatic switch in the expansivedeposition of the ECM. Future work will further estab-

lish the effect of TN-C expression/modification on the

metastasis of K1735 melanoma.

Acknowledgments

This work was supported by grants from the Na-tional Institute of Craniofacial Dental Research R01

DE12856, R01 DE11930, and P01DE13904 to D.M.R.

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Tenascin-C deposition requires β3 integrin and Src

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