©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Susceptibility of Small and Large Human Tenascin-C Isoforms to Degradation by Matrix Metalloproteinases (*)

Annalisa Siri (1), Vera Knäuper (2)(§), Natalia Veirana (1), Fabio Caocci (1), Gillian Murphy (2)(¶), Luciano Zardi (1)(**)

From the (1) Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV,10, 16132 Genoa, Italy and (2) Strangeways Research Laboratory, Worts' Causeway, Cambridge, CB1 4RN, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two major tenascin-C (TN-C) isoforms are generated by the alternative splicing of the pre-mRNA. The large isoform contains seven extra type three repeats that, by contrast, are omitted in the small TN-C isoform. The large TN-C isoform is mainly expressed at the onset of cellular processes that entail active cell migration, proliferation, or tissue remodeling such as occur in neoplasia, wound healing, and during development. Thus, the large TN-C isoform seems to be a specific component of the provisional extracellular matrix. Here we have studied the degradation of the large and small TN-C isoforms by matrix metalloproteinases (MMPs) 2, 3, 7, and 9. Among these proteolytic enzymes only MMP-7 can degrade the small TN-C isoform removing the NH-terminal knob. The large TN-C isoform shows the same MMP-7-sensitive site adjacent to the NH-terminal sequence, but is further degraded in the splicing area where three fibronectin-like type III repeats are completely digested. Moreover, the large TN-C isoform is degraded by MMP-2 and MMP-3 which completely digest a single type III repeat inside the splicing area. By contrast, the large TN-C isoform is resistant to MMP-9 digestion. The results show that the presence of the spliced sequence introduces new protease-sensitive sites in the large TN-C isoform.


INTRODUCTION

Human tenascin-C (TN-C)() is an extracellular matrix glycoprotein composed of six similar subunits joined at their NH-terminal by disulfide bonds (1, 2, 3, 4, 5, 6, 7) . During development, TN-C displays a time and space-dependent tissue distribution with morphogenetically significant boundaries. Each human TN-C subunit includes three types of structural modules: 14 epidermal growth factor-like repeats, 15 fibronectin (FN) type three homology repeats, and a COOH-terminal knob made up of a sequence with homology to the globular domain of the and chains of human fibrinogen (see Fig. 3). TN-C is coded for by a single gene and its expression is regulated by a single promoter (8, 9) . Structurally and functionally different human TN-C isoforms are generated by alternative splicing of the TN-C transcript, seven type three repeats being included or omitted in the mRNA (see Fig. 3) (10, 11) generating two major TN-C isoforms with molecular masses of about 300 and 200 kDa, respectively. Analysis of TN-C isoform ratio has shown that, in human malignantly transformed cultured cells as well as in breast carcinoma and in some cases of lung carcinoma, the higher molecular mass isoform is preferentially expressed (12, 13, 14, 15, 16, 17) . More recently it has been shown that mitogenic stimulation of cultured fibroblasts induces a modification of the ratio between the two TN-C isoforms, the expression of the larger isoform being increased to a much higher extent (18) . Differences in biological functions and sites of expression between TN-C isoforms have been reported (19, 20, 21, 22, 23, 24, 25) . Murphy-Ullrich et al. (26) have demonstrated that focal adhesion integrity is down-regulated by the alternative spliced domain of human TN-C. Chiquet-Ehrismann et al. (27) have shown that the chicken TN-C isoform lacking the alternatively spliced domain binds more strongly to FN than the isoform in which this domain is included, and Kaplony et al. (28) reported that expression of high molecular mass TN-C correlates with corneal cell migration. Moreover, recent reports have shown that the two TN-C isoforms bind to the cell surface through different receptors; in fact, the large TN-C isoform strongly interacts with annexin II in glioma and endothelial cells (29) while the neuronal cell adhesion molecule contactin/F11 mainly binds the small TN-C isoform (30, 31) .


Figure 3: Model of the domain structure of the large isoform subunit of human TN-C and MMP resistant fragments. The ovals and the squares represent the EGF-like and FN-like repeats, respectively. The globular NH-terminal and fibrinogen-like COOH-terminal domains are also represented. The FN-like repeats A1 to D, whose expression is regulated by alternative splicing of pre-mRNA, are shaded. The upper part of the figure reports the mAbs used and the brackets show the shortest sequence in which each epitope was localized. The lower part represents the resistant fragments obtained by large TN-C digestion with MMP-7 and MMP-2, respectively, the mAbs reacting with the various fragments, as well as the amino-terminal sequences of the resistant fragments.



Since extracellular matrix degradation is a crucial step in tumor invasion, a differential susceptibility of the different TN-C isoforms to the proteolytic enzymes released by neoplastic cells may take part in this mechanism. In particular, matrix metalloproteinases (MMPs) are known to be produced by several tumor tissues and are involved in proteolytic degradation of tumor extracellular matrix. In fact, it has been reported that various murine and human tumor-derived cell lines transcribe, synthesize and secrete MMP enzymes (32, 33) . These enzymes are also synthesized and secreted by normal cells under conditions that may be associated with physiologic tissue remodeling.

Here, we have compared the susceptibility of small and large TN-C isoforms to degradation by MMP-2, MMP-3, MMP-7, and MMP-9. Results show that the large TN-C isoform is much more sensitive to MMP proteolytic activity.


MATERIALS AND METHODS

Cells and Monoclonal Antibodies

Baby hamster kidney (BHK) cells transfected with TN-C cDNA constructs (11) in the pNUT expression vector were a gift of Dr H. P. Erickson (34) . Two clones, HxB.L and HxB.S produce the large and the small TN-C splice variants, respectively (large TN-C and small TN-C). Cells were grown in Dulbecco's modified Eagle's medium (HyClone Europe Ltd. Cramlington, UK) containing 10% fetal calf serum obtained from Boehringer (Mannheim, Germany). Preparation and characterization of seven mAbs used has already been reported (35) . Three new mAbs, BC-8, BC-9, and BC-10, not previously described, have been characterized in this study using the procedure previously described (35) based on analysis of mAb reaction in immunoblotting with different TN-C--galactosidase fusion proteins.

Purification and Activation of Recombinant MMPs

Recombinant MMP-2, MMP-3, MMP-7, and MMP-9 were purified from conditioned culture medium of transfected NSO mouse myeloma cells essentially as previously described (36, 37, 38) . The purified proenzymes were activated by treatment with 1 m M aminophenyl-mercuric acetate for 1 h at 25 °C (MMP-2) or 37 °C (MMP-3, -7, and -9).

Purification and Digestion of Large and Small TN-C Isoforms by MMP-2, -3, -7, and -9

Small and large TN-C splicing variants were purified from conditioned culture medium of BHK-transfected cell lines using an immunoadsorbent prepared with the mAb BC-4 as already reported (12) . Each isoform was more than 95% pure as judged by SDS-polyacrylamide gel electrophoresis (PAGE) and the amount of TN-C was evaluated using the absorption coefficient A(1 mg/ml, 1 cm) of 0.97 (1) . 300 µg of the two TN-C isoforms were incubated with either 5 µg of active MMP-7, 13 µg of active MMP-2, or 15 µg of active MMP-3 or MMP-9 in 100 m M Tris/HCl, pH 7.5, 100 m M NaCl, 5 m M CaClfor 24 h at 37 °C. The digestion products were analyzed by SDS-PAGE under reducing conditions followed by Coomassie Blue staining or immunoblotting carried out as previously reported (39) . Amino-terminal sequencing of the fragments was performed by TIB MolBiol (Genoa, Italy). Fragments to be analyzed were run on SDS-PAGE under reducing conditions and blotted on an Immobilon-P membrane (Millipore, Bedford, MA). The bands were revealed by staining with Ponceau S (Gelman Sciences, Ann Arbor, MI) and isolated by cutting the membrane.


RESULTS

Large TN-C Isoform Digestion

MMP-2, MMP-3, MMP-7, and MMP-9 were used to digest the large TN-C isoform under the conditions described under ``Materials and Methods.'' The products obtained were analyzed by SDS-PAGE under reducing conditions and are shown in Fig. 1 A. The digests were also analyzed by immunoblotting using 10 mAbs specific for different epitopes of TN-C (see Fig. 3) and the NH-terminal sequences of the major fragments were determined. Three different patterns were observed.


Figure 1: Analysis on SDS-PAGE of the large and small TN-C digested by MMP-7 and MMP-2. A, large TN-C undigested ( lanes 1 and 3) and digested by MMP-7 ( lane 2) and MMP-2 ( lane 4). In the MMP-7 digest, three major resistant fragments were detectable: according to the molecular mass L-170, L-65, and L-16. In the MMP-2 digest two resistant fragments were detectable: L-190 and L-120. The values on the left are the molecular masses (in kilodaltons) of the standards. B, undigested small TN-C isoform ( lanes 1 and 3) and digested by MMP-7 ( lane 2) or by MMP-2 ( lane 4) were run in 4-18% SDS-PAGE in reducing conditions and stained with Coomassie Blue. Two major resistant fragments were identified in the MMP-7 digest ( S-170 and S-16), while degradation was not observed using MMP-2. The values on the left are the molecular masses (in kilodaltons) of the standards.



Pattern 1

MMP-7 degraded 100% of large TN-C molecules and three major fragments with molecular masses of 170, 65, and 16 kDa, were generated (fragments L-170, L-65, and L-16) (Fig. 1). Analysis of these bands by immunoblotting with specific mAbs is shown in Fig. 2and . According to molecular mass and reaction with the different mAbs, the L-170 band was identified as a fragment including the EGF-like repeats and the FN like repeats 1 to A2. From the molecular mass and mAb reactivity we deduced that the L-65 fragment contains the fibrinogen-like COOH-terminal knob plus the FN like repeats 6 to 8. The mAb BC-7, specific for an epitope located in the amino-terminal portion of the molecule, reacted with the L-16 band. Since no other band with a higher molecular mass was stained by this mAb, the results show that the cleavage of the amino-terminal sequence occurred in 100% of the large TN-C molecules.


Figure 2: Analysis using different mAbs of large TN-C digested by MMP-7. To the left, an SDS-PAGE in which undigested ( lane U) and digested with MMP-7 ( lane D) large TN-C isoform were run under the conditions described under ``Materials and Methods.'' To the right, immunoblottings of samples as in lane D stained with the mAbs indicated above the lanes.



Three mAbs reacting with epitopes located in the splicing areas, -A3, -B, -D, were negative both with fragment L-170 and with fragment L-65 (), indicating that the MMP-7 completely degraded the FN-like repeats A3 to D included in the splicing area under these conditions. The model of the fragment generated by MMP-7 is depicted in Fig. 3 . Amino-terminal sequences of the fragments L-170 and L-65 confirmed this degradation pattern (Fig. 3).

Pattern 2

MMP-2 and MMP-3 cleaved about 60% of the large TN-C isoform into two major fragments of about 190 and 120 kDa (fragments L-190 and L-120), respectively. The polypeptide pattern of the large TN-C digested by MMP-2 was analyzed on a 4-18% SDS-PAGE gradient using the conditions described under ``Materials and Methods'' and is shown in Fig. 1 A. Analysis of these fragments with specific mAbs () has demonstrated that the L-190 includes the amino-terminal end of the molecule, the EGF-like and the FN-like repeats 1 to A2. The 120-kDa fragment includes the FN-like repeats A4 to D and 6 to 8 as shown by mAb reaction and, according to the molecular mass, the fibrinogen like COOH-terminal sequence. The absence of reaction of mAb -A3 with either L-190 or L-120 fragments suggests that its epitope in the A3 repeat is completely digested by these MMPs. The model of the fragment generated by MMP-2 is depicted in Fig. 3. Amino-terminal sequences of the fragment L-120 confirmed the pattern of degradation (Fig. 3).

Pattern 3

MMP-9 did not show detectable degradation of the large TN-C isoform (data not shown).

All of the results of large TN-C-resistant fragment analysis by immunoblotting are summarized in and in Fig. 3, which also shows the localization within the TN-C molecules of the epitopes recognized by the various mAbs and the amino-terminal sequences of the fragments. Small TN-C Isoform Digestion

Digestion of the small TN-C isoform was carried out using the same enzymes and under the same conditions as for large TN-C. The results have shown that small TN-C is much more resistant than the large isoform. MMP-2, MMP-3 (small and large form) and MMP-9 do not cleave small TN-C. Only MMP-7 cleaves 100% of the molecules, giving two fragments detectable in SDS-PAGE (Fig. 1 A). One has an apparent molecular mass of 170 kDa and the second of about 16 kDa (S-170 and S-16), respectively. Analysis with specific mAbs showed that the S-16 kDa is the NH-terminal knob, while the S-170 fragment includes all the remaining part of the molecule from the beginning of the EGF-like repeats to the fibrinogen like COOH-terminal sequence. The model of the fragment generated by MMP-7 is depicted in Fig. 4 . Amino-terminal sequence of the fragment S-170 confirmed this pattern of degradation (Fig. 4). The results are summarized in .


Figure 4: Model of the domain structure of the small isoform subunit of human TN-C and MMP-7 resistant fragments. Symbols are as in Fig. 3 and show the small TN-C isoform in which all the repeats undergoing alternative splicing are omitted. Fragments obtained in MMP-7 digestion are shown, as are NH-terminal sequence and mAbs reacting with the various fragments.




DISCUSSION

Degradation of the extracellular matrix (ECM) is a crucial step of tumor invasion and progression (33) . Although many proteases can cleave ECM molecules, MMPs are believed to be the main physiologically relevant mediators of matrix degradation (33, 40, 41) . The human MMP family consists of at least 11 highly homologous zinc endopeptidases which are active at neutral pH and collectively cleave most, if not all, constituents of the ECM. More recently, it has been elegantly demonstrated that a mixed substrate of TN-C and FN collaborate in the up-regulation of collagenase (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9) gene expression by rabbit fibroblasts (42) , this phenomenon being dependent on an epitope in the carboxyl-terminal fibronectin-like type III repeats. Thus, transient alteration of the composition of the ECM by TN-C presence up-regulates the expression of genes involved in cell migration, tissue remodeling and invasion in regions of tissues undergoing morphological modifications.

TN-C expression has been documented in a wide variety of solid tumor tissues (1, 17, 43, 44) . Moreover, much evidence has shown an increased expression of the TN-C isoform with a higher molecular mass with the onset of neoplastic transformation both in vitro and in vivo (12, 13, 14, 15) . Furthermore, we have recently shown that proliferating cells produce only the large TN-C isoform, while resting cells mainly produce the small isoform (18) .

Analysis of TN-C sensitivity to proteolytic degradation may provide information about the mechanisms involved in ECM degradation. Little information is available about this topic. Friedlander et al. (45) digested TN-C with chymotrypsin and separated the resistant fragments by gel filtration. Taylor et al. (46) described TN-C digestion by -chymotrypsin and trypsin. However, the exact location of cleavage sites in the TN-C structure were not determined in these reports. More recently, Chiquet et al. (47) analyzed resistant fragments obtained by digestion with pepsin and Pronase of chicken TN-C preparations enriched in small or large splicing variants. These authors have localized the resistant fragment in the TN-C molecule using specific mAbs and have shown that the large isoform is cleaved inside the spliced region.

We have studied the degradation patterns of small and large human TN-C isoforms obtained with different MMPs, a group of enzymes which are known to be more highly expressed in neoplastic and remodeling tissues than in the corresponding normal adult tissues (33) . We have been able to precisely localize the cleavage sites through the analysis of resistant fragments with mAbs whose epitope localization within the TN-C molecule was already known. Results were confirmed by determination of the NH-terminal sequences of the major fragments and have shown that the presence of the spliced sequence strikingly modifies TN-C sensitivity to these enzymes. In fact, while large TN-C can be cleaved by MMP-7, -2, and -3 inside this sequence, small TN-C contains only a sensitive site close to the NH-terminal knob that is cleaved by MMP-7.

Degradation by MMPs of TN-C purified from human melanoma cells has recently been reported by Imai et al. (48) . They found that TN-C produced by melanoma cells (essentially the large isoform) is sensitive to MMP-1, 3 and 7 degradation while it is resistant to MMP-2 and MMP-9. The data on MMP-2 digestion do not entirely agree with ours, even though degradation by this enzyme is only partial in our hands as well. However, these authors neither report information about the localization within the TN-C molecule of the cleavage sites nor compare the sensitivity of the two major TN-C isoforms to these enzymes.

The greater sensitivity of the large TN-C isoform to MMP degradation, together with the higher expression of this isoform observed in neoplastic and remodeling tissues (25, 49) , may support the hypothesis that the large TN-C isoform is a specific component of the provisional extracellular matrix and may contribute to the generation of a more suitable environment for cellular proliferation and migration. Furthermore, degradation by MMPs of TN-C could modify its biological activities. In fact, proteolytic cleavage may represent a tool to either abolish or unmask specific TN-C functional sites.

  
Table: 1696613199p4in ND, not determined.


FOOTNOTES

*
This study was supported in part by funds of the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Consiglio Nazionale delle Ricerche (CNR), ``Progetto Finalizzato: Applicazioni Cliniche Della Ricerca Oncologica.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Wellcome Trust Travelling Fellowship.

Supported by the Arthritis and Rheumatism Council United Kingdom.

**
To whom correspondence should be addressed. Tel.: 39-10-3534901; Fax: 39-10-352855; E-mail: lzardi@CISI.unige.it.

The abbreviations used are: TN-C, tenascin-C; FN, fibronectin; MMP, matrix metalloproteinase; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; ECM, extracellular matrix; mAb, monoclonal antibody; BHK, baby hamster kidney.


ACKNOWLEDGEMENTS

We thank Antonella Gessaga for skilful secretarial assistance and Mr. Thomas Wiley for manuscript revision. We are indebted to Prof. L. Santi for his support and encouragement.


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