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Differential Regulation of Fibulin, Tenascin-C, and Nidogen Expression during Wound Healing of Normal and Glucocorticoid-Treated Mice
EXPERIMENTAL CELL RESEARCH
222, 111–116 (1996)
Article No. 0014
Differential Regulation of Fibulin, Tenascin-C, and Nidogen Expression during Wound Healing of Normal and Glucocorticoid-Treated Mice REINHARD FA¨SSLER,1 TAKAKO SASAKI, RUPERT TIMPL, MON-LI CHU,*
AND
SABINE WERNER
Max-Planck-Institut fu¨r Biochemie, D-82152 Martinsried, Federal Republic of Germany; and *Departments of Biochemistry and Molecular Biology and of Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Expression and distribution of fibulins, nidogen, and tenascin-C were analyzed in healing skin wounds of normal and dexamethasone-treated mice. In normal mice both tenascin-C and fibulin-2 showed a marked increase in mRNA expression, which declined to normal levels after completion of skin repair. These two matrix proteins are found throughout the granulation tissue and persisted there after mRNA expression had ceased. Fibulin-1 is present in normal skin and in wounds but is not distinctly upregulated during the healing process. Nidogen, however, is expressed uniformly throughout the granulation tissue early in wound healing, has a peak expression around Day 7, and selectively localizes to basement membranes after healing is accomplished. Dexamethasone treatment led to a decreased expression of tenascin-C in healing wounds but had no effect on fibulin-2 expression. In vitro experiments revealed that growth factors like TGF-b1 can partly counteract glucocorticoid action. These data therefore provide some molecular interpretations for the well-known glucocorticoid suppression of wound healing. They also indicate that repair involves complex regulatory processes which are obviously different for each of the four proteins studied. q 1996 Academic Press, Inc.
INTRODUCTION
In the course of normal wound healing, many cellular events such as proliferation, migration, and differentiation as well as tissue remodeling have to progress in a coordinated manner [1]. Several growth factors, including TGF-b1, are potent promoters of the healing process. They induce angiogenesis and formation of granulation tissue [2], increase chemotaxis of fibroblasts [3] and extracellular matrix deposition [4], and modulate 1 To whom correspondence and reprint requests should be addressed at Max-Planck-Institut fu¨r Biochemie, Abteilung Proteinchemie, Am Klopferspitz 18A, D-82152 Martinsried. Fax: (089) 8578 2422.
tissue remodeling by matrix metalloproteinases [5, 6]. To the contrary, glucocorticoids have detrimental effects on wound healing [7], presumably caused by a decrease in cell proliferation, neovascularization, and matrix production [8]. Increased matrix deposition in healing skin wounds involves several collagen types, fibronectin and a few other components [1], in particular tenascin-C, which is expressed at high levels [9] despite its low abundance in normal adult tissues [10]. Interestingly, tenascin-C production in cultured cells is upregulated by TGF-b1 [11] but downregulated by dexamethasone [12], which correlates with the general effects observed during wound healing. In the present study we have analyzed the expression of three more extracellular matrix proteins during cutaneous wound healing in mice in comparison with that of tenascin-C. These proteins included nidogen, which is a versatile binding protein in basement membranes [13] and during embryonic development is often synthesized by mesenchymal compartments [14, 15], as well as the more novel proteins fibulin-1 [16, 17] and fibulin-2 [18]. Little is known about the biological properties of the fibulins except that they are found in skin and are prominently expressed during mouse heart development [19, 20]. The data obtained with normal and dexamethasone-treated mice demonstrated quite different regulation patterns for each of the four proteins, indicating that their involvement differs during wound repair. MATERIAL AND METHODS Preparation of wound tissue from normal and dexamethasonetreated mice. In each of the two experiments, 22 female Balb/c mice, 4 weeks old, were anesthetized with a single intraperitoneal injection of Avertin. Six full-thickness wounds (6 mm in diameter, 3–4 mm apart) were made on 20 mice by excising the skin and panniculus carnosus. The wounds were allowed to dry in order to form a scab. Four animals were sacrificed and their wounds harvested at 1, 3, 5, 7, and 13 days after wounding. An area of 7 mm in diameter, which included the complete epithelial margins, was excised at each time point. A similar amount of skin from the backs of two nonwounded animals was combined and used as a control. Wound tissue was immediately frozen in liquid nitrogen and stored at 0707C. Three
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wound healing experiments were performed with dexamethasonetreated animals. During the week prior to wounding and for 5 days thereafter, 8 mice were injected subcutaneously at 9 AM every day with either 1 or 0.2 mg dexamethasone/kg body wt. Control animals were treated with phosphate-buffered saline (10 mM phosphate buffer, pH 7.3, 0.15 M NaCl, 0.2 mM EDTA). Five days after injury animals were sacrificed and wound tissue was harvested as described above. Analysis of tenascin-C regulation in cultured fibroblasts. Murine fibroblasts (NIH3T3) were grown to approximately 70% confluency in Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal calf serum. They were subsequently treated for 15 h with 10 mM or 100 nM dexamethasone, the latter in the presence or absence of purified transforming growth factor TGF-b1 (0.1 or 1 ng/ml). RNase protection assay. Total cellular RNA from cultured cells and from mouse skin was isolated as described [21]. For RNase protection mapping, DNA probes were cloned into the transcription vector pBluescript II(KS/) (Stratagene) and linearized. An antisense transcript was synthesized in vitro by using T3 or T7 RNA polymerase and [32P]UTP (800 Ci/mmol, Amersham). Samples of total cellular RNA from tissues (50 mg) or cells (20 mg) were hybridized at 427C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested for 60 min at 307C with RNase A and T1 [22]. Protected fragments were separated on 5% polyacrylamide/8 M urea gels and analyzed by autoradiography. Loading of the gels with equivalent amounts of RNA was controlled in separate runs by ethidium bromide staining with material prior to RNase treatment. Fifty micrograms tRNA was used as a negative control. The same RNA preparations were used for all protection assays. The DNA templates used for RNAse protection assay were as follows: a fragment corresponding to nucleotides 1094–1383 of murine tenascin-C cDNA [23]; a fragment corresponding to nucleotides 980–1350 of murine fibulin1 cDNA [17]; a fragment corresponding to nucleotides 2483–2800 of murine fibulin-2 cDNA [18]; and a fragment corresponding to nucleotides 3370–3763 of murine nidogen cDNA [24]. Indirect immunofluorescence. Double-label immunofluorescence analysis was carried out using affinity-purified rabbit antibodies against fibulin-1 [25] or a rabbit antiserum against fibulin-2 [18] and monoclonal rat antibodies against tenascin-C (MTN-12) [26] or nidogen [27]. All tissue specimens were frozen in liquid nitrogen and sections (6 mm) were prepared on a Reichert–Jung cryostat. Sections were fixed in acetone for 10 min at 0207C and washed in phosphatebuffered saline. Antibodies against fibulin-1 (1:30), fibulin-2 (1:150), tenascin-C (1:200), and nidogen (1:150) were diluted as indicated in phosphate-buffered saline containing 1% ovalbumin. Normal rabbit serum (1:150) served as a negative control. Fluorescence labeling was performed with FITC-conjugated swine anti-rabbit immunoglobulins and Cy3-conjugated goat anti-rat immunoglobulins (Dakopatt, Co-
FIG. 2. Time course study of mRNA expression of fibulin-1, fibulin-2, nidogen, and tenascin-C during wound healing by RNase protection assay. The gels were exposed for 10 h (fibulin-1), 2 h (fibulin-2), 7 h (nidogen), or 8 h (tenascin-C). Expression is compared with a nonwounded control (ctr.), and tRNA was used as a negative control. In each case, the first lane was loaded with the hybridization probe (1000 cpm).
penhagen). Double labeling was performed with antibodies against fibulin-2 and tenascin-C or with antibodies against fibulin-1 and nidogen. All the fluorescent specimens were mounted in 90% glycerol containing 1 mg/ml b-phenylenediamine. Microscopy was carried out with a Axiophot fluorescence microscope (Zeiss).
RESULTS
Expression of Fibulins, Nidogen, and Tenascin-C in Normal Skin and during Wound Repair
FIG. 1. Expression of mRNA encoding fibulin-1, fibulin-2, tenascin-C, and nidogen in the dermis (D) and epidermis (E) of Balb/c mouse tail skin. The two tissues were separated after incubation in 2 M NaBr (377C, 30 min), and purified total RNA (50 mg) was used in a RNase protection assay. Exposure times were varied for fibulin1 (16 h), fibulin-2 (12 h), tenascin-C (60 h), and nidogen (18 h) in order to obtain signals of about equal strength.
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Basic expression levels of mRNAs encoding these proteins were initially examined by RNase protection assay using dermis and epidermis samples obtained separately from normal mouse skin (Fig. 1). This demonstrated expression of mRNA encoding fibulin-1, fibulin-2, nidogen, and tenascin-C almost exclusively in the dermis, although in most cases, at rather low levels when compared to wound samples (Fig. 2). Epidermal expression could only be detected for fibulin-1 mRNA, although it was about 50-fold lower than in the dermis as judged by densitometry (Fig. 1). This indicated that
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FIG. 3. Immunofluorescence analysis of fibulin-1, nidogen, fibulin-2, and tenascin-C during wound healing. Identical tissue sections were used for fibulin-1/nidogen and fibulin-2/tenascin-C in double staining experiments. In each section from 5 and 13d, a piece of nonwounded skin is to the right of newly formed granulation tissue, and the borders are marked by arrows. Various histological details are marked: cl, clot; e, hyperproliferative epithelium; es, eschar; g, granulation tissue; h, hair follicle.
the four proteins examined are primarily of mesenchymal origin. The same analysis of tissues obtained from skin wounds over a healing period of 13 days revealed different time courses and levels of mRNA expression (Fig. 2). Fibulin-1 expression showed the least change compared to normal skin, with a small decrease observed at Day 1 after wounding and marginal increases at later stages. Fibulin-2 mRNA, however, rose considerably between Days 3 and 7 and declined almost to normal levels at Day 13, by which time wound repair and contraction was almost completed. A similar temporal pattern with an earlier onset and an even more distinct increase was observed for tenascin-C mRNA. The nidogen mRNA levels, however, remained low or moderately elevated at early stages, increasing strongly at Day 7 and declining again at Day 13. High nidogen expression correlated with the onset of basement mem-
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brane formation in the wound. These patterns could be reproduced in two different sets of experiments. Immunohistological analysis of extracellular matrix deposition of newly synthesized proteins showed some differences in temporal and spatial patterns (Fig. 3). At Day 1, staining for fibulins and nidogen was weak in the wound bed but distinct in the scab. A stronger tissue reaction was, however, observed for tenascin-C, in agreement with an early rise in mRNA levels. The staining for all four proteins was much more intense and uniform in the granulation tissue of 5-day-old wounds. These patterns were clearly different from those observed in adjacent uninjured skin, where restriction to basement membrane regions is a dominant but not exclusive feature. With the exception of nidogen, these differences persisted until Day 13, even though mRNA expression had returned to nearly normal values. These findings indicate that remodeling of
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dose-dependent increase in expression above threshold levels of detection (Fig. 5). As in wound healing, no downregulation of fibulin-2 mRNA was achieved with dexamethasone (data not shown). This indicates a general difference in the control of synthesis of the two proteins. DISCUSSION
FIG. 4. Different expression of tenascin-C and fibulin-2 mRNA in skin wounds of glucocorticoid-treated and control mice. Daily subcutaneous injections with dexamethasone (1 or 0.2 mg/kg body wt) were for 7 d before and 5 d after wounding. Wound control animals received only solvent injections. Exposure times were 6 h (tenascinC) or 2 h (fibulin-2). For controls (probe, tRNA) see Fig. 2.
the interstitial matrix was not completed by this time. At Day 13, nidogen was mainly restricted to basement membrane zones at the dermal-epidermal junction and around small blood vessels, as is typical for normal skin. Glucocorticoid Effect on Tenascin-C and Fibulin-2 Expression during Wound Healing Dexamethasone was previously shown to suppress tenascin-C expression in cultured cells [12] and to interfere with wound healing [7]. We therefore studied the expression of tenascin-C and fibulin-2 in wound tissue of dexamethasone-treated mice (Fig. 4). This demonstrated a strong and dose-dependent reduction of tenascin-C mRNA, while no effect was observed for fibulin-2 mRNA. Similarly, neither fibulin-1 nor nidogen expression was affected by glucocorticoids (data not shown). Since tenascin-C mRNA expression in wounds is not completely downregulated by dexamethasone, in contrast to cell cultures [12], we considered the possibility that tissue cytokines and in particular TGF-b1 [11] may compete in its regulation. This was examined with cultured 3T3 fibroblasts in order to avoid complication by a complex tissue repertoire of cytokines. These cells express distinct levels of tenascin-C and fibulin-2 mRNA but almost no fibulin-1 mRNA. Expression of tenascin-C mRNA was abolished by dexamethasone used at concentrations of 10 mM or 100 nM, but addition of TGF-b1 with 100 nM dexamethasone led to a
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Wound healing is characterized by the appearance of several extracellular matrix components in distinct sequential and spatial patterns [1, 28]. Most remarkable is the strong expression of tenascin-C [9], which is usually scarce in adult tissues but very abundant in embryonic and tumor tissues [10]. Most of the observations so far have been based on immunohistological data. Transcriptional dynamics have, however, been studied for collagens I, III, and VI and showed a strong rise in mRNA levels which differed in persistence depending on the collagen chain analyzed [29]. Here we have demonstrated a very early and strong increase in the level of tenascin-C mRNA, which remained elevated for several days before returning to low levels when wound repair was complete. Matrix deposition of tenascin at this stage was, however, still very strong, in agreement with previous data [9], indicating that tissue remodeling was not yet finished. Tenascin-C has been implicated as a substrate for cell adhesion and migration, while less is known about its role in supramolecular matrix organization [30, 31]. The long persistence of tenascin-C in cutaneous injuries suggests a role in cellular interactions, but other potential functions remain to be elucidated. In this context, it is of interest that transgenic mice which lack tenascin-C show an apparently normal phenotype [32]. Wound healing studies in such mutants will therefore be a crucial test for the actual role of tenascin-C.
FIG. 5. Effect of TGF-b1 on the regulation of tenascin-C mRNA expression by dexamethasone in 3T3 fibroblasts. Semiconfluent cultured cells were treated for 15 h with different agents as indicated on top, and 20 mg total RNA isolated from these cells were analyzed by RNase protection assay. The gel was exposed for 48 h.
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FIBULIN, TENASCIN-C, AND NIDOGEN IN SKIN WOUNDS
These observations led us to examine a few more extracellular matrix proteins which have so far not been studied in wound healing. Nidogen was chosen because of its ubiquitous occurence in basement membranes and obvious central role in their molecular architecture [13]. Expression of nidogen mRNA showed an increase similar to tenascin-C but over a shorter time-span. The final decline in mRNA levels was, however, accompanied by a change from a very uniform tissue distribution to a distinct basement membrane localization of nidogen. This indicates a mesenchymal origin for nidogen, as shown previously for embryonic rat skin [33], and emphasizes that developmental and repair processes share common features. The recruitment of mesenchymal nidogen for the formation of basement membranes seems to be a more general phenomenon, as demonstrated recently for several other embryonic tissues [14, 15]. Similar epithelial–mesenchymal interactions may therefore occur during wound healing and also play a role in the homeostatic control of adult tissues since, as shown here, nidogen mRNA could only be detected in the dermis of uninjured skin. The functional significance of such interactions has been shown in experiments with antibodies that block laminin–nidogen interaction [34]. These antibodies inhibit kidney tubulogenesis and lung branching by interfering with the formation of novel basement membranes [15]. The most likely explanation at the molecular level is the prevention of nidogen-mediated connections between the networks of collagen IV and laminin [13] that could be crucial for basement membrane stability. The fibulins studied here represent a novel family of extracellular matrix proteins characterized by elongated shapes and many consensus sequences for calcium-binding [16–18, 25, 35]. The two isoforms of the protein known so far showed a different behavior during wound healing. Fibulin-1 mRNA was only moderately modulated during wound repair but nevertheless a distinct deposition of the protein was observed in the granulation tissue, in contrast to the dominant basement membrane localization in uninjured skin [18, 25]. Fibulin-2 has been localized to some basement membranes but also to interstitial spaces of the dermis [18]. Its high mRNA expression during wound healing and the persistence of fibulin-2 in wound tissue at Day 13 was almost identical to that of tenascin. These two fibulins are, however, expressed in a similar way during heart development with a very high expression in valves and septa which even persists at postnatal stages [19, 20]. The precise functions of these proteins are still unclear. The binding repertoires of the two isoforms for other extracellular ligands are not completely identical but include in particular high-affinity binding for nidogen and fibronectin [18, 35]. How such
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binding similarities or differences correlate to the biological observations has yet to be elucidated. Wound repair apparently requires a complex control by positive and negative signals in order to assure a fine balance in the temporal and spatial expression of extracellular proteins [28]. Possible differences in such controls were shown with dexamethasone, which diminished tenascin-C but not fibulin-2 expression both in vivo and in cell culture. This is of particular interest since glucocorticoids are known to delay wound healing, as evident from the reduced formation of granulation tissue [7]. The data shown here therefore possibly provide the first molecular explanation for such effects but also emphasize specificity for distinct extracellular matrix components. Furthermore, TGF-b1 could partially counteract dexamethasone inhibition in cell culture, which indicates that similar processes may occur in situ. Recent studies in fact demonstrated that administration of TGF-b1 reversed impaired wound healing caused by aging or glucocorticoid treatment [36, 37]. Since several more growth factors are likely to be involved in the control of wound healing [1, 28], we almost certainly face a rather complex pattern of regulations. Our data in addition emphasize that the target proteins involved may respond in quite different ways. The characterization of the functional properties of the extracellular proteins involved as well as their transcriptional control will therefore be instrumental for further progress in our understanding of the whole repair process at a molecular level. We thank Drs. Marie Dziadek and Peter Ekblom for monoclonal antibodies, Mrs. Marlene Gru¨ne and Mrs. Karin Angermeyer for technical assistance, and Dr. Judith Brown for critical reading of the manuscript. S.W. thanks Dr. P. H. Hofschneider for continuous support. Supported by grants from the Deutsche Forschungsgemeinschaft (Fa 296/1-1; Ti 95/7-3), the National Institutes of Health (AR 38923), the Fritz-Thyssen-Stiftung (to S.W.), and by a MaxPlanck Research Award.
REFERENCES 1. Gailit, J., and Clark, R. A. F. (1994) Curr. Opin. Cell Biol. 6, 717–725. 2. Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H., and Fauci, A. S. (1986) Proc. Natl. Acad. Sci. USA 83, 4167–4171. 3. Wahl, S. M., Hunt, D. A., Wakefield, L. M., McCartney-Francis, N., Wahl, L. M., Roberts, A. B., and Sporn, M. B. (1987) Proc. Natl. Acad. Sci. USA 84, 5788–5792. 4. Ignotz, R. A., Endo, T., and Massague, J. (1987) J. Biol. Chem. 262, 6443–6446. 5. Edwards, D. R., Murphy, G., Reynolds, J. J., Whitham, S. E., Docherty, J. P., Angel, P., and Heath, J. K. (1987) EMBO J. 6, 1899–1904. 6. Overall, C. M., Wrana, J. L., and Sodek, J. (1989) J. Biol. Chem. 264, 1860–1869.
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7. Ehrlich, H. P., and Hunt, T. K. (1968) Ann. Surg. 167, 324– 328. 8. Wahl, S. M. (1989) Antiinflammatory Steroid Action: Basic and Clinical Aspects, p. 280, Academic Press, New York. 9. Mackie, A. J., Halfter, W., and Liverani, D. (1988) J. Cell Biol. 107, 2757–2767. 10. Erickson, H. P., and Bourdon, M. A. (1989) Annu. Rev. Cell Biol. 5, 71–92. 11. Pearson, C. A., Pearson, D., Shibahara, S., Hofsteenge, J., and Chiquet-Ehrismann, R. (1988) EMBO J. 7, 2977–2981. 12. Ekblom, M., Fa¨ssler, R., Tomasini-Johansson, B., Nilsson, K., and Ekblom, P. (1993) J. Cell. Biol. 123, 1037–1045. 13. Mayer, U., and Timpl, R. (1994) Nidogen: A Versatile Binding Protein of Basement Membranes, p. 389, Academic Press, Orlando, FL. 14. Thomas, T., and Dziadek, M. (1993) Exp. Cell Res. 208, 54–67. 15. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer, U., and Timpl, R. (1994) Development 120, 2003–2014. 16. Argraves, W. S., Tran, H., Burgess, W. H., and, Dickerson K. (1990) J. Cell. Biol. 111, 3155–3164. 17. Pan, C., Kluge, M., Zhang, R.-Z., Mayer, U., Timpl, R., and Chu, M.-L. (1993) Eur. J. Biochem. 215, 733–740. 18. Pan, T.-C., Sasaki, T., Zhang, R.-Z., Fa¨ssler, R., Timpl, R., and Chu, M.-L. (1993) J. Cell. Biol. 123, 1269–1277. 19. Zhang, H.-Y., Kluge, M., Timpl, R., Chu, M.-L., and Ekblom, P. (1993) Differentiation 52, 211–220. 20. Zhang, H.-Y., Chu, M.-L., Pan, T.-C., Sasaki, T., Timpl, R., and Ekblom, P. (1994) Dev. Biol., in press. 21. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159.
22. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acid Res. 12, 7035–7056. 23. Weller, A., Beck, S., and Ekblom, P. (1991) J. Cell Biol. 112, 355–362. 24. Mann, K., Deutzmann, R., Aumailley, M., Timpl, R., Raimondi, L., Yamada, Y., Pan, T., Conway, D., and Chu, M.-L. (1989) EMBO J. 8, 65–72. 25. Kluge, M., Mann, K., Dziadek, M., and Timpl, R. (1990) Eur. J. Biochem. 193, 651–659. 26. Aufderheide, E., and Ekblom, P. (1988) J. Cell Biol. 107, 341– 1349. 27. Dziadek, M., Clements, R., Mitrangas, K., Reiter, H., and Fowler, K. (1988) Eur. J. Biochem. 172, 219–225. 28. Raghow, R. (1994) FASEB J. 8, 823–831. 29. Oono, T., Specks, U., Eckes, B., Majewski, S., Hunzelmann, N., Timpl, R., and Krieg, T. (1993) J. Invest. Dermatol. 100, 329– 334. 30. Riou, J. F., Umbhauer, M., Shi, D. L., and Boucaut, J.-C. (1992) Biol. Cell 75, 1–9. 31. Erickson, H. P. (1993) Curr. Opin. Cell Biol. 5, 869–876. 32. Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T., and Aizawa, S. (1992) Genes Dev. 6, 1821–1831. 33. Hogan, B. L. M., Taylor, A., Kurkinen, M., and Couchman, J. R. (1992) J. Cell. Biol. 95, 197–204. 34. Mayer, U., Nischt, R., Po¨schl, E., Mann, K., Fukuda, K., Gerl, M. Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879–1885. 35. Sasaki, T., Kostka, G., Go¨hring, W., Wiedemann, H., Mann, K., Chu, M.-L., and Timpl, R. (1995) J. Mol. Biol. 245, 241–250. 36. Pierce, G. F., Mustoe, T. A., Lingelbach, J., Masakowski, V. R., Gramates, P., and Deuel, T. F. (1989) Proc. Natl. Acad. Sci. USA 86, 2229–2233. 37. Beck, L. S., DeGuzman, L., Lee, W. P., Xu, Y., Siegel, M. W., and Amento, E. P. (1993) J. Clin. Invest. 92, 2841–2849.
Received July 28, 1995 Revised version received September 22, 1995
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AP: Exp Cell
222, 111–116 (1996)
Article No. 0014
Differential Regulation of Fibulin, Tenascin-C, and Nidogen Expression during Wound Healing of Normal and Glucocorticoid-Treated Mice REINHARD FA¨SSLER,1 TAKAKO SASAKI, RUPERT TIMPL, MON-LI CHU,*
AND
SABINE WERNER
Max-Planck-Institut fu¨r Biochemie, D-82152 Martinsried, Federal Republic of Germany; and *Departments of Biochemistry and Molecular Biology and of Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Expression and distribution of fibulins, nidogen, and tenascin-C were analyzed in healing skin wounds of normal and dexamethasone-treated mice. In normal mice both tenascin-C and fibulin-2 showed a marked increase in mRNA expression, which declined to normal levels after completion of skin repair. These two matrix proteins are found throughout the granulation tissue and persisted there after mRNA expression had ceased. Fibulin-1 is present in normal skin and in wounds but is not distinctly upregulated during the healing process. Nidogen, however, is expressed uniformly throughout the granulation tissue early in wound healing, has a peak expression around Day 7, and selectively localizes to basement membranes after healing is accomplished. Dexamethasone treatment led to a decreased expression of tenascin-C in healing wounds but had no effect on fibulin-2 expression. In vitro experiments revealed that growth factors like TGF-b1 can partly counteract glucocorticoid action. These data therefore provide some molecular interpretations for the well-known glucocorticoid suppression of wound healing. They also indicate that repair involves complex regulatory processes which are obviously different for each of the four proteins studied. q 1996 Academic Press, Inc.
INTRODUCTION
In the course of normal wound healing, many cellular events such as proliferation, migration, and differentiation as well as tissue remodeling have to progress in a coordinated manner [1]. Several growth factors, including TGF-b1, are potent promoters of the healing process. They induce angiogenesis and formation of granulation tissue [2], increase chemotaxis of fibroblasts [3] and extracellular matrix deposition [4], and modulate 1 To whom correspondence and reprint requests should be addressed at Max-Planck-Institut fu¨r Biochemie, Abteilung Proteinchemie, Am Klopferspitz 18A, D-82152 Martinsried. Fax: (089) 8578 2422.
tissue remodeling by matrix metalloproteinases [5, 6]. To the contrary, glucocorticoids have detrimental effects on wound healing [7], presumably caused by a decrease in cell proliferation, neovascularization, and matrix production [8]. Increased matrix deposition in healing skin wounds involves several collagen types, fibronectin and a few other components [1], in particular tenascin-C, which is expressed at high levels [9] despite its low abundance in normal adult tissues [10]. Interestingly, tenascin-C production in cultured cells is upregulated by TGF-b1 [11] but downregulated by dexamethasone [12], which correlates with the general effects observed during wound healing. In the present study we have analyzed the expression of three more extracellular matrix proteins during cutaneous wound healing in mice in comparison with that of tenascin-C. These proteins included nidogen, which is a versatile binding protein in basement membranes [13] and during embryonic development is often synthesized by mesenchymal compartments [14, 15], as well as the more novel proteins fibulin-1 [16, 17] and fibulin-2 [18]. Little is known about the biological properties of the fibulins except that they are found in skin and are prominently expressed during mouse heart development [19, 20]. The data obtained with normal and dexamethasone-treated mice demonstrated quite different regulation patterns for each of the four proteins, indicating that their involvement differs during wound repair. MATERIAL AND METHODS Preparation of wound tissue from normal and dexamethasonetreated mice. In each of the two experiments, 22 female Balb/c mice, 4 weeks old, were anesthetized with a single intraperitoneal injection of Avertin. Six full-thickness wounds (6 mm in diameter, 3–4 mm apart) were made on 20 mice by excising the skin and panniculus carnosus. The wounds were allowed to dry in order to form a scab. Four animals were sacrificed and their wounds harvested at 1, 3, 5, 7, and 13 days after wounding. An area of 7 mm in diameter, which included the complete epithelial margins, was excised at each time point. A similar amount of skin from the backs of two nonwounded animals was combined and used as a control. Wound tissue was immediately frozen in liquid nitrogen and stored at 0707C. Three
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wound healing experiments were performed with dexamethasonetreated animals. During the week prior to wounding and for 5 days thereafter, 8 mice were injected subcutaneously at 9 AM every day with either 1 or 0.2 mg dexamethasone/kg body wt. Control animals were treated with phosphate-buffered saline (10 mM phosphate buffer, pH 7.3, 0.15 M NaCl, 0.2 mM EDTA). Five days after injury animals were sacrificed and wound tissue was harvested as described above. Analysis of tenascin-C regulation in cultured fibroblasts. Murine fibroblasts (NIH3T3) were grown to approximately 70% confluency in Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal calf serum. They were subsequently treated for 15 h with 10 mM or 100 nM dexamethasone, the latter in the presence or absence of purified transforming growth factor TGF-b1 (0.1 or 1 ng/ml). RNase protection assay. Total cellular RNA from cultured cells and from mouse skin was isolated as described [21]. For RNase protection mapping, DNA probes were cloned into the transcription vector pBluescript II(KS/) (Stratagene) and linearized. An antisense transcript was synthesized in vitro by using T3 or T7 RNA polymerase and [32P]UTP (800 Ci/mmol, Amersham). Samples of total cellular RNA from tissues (50 mg) or cells (20 mg) were hybridized at 427C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested for 60 min at 307C with RNase A and T1 [22]. Protected fragments were separated on 5% polyacrylamide/8 M urea gels and analyzed by autoradiography. Loading of the gels with equivalent amounts of RNA was controlled in separate runs by ethidium bromide staining with material prior to RNase treatment. Fifty micrograms tRNA was used as a negative control. The same RNA preparations were used for all protection assays. The DNA templates used for RNAse protection assay were as follows: a fragment corresponding to nucleotides 1094–1383 of murine tenascin-C cDNA [23]; a fragment corresponding to nucleotides 980–1350 of murine fibulin1 cDNA [17]; a fragment corresponding to nucleotides 2483–2800 of murine fibulin-2 cDNA [18]; and a fragment corresponding to nucleotides 3370–3763 of murine nidogen cDNA [24]. Indirect immunofluorescence. Double-label immunofluorescence analysis was carried out using affinity-purified rabbit antibodies against fibulin-1 [25] or a rabbit antiserum against fibulin-2 [18] and monoclonal rat antibodies against tenascin-C (MTN-12) [26] or nidogen [27]. All tissue specimens were frozen in liquid nitrogen and sections (6 mm) were prepared on a Reichert–Jung cryostat. Sections were fixed in acetone for 10 min at 0207C and washed in phosphatebuffered saline. Antibodies against fibulin-1 (1:30), fibulin-2 (1:150), tenascin-C (1:200), and nidogen (1:150) were diluted as indicated in phosphate-buffered saline containing 1% ovalbumin. Normal rabbit serum (1:150) served as a negative control. Fluorescence labeling was performed with FITC-conjugated swine anti-rabbit immunoglobulins and Cy3-conjugated goat anti-rat immunoglobulins (Dakopatt, Co-
FIG. 2. Time course study of mRNA expression of fibulin-1, fibulin-2, nidogen, and tenascin-C during wound healing by RNase protection assay. The gels were exposed for 10 h (fibulin-1), 2 h (fibulin-2), 7 h (nidogen), or 8 h (tenascin-C). Expression is compared with a nonwounded control (ctr.), and tRNA was used as a negative control. In each case, the first lane was loaded with the hybridization probe (1000 cpm).
penhagen). Double labeling was performed with antibodies against fibulin-2 and tenascin-C or with antibodies against fibulin-1 and nidogen. All the fluorescent specimens were mounted in 90% glycerol containing 1 mg/ml b-phenylenediamine. Microscopy was carried out with a Axiophot fluorescence microscope (Zeiss).
RESULTS
Expression of Fibulins, Nidogen, and Tenascin-C in Normal Skin and during Wound Repair
FIG. 1. Expression of mRNA encoding fibulin-1, fibulin-2, tenascin-C, and nidogen in the dermis (D) and epidermis (E) of Balb/c mouse tail skin. The two tissues were separated after incubation in 2 M NaBr (377C, 30 min), and purified total RNA (50 mg) was used in a RNase protection assay. Exposure times were varied for fibulin1 (16 h), fibulin-2 (12 h), tenascin-C (60 h), and nidogen (18 h) in order to obtain signals of about equal strength.
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Basic expression levels of mRNAs encoding these proteins were initially examined by RNase protection assay using dermis and epidermis samples obtained separately from normal mouse skin (Fig. 1). This demonstrated expression of mRNA encoding fibulin-1, fibulin-2, nidogen, and tenascin-C almost exclusively in the dermis, although in most cases, at rather low levels when compared to wound samples (Fig. 2). Epidermal expression could only be detected for fibulin-1 mRNA, although it was about 50-fold lower than in the dermis as judged by densitometry (Fig. 1). This indicated that
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FIG. 3. Immunofluorescence analysis of fibulin-1, nidogen, fibulin-2, and tenascin-C during wound healing. Identical tissue sections were used for fibulin-1/nidogen and fibulin-2/tenascin-C in double staining experiments. In each section from 5 and 13d, a piece of nonwounded skin is to the right of newly formed granulation tissue, and the borders are marked by arrows. Various histological details are marked: cl, clot; e, hyperproliferative epithelium; es, eschar; g, granulation tissue; h, hair follicle.
the four proteins examined are primarily of mesenchymal origin. The same analysis of tissues obtained from skin wounds over a healing period of 13 days revealed different time courses and levels of mRNA expression (Fig. 2). Fibulin-1 expression showed the least change compared to normal skin, with a small decrease observed at Day 1 after wounding and marginal increases at later stages. Fibulin-2 mRNA, however, rose considerably between Days 3 and 7 and declined almost to normal levels at Day 13, by which time wound repair and contraction was almost completed. A similar temporal pattern with an earlier onset and an even more distinct increase was observed for tenascin-C mRNA. The nidogen mRNA levels, however, remained low or moderately elevated at early stages, increasing strongly at Day 7 and declining again at Day 13. High nidogen expression correlated with the onset of basement mem-
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brane formation in the wound. These patterns could be reproduced in two different sets of experiments. Immunohistological analysis of extracellular matrix deposition of newly synthesized proteins showed some differences in temporal and spatial patterns (Fig. 3). At Day 1, staining for fibulins and nidogen was weak in the wound bed but distinct in the scab. A stronger tissue reaction was, however, observed for tenascin-C, in agreement with an early rise in mRNA levels. The staining for all four proteins was much more intense and uniform in the granulation tissue of 5-day-old wounds. These patterns were clearly different from those observed in adjacent uninjured skin, where restriction to basement membrane regions is a dominant but not exclusive feature. With the exception of nidogen, these differences persisted until Day 13, even though mRNA expression had returned to nearly normal values. These findings indicate that remodeling of
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dose-dependent increase in expression above threshold levels of detection (Fig. 5). As in wound healing, no downregulation of fibulin-2 mRNA was achieved with dexamethasone (data not shown). This indicates a general difference in the control of synthesis of the two proteins. DISCUSSION
FIG. 4. Different expression of tenascin-C and fibulin-2 mRNA in skin wounds of glucocorticoid-treated and control mice. Daily subcutaneous injections with dexamethasone (1 or 0.2 mg/kg body wt) were for 7 d before and 5 d after wounding. Wound control animals received only solvent injections. Exposure times were 6 h (tenascinC) or 2 h (fibulin-2). For controls (probe, tRNA) see Fig. 2.
the interstitial matrix was not completed by this time. At Day 13, nidogen was mainly restricted to basement membrane zones at the dermal-epidermal junction and around small blood vessels, as is typical for normal skin. Glucocorticoid Effect on Tenascin-C and Fibulin-2 Expression during Wound Healing Dexamethasone was previously shown to suppress tenascin-C expression in cultured cells [12] and to interfere with wound healing [7]. We therefore studied the expression of tenascin-C and fibulin-2 in wound tissue of dexamethasone-treated mice (Fig. 4). This demonstrated a strong and dose-dependent reduction of tenascin-C mRNA, while no effect was observed for fibulin-2 mRNA. Similarly, neither fibulin-1 nor nidogen expression was affected by glucocorticoids (data not shown). Since tenascin-C mRNA expression in wounds is not completely downregulated by dexamethasone, in contrast to cell cultures [12], we considered the possibility that tissue cytokines and in particular TGF-b1 [11] may compete in its regulation. This was examined with cultured 3T3 fibroblasts in order to avoid complication by a complex tissue repertoire of cytokines. These cells express distinct levels of tenascin-C and fibulin-2 mRNA but almost no fibulin-1 mRNA. Expression of tenascin-C mRNA was abolished by dexamethasone used at concentrations of 10 mM or 100 nM, but addition of TGF-b1 with 100 nM dexamethasone led to a
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Wound healing is characterized by the appearance of several extracellular matrix components in distinct sequential and spatial patterns [1, 28]. Most remarkable is the strong expression of tenascin-C [9], which is usually scarce in adult tissues but very abundant in embryonic and tumor tissues [10]. Most of the observations so far have been based on immunohistological data. Transcriptional dynamics have, however, been studied for collagens I, III, and VI and showed a strong rise in mRNA levels which differed in persistence depending on the collagen chain analyzed [29]. Here we have demonstrated a very early and strong increase in the level of tenascin-C mRNA, which remained elevated for several days before returning to low levels when wound repair was complete. Matrix deposition of tenascin at this stage was, however, still very strong, in agreement with previous data [9], indicating that tissue remodeling was not yet finished. Tenascin-C has been implicated as a substrate for cell adhesion and migration, while less is known about its role in supramolecular matrix organization [30, 31]. The long persistence of tenascin-C in cutaneous injuries suggests a role in cellular interactions, but other potential functions remain to be elucidated. In this context, it is of interest that transgenic mice which lack tenascin-C show an apparently normal phenotype [32]. Wound healing studies in such mutants will therefore be a crucial test for the actual role of tenascin-C.
FIG. 5. Effect of TGF-b1 on the regulation of tenascin-C mRNA expression by dexamethasone in 3T3 fibroblasts. Semiconfluent cultured cells were treated for 15 h with different agents as indicated on top, and 20 mg total RNA isolated from these cells were analyzed by RNase protection assay. The gel was exposed for 48 h.
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These observations led us to examine a few more extracellular matrix proteins which have so far not been studied in wound healing. Nidogen was chosen because of its ubiquitous occurence in basement membranes and obvious central role in their molecular architecture [13]. Expression of nidogen mRNA showed an increase similar to tenascin-C but over a shorter time-span. The final decline in mRNA levels was, however, accompanied by a change from a very uniform tissue distribution to a distinct basement membrane localization of nidogen. This indicates a mesenchymal origin for nidogen, as shown previously for embryonic rat skin [33], and emphasizes that developmental and repair processes share common features. The recruitment of mesenchymal nidogen for the formation of basement membranes seems to be a more general phenomenon, as demonstrated recently for several other embryonic tissues [14, 15]. Similar epithelial–mesenchymal interactions may therefore occur during wound healing and also play a role in the homeostatic control of adult tissues since, as shown here, nidogen mRNA could only be detected in the dermis of uninjured skin. The functional significance of such interactions has been shown in experiments with antibodies that block laminin–nidogen interaction [34]. These antibodies inhibit kidney tubulogenesis and lung branching by interfering with the formation of novel basement membranes [15]. The most likely explanation at the molecular level is the prevention of nidogen-mediated connections between the networks of collagen IV and laminin [13] that could be crucial for basement membrane stability. The fibulins studied here represent a novel family of extracellular matrix proteins characterized by elongated shapes and many consensus sequences for calcium-binding [16–18, 25, 35]. The two isoforms of the protein known so far showed a different behavior during wound healing. Fibulin-1 mRNA was only moderately modulated during wound repair but nevertheless a distinct deposition of the protein was observed in the granulation tissue, in contrast to the dominant basement membrane localization in uninjured skin [18, 25]. Fibulin-2 has been localized to some basement membranes but also to interstitial spaces of the dermis [18]. Its high mRNA expression during wound healing and the persistence of fibulin-2 in wound tissue at Day 13 was almost identical to that of tenascin. These two fibulins are, however, expressed in a similar way during heart development with a very high expression in valves and septa which even persists at postnatal stages [19, 20]. The precise functions of these proteins are still unclear. The binding repertoires of the two isoforms for other extracellular ligands are not completely identical but include in particular high-affinity binding for nidogen and fibronectin [18, 35]. How such
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binding similarities or differences correlate to the biological observations has yet to be elucidated. Wound repair apparently requires a complex control by positive and negative signals in order to assure a fine balance in the temporal and spatial expression of extracellular proteins [28]. Possible differences in such controls were shown with dexamethasone, which diminished tenascin-C but not fibulin-2 expression both in vivo and in cell culture. This is of particular interest since glucocorticoids are known to delay wound healing, as evident from the reduced formation of granulation tissue [7]. The data shown here therefore possibly provide the first molecular explanation for such effects but also emphasize specificity for distinct extracellular matrix components. Furthermore, TGF-b1 could partially counteract dexamethasone inhibition in cell culture, which indicates that similar processes may occur in situ. Recent studies in fact demonstrated that administration of TGF-b1 reversed impaired wound healing caused by aging or glucocorticoid treatment [36, 37]. Since several more growth factors are likely to be involved in the control of wound healing [1, 28], we almost certainly face a rather complex pattern of regulations. Our data in addition emphasize that the target proteins involved may respond in quite different ways. The characterization of the functional properties of the extracellular proteins involved as well as their transcriptional control will therefore be instrumental for further progress in our understanding of the whole repair process at a molecular level. We thank Drs. Marie Dziadek and Peter Ekblom for monoclonal antibodies, Mrs. Marlene Gru¨ne and Mrs. Karin Angermeyer for technical assistance, and Dr. Judith Brown for critical reading of the manuscript. S.W. thanks Dr. P. H. Hofschneider for continuous support. Supported by grants from the Deutsche Forschungsgemeinschaft (Fa 296/1-1; Ti 95/7-3), the National Institutes of Health (AR 38923), the Fritz-Thyssen-Stiftung (to S.W.), and by a MaxPlanck Research Award.
REFERENCES 1. Gailit, J., and Clark, R. A. F. (1994) Curr. Opin. Cell Biol. 6, 717–725. 2. Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H., and Fauci, A. S. (1986) Proc. Natl. Acad. Sci. USA 83, 4167–4171. 3. Wahl, S. M., Hunt, D. A., Wakefield, L. M., McCartney-Francis, N., Wahl, L. M., Roberts, A. B., and Sporn, M. B. (1987) Proc. Natl. Acad. Sci. USA 84, 5788–5792. 4. Ignotz, R. A., Endo, T., and Massague, J. (1987) J. Biol. Chem. 262, 6443–6446. 5. Edwards, D. R., Murphy, G., Reynolds, J. J., Whitham, S. E., Docherty, J. P., Angel, P., and Heath, J. K. (1987) EMBO J. 6, 1899–1904. 6. Overall, C. M., Wrana, J. L., and Sodek, J. (1989) J. Biol. Chem. 264, 1860–1869.
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7. Ehrlich, H. P., and Hunt, T. K. (1968) Ann. Surg. 167, 324– 328. 8. Wahl, S. M. (1989) Antiinflammatory Steroid Action: Basic and Clinical Aspects, p. 280, Academic Press, New York. 9. Mackie, A. J., Halfter, W., and Liverani, D. (1988) J. Cell Biol. 107, 2757–2767. 10. Erickson, H. P., and Bourdon, M. A. (1989) Annu. Rev. Cell Biol. 5, 71–92. 11. Pearson, C. A., Pearson, D., Shibahara, S., Hofsteenge, J., and Chiquet-Ehrismann, R. (1988) EMBO J. 7, 2977–2981. 12. Ekblom, M., Fa¨ssler, R., Tomasini-Johansson, B., Nilsson, K., and Ekblom, P. (1993) J. Cell. Biol. 123, 1037–1045. 13. Mayer, U., and Timpl, R. (1994) Nidogen: A Versatile Binding Protein of Basement Membranes, p. 389, Academic Press, Orlando, FL. 14. Thomas, T., and Dziadek, M. (1993) Exp. Cell Res. 208, 54–67. 15. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer, U., and Timpl, R. (1994) Development 120, 2003–2014. 16. Argraves, W. S., Tran, H., Burgess, W. H., and, Dickerson K. (1990) J. Cell. Biol. 111, 3155–3164. 17. Pan, C., Kluge, M., Zhang, R.-Z., Mayer, U., Timpl, R., and Chu, M.-L. (1993) Eur. J. Biochem. 215, 733–740. 18. Pan, T.-C., Sasaki, T., Zhang, R.-Z., Fa¨ssler, R., Timpl, R., and Chu, M.-L. (1993) J. Cell. Biol. 123, 1269–1277. 19. Zhang, H.-Y., Kluge, M., Timpl, R., Chu, M.-L., and Ekblom, P. (1993) Differentiation 52, 211–220. 20. Zhang, H.-Y., Chu, M.-L., Pan, T.-C., Sasaki, T., Timpl, R., and Ekblom, P. (1994) Dev. Biol., in press. 21. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159.
22. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acid Res. 12, 7035–7056. 23. Weller, A., Beck, S., and Ekblom, P. (1991) J. Cell Biol. 112, 355–362. 24. Mann, K., Deutzmann, R., Aumailley, M., Timpl, R., Raimondi, L., Yamada, Y., Pan, T., Conway, D., and Chu, M.-L. (1989) EMBO J. 8, 65–72. 25. Kluge, M., Mann, K., Dziadek, M., and Timpl, R. (1990) Eur. J. Biochem. 193, 651–659. 26. Aufderheide, E., and Ekblom, P. (1988) J. Cell Biol. 107, 341– 1349. 27. Dziadek, M., Clements, R., Mitrangas, K., Reiter, H., and Fowler, K. (1988) Eur. J. Biochem. 172, 219–225. 28. Raghow, R. (1994) FASEB J. 8, 823–831. 29. Oono, T., Specks, U., Eckes, B., Majewski, S., Hunzelmann, N., Timpl, R., and Krieg, T. (1993) J. Invest. Dermatol. 100, 329– 334. 30. Riou, J. F., Umbhauer, M., Shi, D. L., and Boucaut, J.-C. (1992) Biol. Cell 75, 1–9. 31. Erickson, H. P. (1993) Curr. Opin. Cell Biol. 5, 869–876. 32. Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T., and Aizawa, S. (1992) Genes Dev. 6, 1821–1831. 33. Hogan, B. L. M., Taylor, A., Kurkinen, M., and Couchman, J. R. (1992) J. Cell. Biol. 95, 197–204. 34. Mayer, U., Nischt, R., Po¨schl, E., Mann, K., Fukuda, K., Gerl, M. Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879–1885. 35. Sasaki, T., Kostka, G., Go¨hring, W., Wiedemann, H., Mann, K., Chu, M.-L., and Timpl, R. (1995) J. Mol. Biol. 245, 241–250. 36. Pierce, G. F., Mustoe, T. A., Lingelbach, J., Masakowski, V. R., Gramates, P., and Deuel, T. F. (1989) Proc. Natl. Acad. Sci. USA 86, 2229–2233. 37. Beck, L. S., DeGuzman, L., Lee, W. P., Xu, Y., Siegel, M. W., and Amento, E. P. (1993) J. Clin. Invest. 92, 2841–2849.
Received July 28, 1995 Revised version received September 22, 1995
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