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Role of fibronectin in collagenous matrix-induced mesenchymal cell proliferation and differentiation in vivo
OLE
OF FIBRONECTIN
I
ROY E. WEIS5P2~ * and A. H. REDDI’ ‘Laboratory of Biological Structure, National Institute of Dental Research, National insfitute qf Health, Bethesda, MD 20205, and ZDepartment ojOrthopaedics, University of Southern California Medical School, Orthopaedic Hospital, Los Angeles, CA 90007, USA
SUMMARY The importance of fibronectin in in vivo collagenous matrix-mesenchyme cell interaction was investigated using purified antibodies to rat plasma fibronectin. Subcutaneous ~m~lant~t~o~ of demineralized bone matrix normally resulted in de nova local endochondral ossification. Iocal injections of the purified antibodies apparently inhibited collagenous mat~x-mese~cbyme cell interaction by inhibiting the action of endogenous fibronectin. Anti-fibronectin treatment resulted in reduced cell proliferation as assessed by [3H]thymidine incoporation (.59%, reduction) and omithine decarboxylase activity (66%, reduction); and chondrogenesis as measured by proteoglycan synthesis (43 %, reduction). Neutralization of fibronectin’s biological activity by antibodies also resulted in a qualitative change in the proteoglycan type synthesized. The physiological role of fibronectin in tissue morphogenesis appears to allow for initial extracellular matrix-cell attachment.
~ib~onect~n is a major cell surface glycoprotein and functions as a cell attachment factor for cell-substratum (usually collagen) interaction [2, 5,9, 11, 18,241. A structuralHy related and immunologically cross-reactive protein, previously known as cold insohrble globulin, is present in the blood asma [IO, 231. The role of fibronectin in VIVQ is not known. In the present study, a potential role for fibronectin in cell attachent and matrix-cell interaction was sugnts involving subcutaneof demineralized colix into rats which results pro~~era~~on of mesenchymal cells eir differentiation into cartilage and e [14-17,201. Upon implantation, one of early events is the binding of circulating to the collagenous ma-
trix [21) 221. This is follovve synthesis of tissue
action was unventilated usi bodies to plasma fibronect matrix and c~~~n~~ Bocal. ‘-f~bronectin were used to activBty
of iooth
ctins, as tlaey are immunologically ind~$t~ngn~s Our results revealed the ~n~i~~~~ry influence of fibronect~n antibodies on matrix~e~t~5n i3Jld * To whom offprint requests should be sent addressed to Orthopaedic Hospital, 2400 So. Flower str, Los Angeles, GA 90007, ZrSA.
248
Weiss and Reddi MATERIALS
Preparation antibodies
AND
METHODS
of anti-fibronectin
Rat plasma fibronectin was isolated from freshlv obtained plasma, using EDTA as an anticoagulant, by gelatin-Sepharose affinity chromatoaranhv T31. Plasma fibronectin was further - purified bi prep&ative gel electrophoresis under reducing conditions [21]. The fibronectin band was sliced from polyactylamide gels homogenized in Incomplete Fret&l’s -Adjuvant andinjetted into rabbits to obtain fibronectin antiserum. Fibronectin antibodies were isolated from the antiserum by fibronectin-Sepharose affinity chromatography [21]. The antibodies did not cross-react with type I and II collagens, albumin, or fibrin, as determined by immunodiffusion. There was weak crossreactivity with plasma fibronectins of human, chicken. and horse.
Preparation
of matrix and implantation
Demineralized powdered bone matrix [14-171 was washed with 0.02 M NaHZPGI, 0.15 M NaCl, pH 7.4, and then one aliquot (250 mn) was metreated with 15 ml of the fibronectin antibody (7 ig antibody/ml; 1: 320 titer determined bv ELISA assav) for 1 h at 22°C with gentle agitation-. Another aliquot served as a control and was treated with rabbit antibodies (IgG fraction) to rat albumin (7 pg/ml) under identical conditions. Rats of the Long Evans strain (6 per experiment) were anesthetized and implanted subcutaneously over the gastrocnemius musile with 20-25 mg of the fibronectin antibody-pretreated matrix on one side and a similar amount of control matrix treated with the albumin antibody on the contralateral side. Two dailv subcutaneous injections of rabbit anti-rat plasma fibronectin antibodies (0.2 ml) were given locally in the antibody pretreated site and of rabbit anti-rat albumin IgG (0.2 ml) on the control site. Rabbit InG anti-rat albumin was found to have no effect on the parameters measured when compared with a non-injected control or a control group treated and injected with normal rabbut IgG.
weighed, and homogenized in ice-cold 10% (wtlvol) trichloroacetic acid (TCA) containing 1 mM thymidine (non-reactive). The tubes were kept cold for 30 min and subsequently centrifuged at 4500 g for 15 min. The supernatantwas saved for determination of acidsoluble radioactivity by liquid-scintillation spectrometry, and the precipitate was washed twice with icecold 10% (wtlvol) TCA without thymidine. The washed precipitate was hydrolysed in 2 ml 10% TCA at 90°C for 20 min and then immediately placed in an ice bath for 30 min. Samples were then centrifuged for 15 min at 4500 a. and the radioactivitv in the aliquots of the supematants was determined. The deoxyribonucleic acid (DNA) content of the hydrolvsed supematant was determined bv the dinhenvlamine procedure [l]. The results were expressed -as hugs DNA/ma tissue, cnm/ng DNA in acid-soluble supematant and acid-insolubk precipitate. The percentage inhibition caused by anti-fibronectin antibody compared with the control site was determined in each rat.
Chondrogenesis characterization
and proteoglycan
As a marker for cartilage differentiation, proteoglycan biosynthesis was examined by monitoring Y30, incorporation per pg DNA in the matrix implants. Seven days following implantation, tissues were excised 2 h after an iv injection of 1 &i 35S0, (960 mCi/mmol) per gram body weight [18]. Molecular sieve chromatography of the radio-labelled proteoglycans was done. The plaques were extracted in 4.0 guanidineHCI, 0.05 M Na acetate, pH 5.8, which contained a mixture of protease inhibitors (50 mM EDTA, 5 mM benzamidine, 0.1 M 6-aminohexanoic acid, 0.5 mM phenylmethylsulfonylfluoride). The extracts were placed on a Pharmacia PD-10 column containing Sephadex G-25M to separate the macromolecular fraction from the unincorporated isotope. The eluent from the G-25M column was chromatographed in a Sepharose 2B-Cl column which was equilibrated with the extraction buffer described above. The void volume of the column was determined using labelled bovine nasal cartilage and the total volume was determined using 3H,0.
Cell proliferation Three days following imalantation, develoninn implant tissues were excised-2 h after ‘an iv inject& of 1 &i [3H]thymidine (6.7 mCi/mmol) per gram body weight. The amount of fH]thymidine incorporated per pg DNA and the activity of ornithine decarboxylase (EC 4.1.17) were determined. These parameters have been useful markers of cell proliferation [7, 12, 13, 201. Tissues were dissected out and homogenized with the aid of a Polytron homogenizer in ice-cold buffer containing 50 mM Tris-HCl;S mM dithiothreitol, and 1 mM EDTA at pH 7.4 and centrifuged at 30000 g for 30 min. and the sunernatant was assaved as described by IBnne & Williams-Ashman [7] and Rath & Reddi [12]. Omithine decarboxylase activity was expressed as pmoles YO, released from [1-r4C]L-omithine (56.6 mCilmmo1) per hour per mg of protein. The subcutaneous button-like plaques were removed,
Osteogenesis 45CaCl, (15-20 Cilg) at a dose of 1 &i/g body weight was iniected intraueritoneallv in 0.15 M NaCl. 12 davs following implant&ion. The plaques were dissected 2h later, weighed, homogenized in ice-cold 0.15 M NaCl and 0.003 M NaHCO, (pH 7.4), and centrifuged at 20000 g for 15 min at 4°C. The supematants were assayed for alkaline phosphatase and acid phosphatase using p-nitrophenol phosphate (Sigma Chemical Co.) as a substrate with 0.1 M barbital buffer (pH 9.3) for alkaline phosphatase and a 0.1 M acetate buffer (PH 5.0) for acid phosphatase, as described previously [15-171. Soluble protein was also assayed in the supematant by the Lowry method. The sediment was stirred with 0.1 M CaCl, in .5 mM Tris-HCl (pH 7.5) for 30 min, centrifuged, and washed twice in
5 mM Tris-HC1 (pH 7.5). The washed sediment was then stirred in 0.5 M HCI for 16 h and centrifuged, and aliquots were taken for determination of radioactivity and for determination of calcium by atomic absorption spectrometry. The 45Ca uptake data were used to measure the rate of calcification during the pulse of label, and total calcium was a monitor of total calcification. A detailed description of this method for concurrent determination of phosphatases and “YZa incorporation will be published elsewhere (unpublished observations).
DA?’
3
j
60
r
Localization of ar,ti-fibronectin by im~~no~~o~e~cence Tissues from rats 3 days after imulantation were immediately placed on dry ice and then frozen, unfixed in GCTTM Embedding Medium (miles). Frozen sections 6 pm thick were cut in a cryostat’at -20°C within 3 h of autopsy. Sections of unfixed developing matrix were incubated with fluoresceinisothiocyanate (FIT0conjugated goat antibodies to rabbit IgG (diluted 1:20 with 0.02 M NaH2POI, 0.15 M iles). The sections were washed three times in saline. A control was observed using tissue not injected in rabbit antibodies, and preabsorbing the anti-rabbit IgG with the rabbit anti-fibronectin, neither of which Tielded fluorescence,
Scanning Electron
Microscopy
(SEM)
eveloping plaques were dissected out 3 days after implantation from the anti-fibronectin injected site and the anti-albumin (control site). Immediately upon surgically opening the site of implantation the area was flushed with primary fixative containing 2.0 % glutaraldehyde, 2.0% formaldehyde in 0.1 M sodium cacodylate buffer with 7.0% sucrose, pH 7.4. Samples were minced to 5 mm in the fixative and processed by standard procedures for SEM [20].
ESULTS The influence of anti-fibronectin antibodies on cell proliferation 3 days following im~a~~at~o~ of demineralized bone matrix particles are illustrated in fig. 1. The incorporation of [3H]thymidine and the acof ornithine decarboxylase were reby 59 and 66% respectively. The of inhibition was not pronounced ox. 30%) when the fibronectin antiwas injected without pretreatment of the matrix prior to implantation; or if the matrix was pretreated but not followed by twice daily injections. The total DNA con-
!dR
1. Effect of anti-fibronectin antibodies on [HIthvmidine incoruoraiion and ornithine decarb~~xylase aciivity in day 3plaques (matrix implants). The thymidine incorporation was reported 2 h after injection of isotope (I &i/g body weight) as dpmlpg DNA. Omithine decarboxyiase activity (OX’) is reported as pmoles of WO, released per hour/mg protein. 0, Control site injected with anti-rat albumin; site treated with antibodies to fibronectin. Anlialbumin antibodies were found to have no effect on tkymidine incorporation or ODC activity when compared to a non-injected control or a controi injected with pre-immune IgG.
Fig.
tent of the a~t~-a~~~rni~ 1 .l pg/mg tissue compared to tissue of the side treate nectin ~ ause i-fibronectin antib to in it cell ~ro~~erati~~ cellularity of the matrix was assessed by observation under the scanning electron microscope. Fig. 24, tory influence of ~ibronect~~ ber of fibroblas
cells. Anti-f~b~o~ect~~
tre
esulted in a
250
Weiss and Reddi
SEMs of day 3 plaques treated with (a) zzntirat albumin; or (b) anti-r ‘at fibronectin. Note the reduced number of cells at tached to the matrix trearted with anti-fibronectin. x 3 000.
Fig. 2.
DAY 7
3. Effect of anti-fibronectin antibodies on ?S04 incorporation into proteoglycans ii, day 7 plaque tissues. The results are reported as dpm of ?SO, per fig DNA, 2 h following an iv injection of the isotope. 0, Control site injected with antibodies to rat albumin, IgG fraction, made in rabbits; n , site treated with fibronectin antibodies. Anti-albumin antibodies were found to have no effect on label incorporation compared with a non-injected control, or a control injected with preimmune IgG. Fig.
Table 1. Influence of anti-fibranectin body on mineralization (day 12)
Wa cpm/mg tissue pg Calmg tissue Alkaline phosphatase IJ/mg tissue
anti-
Control
Antifibronectin
960+22 78f25
624+ 15 515 2
325 2
21* 2
measured by the incorporation of 35S0, into groteoglycans (fig. 3). The results of molecular sieve chromatography of the proteoglycans synthesized 7 days after matrix implantation is shown in fig. 4. The upper curve represents the profile from the site injected with anti-rat albumin and the major eak corresponds to the proteoglycan monomer. The lower curve represents the profile for the site treated with anti-fibronectin and shows 65 % of the total activity present in the monomer peak while 35 %
5 3 1
5415
25
vo
35
45 L 55
FRACTIQNS
IV t
65
-L.-d 75
Fig. 4. Molecular
sieve chromatography of proteoglycans. A 4.0 M guanidine extract (cf ~~ate~als and Methods) was chromatographed on a S CL column which was equilibrated wit tion buffer. The void volmne of the column was determined using labehed bovine nasal cartilage proteoglycan aggregate (avow, V,) and the total volume was determined using 3Hz0 (ar-vow, V,). The upper curve represents the profile from the site injected with anti-rat albumin and the major peak corresponds to the proteoglycan monomer. The lower curve represents the profde for the site treated with anti-fibronectin and shows 65% of the total activity was present in the monomer peak, while 35% of the activity was present in a lower molecular weight component.
was present in a Bower
tive nature of the macr~~o~ec~le in the absence of fibronectin. poration
of 45Ca a
0-
is affected
252
Weiss and Reddi
Fig. 5. Immunofluorescent localization of rabbit IgG in developing implants 3 days after implantation. Rabbit IgG wzs localized to determine the distribution of
the rabbit anti-rat fibronectin antibody. Tissue treated with (left) anti-rat albumin; (right) anti-rat fibronectin. x 150.
line and acid phosphatases on day 12 after implantation was inhibited, but to a lesser extent when compared with chondrogenesis (table 1). The inhibitory influence of the antibody declined progressively with the development of the tissue despite daily administrations of the antibodies. The alkaline and acid phosphatases exhibited a 35% reduction in activity when injected with the anti-rat fibronectin and similarly the incorporation of 45Ca was significantly (p< 0.02) albeit moderately reduced. In order to assess the location of the fibronectin antibody after injection and matrix pretreatment, indirect immuno-localization was done. Fig. 5 illustrates that the rabbit anti;rat fibronectin was present both on the matrix and in the presumptive pro-
liferating mesenchyme. The site injected with rabbit anti-rat albumin and then stained with FITC-conjugated goat antirabbit IgG showed a negative reaction in the matrix and surrounding mesenchymal tissue (fig. 5, left). DISCUSSION The in vivo role of fibronectin has been clarified by the use of specific antibodies to neutralize its biological effect. Injection of fibronectin antibodies into the site of in vivo mesenchymal cell-collagen matrix interaction inhibited the differentiation of these tissues. Upon implantation we have previously shown that plasma fibronectin binds to the collagenous matrix [21, 221.
In a separate experiment we have shown that injection of exogenous fibronectin into a normal rat was without effect on cell proliferation or the cellularity of the matrix. Treatment of the developing plaque with fibronectin antibodies result in the binding of the antibody to the matrix particle and the interparticle matrix. As observed by , cell attachment was inhibited in the sence of fibronectin. It has long been nown that cell attachment to a suitable substrate is a prerequisite to cell proliferation and differentiation. Therefore it folIows that our observations of inhibited cell proliferation and differentiation following removal of fibronectin are due to failure of initial cell adhesion. Inhibition of cell proliferation by anti-fibronectin was observed decreased ornithine decarboxylase acity and t~ymid~e incorporation. These e importance of cell attachent in vivo for cell proliferation. Proteouring treatment with fibroantibodies were in the molecular biquitous’ proteoglycans, dicating a non-cartilaginous nature, pers bin-d~fere~tiated~ This further indicates the importance of fibronectin in cell attachment and subsequently cell differentiation. e inhibitory influence of the fibronectm antibody was seen to decrease with deve~~~rne~t of the tissue despite continued treatment. This is presumably due to the ex s of endogenous plasma fibronectin an r tissue fibronectins. Alternatively, ses may be less dependent ossible development of imnmnity to the rabbit anti-fibronectin was not likely in the short time course of these experiments. The use of anti-rat albumin as a control supports the specificity of the observed i~~ibiti~~ of cell proliferation and y anti-fibronectin. We can-
[6] using normal rat kidney cells in vitro concluded that f~bro~e~t~ may serve as a building block for extracellular matrix resent novel in vivo evidence that fibronectin plays a crucial role in initial extra~e~l~~ar matrix-cell interacti
R. E.W. was supported by a USPHS Postdoctoral Fellowship from the National Institute of Arthritis, Metabolism and Digestive Disease (~o.~32A~~6~~7) and funds from the Orthooaedic 0sDitai of Los Angeles. We wou Carol Itatani and scanning electron kawa for excellent photographic assist Calbes for her help in preparation of the manuscript.
1. Burton, K, Biochem j 62 (19%) 315. 2. Chen, LB, Murray, A, Segal, R A, Walsh, M L, Cell 14 (1978) 377. 3. Engvail, E & Ruoslahti. E, Int j cancer 20 (1977) Z. 4. Goetinck, D F: Pennypacker, 3 P & Royal. P D, Exp cell res 87 (1974) 241. 5. Grinnell, F, Exp cell res 97 (1976) 265. 6. Hayman, E G B Ruoslahti, E, J ceil bioi 83 (1979) 255. 7. Jgnne, J & Williams-Asbma~, H 6; 9 biol them 246 (1971) 1725. 8. Kimura, J H, Hardingham, T E; Hascall. V C & Solursh, M J, J biol them 254 (1979) 2400. 9. Klebe, R J, Nature 250 (1?74) 248. 10. Mosesson, M W, Ann NY acad sci 312 (1978) 11. Il. Pearlstein, E, Gold, L I & Garcia-Pa.rdo, A, Molec 12.
, Biochem biophys res commun 81 (1978) 106. 13. - Nature 278 (1979) 855. 14. Reddi, A H, Biochemistry of collagen (ed G N Ramachandran BL A H Reddi) p. 449. $ienum Press, New York (1976). 1.5. Reddi. A H & Huggins, C , Proc natl acad sci US 69 (1972) 1601. 16. - ibid $2 (1975) 2212. 17. Reddi. A H & Anderson. W A, 4 cell bioi 69 (1976) 557. 18. Reddi, A H, Hascali, V C & Hascail, G K, $ biol them 253 (1978) 2249. 19. Vaheri, I, uoslahti, E & Masher, D F ted), Fibroblast surface protein. Ann NY acad SCF312 (1978) 456.
254
Weiss and Reddi
20. Weiss, R E & Reddi, A H, Am j physiol238 (1980) E200. 21. - Proc natl acad sci US 77 (1980) 2074. 22. - J cell bio188 (1981) 630. 23. Yamada, K & Kennedy, D W, J cell biol80 (1979) 492. 24. Yamada, K & Olden, K, Nature 275 (1979) 179.
Received September 26, 1980 Revised version received Januarv 15, 1981 Accepted January 16, 1981 -
Printed
in Sweden
OF FIBRONECTIN
I
ROY E. WEIS5P2~ * and A. H. REDDI’ ‘Laboratory of Biological Structure, National Institute of Dental Research, National insfitute qf Health, Bethesda, MD 20205, and ZDepartment ojOrthopaedics, University of Southern California Medical School, Orthopaedic Hospital, Los Angeles, CA 90007, USA
SUMMARY The importance of fibronectin in in vivo collagenous matrix-mesenchyme cell interaction was investigated using purified antibodies to rat plasma fibronectin. Subcutaneous ~m~lant~t~o~ of demineralized bone matrix normally resulted in de nova local endochondral ossification. Iocal injections of the purified antibodies apparently inhibited collagenous mat~x-mese~cbyme cell interaction by inhibiting the action of endogenous fibronectin. Anti-fibronectin treatment resulted in reduced cell proliferation as assessed by [3H]thymidine incoporation (.59%, reduction) and omithine decarboxylase activity (66%, reduction); and chondrogenesis as measured by proteoglycan synthesis (43 %, reduction). Neutralization of fibronectin’s biological activity by antibodies also resulted in a qualitative change in the proteoglycan type synthesized. The physiological role of fibronectin in tissue morphogenesis appears to allow for initial extracellular matrix-cell attachment.
~ib~onect~n is a major cell surface glycoprotein and functions as a cell attachment factor for cell-substratum (usually collagen) interaction [2, 5,9, 11, 18,241. A structuralHy related and immunologically cross-reactive protein, previously known as cold insohrble globulin, is present in the blood asma [IO, 231. The role of fibronectin in VIVQ is not known. In the present study, a potential role for fibronectin in cell attachent and matrix-cell interaction was sugnts involving subcutaneof demineralized colix into rats which results pro~~era~~on of mesenchymal cells eir differentiation into cartilage and e [14-17,201. Upon implantation, one of early events is the binding of circulating to the collagenous ma-
trix [21) 221. This is follovve synthesis of tissue
action was unventilated usi bodies to plasma fibronect matrix and c~~~n~~ Bocal. ‘-f~bronectin were used to activBty
of iooth
ctins, as tlaey are immunologically ind~$t~ngn~s Our results revealed the ~n~i~~~~ry influence of fibronect~n antibodies on matrix~e~t~5n i3Jld * To whom offprint requests should be sent addressed to Orthopaedic Hospital, 2400 So. Flower str, Los Angeles, GA 90007, ZrSA.
248
Weiss and Reddi MATERIALS
Preparation antibodies
AND
METHODS
of anti-fibronectin
Rat plasma fibronectin was isolated from freshlv obtained plasma, using EDTA as an anticoagulant, by gelatin-Sepharose affinity chromatoaranhv T31. Plasma fibronectin was further - purified bi prep&ative gel electrophoresis under reducing conditions [21]. The fibronectin band was sliced from polyactylamide gels homogenized in Incomplete Fret&l’s -Adjuvant andinjetted into rabbits to obtain fibronectin antiserum. Fibronectin antibodies were isolated from the antiserum by fibronectin-Sepharose affinity chromatography [21]. The antibodies did not cross-react with type I and II collagens, albumin, or fibrin, as determined by immunodiffusion. There was weak crossreactivity with plasma fibronectins of human, chicken. and horse.
Preparation
of matrix and implantation
Demineralized powdered bone matrix [14-171 was washed with 0.02 M NaHZPGI, 0.15 M NaCl, pH 7.4, and then one aliquot (250 mn) was metreated with 15 ml of the fibronectin antibody (7 ig antibody/ml; 1: 320 titer determined bv ELISA assav) for 1 h at 22°C with gentle agitation-. Another aliquot served as a control and was treated with rabbit antibodies (IgG fraction) to rat albumin (7 pg/ml) under identical conditions. Rats of the Long Evans strain (6 per experiment) were anesthetized and implanted subcutaneously over the gastrocnemius musile with 20-25 mg of the fibronectin antibody-pretreated matrix on one side and a similar amount of control matrix treated with the albumin antibody on the contralateral side. Two dailv subcutaneous injections of rabbit anti-rat plasma fibronectin antibodies (0.2 ml) were given locally in the antibody pretreated site and of rabbit anti-rat albumin IgG (0.2 ml) on the control site. Rabbit InG anti-rat albumin was found to have no effect on the parameters measured when compared with a non-injected control or a control group treated and injected with normal rabbut IgG.
weighed, and homogenized in ice-cold 10% (wtlvol) trichloroacetic acid (TCA) containing 1 mM thymidine (non-reactive). The tubes were kept cold for 30 min and subsequently centrifuged at 4500 g for 15 min. The supernatantwas saved for determination of acidsoluble radioactivity by liquid-scintillation spectrometry, and the precipitate was washed twice with icecold 10% (wtlvol) TCA without thymidine. The washed precipitate was hydrolysed in 2 ml 10% TCA at 90°C for 20 min and then immediately placed in an ice bath for 30 min. Samples were then centrifuged for 15 min at 4500 a. and the radioactivitv in the aliquots of the supematants was determined. The deoxyribonucleic acid (DNA) content of the hydrolvsed supematant was determined bv the dinhenvlamine procedure [l]. The results were expressed -as hugs DNA/ma tissue, cnm/ng DNA in acid-soluble supematant and acid-insolubk precipitate. The percentage inhibition caused by anti-fibronectin antibody compared with the control site was determined in each rat.
Chondrogenesis characterization
and proteoglycan
As a marker for cartilage differentiation, proteoglycan biosynthesis was examined by monitoring Y30, incorporation per pg DNA in the matrix implants. Seven days following implantation, tissues were excised 2 h after an iv injection of 1 &i 35S0, (960 mCi/mmol) per gram body weight [18]. Molecular sieve chromatography of the radio-labelled proteoglycans was done. The plaques were extracted in 4.0 guanidineHCI, 0.05 M Na acetate, pH 5.8, which contained a mixture of protease inhibitors (50 mM EDTA, 5 mM benzamidine, 0.1 M 6-aminohexanoic acid, 0.5 mM phenylmethylsulfonylfluoride). The extracts were placed on a Pharmacia PD-10 column containing Sephadex G-25M to separate the macromolecular fraction from the unincorporated isotope. The eluent from the G-25M column was chromatographed in a Sepharose 2B-Cl column which was equilibrated with the extraction buffer described above. The void volume of the column was determined using labelled bovine nasal cartilage and the total volume was determined using 3H,0.
Cell proliferation Three days following imalantation, develoninn implant tissues were excised-2 h after ‘an iv inject& of 1 &i [3H]thymidine (6.7 mCi/mmol) per gram body weight. The amount of fH]thymidine incorporated per pg DNA and the activity of ornithine decarboxylase (EC 4.1.17) were determined. These parameters have been useful markers of cell proliferation [7, 12, 13, 201. Tissues were dissected out and homogenized with the aid of a Polytron homogenizer in ice-cold buffer containing 50 mM Tris-HCl;S mM dithiothreitol, and 1 mM EDTA at pH 7.4 and centrifuged at 30000 g for 30 min. and the sunernatant was assaved as described by IBnne & Williams-Ashman [7] and Rath & Reddi [12]. Omithine decarboxylase activity was expressed as pmoles YO, released from [1-r4C]L-omithine (56.6 mCilmmo1) per hour per mg of protein. The subcutaneous button-like plaques were removed,
Osteogenesis 45CaCl, (15-20 Cilg) at a dose of 1 &i/g body weight was iniected intraueritoneallv in 0.15 M NaCl. 12 davs following implant&ion. The plaques were dissected 2h later, weighed, homogenized in ice-cold 0.15 M NaCl and 0.003 M NaHCO, (pH 7.4), and centrifuged at 20000 g for 15 min at 4°C. The supematants were assayed for alkaline phosphatase and acid phosphatase using p-nitrophenol phosphate (Sigma Chemical Co.) as a substrate with 0.1 M barbital buffer (pH 9.3) for alkaline phosphatase and a 0.1 M acetate buffer (PH 5.0) for acid phosphatase, as described previously [15-171. Soluble protein was also assayed in the supematant by the Lowry method. The sediment was stirred with 0.1 M CaCl, in .5 mM Tris-HCl (pH 7.5) for 30 min, centrifuged, and washed twice in
5 mM Tris-HC1 (pH 7.5). The washed sediment was then stirred in 0.5 M HCI for 16 h and centrifuged, and aliquots were taken for determination of radioactivity and for determination of calcium by atomic absorption spectrometry. The 45Ca uptake data were used to measure the rate of calcification during the pulse of label, and total calcium was a monitor of total calcification. A detailed description of this method for concurrent determination of phosphatases and “YZa incorporation will be published elsewhere (unpublished observations).
DA?’
3
j
60
r
Localization of ar,ti-fibronectin by im~~no~~o~e~cence Tissues from rats 3 days after imulantation were immediately placed on dry ice and then frozen, unfixed in GCTTM Embedding Medium (miles). Frozen sections 6 pm thick were cut in a cryostat’at -20°C within 3 h of autopsy. Sections of unfixed developing matrix were incubated with fluoresceinisothiocyanate (FIT0conjugated goat antibodies to rabbit IgG (diluted 1:20 with 0.02 M NaH2POI, 0.15 M iles). The sections were washed three times in saline. A control was observed using tissue not injected in rabbit antibodies, and preabsorbing the anti-rabbit IgG with the rabbit anti-fibronectin, neither of which Tielded fluorescence,
Scanning Electron
Microscopy
(SEM)
eveloping plaques were dissected out 3 days after implantation from the anti-fibronectin injected site and the anti-albumin (control site). Immediately upon surgically opening the site of implantation the area was flushed with primary fixative containing 2.0 % glutaraldehyde, 2.0% formaldehyde in 0.1 M sodium cacodylate buffer with 7.0% sucrose, pH 7.4. Samples were minced to 5 mm in the fixative and processed by standard procedures for SEM [20].
ESULTS The influence of anti-fibronectin antibodies on cell proliferation 3 days following im~a~~at~o~ of demineralized bone matrix particles are illustrated in fig. 1. The incorporation of [3H]thymidine and the acof ornithine decarboxylase were reby 59 and 66% respectively. The of inhibition was not pronounced ox. 30%) when the fibronectin antiwas injected without pretreatment of the matrix prior to implantation; or if the matrix was pretreated but not followed by twice daily injections. The total DNA con-
!dR
1. Effect of anti-fibronectin antibodies on [HIthvmidine incoruoraiion and ornithine decarb~~xylase aciivity in day 3plaques (matrix implants). The thymidine incorporation was reported 2 h after injection of isotope (I &i/g body weight) as dpmlpg DNA. Omithine decarboxyiase activity (OX’) is reported as pmoles of WO, released per hour/mg protein. 0, Control site injected with anti-rat albumin; site treated with antibodies to fibronectin. Anlialbumin antibodies were found to have no effect on tkymidine incorporation or ODC activity when compared to a non-injected control or a controi injected with pre-immune IgG.
Fig.
tent of the a~t~-a~~~rni~ 1 .l pg/mg tissue compared to tissue of the side treate nectin ~ ause i-fibronectin antib to in it cell ~ro~~erati~~ cellularity of the matrix was assessed by observation under the scanning electron microscope. Fig. 24, tory influence of ~ibronect~~ ber of fibroblas
cells. Anti-f~b~o~ect~~
tre
esulted in a
250
Weiss and Reddi
SEMs of day 3 plaques treated with (a) zzntirat albumin; or (b) anti-r ‘at fibronectin. Note the reduced number of cells at tached to the matrix trearted with anti-fibronectin. x 3 000.
Fig. 2.
DAY 7
3. Effect of anti-fibronectin antibodies on ?S04 incorporation into proteoglycans ii, day 7 plaque tissues. The results are reported as dpm of ?SO, per fig DNA, 2 h following an iv injection of the isotope. 0, Control site injected with antibodies to rat albumin, IgG fraction, made in rabbits; n , site treated with fibronectin antibodies. Anti-albumin antibodies were found to have no effect on label incorporation compared with a non-injected control, or a control injected with preimmune IgG. Fig.
Table 1. Influence of anti-fibranectin body on mineralization (day 12)
Wa cpm/mg tissue pg Calmg tissue Alkaline phosphatase IJ/mg tissue
anti-
Control
Antifibronectin
960+22 78f25
624+ 15 515 2
325 2
21* 2
measured by the incorporation of 35S0, into groteoglycans (fig. 3). The results of molecular sieve chromatography of the proteoglycans synthesized 7 days after matrix implantation is shown in fig. 4. The upper curve represents the profile from the site injected with anti-rat albumin and the major eak corresponds to the proteoglycan monomer. The lower curve represents the profile for the site treated with anti-fibronectin and shows 65 % of the total activity present in the monomer peak while 35 %
5 3 1
5415
25
vo
35
45 L 55
FRACTIQNS
IV t
65
-L.-d 75
Fig. 4. Molecular
sieve chromatography of proteoglycans. A 4.0 M guanidine extract (cf ~~ate~als and Methods) was chromatographed on a S CL column which was equilibrated wit tion buffer. The void volmne of the column was determined using labehed bovine nasal cartilage proteoglycan aggregate (avow, V,) and the total volume was determined using 3Hz0 (ar-vow, V,). The upper curve represents the profile from the site injected with anti-rat albumin and the major peak corresponds to the proteoglycan monomer. The lower curve represents the profde for the site treated with anti-fibronectin and shows 65% of the total activity was present in the monomer peak, while 35% of the activity was present in a lower molecular weight component.
was present in a Bower
tive nature of the macr~~o~ec~le in the absence of fibronectin. poration
of 45Ca a
0-
is affected
252
Weiss and Reddi
Fig. 5. Immunofluorescent localization of rabbit IgG in developing implants 3 days after implantation. Rabbit IgG wzs localized to determine the distribution of
the rabbit anti-rat fibronectin antibody. Tissue treated with (left) anti-rat albumin; (right) anti-rat fibronectin. x 150.
line and acid phosphatases on day 12 after implantation was inhibited, but to a lesser extent when compared with chondrogenesis (table 1). The inhibitory influence of the antibody declined progressively with the development of the tissue despite daily administrations of the antibodies. The alkaline and acid phosphatases exhibited a 35% reduction in activity when injected with the anti-rat fibronectin and similarly the incorporation of 45Ca was significantly (p< 0.02) albeit moderately reduced. In order to assess the location of the fibronectin antibody after injection and matrix pretreatment, indirect immuno-localization was done. Fig. 5 illustrates that the rabbit anti;rat fibronectin was present both on the matrix and in the presumptive pro-
liferating mesenchyme. The site injected with rabbit anti-rat albumin and then stained with FITC-conjugated goat antirabbit IgG showed a negative reaction in the matrix and surrounding mesenchymal tissue (fig. 5, left). DISCUSSION The in vivo role of fibronectin has been clarified by the use of specific antibodies to neutralize its biological effect. Injection of fibronectin antibodies into the site of in vivo mesenchymal cell-collagen matrix interaction inhibited the differentiation of these tissues. Upon implantation we have previously shown that plasma fibronectin binds to the collagenous matrix [21, 221.
In a separate experiment we have shown that injection of exogenous fibronectin into a normal rat was without effect on cell proliferation or the cellularity of the matrix. Treatment of the developing plaque with fibronectin antibodies result in the binding of the antibody to the matrix particle and the interparticle matrix. As observed by , cell attachment was inhibited in the sence of fibronectin. It has long been nown that cell attachment to a suitable substrate is a prerequisite to cell proliferation and differentiation. Therefore it folIows that our observations of inhibited cell proliferation and differentiation following removal of fibronectin are due to failure of initial cell adhesion. Inhibition of cell proliferation by anti-fibronectin was observed decreased ornithine decarboxylase acity and t~ymid~e incorporation. These e importance of cell attachent in vivo for cell proliferation. Proteouring treatment with fibroantibodies were in the molecular biquitous’ proteoglycans, dicating a non-cartilaginous nature, pers bin-d~fere~tiated~ This further indicates the importance of fibronectin in cell attachment and subsequently cell differentiation. e inhibitory influence of the fibronectm antibody was seen to decrease with deve~~~rne~t of the tissue despite continued treatment. This is presumably due to the ex s of endogenous plasma fibronectin an r tissue fibronectins. Alternatively, ses may be less dependent ossible development of imnmnity to the rabbit anti-fibronectin was not likely in the short time course of these experiments. The use of anti-rat albumin as a control supports the specificity of the observed i~~ibiti~~ of cell proliferation and y anti-fibronectin. We can-
[6] using normal rat kidney cells in vitro concluded that f~bro~e~t~ may serve as a building block for extracellular matrix resent novel in vivo evidence that fibronectin plays a crucial role in initial extra~e~l~~ar matrix-cell interacti
R. E.W. was supported by a USPHS Postdoctoral Fellowship from the National Institute of Arthritis, Metabolism and Digestive Disease (~o.~32A~~6~~7) and funds from the Orthooaedic 0sDitai of Los Angeles. We wou Carol Itatani and scanning electron kawa for excellent photographic assist Calbes for her help in preparation of the manuscript.
1. Burton, K, Biochem j 62 (19%) 315. 2. Chen, LB, Murray, A, Segal, R A, Walsh, M L, Cell 14 (1978) 377. 3. Engvail, E & Ruoslahti. E, Int j cancer 20 (1977) Z. 4. Goetinck, D F: Pennypacker, 3 P & Royal. P D, Exp cell res 87 (1974) 241. 5. Grinnell, F, Exp cell res 97 (1976) 265. 6. Hayman, E G B Ruoslahti, E, J ceil bioi 83 (1979) 255. 7. Jgnne, J & Williams-Asbma~, H 6; 9 biol them 246 (1971) 1725. 8. Kimura, J H, Hardingham, T E; Hascall. V C & Solursh, M J, J biol them 254 (1979) 2400. 9. Klebe, R J, Nature 250 (1?74) 248. 10. Mosesson, M W, Ann NY acad sci 312 (1978) 11. Il. Pearlstein, E, Gold, L I & Garcia-Pa.rdo, A, Molec 12.
, Biochem biophys res commun 81 (1978) 106. 13. - Nature 278 (1979) 855. 14. Reddi, A H, Biochemistry of collagen (ed G N Ramachandran BL A H Reddi) p. 449. $ienum Press, New York (1976). 1.5. Reddi. A H & Huggins, C , Proc natl acad sci US 69 (1972) 1601. 16. - ibid $2 (1975) 2212. 17. Reddi. A H & Anderson. W A, 4 cell bioi 69 (1976) 557. 18. Reddi, A H, Hascali, V C & Hascail, G K, $ biol them 253 (1978) 2249. 19. Vaheri, I, uoslahti, E & Masher, D F ted), Fibroblast surface protein. Ann NY acad SCF312 (1978) 456.
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20. Weiss, R E & Reddi, A H, Am j physiol238 (1980) E200. 21. - Proc natl acad sci US 77 (1980) 2074. 22. - J cell bio188 (1981) 630. 23. Yamada, K & Kennedy, D W, J cell biol80 (1979) 492. 24. Yamada, K & Olden, K, Nature 275 (1979) 179.
Received September 26, 1980 Revised version received Januarv 15, 1981 Accepted January 16, 1981 -
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