8,5
lliochimica et RkJph.v.~ica ,4ct~t. I 1 I5 ( 1991 ) 85-S~
~5 1991 Elsevier Science Publishers B.V. All rights reserved I}3i}4-4165/t}i/$1)3,5()
Rapid Report
B B A G E N 2(}287
Thrombospondin in ligament, meniscus and intervertebral disc R a y m o n d R. M i l l e r a n d C a h i r A. M c D e v i t t Department t~l"Ahtsculoskeletal Research, The ("h'cchtnd ("lime ['~mndari~m Rc~c:trch Institt~te, ('h'cefand, 0 t t ~'U.S.,4, )
(Received 28 August 1991)
Key words: 3hrombospondin: Ligament: Mcni.~cu.,,: intcrvcrtebral disc: Matrix
The presence of thrombospondin in extracts of ligament, meniscus and human nucleus pulposus was demonstrated by Western blot analysis using an anti-human thrombospondin antibody. Metabolic labeling of ligament fibroblast and meniscal fibrochondrocyte cultures followed by immunoprecipitation with anti-human thrombospondin revealed a single band corresponding to the molecular weight of thrombospondin when analyzed by SDS-PAGE and fluorography under reduced and non-reduced conditions. Thrombospondin, therefore, is a matrix constituent of ligament, knee joint meniscus and the nucleus pulposus and is synthesized by ligament fibroblasts and meniscal fibrochondrocytes in vitro.
Connective tissues such as ligaments [I], the knee joint meniscus [2], and the intervertebral disc [3] are characterized by an abundant extracellular matrix with relatively few cells. While type ! or type I! collagen constitutes the major extracellular fibrillar protein in these tissues [4], minor collagens and matrix glycoproteins are beginning to be recognized for their potential importance in the development, maintenance and repair of these tissues. We have recently reported that the high molecular weight, multidomain glycoprotein, thrombospondin, is present in articular cartilage and is synthesized by articular chondrocytes in culture [5]. Thrombospondin was first identified as a major protein of platelets [6], where it appears to participate in platelet aggregation [7]. The protein is composed or" three identical peptide chains, each of absolute molecular mass 140 kDa [8,9], that migrate in most electrophoretie gels under reduced conditions as a 180 kDa species [9,10]. Electron microscopic studies have demonstrated that each polypeptide is in the fo:m of a thin strand that bears a globular domain at each extremity [8,11,12]. Like t'ibronectin and laminin, thrombospondin contains discrete functional domains that can interact with other molecules, including fibronectin, fibrinogen, plasminogen, sulfated glycolipids, type V collagen, and calcium [8,11,12]. Throm-
Correspondence: C.A. McDevitt, Department of Musculoskeletal
Research, Cleveland Clinic Foundation Research Inslilute. 95111) Euclid Avenue, Cleveland, O H 44195-5254, U.S.A.
b¢)spondin also contains an RGD sequence that could bind to integrin receptors on cell surfaces, The protein, therefore, is an excellent candidate for mediating cellmatrix and intcrmolecular matrix processes in normal tissue homeostasis as well as repair processes in patbological tissues. In the present study we demonstrate that thrombospondin is a constituent of intervertebral disc, ligament and meniscus. The anterior and posterior cruciate ligaments and the lateral and medial menisci were dissected from an adult sheep knee joint. The nucleus pulposus from lumbar discs (LJL,_-L,JL~) were dissected from a radioiogically normal 20-year-old human spine obtained at autopsy. Tissues were minced with a scalpel blade and extracted with 4 M guanidine-HCl, 0.05 M sodium acetate (pH 5.8) containing proteinase inhibitors: 0.01 M EDTA, 0.1 M 6-aminocaproic acid, 5 mM benzamidine, 0.01 M N-ethylmaleimide and l mM phenylmethylsulfonyt fluoride (10 ml per I g of tissue). The extracts were clarified by centrifugation at 18000 × g and the supernatant was dialyzed exhaustively at 4 o C against 0.tl5 M sodium acetate (pH 6.8) containing proteinase inhibitors. Canine mcniscal fibrochondrocytes were isolated essentially by the methods of Green [13] and Webber et al. [14]. Menisci from the knee joint were dissected carefully to remove any adhering tissue. The dissected tissue was sequentially digested with testicular hyaluronidase (5 m g / g tissue), trypsin (0.2%) and collagenase (0.2%). The final celi pellet was resuspended, counted with a hemocytometer and plated in DMEM containing 25 mM Hepes (pH 7.4), sodium ascorbate
86 (50 /,tg/ml) supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 l,J/mI and streptomycin 100/xg/ml). Ligament tissue from a bovine ankle joint was dissected free of adhering tissue, minced into pieces (1-2 mm 3) and the explants were cultured in 60 mm dishes with 4 ml of Ham's F-12 medium containing 25 mM Hepes (pH 7.4), sodium ascorbatc (50 p,g/ml) supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 U/ml and streptomycin, 100 /.tg/ml). After 1 week, the medium was changed every 4 days, After 3 weeks, cells that grew from the explants were detached with 0.025% trypsin/0.02% EDTA and replated in 60 mm dishes for labeling experiments. Culture medium was changed cvery 2 c~ys. Newly synthesized proteins were radiolabeled in confluent monolayer cultures of meniscal fibrochondrocytes and ligament fibroblasts. Medium was removed and the monolayers were preincubated with either proline-free medium {ligament fibroblasts)or methionine-free medium (menisc~d fibrochondrocytes) for 30 rain. The medium was then replaced with l resh medium containing [~H]proline, 50 # C i / m l (ligament fibroblasts) or [~SS]methionine 50 /.tCi/ml (mcniscal fibrochondrocytes). Aftcr a 24-h incubation, proteinase inhibitors were added to the medium• Aliquots of the medium (I ml) were incubated with 2 ul of anti-human platelet thrombospondin monocional antibody for 2 h at room temperature and overnight at 4 ° C. The antibody was generously supplied by Dr. Dean Mosher, University of Wisconsin. Separate aliquots were incubated with control mouse ascites fluid as a negative control. A suspension of protein A agarose (Affi-Gel Protein A, Bio-Rad)was incubated with goat anti.mouse IgG as described above. The protein A agarose-lgG suspension was then centrifuged, the supernatant removed and the pellet resuspended in phosphate buffered saline. An aliquot (0.025 ml) of this suspension was added to the above specimens. Following a 2-h incubation at room temperature, the immunoprecipltates were centrifuged at 11 000 × g for 30 s. The supernatant was removed and the pellet was washed three times with immunoprecipitation buffer (50 mM Tris-HC! (pH 7.5), 0.15 M NaCI, 0.1% SDS, 0.5% Tween 20 and 0.2% NaN 3) and once with immunoprecipitation buffer without detergents. The final pellets were solubilized in electrophoresis sample buffer, boiled for 5 min and electrophoresed as described below. Samples were electrophoresed on either slab gels composed of a gradient of polyacrylamide and agarose or 7.5% polyacrylamide slab gels. Specimens were boiled in electrophoresis sample buffer (50 mM Tris; 2% SDS; 15% glycerol) and run under reduced or non-reduced conditions in the presence of SDS. For immunoblotting, proteins were transferred to nitro-
I
3
,¢
~. Lr. !
..
180 ~I16 "84 ...,.
~ '
"~,
"
48.5
"I 3 6 . 5
Fig• I. Immunob|otting of thrombospondin from tissue extracts electrophoresed on a polyacrylamide.agarose gradient gel. Lane 1: human interverlebral disc. Lane 2: ovine meniscus. Lane 3: ovine ligament, Arrow heads indicate the migration of reduced protein standards (Sigma) and their respective molecular masses in kDa;
•~-macroglobutin./3 galactosidase, fructose-6.phosphate kinase, pyrurate kinase, and fumarase. Large arrow indicates top of gel.
cellulose and probed for the presence of thrombospondin with the anti-human platelet thrombospondin monoelonal antibody (1:5000) and an alkaline phosphatase conjugated goat anti-mouse IgG (1:3000). Radiolabeled proteins were detected by impregnating the gels with 3En Hance (New England Nuclear, Boston, MA), drying the gels and exposing them to X-Omat X-ray film (Kodak, Rochester, NY) at 70°C. Eleetrophoresis under non-reduced conditions and Western blotting using the specific monoelonal antibody revealed the presence of thrombospondin in extracts of nucleus pulposus (Fig. 1, lane 1), meniscus (lane 2) and ligament (lane 3). A single, major band was obtained with all three specimens, with a minor band migrating just ahead of the major band in the meniscus and ligament extracts. Extrapolation of the mobilities of the reduced globular protein standards yielded a molecular mass of 420 kDa for the major thrombospondin band, consistent with our previous observations for this protein in articular cartilage [5] and the known apparent molecular mass in dec-
~7 trophoresis of the non-reduced molecule [9.I0]. Thrombospondin isolated from human ptatelets migrates in this gel system under identical conditions as a band with an apparent molecular mass of 420 kDa (5). No immunoreactive material was evident in Western blots when non-immune mouse ascites was substituted for the anti-thrombospondin antibody. Moreover, dot blot analysis demonstrated that thrombospondin in ligament and meniscal extracts bound to heparin-agarose columns in buffer containing 0.15 M sodium chloride and was eluted from the column with 1.0 M sodium chloride. We wished to establish whether the thrombospondin observed by Western blot was a biosynthetic product of the cells of these tissues. Cultures of ligament fibroblasts and meniscal fibroehondroeytes were established, radioiabeled separately with [3H]proline and
1
2
3
3
Fig. 3. lmmunoprecipitationof Ihrombospondia from the culture medium of [~:Slmc',hioninelabeled meniscal fibrochondroc)'teswilh anti-human thrombospondin. Samples v.ere electrophoresed on a 7.5c;- polyacrylamidc slab gel. Lane 1: culture medium, non-reduced.
Lane 2: cullure medium, reduced, Lane 3: radiolabeled protein slandard.~(see Fig. 2).
[3SS]methionine, respectively, and the culture media subjected to immunopreeipitation with the anti-thrombospondin monocionai antibody. When analyzed by SDS-PAGE and autoradiography, the immunoprecip-
itatt:s ~f ligament fibr0blast medium (Fig. 2) and meniscal fibroch~,ndrocyte medium (Fig. 3) migrated as a single band when tither non-reduced (lane 1) or reduced (lane 2). The molecular mass of the reduced forms was 180 kDa, which is similar to previously published values for reduced thrombospondin in eiectrophoretic gels [9,10]. No radioactivity was immuno-
precipitated when a control mouse ascites fluid was substituted for the anti-thrombospondin. Thus, the ligament fibroblasts and meniscal fibrochondrocytes synthesize thrombospondin in vitro. Fig. 2. I m m u n o p r e e i p i t a t i o n of t h r o m b o s p o n d i n from the culture
medium of ['aH]prolinelabeled ligament fibroblastswilh anti-human thrombos~ndin. Samples were electrophoresed on a gradient gel. Lane 1: culture medium, non-reduced. Lane 2: culture medium, reduced Lane 3: radiolabeledprotein standards (Amersham); myosin. phospho~lase b, bovine serum albumin, ovalbumin, carbonic anhydrase and lysoz,yme.
This study establishes that thrombospondin is present in the knee joint meniscus, ligaments and the nucleus pulposus of the disc aod is synthesized by
meniscal fibrochondrocytes and ligament fibroblasts in vitro. This study, therefore, extends previous observations on the presence of thrombospondin in articular cartilage [5] and mineralized bone [15]. and its synthe-
88 sis by c h o n d r o c y t e s [5] a n d o s t c o b l a s t s [16], r e s p e c tively, in culture. T h r o m b o s p o n d i n , l h e r e f o r e , m a y be a u b i q u i t o u s c o n s t i t u e n t o f c o n n e c t i v e tissues. T h e physiological f u n c t i o n o f t h r o m b o s p o n d i n in c o n n e c t i v e tissues is u n k n o w n . P r o l i f e r a t i n g c u l t u r e s o f e n d o t h e l i a l cells, s m o o t h m u s c l e cells a n d fibroblasts synthesize h i g h e r levels o f t h r o m b o s p o n d i n t h a n d o n o n d i v i d i n g cells [1%18]. M a j a c k et al. [19] have p r o p o s e d f r o m s t u d i e s with c u l t u r e d v a s c u l a r s m o o t h m u s cle cells that ceil-surface t h r o m b o s p o n d i n is f u n c t i o n ally essential for p r o l i f e r a t i o n a n d that t h e reo, u i r c m e n t is l o c a t e d in t h c G~ p h a s e of t h e cell cycle. T h r o m b o s p o n d i n , e i t h e r t h r o u g h its p a r t i c i p a t i o n in cell division or s o m e o t h e r celluhtr or c x t r a c c l l u l a r process, a p p e a r s to f u n c t i o n in tissue development and repair. O ' S h c a a n d Dixit have d e m o n s t r a t e d in t h e d e v c l o p m g m o u s e e m b r y o that t h e r e was c o n s i d e r a b l e i m m u n o s t a i n i n g for t h r o m b o s p o n d i n on the s u r f a c e o f c h o n d r o b l a s t s a n d myoblasI,,i w h i c h s u b s e q u e n t l y dec r e a s e d ,vith diffcrcnti~.tion [2{I]. At early s t a g e s of skin w o u n d s , thron'~bospondin s t a i n i n g is i n t e n s e , while h c a l c d w o u n d s exhibit greatly d i m i n i s h e d s t a i n i n g [21]. T h e p r e c i s e talc of t h r o n l b o s p t m d i n in normal and p a t h o l o g i c a l c o n n e c t i v e tissues r e m a i n s 1o bc e s t a b lishcd. Acknowledgements T h c a u l h n r s w o u l d like to l h a n k Julie Pahl a n d P a m e l a Rydcll for their t e c h n i c a l assistance a n d J u d y C h r i s t o p h e r for typing a n d e d i t o r i a l assistance in t h e p r e p a r a t i o n o f this m a n u s c r i p t . S u p p o r t f r o m N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t s A R 3 9 3 9 6 a n d A R 39569 is acknowledged.
References
I Amid, D., Frank, C., |iarwood, F.. Fronek, J. and Akeson. W. (t9841 J. Orthoo, Re,-;. 1, 257-2fl5, 2 McDevilt, C.A. and Webber, R.J. (1990~ Clin. Orthop. 252, 8-18. 3 McDevitl, C.A, 119881 in Biology of the Inlet'vertebral Disc (Ghosh. P.. ed.), Vol. I. pp, 151-170, CRC Press, Uniscienee Series. New York. 4 Eyre. D.R. (19881 in The Biology of the Inter'vertebral Disc (Ohosh, P., ed.). Vol, I, pp. 171-188, CRC Press, Uniscience Series, New York, 5 Miller. R,R. and McDevitl. C.A. (19881 Biochem. Biophys. Res. Commun. 153, 7118-714. 6 Baenziger, N.L.. Brodie. O.N. aqd M,@ru.~, P.W, (tt]71'l Prec. Natl. Acad. Sci. USA 68, 240-243. 7 keung. L.L.K, 11984)J. Clin. Invest. 74. 1764-1772, 8 Frazier, W.A. (19871J. Cell Biol. 1115.~25-632. 9 Lawler, J.W,, Slayter, iI.S. and Coligan. J.E. (19781J. Biol. Chem, 253. 8609-8616, lit Margossian. S.S., Lawler. J.W. :md Slayter, lt.S. (19811 J. Biol. (?h¢.11, 25h, 7495-75(1t), t I Lawler, J. (19861 Blood 76. I its7- 1209. 12 Frazier. W,A.S. {19871J. Cell Biol. 1115,~'~25-632. 13 Green, W.T. (19711 Clin. Orthop. 75, 248-260. 14 Webber, R.J., ltarris, M.G. and Itough. AJ., Jr. (19851J. Orth~p. Res. 3, 36~42, 15 Gehron Rt~bey, P.. Young, M.F., Fisher, L.W. and MeClain, T.D. (It18~.~1J. Cell Biol. It)8. 71~)-727. 16 Ulezardin, P.. Jouishomme. t!., Chavassieux, P. and Marie, PJ. (IC~89) Eur. J. Biochem. 181. 721-725. 17 Mosher, D.F. 11991)) Annu. Rev. Med. 41.85-97. 18 Mumby, S,M., Abbott-Brown, D., Raugi, G.J. and Bornstein, P. (1984) J, Cell Physiol. 120. 280~288. 19 M:tjack. R,A,, Goodman, L.V. and Dixit. V.M. (19881J. Cell Biol, [lib, 415-422. 211 O'Shca. K.S. and Di×it, V.M. ( 19881J. Cell Biol. 107, 2737-2748. 21 Raugi. G.J.. Olerud, J.E, and Gown, A.M. (t9871 J. Invest. Dermalol. 8tL 551-554.
lliochimica et RkJph.v.~ica ,4ct~t. I 1 I5 ( 1991 ) 85-S~
~5 1991 Elsevier Science Publishers B.V. All rights reserved I}3i}4-4165/t}i/$1)3,5()
Rapid Report
B B A G E N 2(}287
Thrombospondin in ligament, meniscus and intervertebral disc R a y m o n d R. M i l l e r a n d C a h i r A. M c D e v i t t Department t~l"Ahtsculoskeletal Research, The ("h'cchtnd ("lime ['~mndari~m Rc~c:trch Institt~te, ('h'cefand, 0 t t ~'U.S.,4, )
(Received 28 August 1991)
Key words: 3hrombospondin: Ligament: Mcni.~cu.,,: intcrvcrtebral disc: Matrix
The presence of thrombospondin in extracts of ligament, meniscus and human nucleus pulposus was demonstrated by Western blot analysis using an anti-human thrombospondin antibody. Metabolic labeling of ligament fibroblast and meniscal fibrochondrocyte cultures followed by immunoprecipitation with anti-human thrombospondin revealed a single band corresponding to the molecular weight of thrombospondin when analyzed by SDS-PAGE and fluorography under reduced and non-reduced conditions. Thrombospondin, therefore, is a matrix constituent of ligament, knee joint meniscus and the nucleus pulposus and is synthesized by ligament fibroblasts and meniscal fibrochondrocytes in vitro.
Connective tissues such as ligaments [I], the knee joint meniscus [2], and the intervertebral disc [3] are characterized by an abundant extracellular matrix with relatively few cells. While type ! or type I! collagen constitutes the major extracellular fibrillar protein in these tissues [4], minor collagens and matrix glycoproteins are beginning to be recognized for their potential importance in the development, maintenance and repair of these tissues. We have recently reported that the high molecular weight, multidomain glycoprotein, thrombospondin, is present in articular cartilage and is synthesized by articular chondrocytes in culture [5]. Thrombospondin was first identified as a major protein of platelets [6], where it appears to participate in platelet aggregation [7]. The protein is composed or" three identical peptide chains, each of absolute molecular mass 140 kDa [8,9], that migrate in most electrophoretie gels under reduced conditions as a 180 kDa species [9,10]. Electron microscopic studies have demonstrated that each polypeptide is in the fo:m of a thin strand that bears a globular domain at each extremity [8,11,12]. Like t'ibronectin and laminin, thrombospondin contains discrete functional domains that can interact with other molecules, including fibronectin, fibrinogen, plasminogen, sulfated glycolipids, type V collagen, and calcium [8,11,12]. Throm-
Correspondence: C.A. McDevitt, Department of Musculoskeletal
Research, Cleveland Clinic Foundation Research Inslilute. 95111) Euclid Avenue, Cleveland, O H 44195-5254, U.S.A.
b¢)spondin also contains an RGD sequence that could bind to integrin receptors on cell surfaces, The protein, therefore, is an excellent candidate for mediating cellmatrix and intcrmolecular matrix processes in normal tissue homeostasis as well as repair processes in patbological tissues. In the present study we demonstrate that thrombospondin is a constituent of intervertebral disc, ligament and meniscus. The anterior and posterior cruciate ligaments and the lateral and medial menisci were dissected from an adult sheep knee joint. The nucleus pulposus from lumbar discs (LJL,_-L,JL~) were dissected from a radioiogically normal 20-year-old human spine obtained at autopsy. Tissues were minced with a scalpel blade and extracted with 4 M guanidine-HCl, 0.05 M sodium acetate (pH 5.8) containing proteinase inhibitors: 0.01 M EDTA, 0.1 M 6-aminocaproic acid, 5 mM benzamidine, 0.01 M N-ethylmaleimide and l mM phenylmethylsulfonyt fluoride (10 ml per I g of tissue). The extracts were clarified by centrifugation at 18000 × g and the supernatant was dialyzed exhaustively at 4 o C against 0.tl5 M sodium acetate (pH 6.8) containing proteinase inhibitors. Canine mcniscal fibrochondrocytes were isolated essentially by the methods of Green [13] and Webber et al. [14]. Menisci from the knee joint were dissected carefully to remove any adhering tissue. The dissected tissue was sequentially digested with testicular hyaluronidase (5 m g / g tissue), trypsin (0.2%) and collagenase (0.2%). The final celi pellet was resuspended, counted with a hemocytometer and plated in DMEM containing 25 mM Hepes (pH 7.4), sodium ascorbate
86 (50 /,tg/ml) supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 l,J/mI and streptomycin 100/xg/ml). Ligament tissue from a bovine ankle joint was dissected free of adhering tissue, minced into pieces (1-2 mm 3) and the explants were cultured in 60 mm dishes with 4 ml of Ham's F-12 medium containing 25 mM Hepes (pH 7.4), sodium ascorbatc (50 p,g/ml) supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 U/ml and streptomycin, 100 /.tg/ml). After 1 week, the medium was changed every 4 days, After 3 weeks, cells that grew from the explants were detached with 0.025% trypsin/0.02% EDTA and replated in 60 mm dishes for labeling experiments. Culture medium was changed cvery 2 c~ys. Newly synthesized proteins were radiolabeled in confluent monolayer cultures of meniscal fibrochondrocytes and ligament fibroblasts. Medium was removed and the monolayers were preincubated with either proline-free medium {ligament fibroblasts)or methionine-free medium (menisc~d fibrochondrocytes) for 30 rain. The medium was then replaced with l resh medium containing [~H]proline, 50 # C i / m l (ligament fibroblasts) or [~SS]methionine 50 /.tCi/ml (mcniscal fibrochondrocytes). Aftcr a 24-h incubation, proteinase inhibitors were added to the medium• Aliquots of the medium (I ml) were incubated with 2 ul of anti-human platelet thrombospondin monocional antibody for 2 h at room temperature and overnight at 4 ° C. The antibody was generously supplied by Dr. Dean Mosher, University of Wisconsin. Separate aliquots were incubated with control mouse ascites fluid as a negative control. A suspension of protein A agarose (Affi-Gel Protein A, Bio-Rad)was incubated with goat anti.mouse IgG as described above. The protein A agarose-lgG suspension was then centrifuged, the supernatant removed and the pellet resuspended in phosphate buffered saline. An aliquot (0.025 ml) of this suspension was added to the above specimens. Following a 2-h incubation at room temperature, the immunoprecipltates were centrifuged at 11 000 × g for 30 s. The supernatant was removed and the pellet was washed three times with immunoprecipitation buffer (50 mM Tris-HC! (pH 7.5), 0.15 M NaCI, 0.1% SDS, 0.5% Tween 20 and 0.2% NaN 3) and once with immunoprecipitation buffer without detergents. The final pellets were solubilized in electrophoresis sample buffer, boiled for 5 min and electrophoresed as described below. Samples were electrophoresed on either slab gels composed of a gradient of polyacrylamide and agarose or 7.5% polyacrylamide slab gels. Specimens were boiled in electrophoresis sample buffer (50 mM Tris; 2% SDS; 15% glycerol) and run under reduced or non-reduced conditions in the presence of SDS. For immunoblotting, proteins were transferred to nitro-
I
3
,¢
~. Lr. !
..
180 ~I16 "84 ...,.
~ '
"~,
"
48.5
"I 3 6 . 5
Fig• I. Immunob|otting of thrombospondin from tissue extracts electrophoresed on a polyacrylamide.agarose gradient gel. Lane 1: human interverlebral disc. Lane 2: ovine meniscus. Lane 3: ovine ligament, Arrow heads indicate the migration of reduced protein standards (Sigma) and their respective molecular masses in kDa;
•~-macroglobutin./3 galactosidase, fructose-6.phosphate kinase, pyrurate kinase, and fumarase. Large arrow indicates top of gel.
cellulose and probed for the presence of thrombospondin with the anti-human platelet thrombospondin monoelonal antibody (1:5000) and an alkaline phosphatase conjugated goat anti-mouse IgG (1:3000). Radiolabeled proteins were detected by impregnating the gels with 3En Hance (New England Nuclear, Boston, MA), drying the gels and exposing them to X-Omat X-ray film (Kodak, Rochester, NY) at 70°C. Eleetrophoresis under non-reduced conditions and Western blotting using the specific monoelonal antibody revealed the presence of thrombospondin in extracts of nucleus pulposus (Fig. 1, lane 1), meniscus (lane 2) and ligament (lane 3). A single, major band was obtained with all three specimens, with a minor band migrating just ahead of the major band in the meniscus and ligament extracts. Extrapolation of the mobilities of the reduced globular protein standards yielded a molecular mass of 420 kDa for the major thrombospondin band, consistent with our previous observations for this protein in articular cartilage [5] and the known apparent molecular mass in dec-
~7 trophoresis of the non-reduced molecule [9.I0]. Thrombospondin isolated from human ptatelets migrates in this gel system under identical conditions as a band with an apparent molecular mass of 420 kDa (5). No immunoreactive material was evident in Western blots when non-immune mouse ascites was substituted for the anti-thrombospondin antibody. Moreover, dot blot analysis demonstrated that thrombospondin in ligament and meniscal extracts bound to heparin-agarose columns in buffer containing 0.15 M sodium chloride and was eluted from the column with 1.0 M sodium chloride. We wished to establish whether the thrombospondin observed by Western blot was a biosynthetic product of the cells of these tissues. Cultures of ligament fibroblasts and meniscal fibroehondroeytes were established, radioiabeled separately with [3H]proline and
1
2
3
3
Fig. 3. lmmunoprecipitationof Ihrombospondia from the culture medium of [~:Slmc',hioninelabeled meniscal fibrochondroc)'teswilh anti-human thrombospondin. Samples v.ere electrophoresed on a 7.5c;- polyacrylamidc slab gel. Lane 1: culture medium, non-reduced.
Lane 2: cullure medium, reduced, Lane 3: radiolabeled protein slandard.~(see Fig. 2).
[3SS]methionine, respectively, and the culture media subjected to immunopreeipitation with the anti-thrombospondin monocionai antibody. When analyzed by SDS-PAGE and autoradiography, the immunoprecip-
itatt:s ~f ligament fibr0blast medium (Fig. 2) and meniscal fibroch~,ndrocyte medium (Fig. 3) migrated as a single band when tither non-reduced (lane 1) or reduced (lane 2). The molecular mass of the reduced forms was 180 kDa, which is similar to previously published values for reduced thrombospondin in eiectrophoretic gels [9,10]. No radioactivity was immuno-
precipitated when a control mouse ascites fluid was substituted for the anti-thrombospondin. Thus, the ligament fibroblasts and meniscal fibrochondrocytes synthesize thrombospondin in vitro. Fig. 2. I m m u n o p r e e i p i t a t i o n of t h r o m b o s p o n d i n from the culture
medium of ['aH]prolinelabeled ligament fibroblastswilh anti-human thrombos~ndin. Samples were electrophoresed on a gradient gel. Lane 1: culture medium, non-reduced. Lane 2: culture medium, reduced Lane 3: radiolabeledprotein standards (Amersham); myosin. phospho~lase b, bovine serum albumin, ovalbumin, carbonic anhydrase and lysoz,yme.
This study establishes that thrombospondin is present in the knee joint meniscus, ligaments and the nucleus pulposus of the disc aod is synthesized by
meniscal fibrochondrocytes and ligament fibroblasts in vitro. This study, therefore, extends previous observations on the presence of thrombospondin in articular cartilage [5] and mineralized bone [15]. and its synthe-
88 sis by c h o n d r o c y t e s [5] a n d o s t c o b l a s t s [16], r e s p e c tively, in culture. T h r o m b o s p o n d i n , l h e r e f o r e , m a y be a u b i q u i t o u s c o n s t i t u e n t o f c o n n e c t i v e tissues. T h e physiological f u n c t i o n o f t h r o m b o s p o n d i n in c o n n e c t i v e tissues is u n k n o w n . P r o l i f e r a t i n g c u l t u r e s o f e n d o t h e l i a l cells, s m o o t h m u s c l e cells a n d fibroblasts synthesize h i g h e r levels o f t h r o m b o s p o n d i n t h a n d o n o n d i v i d i n g cells [1%18]. M a j a c k et al. [19] have p r o p o s e d f r o m s t u d i e s with c u l t u r e d v a s c u l a r s m o o t h m u s cle cells that ceil-surface t h r o m b o s p o n d i n is f u n c t i o n ally essential for p r o l i f e r a t i o n a n d that t h e reo, u i r c m e n t is l o c a t e d in t h c G~ p h a s e of t h e cell cycle. T h r o m b o s p o n d i n , e i t h e r t h r o u g h its p a r t i c i p a t i o n in cell division or s o m e o t h e r celluhtr or c x t r a c c l l u l a r process, a p p e a r s to f u n c t i o n in tissue development and repair. O ' S h c a a n d Dixit have d e m o n s t r a t e d in t h e d e v c l o p m g m o u s e e m b r y o that t h e r e was c o n s i d e r a b l e i m m u n o s t a i n i n g for t h r o m b o s p o n d i n on the s u r f a c e o f c h o n d r o b l a s t s a n d myoblasI,,i w h i c h s u b s e q u e n t l y dec r e a s e d ,vith diffcrcnti~.tion [2{I]. At early s t a g e s of skin w o u n d s , thron'~bospondin s t a i n i n g is i n t e n s e , while h c a l c d w o u n d s exhibit greatly d i m i n i s h e d s t a i n i n g [21]. T h e p r e c i s e talc of t h r o n l b o s p t m d i n in normal and p a t h o l o g i c a l c o n n e c t i v e tissues r e m a i n s 1o bc e s t a b lishcd. Acknowledgements T h c a u l h n r s w o u l d like to l h a n k Julie Pahl a n d P a m e l a Rydcll for their t e c h n i c a l assistance a n d J u d y C h r i s t o p h e r for typing a n d e d i t o r i a l assistance in t h e p r e p a r a t i o n o f this m a n u s c r i p t . S u p p o r t f r o m N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t s A R 3 9 3 9 6 a n d A R 39569 is acknowledged.
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