0013.7227/92/1305-2565$03.00/O Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 5 Society

Printed

Differentiation-Controlled Synthesis Thrombospondin to Granulosa Cells* M. DREYFUSt,

R. DARDIK,

B. S. SUH,

A. AMSTERDAMS,

AND

and Binding

in U.S.A.

of

J. LAHAV

Departments of Polymer Research (M.D., J.L.) and Hormone Research (B.S.S., A.A.), Weizmann Institute of Science, Rehovot; the National Center for Hemophilia, Sheba Hospital (R.D.), Tel Hashomer; and the Institute of Hematology (J.L.), Beilinson Medical Center, Petah Tiqva, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

TSP production dramatically, and forskolin completely inhibits it. In parallel, we observed that the undifferentiated cells bind TSP specifically with a Kd of 1.8 nM, and the number of binding sites per cell is 1.7 X 105. This binding can be prevented by excess TSP or an anti-TSP monoclonal antibody (B7-3). This ability to bind TSP is completely lost after induction of differentiation by FSH or 8-Br-CAMP. Our findings show that both the production and binding of TSP to granulosa cells are tightly controlled by normal cell differentiation and indicate that changes in TSP are correlated with the passage of the cell through the stages of maturation, a passage that also involves changes in cell shape and extracellular interactions and in the steroidogenic capacity of these cells. (Endocrinology 130: 25652570, 1992)

ABSTRACT. Thrombospondin (TSP) is a large glycoprotein, synthesized by several matrix-forming cells and incorporated into their extracellular matrix. In several cell types its presence supports cell growth and proliferation. To investigate the role of this protein in cell differentiation, we studied the hormonal effect of TSP production and receptor-mediated binding to primary granulosa cells prepared from diethylstilbestrol-treated immature female rats. These cells can be induced to differentiate by FSH, b-bromo-CAMP (8-Br-CAMP), or forskolin. Progesterone production is induced during differentiation, and its level of synthesis is an important manifestation of the differentiated phenotype. We find that undifferentiated granulosa cells synthesize and secrete TSP. The protein comprises about 0.5% of the total cell protein, and it is the major protein secreted in culture. Treatment of the cells with FSH or 8-Br-CAMP reduces

T

(TSP) is a multifunctions HROMBOSPONDIN glycoprotein synthesized by several cell types in culture (l-5) and incorporated into the extracellular matrix (ECM) of these cells (2, 6, 7). In uiuo it has been detected in most interstitial spaces (8). It can bind to the surface of cells (9, 10) and control cell adhesion (11-15) and growth (6, 16, 17). Several lines of evidence indicate that TSP is involved in development. Fetal endothelial cells in culture produce 5-fold more TSP than their adult counterparts, and its incorporation into the ECM is only observed in fetal cells (6). TSP production is regulated by growth factors (16, 18), and it has an autocrine role in the growth regulation of these cells (19). TSP is involved in cell migration, particularly during development, as shown for cerebral granule cells (20) and neural crest cells (21). Its

distribution in the various embryonal tissues is developmentally regulated (22,23). We have previously shown that incorporation of TSP into the ECM is achieved by TSP-producing cells only (7), and we, therefore, suggested that TSP production and binding by the cell can control a specific stage in the development of a tissue. Control of cell differentiation by the ECM has indeed been demonstrated (24-26). Direct linkage between cell differentiation and TSP production and binding, however, has not been investigated. To study the effect of cell differentiation on TSP production and binding capacity by the cell, we used rat granulosa cells grown in culture. This cell system is particularly attractive for our study, since it plays an integral role in follicular development (27, 28). In response to stimulation with FSH, granulosa cells increase CAMP production and induce the expression of steroidogenic enzymes and receptors for LH (27-32). These changes can also be obtained with 8bromo-CAMP (8-Br-CAMP) or compounds that elevate intracellular CAMP levels (33). In addition, human and rat granulosa cell differentiation was recently demonstrated to be stimulated by culturing these cells on a native basement membrane (34-36), and an inverse correlation between granulosa cell differentiation and synthesis of fibronectin (FN) was reported (37, 38). In this

Received September 23, 1991. Address requests for reprints to: A. Amsterdam, Ph.D., Department of Hormone Research, Weizmann Institute of Science, Rehovot 76100, Israel. *This work was supported in part by Grant 84-00250 from the United States-Israel Binational Science Foundation (Jerusalem, Israel) and a grant from the Minerva Foundation (Munich, Germany). t Present address: Laboratory of Hematology, Hopital Antoine Beclere, 157, rue de la Porte de Trivaux, F-92141 Clamart, France. $ Incumbent of the Joyce and Ben B. Eisenberg Professional Chair of Molecular Endocrinology and Cancer Research at the Weizmann Institute of Science.

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2566

SYNTHESIS

AND

report we used granulosa cells to study the effect of hormonally induced differentiation on the synthesis and secretion of TSP by the cells and the expression of TSP receptors on the cell membrane. We found that granulosa cells can both produce TSP and bind it on their surface. Upon hormonally induced differentiation, the cells lose both synthesis and binding capacities. Materials Cell culture and radioactive

and Methods labeling

Granulosa cells were prepared from 26-day-old immature rats injected with 1.5 mg/rat .day diethylstilbestrol (DES) for the last 3 days (39). The ovaries of these rats were removed into Dulbecco’sModified Eagle’sMedium-Ham’s F-12 medium (DMEM/F12; l:l, vol/vol) containing 4 mM L-glutamine, penicillin (100U//ml), and streptomycin sulfate (100 pg/ml). They were incubated for 45 min at 37 C with 6.8 mM EGTA-0.5 M sucrose and for an additional 45 min in fresh DMEM/F12 mediumto disrupt the intercellular gapjunctions (28). Granulosa cells were releasedfrom the ovaries by forcing the tissue through a steel mesh(pore size, 0.4 mm) and were sedimented by centrifugation at 500 x g for 10 min. About lo6 viable cells were seededin 1 ml medium (without sucrose and EGTA) supplementedwith 5% fetal calf serum or 2 Fg/ml insulin in 35-mm diameter dishes(Nunc, Roskilde, Denmark). After 24 h in culture, the cells in someof the disheswere treated for 24 h with 250 rig/ml ovine FSH (oFSH NIH13), 1 mM 8-BrCAMP, or 0.1 mM forskolin, which are saturating dosesfor the in uitro induction of their differentiation (30, 33, 37, 39). Other dishes were incubated without the stimulants for the same period of time. The cells were subsequently labeled with 25 &i/ml [““Slmethionine for an additional 24 h in the same medium. At the end of the incubation period, culture medium was removed and centrifuged at 10,000X g for 10 min, EDTA and phenylmethylsulfonylfluoride were addedat a final concentration of 2 mM each, and the culture medium was stored at -80 C until further use (40). The cells were rinsed three times in serum-freemedium,harvested, solubilized in electrophoresis samplebuffer (see below), and kept frozen at -80 C until analyzed. The number of cells at the end of the different treatments did not deviate by more than 13.5%from the mean. Bovine aortic endothelial cells were usedfor the production of TSP. Radiolabeling of secreted material was performed by metabolic incorporation of radioactive methionine under conditions previously described(40). The cells were grown to near confluencein lo-cm dishesin DMEM supplementedwith 10% bovine serum. Cultures were then labeled by incubation for 24 h in DMEM containing 25 &i/ml [“5S]methionine (Amersham, Arlington Heights, IL), one tenth the concentration of unlabeled methionine, and 10% bovine serum.The culture medium wasthen collected, centrifuged for 15 min at 5000 x g at 4 C, phenylmethylsulfonylfluoride was added to 2 mM, and the mediumwaskept frozen at -80 C until further use. Isolation TSP)

of

metabolically

labeled endothelial

cell TSP (EC-

Isolation of TSP wasperformed aspreviously described(41). Briefly, culture medium containing labeled secreted proteins

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OF TSP

Endo. Voll30.

1992 No 5

was first depleted of FN by passage through a gelatin-Sepharose column (42) at a ratio of 5 ml conditioned medium/ml gelatinSepharose. The effluant was collected and passed over a heparin-Sepharose column (5 ml effluant/ml), pr-equilibrated in Ca2+ containing Tris-buffered saline (Tris-NaCl; 0.15 M NaCl, 2 mM CaCl,, 10 mM NaN,, and 20 mM Tris-HCl, pH 7.6). The heparin column was then washed with Tris-NaCl, followed by 0.25 M NaCl in the same buffer. Finally, [““SITSP was eluted with 0.55 M NaCl in the same buffer, frozen, and stored at -80 c (43). Sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis

gel

Discontinuous electrophoresiswas carried out on slab gels according to the method of Laemmli (44). Unless otherwise stated, samplesto be analyzed were boiled for 3 min in electrophoresissamplebuffer containing 2% SDS, 2 mM EDTA, 10% glycerol, and 80 mM Tris, pH 6.8; 0.1 M dithiothreitol wasadded to samplesto be analyzed in the reducedform. Mol wt calibration was performed using reduced FN (220,000), myosin (200,000), and the low mol wt marker kit (Pharmacia Fine Chemicals,Piscataway, NJ) containing polypeptides of 94,000, 68,000,43,000,30,000,20,100,and 14,400.After electrophoresis, protein bands were detected by staining the gels with 1% Coomassiebrilliant blue R (Sigma, St. Louis, MO) in 50% methanol and 10% acetic acid. For detection of radiolabeled proteins, gels were impregnated with Autofluor (National Diagnostics, Somerville, NJ), dried, and incubated with AGFA Gevaert x-ray film (AGFA, Leverkussen, Germany) at -80 C, according to the method of Bonner and Laskey (45). Cell binding

assay for TSP

The cell binding assaywasperformed aspreviously described with several modifications (10). Briefly, rat granulosacellswere harvested with PBS containing 5 mM EDTA, washed with DMEM, resuspendedin DMEM containing 0.2% BSA, and divided into 200~~1samplescontaining 1 X lo5 cells. In those experiments in which the effect of serumwas studied, the cells were resuspendedin DMEM supplementedwith either 10% bovine serum or 10% of the sameserum depleted of FN and TSP by affinity adsorption on heparin-Sepharose.In dosedependenceexperiments, equal number of cellswere incubated with varying amounts of [““SIEC-TSP for 60 min at 22 C. In inhibition experiments, eachsamplewasincubated with 0.5 pg/ ml [“‘S]EC-TSP and inhibitor. After incubation, the cells were washedtwice with PBS by centrifugation for 1 min at 10,000 x g and resuspendedin 0.2 ml PBS, and the cell-bound radioactivity was determined in the scintillation spectrometer.Nonspecific binding of radiolabeledligands was determined in the presenceof a lOO-fold excess(50 pg/ml) of unlabeledEC-TSP. Unless stated otherwise, data shown in the figures represent specific binding, i.e. the difference between total binding and nonspecific binding, which varied between 20-40% of the total binding. Statistical

analysis

Analysis of binding of [iZ51]TSP was performed using the t test for comparison of the means.Differences between treatment groups were considered statistically significant at P < 0.05.

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SYNTHESIS

AND

BINDING

OF TSP A

Results Production

Primary rat granulosa cells grown in the presence of serum secrete into their culture medium a high mol wt protein that migrates at 450,000 mol wt (Fig. 1A). Under the culture conditions used, this band is the major protein secreted by the cells, constituting over 90% of the secreted protein, as judged by densitometric analysis. After reduction of disulfide bonds, this protein migrates at 180,000 mol wt (Fig. 1B). In both forms it has the characteristic mol wt of the glycoprotein TSP, which is synthesized and secreted by many mesenchymal cells in culture. Comparison of its migration with that of TSP secreted by endothelial cells (Fig. 1, lanes 1 and 4) showed that the protein secreted by granulosa cells comigrates with EC-TSP in both the nonreduced and the reduced state (Fig. l), further confirming the identity of this protein with TSP. Analysis of cellular radiolabeled proteins showed that TSP constitutes about 0.5% of the total cell protein, as determined by densitometric analysis of the electrophoretie radiogram of a whole cell lysate (data not shown).

2

TSPb

I23 TSP bs i

b \\’ \

:

FIG. 2. Effect of differentiation-inducing agents on TSP production by granulosa cells. Two sets of experiments were conducted in which the amounts of TSP produced by an equal number of cells grown in serum (lanes l), FSH in A2, 8-Br-CAMP in B2, and the more potent differentiation inducing-agent forskolin (lanes 3) were compared. Bovine EC-TSP was used as a marker (lane 4).

on TSP synthesis

Replacing serum with insulin in the granulosa cell culture medium resulted in lower production of TSP per cell (Fig. 1). Consequently, the effect of hormonally induced cell differentiation on TSP production was further studied by culturing granulosa cells in the presence of FSH and other agents that elevate intracellular CAMP levels. We found that in response to the addition of FSH in serum-free medium, TSP production per cell decreased (Fig. 2A). In the presence of forskolin, an agent that elevates intracellular CAMP levels and stimulates granI

B

I234

of TSP by granulosa cells

Effect of differentiation

2567

3

4

56 x ** *

TSPb

FIG. 3. Specificity of TSP binding to granulosa cells. Competition for binding of radiolabeled TSP to granulosa cells was observed with 50 pg/ml unlabeled EC-TSP and with our monoclonal antiTSP antibody B7-3 as well as with 100 rg/ml FN. No inhibition was observed with 100 pg/ml heparin or 1 mg/ml of the cell adhesion peptide of the integrin family Gly-Arg-Gly-Asp acid-Ser (GRGDS).

ulosa cell differentiation (for review, see Ref. 46), the production of TSP was completely inhibited (Fig. 2A). In a different set of experiments, we observed that 8-BrCAMP inhibited TSP production, albeit to a lesser extent than forskolin (Fig. 2B). Binding



of TSP to granulosa cells

\

I

1

I

I

Non Reduced Reduced FIG. 1. Production of TSP by rat granulosa cells. Affinity-purified ECTSP (lanes 1 and 4) was used as a marker in the SDS-polyacrylamide gel analysis of proteins secreted by immature rat granulosa cells grown in culture in the presence of 5% serum (lanes 2 and 5) or 2 pg/ml insulin (lanes 3 and 6) in a serum-free medium. The typical migration pattern of nonreduced/reduced TSP is clearly visible in the granulosa cell-conditioned medium (arrowheads).

The binding of TSP to granulosa cells was studied with TSP purified from metabolically labeled endothelial cell culture medium. This provided a radiolabeled protein that was not chemically modified. We found that TSP binds to the cells in a specific manner (Fig. 3); its binding was inhibited by a loo-fold excess of unlabeled TSP (P < 0.001) and by the monoclonal anti-TSP antibody B73 (P < 0.001). We also found that FN at a loo-fold excess

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SYNTHESIS

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AND

could inhibit TSP binding to the cells. The cell attachment fragment GRGDS (the cell adhesion peptide of the integrin family Gly-Arg-Gly-Asp acid-Ser), however, did not inhibit TSP binding, nor did heparin, another compound that binds to TSP at multiple sites (Fig. 3). The effect of differentiation on the expression of the receptor for TSP on the surface of the cells was studied by measuring TSP binding after induction of differentiation by addition of FSH, 8-Br-CAMP, or forskolin. We found that TSP binding to the differentiated cells was completely eliminated (Fig. 4). Similar results were observed with FSH, forskolin, and 8Br-CAMP (Fig. 4). Based on these observations, we determined TSP binding kinetics to the undifferentiated cells. In this state, binding of TSP was concentration dependent (Fig. 5). Specific binding was calculated from the difference between total binding of radiolabled TSP and binding in the presence of a loo-fold excess of unlabeled TSP. Graphical analysis of these values in a double reciprocal plot yielded a straight line (Fig. 5, inset). This permitted determination of the dissociation constant (Kd) and the number of binding sites per cell (10). The Kd determined for the binding of TSP to undifferentiated granulosa cells was 1.8 f 0.7 nM, and the number of binding sites was 1.7 f 0.4 X 105/Cell (mean f SD; n = 4). Discussion

In this study we show for the first time that granulosa cells synthesize and secrete the glycoprotein TSP. Using primary rat granulosa cells from immature rats injected with DES and grown in culture, the protein was identified by its unique electrophoretic migration under nonreduced and reduced conditions (1, 40, 47) and by its comigration with purified EC-TSP under both condi-

5

15

IO Time

20 (min)

25

30

4. Binding of radiolabled TSP by granulosa cells. Two hundredmicroliter samples of granulosa cells (2 X lo5 cells/sample), mature (0) or stimulated to differentiate [by FSH (O), SBr-CAMP (A), or forsklin (O)], were incubated in DMEM containing 0.2% BSA and 0.5 rg/ml radiolabeled TSP at 22 C. At increasing periods, the cells were washed, and bound radioactivity was determined, as described in Materials and Methods. The values shown are specific binding, obtained by subtraction of the amount of radiolabeled TSP bound in the presence of 50 pg/ml unlabeled TSP (nonspecific binding) from the total binding. FIG.

BINDING

OF TSP

G

Endo. Voll30.

1992 No 5

0.03

z E : 73 E a E c

0.02 0.01

73 c 2

m

v!

0

0.22

0.44

0.66

Applied TSP (pmole/sample) FIG. 5. Dose dependence of [“‘IJEC-TSP binding by immature granulosa cells. Two hundred-microliter samples of immature granulosa cells (2 x lo5 cells/sample) were incubated with increasing concentrations of [‘251]EC-TSP for 1 h at 22 C. After incubation, the samples were treated as describedin Materials and Methods. The values shown represent specific binding determined by subtraction of the radioactivity bound in the presence of 50 @g/ml unlabeled EC-TSP (nonspecific binding) from the total cell-bound radioactivity. Double reciprocal plot analysis of the data is shown in inset.

tions. In serum-free medium, the glycoprotein constituted 0.5% of the total cell protein. It was, however, the major secreted protein under these conditions. The protein was also identified in the culture medium of human granulosa cells by specific immunoprecipitation (Lahav, J. and M. Dreyfus, manuscript in preparation), using the monoclonal antibody B7-3 directed against human TSP (7). Secretion of TSP has been shown in various mesenchymal and epithelial cells (for review, see Refs. 4752). It appears in the interstitial spaces of many tissues (8) and has an intriguing regional and temporal distribution in the developing embryo (22, 23). In addition, it is found in the basement membrane separating granulosa cells from theta internal cells in developing ovarian follicles (22). It is detected in the ECM produced by many TSP-secreting cells in culture (see reviews in Refs. 47-52). Its incorporation into the ECM, however, seems to be dependent on prior formation of a fibrillar FN network (7). Granulosa cells in culture were shown to synthesize and secrete FN (37, 53,54). Our observations show that FN secretion is very low when TSP production is readily detectable. These observations indicate that TSP production in granulosa cells is under a different control mechanism than that of FN. Similar differences in early levels of synthesis between TSP and FN were previously reported in endothelial cells (7, 47), further corroborating the distinction between control mechanisms of synthesis in the two ECM proteins. Rat granulosa cells collected after treatment with DES represent an immature state which is further maintained in culture in the presence of serum (55). Growth in serum-free medium in the presence of insulin, however,

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SYNTHESIS

AND

induces the expression of several markers for differentiation (54, 56). Insulin also mimics the effect of FSH on FN production (57). In view of these observations we compared TSP production by immature granulosa cells grown in the presence of serum and those grown in the presence of insulin. Indeed, we found that production of TSP was markedly decreased by insulin. Induction of differentiation with FSH or directly by elevating intracellular levels of CAMP similarly demonstrated suppression of TSP synthesis. Forskolin at 10m4 M was more potent than 250 mg/ml FSH or 1 mM 8-BrCAMP. These observations indicate that 1) TSP production is inversely correlated with differentiation [the secretion of FN has similary been shown to be arrested by differentiation (57)]; and 2) the action of FSH on TSP production is mediated via the elevation of CAMP levels. We are apparently the first to find that granulosa cells express a specific membrane receptor for TSP. Binding of TSP was concentration dependent, saturable, and inhibitable by excess unlabeled protein. It is interesting to note that FN can compete with TSP for binding to granulosa cells. We observed similar effects with endothelial cells. Our monoclonal anti-TSP antibody B7-3 also competes with TSP binding in both cell systems. It is probable that the cell-binding domain on TSP is close to its FN-binding demain, resulting in steric hindrance to binding in the presence of FN. Another macromolecule that binds at or close to the FN domain is heparin (41), which, however, does not compete with TSP binding to granulosa cells, probably because of the relatively low affinity of its binding (41). The peptide GRGDS, which contains the RGD sequence recognized by receptors for cell adhesion molecules of the integrin family (58), does not compete with TSP binding to granulosa cells. This observation suggests that binding does not depend on the RGD sequence. Indeed, we have previously shown that the FN-binding domain for TSP does not include the RGD sequence (59), further corroborating this contention. The expression of the TSP receptor on granulosa cells, like the synthesis of TSP itself, is differentiation controlled. Induction of differentiation by the addition of FSH reduced TSP binding to granulosa cells. Direct elevation of cellular CAMP levels with 8-Br-CAMP or forskolin completely abolished EC-TSP binding. Thus, expression of both the protein and its receptor is turned off in parallel. This observation is a striking manifestation in a single cell type of an observation we made previously using different cell types (7), where we found that cells that synthesize TSP possess the ability to incorporate it into their ECM, whereas cells that do not synthesize it do not incorporate it into the ECM they form even though exogenous TSP is added. It has been suggested that TSP has an autocrine effect (19), and smooth muscle cells that synthesize the protein, depend

BINDING

OF

TSP

2569

on binding it for proliferation (17). There is also a direct correlation between the rate of cell proliferation and the amount of TSP synthesized (47, 60). The correlation between TSP synthesis and receptor expression on granulosa cells and their common control mechanism further support the idea of an autocrine effect of TSP in the regulation of the growth and maturation of granulosa cells. Acknowledgments We would like to thank secretarial assistance, and of the manuscript.

Mrs. M. Kopelowitz Dr. A. M. Kaye for

for critical

excellent reading

References 1. McPherson J, Sage H, Bornstein P 1981 Isolation and characterization of a glycoprotein secreted by aortic endothelial cells in culture. Apparent identity with platelet thrombospondin. J Biol Chem 256:11330-11336 2. Jaffe EA, Ruggiero JT, Leung LLK, Doyle MY, McKeown-Longo PJ, Mosher DF 1983 Cultured human fibroblasts synthesize and secrete thrombospondin and incoporate it into extracellular matrix. Proc Nat1 Acad Sci USA 80:998-1002 3. Asch AS. Leune LLK. Shauiro J. Nachman RL 1986 Human alial cells synthesizeihrombosp&din.‘Proc Nat1 Acad Sci USA 83:29042908 4. Canfield AE, Schor AM, Loskutoff DJ, Schor SL, Grant ME 1989 Plasminogen activator inhibitor type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture. Biochem J 259:529-535 5. Clezardin P, Jouishomme H, Chavassieux P, Marie PJ 1989 Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells. A model to study the different effects of thrombospondin in cell adhesion. Eur J Biochem 181:721-726 6. Kramer RH, Fuh GM, Beusch KG, Karasek MA 1985 Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from dermis of neonatal and adult skin. J Cell Physiol 123:1-g M, Lahav J 1988 The build up of the thrombospondin I. Dreyfus extracellular matrix. Eur J Cell Biol 47:275-282 SM, Bornstein P 1985 Light micro8. Wight TN, Raugi GJ, Mumby scopic immunolocation of thrombospondin in human tissues. J Histochem Cytochem 32295-302 JE, Mosher DF 1987 Interaction of thrombospon9. Murphy-Ullrich din with endothelial cells: receptor mediated binding and degradation. J Cell Biol 105:1603-1611 10. Dardik R, Lahav J 1991 The cell binding domain of endothelial cell thrombospondin. Localization to the 70 kD core fragment and determination of binding characteristics. Biochemistry 30:93789386 11. Varani J, Dixit VM, Fligiel SEG, McKeever PE, Carey TE 1986 Thrombospondin-induced attachment and spreading of human squamous carcinoma cells. Exp Cell Res 167:376-390 12. Robert DD, Sherwood JA, Ginsburg V 1987 Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J Cell Biol 104:131-137 13. Tuszynski GP, Gasic TB, Rothman VI, Knudsen KA, Gasic GH 1987 Thrombospondin, a potentiator of tumor cell metastasis. Cancer Res 47:4130-4133 14. Lahav J 1988 Thrombospondin inhibits adhesion of platelets to glass and protein-covered substrata. Blood 71:1096-1099 15. Lahav J 1988 Thrombospondin inhibits endothelial cell adhesion. Exp Cell Res 177:199-204 16. Majak RA, Coats-Cook S, Bornstein P 1985 Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J Cell Biol 101:1059-1070 17. Majack RA, Goodman LV, Dixit VM 1988 Cell surface thrombos-

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AND

pondin is functionally essential for smooth muscle cell proliferation. J Cell Biol 106:415-422 Donoviel DB, Amacher SL, Judge KW, Bornstein P 1990 Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor. J Cell Physiol 145:16-23 Majack RA, Cook SC, Bornstein P 1986 Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Prot Nat1 Acad Sci USA 83:9050-9054 O’Shea KS, Rheinheimer JST, Dixit VM 1990 Deposition and role of thrombospondin in the histogenesis of the cerebellar cortex. J Cell Biol 110:1275-1283 Boyne LJ, O’Shea KS, Dixit VM 1989 Neural crest migration on an extracellular matrix protein thrombospondin. J Cell Biol 109:112a O’Shea KS, Dixit VM 1988 Unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo. J Cell Biol 107:2737-2748 O’Shea KS, Liu L-HJ, Kinnunen LH, Dixit VM 1990 Role of the extracellular protein thrombospondin in the early development of the mouse embryo. J Cell Biol 111:2713-2723 Gospodarowicz D, Delgado D, Vlodavsky I 1980 Permissive effect of the extracellular matrix on cell proliferation in uitro. Proc Nat1 Acad Sci USA 77:4044-4048 Sanes JR 1983 Role of extracellular matrix in neural development. Annu Rev Physlol45:581-594 Snira 0.-. Vlodavskv I. Ulmansku R. Atzmon R. Fuks Z. Gordon A, -r--m ~~~ Gross J 1983 Thyrotropin and growth hormones secretion and cell morphology in hypothyroid pituitary cells cultured on a natural extra cellular matrix. Acta Endocrinol (Copenh) 104:279-291 Hsueh AJW, Adashi EY, Jones PBC, Welsh TH 1984 Hormonal regulation of the differntiation of cultured ovarian granulosa cells. Endocr Rev 5:76-126 Amsterdam A, Rotmensch S 1987 Structure-function relationship during granulosa cell differentiation. Endocr Rev 8:309-338 Orly J, Sato G 1979 Fibronectin mediates cytokinesis and growth of rat follicular cells in serum free medium. Cell 17:295-305 Amsterdam A, Knecht M, Catt KJ 1981 Hormonal regulation of cytodifferentiation and intercellular communication in cultured granulosa cells. Prot Nat1 Acad Sci USA 78:3000-3004 Amsterdam A, Naor Z, Knecht M, Dufau ML, Catt KJ 1981 Hormone action and receptor redistribution in endocrine target cells: gonadotropins and gonadotropin-releasing hormone. In: Middlebrook JL, Kohn LD (eds) Receptor Mediated Binding and Internalization of Toxins and Hormones. Academic Press, New York, pp 283-310 Amsterdam A, Lindner HR 1984 Localization of gonadotropin receptors in the gonads. In: Motta PM (ed) Ultrastructure of Endocrine Cells and Tissues. Martinus Nijhoff, Boston, pp 255264 Knecht M, Amsterdam A, Catt KJ 1981 The regulatory role of cyclic AMP in hormone induced granulosa cell differentiation. J Biol Chem 25610628-10633 Furman A, Rotmensch S, Kohen F, Mashiach S, Amsterdam A 1986 Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine cornea1 endothelial cells. Endocrinology 1181878-1885 Ben-Ze’ev A, Amsterdam A 1986 Down regulation in the expression of cytoskeletal proteins involved in cell contact formation during differentiation of eranulosa cells on ECM coated surfaces. Proc Nat1 Acad Sci USA 83:2894-2898 Amsterdam A, Rotmensch S, Furman A, Venter EA, Vlodavsky I 1989 Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology 124:1956-1964

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OF TSP

Endo. 1992 Vol 130. No 5

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Differentiation-controlled synthesis and binding of thrombospondin to granulosa cells.

Thrombospondin (TSP) is a large glycoprotein, synthesized by several matrix-forming cells and incorporated into their extracellular matrix. In several...
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