Vol. 11, No. 9
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1991, p. 44904496 0270-7306/91/094490-07$02.00/0 Copyright © 1991, American Society for Microbiology
Glucocorticoid Regulation of Transforming Growth Factor Activity and Binding in Osteoblast-Enriched Cultures from Fetal Rat Bone
31
MICHAEL CENTRELLA,* THOMAS L. McCARTHY, AND ERNESTO CANALIS Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105, and the University of Connecticut School of Medicine, Farmington, Connecticut 06030 Received 26 April 1991/Accepted 20 June 1991
Transforming growth factor (TGF-I) enhances replication and bone matrix protein synthesis and associates with distinct binding sites in osteoblast-enriched cultures from fetal rat bone. In the organism high levels of or sustained exposure to glucocorticoids alters bone cell activity and decreases bone mass, effects that may be mediated in part by changes in local TGF-I actions in skeletal tissue. Preexposure of osteoblastenriched cultures to 100 nM cortisol reduced the stimulatory effects of TGF-0I1 on DNA and collagen synthesis by 40 to 50%. Binding studies showed that cortisol moderately enhanced total TGF-fr1 binding, but chemical cross-linking and polyacrylamide gel electrophoretic analysis revealed an increase only within Mr 250,000 (type III) TGF-4-binding complexes, which are thought to represent extracellular TGF-,I storage sites. In contrast, a decrease in TGF-jI1 binding was detected in Mr 65,000 (type I) and 85,000 (type II) complexes, which have been implicated as signal-transducing TGF-,I receptors. Our present studies therefore indicate that glucocorticoids can decrease the anabolic effects of TGF-131 in bone, and these may occur in part by a redistribution of its binding toward extracellular matrix storage sites. Alterations of this sort could contribute to bone loss associated with glucocorticoid excess.
part of which may be related to decreased bone matrix collagen synthesis (3). In the present study we have examined the effect of cortisol, the prototypic natural glucocorticoid (13), on TGF-P activity in osteoblast-enriched cultures from growing rat bone and have observed changes in cell replication and collagen synthesis that may be related to a redistribution in the pattern of TGF-1 binding among various sites on the surface of bone cells.
Transforming growth factor 1 (TGF-P) is produced by bone cells, is abundant in bone matrix, and regulates replication and differentiated cell function in normal or immortalized cultures of osteoblastlike cells from fetal and mature bone (6-10, 14, 15, 23, 25, 29, 30). Our previous studies established that three TGF-P isoforms (TGF-1l, TGF-P2, and TGF-P3) have a biphasic stimulatory effect on replication and increase the synthesis of type I collagen, the principal organic component of bone matrix, in osteoblastenriched cultures from fetal rat parietal bone (8, 9, 30). Chemical cross-linking demonstrates that these cultures contain three distinct TGF-1-binding complexes of relative molecular mass (Mr) 65,000, 85,000, and 250,000, corresponding to types I, II, and III TGF-f-binding sites found on a variety of tissue-derived cells (1, 4, 9, 11, 12, 17, 27, 28, 30, 31). Competition binding indicates that bone cell receptors found within the Mr 65,000 and 85,000 (type I and II) complexes bind TGF-13 with high affinity relative to the Mr 250,000 (type III) complex (9, 30). Experiments with mutated mink lung cell cultures predict that type I and II TGF-1-binding complexes are necessary for intracellular signal transduction and many of the biochemical effects of TGF-1 (4, 17). Other studies indicate that the type III TGF-,B-binding complex contains a proteoglycan that is found attached to and dissociated from the cell surface and may represent extracellular TGF-1 storage (1, 11, 28). Glucocorticoid-induced bone loss is a significant clinical problem that may be manifested in both pathophysiological and pharmacological situations, but the underlying mechanisms by which this occurs are not well established. Experimental and clinical studies indicate that chronic glucocorticoid administration has deleterious effects on bone integrity, *
MATERIALS AND METHODS Cell cultures. Parietal bones from 22-day-old rat fetuses were dissected free of sutures and subjected to five sequential collagenase digestions. Although overlap among cells at various stages of osteoblast differentiation is likely, the populations released during the last three digestions are enriched in cells exhibiting osteoblast characteristics (21, 32). Cells from a pool of the last three populations were plated at 12,500 cells per cm2 in Dulbecco modified Eagle medium containing 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer (pH 7.2), 100 ,ug of ascorbic acid per ml, penicillin, and streptomycin (all from GIBCO) and 10% fetal bovine serum (Sigma). Confluent cultures (about 6 x 104 cells per cm2) were serum deprived, without or with cortisol, for 20 h. TGF-1l was then added in serum-free medium, and the cultures were incubated for an additional 23 h. All experiments were performed with primary cultures. Test agents. Recombinant human TGF-1l, obtained from Genentech, Inc., was dissolved in 0.05 M HCl containing 4 mg of bovine serum albumin per ml to obtain a concentration of 800 nM and was stored at 4°C. Recombinant human platelet-derived growth factor BB (PDGF-BB), containing amino acid substitutions in positions 1 (serine to alanine) and 12 (methionine to valine), which did not alter mitogenic activity (5), was obtained from Creative Biomolecules and
Corresponding author. 4490
GLUCOCORTICOID REGULATION OF TGF-l ACTIVITY IN BONE
VOL . 1 l, 1991
was dissolved in the same vehicle to obtain a concentration of 333 nM. 125I-TGF-13 (native human platelet TGF-,1; 1,500 to 3,100 Ci/mmol) and 125I-PDGF-BB (recombinant human PDGF-BB; 1,300 to 4,500 Ci/mmol) were obtained from Biomedical Technologies Inc. and stored at -20°C until use. Cortisol (hydroxycortisone [Sigma]) was dissolved in 95% ethanol to obtain a concentration of 10 mM and diluted in serum-free culture medium immediately prior to use. Cycloheximide (Sigma) was dissolved and diluted in serum-free medium. In control studies, vehicle dilutions equivalent to the amounts found in the highest concentration of TGF-p1 (1:2,000), PDGF-BB (1:333), or cortisol (1:33,300) had no effect in the cell culture assays (5). Cell replications. Effects on DNA synthesis were measured by pulse-labeling cultures with [methyl-3H]thymidine (5 ,Ci/ml, 80 Ci/mmol; Dupont NEN) during the last 2 h of treatment. [3H]thymidine incorporation into DNA was determined by cell lysis with 0.1 M sodium dodecyl sulfate- 0.1 N sodium hydroxide, collection of the material precipitated by 10% trichloroacetic acid, and scintillation counting. Data are shown as the total amount of acid-insoluble [3H]thymidine incorporated per culture well. Cell numbers were determined by counting an aliquot of a trypsinized cell suspension in a fixed-volume hemacytometer and are expressed as cell number per square centimeter.
Protein synthesis. Effects on collagen and noncollagen protein (NCP) synthesis were measured by pulse-labeling cultures with 12.5 ,uCi of [2,3-3Hlproline (2.5 Ci/mmol; Dupont NEN) per ml for the last 2 h of treatment. Cells were lysed by freeze-thawing; homogenates were collected in 0.5% Triton X-100 (Sigma), precipitated with 10% trichloroacetic acid, and chilled; and the acid-precipitable material was collected by centrifugation. The precipitates were acetone extracted, dried, resolubilized in 0.5 M acetic acid, and neutralized with 0.5 M sodium hydroxide. [3H]proline incorporation into collagenase-digestible protein (CDP) and NCP was measured by using collagenase purified free of nonspecific protease activity (Worthington Biochemicals) (24) and shown as the total amount of [3H]proline incorporated per culture. Percent collagen synthesis (PCS) was calculated after correcting for the relative abundance of proline in CDP and NCP. Acid-soluble incorporation was determined in the trichloroacetic acid supernatants. Binding studies. Effects on
TGF-pl binding were exam-
ined by incubation with serum-free medium for 3 h at 4°C containing 4 mg of bovine serum albumin per ml (binding medium) and 125I-TGF-pl. Cultures were extracted with 1% Triton X-100, and the amount of bound label was measured
in a gamma spectrometer. Nonspecific binding was determined in the presence of 100-fold molar excess unlabeled recombinant human TGF-pl. To visualize TGF-p-binding complexes, cultures were incubated with 125I-TGF-pl, without or with unlabeled TGF-pl, rinsed with chilled binding medium, cross-linked with 0.2 mM disuccinimidyl suberate, extracted, and fractionated by electrophoresis on a 7.5%
polyacrylamide gel, and bound 125I-TGF-1l was visualized
by autoradiography as previously reported (9, 30). 1251_ PDGF-BB binding was evaluated by analogous techniques. Statistical methods. Data are shown as means + standard errors (SE); when comparing multiple concentrations of variables, statistical differences between groups were assessed by analysis of variance, with limits set by the methods of Bonferroni or Dunnet (14a).
4491
12. 4
z
a c
z
12 pM
i
w
7TGFP
8-
a
I--
I
x
0~IL O
nM r \
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1991, p. 44904496 0270-7306/91/094490-07$02.00/0 Copyright © 1991, American Society for Microbiology
Glucocorticoid Regulation of Transforming Growth Factor Activity and Binding in Osteoblast-Enriched Cultures from Fetal Rat Bone
31
MICHAEL CENTRELLA,* THOMAS L. McCARTHY, AND ERNESTO CANALIS Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105, and the University of Connecticut School of Medicine, Farmington, Connecticut 06030 Received 26 April 1991/Accepted 20 June 1991
Transforming growth factor (TGF-I) enhances replication and bone matrix protein synthesis and associates with distinct binding sites in osteoblast-enriched cultures from fetal rat bone. In the organism high levels of or sustained exposure to glucocorticoids alters bone cell activity and decreases bone mass, effects that may be mediated in part by changes in local TGF-I actions in skeletal tissue. Preexposure of osteoblastenriched cultures to 100 nM cortisol reduced the stimulatory effects of TGF-0I1 on DNA and collagen synthesis by 40 to 50%. Binding studies showed that cortisol moderately enhanced total TGF-fr1 binding, but chemical cross-linking and polyacrylamide gel electrophoretic analysis revealed an increase only within Mr 250,000 (type III) TGF-4-binding complexes, which are thought to represent extracellular TGF-,I storage sites. In contrast, a decrease in TGF-jI1 binding was detected in Mr 65,000 (type I) and 85,000 (type II) complexes, which have been implicated as signal-transducing TGF-,I receptors. Our present studies therefore indicate that glucocorticoids can decrease the anabolic effects of TGF-131 in bone, and these may occur in part by a redistribution of its binding toward extracellular matrix storage sites. Alterations of this sort could contribute to bone loss associated with glucocorticoid excess.
part of which may be related to decreased bone matrix collagen synthesis (3). In the present study we have examined the effect of cortisol, the prototypic natural glucocorticoid (13), on TGF-P activity in osteoblast-enriched cultures from growing rat bone and have observed changes in cell replication and collagen synthesis that may be related to a redistribution in the pattern of TGF-1 binding among various sites on the surface of bone cells.
Transforming growth factor 1 (TGF-P) is produced by bone cells, is abundant in bone matrix, and regulates replication and differentiated cell function in normal or immortalized cultures of osteoblastlike cells from fetal and mature bone (6-10, 14, 15, 23, 25, 29, 30). Our previous studies established that three TGF-P isoforms (TGF-1l, TGF-P2, and TGF-P3) have a biphasic stimulatory effect on replication and increase the synthesis of type I collagen, the principal organic component of bone matrix, in osteoblastenriched cultures from fetal rat parietal bone (8, 9, 30). Chemical cross-linking demonstrates that these cultures contain three distinct TGF-1-binding complexes of relative molecular mass (Mr) 65,000, 85,000, and 250,000, corresponding to types I, II, and III TGF-f-binding sites found on a variety of tissue-derived cells (1, 4, 9, 11, 12, 17, 27, 28, 30, 31). Competition binding indicates that bone cell receptors found within the Mr 65,000 and 85,000 (type I and II) complexes bind TGF-13 with high affinity relative to the Mr 250,000 (type III) complex (9, 30). Experiments with mutated mink lung cell cultures predict that type I and II TGF-1-binding complexes are necessary for intracellular signal transduction and many of the biochemical effects of TGF-1 (4, 17). Other studies indicate that the type III TGF-,B-binding complex contains a proteoglycan that is found attached to and dissociated from the cell surface and may represent extracellular TGF-1 storage (1, 11, 28). Glucocorticoid-induced bone loss is a significant clinical problem that may be manifested in both pathophysiological and pharmacological situations, but the underlying mechanisms by which this occurs are not well established. Experimental and clinical studies indicate that chronic glucocorticoid administration has deleterious effects on bone integrity, *
MATERIALS AND METHODS Cell cultures. Parietal bones from 22-day-old rat fetuses were dissected free of sutures and subjected to five sequential collagenase digestions. Although overlap among cells at various stages of osteoblast differentiation is likely, the populations released during the last three digestions are enriched in cells exhibiting osteoblast characteristics (21, 32). Cells from a pool of the last three populations were plated at 12,500 cells per cm2 in Dulbecco modified Eagle medium containing 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer (pH 7.2), 100 ,ug of ascorbic acid per ml, penicillin, and streptomycin (all from GIBCO) and 10% fetal bovine serum (Sigma). Confluent cultures (about 6 x 104 cells per cm2) were serum deprived, without or with cortisol, for 20 h. TGF-1l was then added in serum-free medium, and the cultures were incubated for an additional 23 h. All experiments were performed with primary cultures. Test agents. Recombinant human TGF-1l, obtained from Genentech, Inc., was dissolved in 0.05 M HCl containing 4 mg of bovine serum albumin per ml to obtain a concentration of 800 nM and was stored at 4°C. Recombinant human platelet-derived growth factor BB (PDGF-BB), containing amino acid substitutions in positions 1 (serine to alanine) and 12 (methionine to valine), which did not alter mitogenic activity (5), was obtained from Creative Biomolecules and
Corresponding author. 4490
GLUCOCORTICOID REGULATION OF TGF-l ACTIVITY IN BONE
VOL . 1 l, 1991
was dissolved in the same vehicle to obtain a concentration of 333 nM. 125I-TGF-13 (native human platelet TGF-,1; 1,500 to 3,100 Ci/mmol) and 125I-PDGF-BB (recombinant human PDGF-BB; 1,300 to 4,500 Ci/mmol) were obtained from Biomedical Technologies Inc. and stored at -20°C until use. Cortisol (hydroxycortisone [Sigma]) was dissolved in 95% ethanol to obtain a concentration of 10 mM and diluted in serum-free culture medium immediately prior to use. Cycloheximide (Sigma) was dissolved and diluted in serum-free medium. In control studies, vehicle dilutions equivalent to the amounts found in the highest concentration of TGF-p1 (1:2,000), PDGF-BB (1:333), or cortisol (1:33,300) had no effect in the cell culture assays (5). Cell replications. Effects on DNA synthesis were measured by pulse-labeling cultures with [methyl-3H]thymidine (5 ,Ci/ml, 80 Ci/mmol; Dupont NEN) during the last 2 h of treatment. [3H]thymidine incorporation into DNA was determined by cell lysis with 0.1 M sodium dodecyl sulfate- 0.1 N sodium hydroxide, collection of the material precipitated by 10% trichloroacetic acid, and scintillation counting. Data are shown as the total amount of acid-insoluble [3H]thymidine incorporated per culture well. Cell numbers were determined by counting an aliquot of a trypsinized cell suspension in a fixed-volume hemacytometer and are expressed as cell number per square centimeter.
Protein synthesis. Effects on collagen and noncollagen protein (NCP) synthesis were measured by pulse-labeling cultures with 12.5 ,uCi of [2,3-3Hlproline (2.5 Ci/mmol; Dupont NEN) per ml for the last 2 h of treatment. Cells were lysed by freeze-thawing; homogenates were collected in 0.5% Triton X-100 (Sigma), precipitated with 10% trichloroacetic acid, and chilled; and the acid-precipitable material was collected by centrifugation. The precipitates were acetone extracted, dried, resolubilized in 0.5 M acetic acid, and neutralized with 0.5 M sodium hydroxide. [3H]proline incorporation into collagenase-digestible protein (CDP) and NCP was measured by using collagenase purified free of nonspecific protease activity (Worthington Biochemicals) (24) and shown as the total amount of [3H]proline incorporated per culture. Percent collagen synthesis (PCS) was calculated after correcting for the relative abundance of proline in CDP and NCP. Acid-soluble incorporation was determined in the trichloroacetic acid supernatants. Binding studies. Effects on
TGF-pl binding were exam-
ined by incubation with serum-free medium for 3 h at 4°C containing 4 mg of bovine serum albumin per ml (binding medium) and 125I-TGF-pl. Cultures were extracted with 1% Triton X-100, and the amount of bound label was measured
in a gamma spectrometer. Nonspecific binding was determined in the presence of 100-fold molar excess unlabeled recombinant human TGF-pl. To visualize TGF-p-binding complexes, cultures were incubated with 125I-TGF-pl, without or with unlabeled TGF-pl, rinsed with chilled binding medium, cross-linked with 0.2 mM disuccinimidyl suberate, extracted, and fractionated by electrophoresis on a 7.5%
polyacrylamide gel, and bound 125I-TGF-1l was visualized
by autoradiography as previously reported (9, 30). 1251_ PDGF-BB binding was evaluated by analogous techniques. Statistical methods. Data are shown as means + standard errors (SE); when comparing multiple concentrations of variables, statistical differences between groups were assessed by analysis of variance, with limits set by the methods of Bonferroni or Dunnet (14a).
4491
12. 4
z
a c
z
12 pM
i
w
7TGFP
8-
a
I--
I
x
0~IL O
nM r \
