JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 8, 1990 Mary Ann Liebert, Inc., Publishers

Chemotactic Response of Osteoblastlike Cells to Transforming Growth Factor ,6 JOHANNES PFEILSCHIFTER,' OLIVER WOLF,' ANTJE NAUMANN,' HELMUT W. MINNE,' GREGORY R. MUNDY,' and REINHARD ZIEGLER'

ABSTRACT Transforming growth factor @ (TGF-@)has multiple effects on bone cell metabolism in vitro but its exact role in bone remodeling still needs to be defined. Here we demonstrate that TGF-@is chemotactic for osteoblastlike cells from fetal rat calvariae and osteoblastlike ROS 17/2.8 osteosarcoma cells. Maximal chemotaxis occurred at 5-15 pg/ml of TGF-P and was observed with TGF-@,and TGF-@,at equivalent concentrations. Conditioned medium from osteoblastlike cells containing latent TGF-/3 failed to stimulate chemotactic migration. However, chemotactic activity was observed in conditioned medium that had been transiently acidified. Since acidification is known to activate TGF-P, these results suggest that only active TGF-@is capable of inducing a chemotactic response. Preincubation of osteoblastlike cells with TGF-P in concentrations from 10 pg/ml to 1 ng/ml for 48 h abolished a subsequent chemotactic response of these cells to TGFP, indicating that TGF-@-inducedchemotaxis is a transient phenomenon. Since TGF-/3 may be released from the bone matrix and/or activated during bone resorption, the chemotactic activity of TGF-@for osteoblastlike cells may be important for the recruitment of osteoblastlike cells to sites of bone remodeling.

INTRODUCTION (TGF-0) is a multifunctional polypeptide that can affect growth and differentiated functions of a variety of cell types.""' Bone matrix is one of the major storage sites for TGF-P,'41and TGF-0 is capable of stimulating bone formation in vitro and in viva.'"') TGF-/3 is, therefore, believed to play a major role in bone remodeling. Bone remodeling is characterized by a phase of bone resorption that is followed by a period of bone formation in which the defect formed by the resorption process is repaired. ( 8 - 1 0 ) This coupling of bone formation to bone resorption is one of the key events of bone cell metabolism. However, its exact mechanism is still unknown. Previous studies have suggested that TGF-/3 activity may be increased at bone resorption sites.'",'2) Since the initial step during the coupling process is likely to be an attraction of

T

RANSFORMING GROWTH FACTOR

osteoblastlike cells to the site of previous bone resorption, we have studied the ability of TGF-0 to stimulate the chemotactic attraction of osteoblastlike cells.

MATERIALS AND METHODS TGR-PI and TGF-0, purified from human platelets were purchased from R&D Systems (Minneapolis, MN). Recombinant human TGF-@, was generously provided by Drs. D.R. Twardzik and A.F. Purchio (Oncogen, Seattle, WA). Primary cultures of fetal rat osteoblastlike cells were obtained from 21-day-old fetal rats by sequential collagenase digestion as described previously. I ) ) Five single-cell suspensions were recovered from each 20 minute collagenase treatment and were numbered as fractions 1-5 conforming to their order of release. The second fraction was discarded, and fractions 3-5 were pooled. ('

'Department of Medicine 1, Endocrinology, University of Heidelberg, Luisenstrasse 5 , 6900 Heidelberg, Federal Republic of Germany. 'Department of Medicine, Endocrinology, University of Texas Health Science Center, San Antonio, TX 78284.

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PFEILSCHIFTER ET AL.

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ROS 17/2.8 cells were obtained from Dr. G. Rodan (Merck, Sharpe and Dohme, West Point, PA). They are clonal rat osteosarcoma cells with many characteristics of cells of the osteoblast phenotype and have been described in detail previously.(14Is) All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS, GIBCO). When the cells reached confluence, they were trypsinized, washed three times in DMEM containing 1 mg/ml of bovine serum albumin (BSA, Sigma), and transferred to the chemotaxis chambers. Chemotaxis was measured in modified blind-well chemotaxis chambers (Nucleopore, Tubingen, FRG) equipped with porous (12 pm) nitrocellulose filters (Schleicher & Schiill, Dassel, FRG).'I6 ''I The osteoblastlike cells were placed in the upper compartment at a concentration of 2 x lo5 cells per ml of DMEM medium. Medium containing various concentrations of TGF-@(200 pl) was placed in the lower compartment, and the chambers were incubated for 4 h at 37°C in 5% CO,. Filters were then washed, fixed, and stained with hematoxylin. Chemotaxis was measured by counting the number of cells in 30 light microscopic high-power fields ( x 400) that had migrated into the membrane to a distance of more than 20 pm. All samples were tested in triplicate or quadruplicate. Conditioned medium from fetal rat calvarial cell cultures was obtained by incubating confluent cells with serum-free DMEM medium containing 1 mg/ml of BSA and collecting the conditioned medium after 48 h. TGF-@ activity in the conditioned medium was determined using colony formation of normal rat kidney fibroblasts as deSince TGF-@is known to be released scribed in an inactive form, the conditioned medium samples were assayed before and after transient acidification to activate latent TGF-@. TGF-@was activated by adding 1 N HCI to the samples to a final pH of 2. After I h incubation the pH was returned to 7.4 by the addition of 1 N NaOH. In experiments in which acid-activated and nonactivated samples were assayed, equal amounts of 1 N NaCl were added to the nonactivated samples and to all TGF-@standard dilutions to avoid differences in volume and osmolarity. To examine the chemotactic activity of the conditioned medium, aliquots of conditioned medium were added either directly to the lower compartment of the chemotaxis chambers or transiently acidified and then added to the chambers. Previous experiments had shown that transient acidification of unconditioned culture medium had no effect on the migration of osteoblastlike cells.

both cell fractions are likely to contain a small percentage of cells unrelated to the osteoblastic lineage. In addition, we therefore examined the response of a clonal osteoblastlike cell population to [email protected] used the ROS 17/2.8 rat osteosarcoma cells, which have several characteristics of osteoblastlike cells. TGF-@ also stimulated the migration of these cells at concentrations similar to those that had been observed for the preosteoblastic and mature osteoblasts (Fig. 1). Maximal migration of all three cell types was observed at concentration gradients of 5-15 pg/ml of TGF-@The dose-response curves were bell shaped, and migration was reduced to background levels at concentration gradients exceeding 100 pg/ml of TGF-@ (Figs. 1 and 2). We used platelet-derived TGF-@ in most of our experiments. However, recombinant TGF-@, had a similar

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FIG. 1. Comparison between the chemotactic activity of human platelet TGF-@, for osteoblastlike cells from fetal rat calvariae (fraction 1, 0;fractions 3-5, W) and ROS 17/2.8 osteosarcoma cells ( 0 ) . Values are means f SEM for four filters.

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RESULTS TGF-@stimulated the migration of fraction 3-5 calvarial cells in a dose-dependent manner (Fig. I). These cells originate from the cell layer next to the bone surface and are enriched for cells with a mature osteoblastic phenotype.'ls) TGF-@ also induced the migration of fraction 1 calvarial cells (Fig. I). These cells are released from the outermost layers of the periosteum during the collagenase digestion and probably represent preosteoblastic cells. However,

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FIG. 2. Chemotactic response of fetal rat calvarial cells (fractions 3-5) to purified TGF-@, (0),purified TGF-@, (o), and recombinant TGF-@, (W). Values are means + SEM for four filters.

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CHEMOTAXIS OF OSTEOBLASTS TO TGF-(3 chemotactic activity for osteoblastlike cells compared to TGF-P purified from human platelets (Fig. 2). Thus, it seems unlikely that the chemotactic activity of platelet-derived TGF-P resulted from impurities in the TGF-P preparation. Two forms of TGF-(3 have been isolated from bone matrix that share a 70% sequence homology but differ in their binding capacity for the various TGF-(3 recept o r ~ .I9-'(l) ~ Although TGF-0, and TGF-P2 may therefore elicit different biologic responses in bone cells, we observed no difference between the chemotactic activity of TGF-(3, and TGF-P,. Both forms of TGF-/3 stimulated the migration of osteoblastlike cells at equivalent concentrations (Fig. 2). Zigmond-Hirsch checkerboard analysis revealed that TGF-(3 affected the migration of osteoblastlike cells only when present in higher concentrations in the lower compartment of the chemotaxis chasmbers (Fig. 3). This indicates that TGF-P induces chemotactic migration, not chemokinesis of osteoblastlike cells."" Osteoblastlike cells are known to release latent TGF-P into the culture medium.('" This latent form of TGF-P can be activated in vitro by transient acidification. T o determine whether latent TGF-(3 has chemotactic activity, we assayed osteoblast-conditioned medium of known latent TGF-6 concentration before and after acidification. We found no chemotactic activity in conditioned medium prior to acidification. However, chemotactic activity was readily observed in conditioned medium after acidification (Fig. 4). Unconditioned medium had no chemotactic activity. Our results therefore suggest that only active TGF-P has chemotactic activity. The observed chemotactic responses of osteoblastlike cells to TGF-8 occurred within 4 h after the addition of TGF-(3. T o examine whether the cells might still be responsive after a prolonged incubation with TGF-13, we preincubated osteoblastlike cells with different concentrations of TGF-(3 for 48 h before the chemotaxis assay. Preincubation with TGF-(3 in concentrations from 10 pg/ml to 1 ng/ ml completely abolished the chemotactic response of osteoblastlike cells to TGF-6 (Table 1).

DISCUSSION TGF-(3 is a stimulator of osteoblastic bone formation in vitro and in vivo. We now show that TGF-(3 is also a chemoattractant for osteoblastlike cells. Maximal stimulation of chemotaxis was observed at concentrations as low as 5-15 pg/ml, regardless of whether TGF-0, or TGF-(3, was used. This makes chemotaxis probaby the most sensitive effect of TGF-0 on osteoblastlike cell function. Primary cultures of calvarial cells may contain a small percentage of nonosteoblastic cells. TGF-(3 is chemotactic for rnonocytes',") and fibroblastic cells('s z b ) at concentration gradients comparable to those observed for osteoblastlike cells. This raises the question of whether some of the cells that migrated into the filter in response to TGF-P may have been cells of other cell species. However, most

TGFp C O N C E N T R A T I O N I N UPPER CHAMBER ( p g m l ) 0

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FIG. 3. Effect of varying concentration gradients of purified TGF-(3, on migration of fetal rat calvarial cells (fractions 3-5). The vertical columns indicate the concentration of TGF-P in the upper compartment; the horizontal rows indicate the concentration in the lower compartment. Note that there is no migration in the absence of a concentration gradient between the upper and lower compartments (values between the parallel lines). Values are means * SEM for three filters.

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FIG. 4. Chemotactic response of fetal rat calvarial cells (fractions 3-5) to 48 h conditioned medium from fetal rat calvarial cells. Aliquots of conditioned medium containing 10 pg/ml of latent TGF-P were added to the chemotaxis chambers. (B) The medium was added untreated; (C) The medium had been transiently acidified to pH 2 to activate latent TGF-0. Unconditioned DMEM medium (A) served as control. Values are means SEM for four filters. of these cells stained positively for alkaline phosphatase activity, a key marker of the osteoblast phenotype (not shown), which makes it unlikely that these cells were unrelated to the osteoblastic lineage. Further proof that TGF-P is chemotactic for osteoblastlike cells is added by the fact that osteoblastlike ROS 1 7 / 2 3 cells had a chemotactic re-

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TABLE1. INHIBITIONOF TGF-(3-INDUCEDCHI-MO I AXI\ HV FETALRATCALVARIAL CELLS W I l H TGk-p,

PREINCUBATION OF

TGF-0, concentrations in upper compartment (Pdml) Experiment 1, pretreatment with 10 pg/ml TGF-(3, 0 5 10 15 40 100

Experiment 2, pretreatment with 100 pg/ml TGF-(3, 0 5 10 15 40 100 Experiment 3, pretreatment with lo00 pg/ml TGF-(3, 0 5 10 25 100

Cells migrated per 30 fields Untreated

Pretreated with TGF-P,

9.5 f 0.5 14.5 i 1.4a 14.2 f 1.la 22.6 f 2.4b 10.3 f 4.6 11.2 f 2.4

8.5 f 0.4 9.0 f 2.0 7.8 f 2.4 9.1 f 1.4 7.6 f 0.9 8.5 f 3.4

8.0 11.2 27.4 10.5 14.0 10.4

f

1.2 1.0 5.8a 3.2 5.4 1.4

9.4 5.4 9.5 7.8 8.8 8.6

6.6 13.3 26.0 8.2 6.7

2.5 3.5 f 1.6c f 1.5 f 0.5

7.5 8.7 8.0 7.7 7.0

f f

f f f

f

f

f f f f f f

f f

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2.3 2.6 0.6 2.0 2.7 2.8

0.6 1.6 0.9 2.7 1.3

< 0.01. < 0.05. cp < 0.001 ap

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sponse to TGF-P that was comparable to that of osteoblastlike calvarial cells. TGF-/3 is released from osteoblastlike cells in vitro as a latent, high-molecular-weight complex."') Activation of latent TGF-P can be accomplished by transient acidification.(z8) All the currently known effects of TGF-P on osteoblastlike cells seem to require previous activation of TGF-6. However, there is evidence that growth factors may exert chemotactic activity independently of their effects on other cell functions and their structural integrity.('pl We therefore examined whether latent TGF-P is chemotactic for osteoblastlike cells. Our results show that conditioned medium from osteoblastlike cells containing the latent form of TGF-P had no chemotactic activity unless it had been transiently acidified. Therefore, our data strongly suggest that only active TGF-@is a chemoattractant for osteoblastlike cells. TGF-P failed to stimulate the migration of calvarial cells that had been preincubated with TGF-P before the chemotaxis assay, indicating that its chemotactic activity may be only transient. Following the incubation with TGF-P, the cell layer was extensively washed with phosphate-buffered

saline and incubation was continued for 12 h with medium that did not contain TGF-(3. The cells were then detached with trypsin, which is known to rapidly degrade active TGF+?.(") It is therefore unlikely that any active TGF-P was still attached to the cell surface. TGF-(3 is known to increase the synthesis of cell adhesion proteins and the number of cell adhesion proteins and the number of cell adhesion protein receptors in fibroblastic cells at picomolar concentration^.^^^-^^) It is possible that these effects interfere with the chemotactic effects of TGF-P, but further studies are necessary to identify the exact mechanisms of the effects of TGF-P on cell movement. TGF-P may account for the chemotactic activity that has been observed in conditioned medium from resorbing bone cultures.[171 However, TGF-/3 may not be the only chemotactic agent that is released o r activated during bone resorption. Previous studies have shown that bone matrix proteins and fragments, such as osteocalcin and collagen, are also chemoattractive for bone cells. ( 1 7 , 3 4 3 1 In addition, other growth factors, such as platelet-derived growth factor and fibroblast growth factor, have been reported to be chemotactic for fibroblastic cells.(26.36) It is therefore possi-

CHEMOTAXIS OF OSTEOBLASTSTO TCF-0 ble that TGF-P acts in concert with one or several of these factors to attract osteoblastlike cells to the sites of bone remodeling.

ACKNOWLEDGMENTS This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We thank Dr. D.R. Twardzik and Dr. A.F. Purchio (Oncogen, Seattle, WA) for the recombinant TGF-@,. The authors are grateful to Dr. A. Habenicht and Dr. R. Gronwald for many helpful comments on the manuscript.

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830 31. lgnotz RA, Massague J 1987 Cell adhesion protein receptors as targets for transforming growth factor-8 action. Cell 51: 189- 197. 32. Heino J , Ignotz RA, Hernler ME, Crouse C, Massague J 1989 Regulation of cell adhesion receptors by transforming growth factor-0. Concomitant regulation of integrins that share a common 0, subunit. J Biol Chem 264:380-388. 33. Ignotz RA, Heino J, Massague J 1989 Regulation of cell adhesion receptors by transforming growth factor-o. Regulation of vitronectin receptor and LFA-I. J Biol Chem 264: 389-392. 34. Mundy GR, Poser JW 1983 Chemotactic activity of the gamma-carboxyglutamic acid containing protein in bone. Calcif Tissue Int 35164-168. 35. Lucas PA, Price PA, Caplan A1 1988 Chemotactic response of mesenchymal cells, fibroblasts and osteoblast-like cells to

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J. Pfeilschifter, M .D. Deparrmenl of Medicine I, Endocrinology University of Heidelberg Luisenstrasse 5 0-6900 Heidelberg, Federal Republic of Germany Received for publication August 19, 1989; in revised form January 3, 1990; accepted February 14, 1990.

Chemotactic response of osteoblastlike cells to transforming growth factor beta.

Transforming growth factor beta (TGF-beta) has multiple effects on bone cell metabolism in vitro but its exact role in bone remodeling still needs to ...
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