JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 7, 1990 Mary Ann Liebert, Inc., Publishers

Insulinlike Growth Factor 1 Regulates mRNA Levels of Osteonectin and Pro-a,(I)-Collagen in Clonal Preosteoblastic Calvarial Cells DANIEL THIEBAUD, KONG WAH NG, DAVID M. FINDLAY, MICHAEL HARKER, and T. JOHN MARTIN

ABSTRACT A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-a,(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-a,(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10-9-10-7 M) or insulin (10-9-10-6M) on mRNA species for osteonectin or pro-a,(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5‘ flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by lo-* and M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor (TGF-0, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-cyl(I)-collagen in a dose-dependent manner. Transforming growth factor a (TGF-a) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-a,(I)-collagen mRNA. The positive stimulatory effect of both IGF-1 and TGF-0 provides insights on the regulation of bone matrix proteins and suggests a major role of these growth factors in the remodeling process of bone.

INTRODUCTION STEONECTIN is a 32 kD glycoprotein secreted by osteoblasts in vivo and by osteoblastlike cells in culture, representing a significant proportion of the noncollagenous bone matrix Osteonectin has a high affinity for calcium, hydroxyapatite, and type I collagen and is thought to be involved in mineralization of bone extracellular matrix, although its exact role has not yet been defined. w UMR-201 is an established nontransformed rat clonal

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cell line derived from neonatal calvariae, with phenotypic characteristics of preosteoblastic cells. ( & ) UMR-201 cells produce type I collagen and other bone matrix proteins consistent with the osteoblastic phenotype. UMR-201 cells differentiate on treatment with retinoic acid, showing an increase in alkaline phosphatase activity and mRNA expression. ( w UMR-201 cells possess specific high-affinity receptors for growth hormone (GH, unpublished observations), and other osteoblastlike cell lines have been shown to possess receptors for insulin, GH, or IGF-l,(9-11)and, further-

Department of Medicine, University of Melbourne and St. Vincent’s Institute of Medical Research, St. Vincent’s Hospital, Fitzroy, 3065 Victoria, Australia.

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more, to produce IGF-I .(9.11.13) According to the somato- a-MEM containing 10% FCS. The medium was changed medin hypothesis, GH promotes early differentiation of 48 h later to a-MEM with 2% (v/v) vitamin A-depleted target cells and systemic, and locally secreted IGF-1 (and (ultraviolet-irradiated) FCS containing the appropriate doses of each treatment with or without added retinoic IGF-2) promotes their m ~ l t i p l i c a t i o n . ( ~ ~However, -~~) IGF-1 was recently shown to stimulate bone collagen syn- acid. UMR-201 cells are not viable for 24 h in the absence thesis in rat calvarial cells, indicating direct effects on dif- of serum. 1 6 ) Unless otherwise specified, all treatments were ferentiation independent of cell replication stimula- for 24 h. For the transfection experiments, UMR-201 cells t i ~ n . ' ~ Since ~ . ~ 'IGF-1 ~ is secreted locally in response to were subcultured into 9.6 cmz six-place multiwell dishes in many hormones or factors (GH, parathyroid hormone, a-MEM containing 10% (v/v) FCS. and ~,-mi~r~globulin),~'~~~*~'~~ its role may be critical in bone remodeling. Similarly, transforming factor p, a 24 kD protein dimer Northern blot analysis that modulates proliferation and differentiation in a variTotal RNA was was isolated with guanidinium thiocyais secreted by osteoblast cells, ety of cells and tissues,(z0~211 nate according to the technique described by Chomczynski particularly after exposure to osteolytic hormones.(22.231 and Sacchi,'z81separated in a 1.5% (w/v) agarose-formalTGF-P stimulates type I collagen production and steady- dehyde gel, and transferred to nylon filters. (29) The nylon state mRNA levels in fetal rat c a l ~ a r i a e ( ~and ~ ~ 'was ~ ) also filters (Amersham Australia Pty., Ltd.) were hybridized shown recently to increase osteonectin mRNA levels in the overnight in 50% (v/v) formamide, 0.6 M NaCI, 0.018 M rat osteoblastic cell line ROS 17/2.8'261as well as other NaH,P04, 0.03 M Na2HP04,0.005 M EDTA, Denhardt's bone matrix proteins.(26~27) solution (200 mg/liter polyvinylpyrrolidone, 200 mg/liter The respective effects of GH, IGF-1, and insulin on bovine serum albumin, and 200 mg/liter Ficoll W ) ,0.5% bone matrix proteins are not yet clear. The purpose of the (w/v) sodium dodecyl sulfate (SDS), 1% skim milk powpresent study was to investigate the effects of these ago- der, and denatured salmon sperm DNA (200 mg/ml) at nists in the UMR-201 cell line and to study the regulation 42°C and washed in 0.1-fold SSC (standard saline citrate of mRNA for osteonectin and pro-a,(I)-collagen. Treat- contains 150 mM NaCl and 15 mM sodium citrate, pH 7.0) ments with transforming growth factors a and p were also with 0.1% SDS at the same temperature. cDNA probes studied in the same osteoblastic model. were nick translated with [cx-~'P]~CTP to a specific activity

MATERIALS AND METHODS Materials a-Modified minimum essential medium (a-MEM) was purchased from Flow Laboratories, Australasia, Pty., Ltd. (Mt. Waverly, Victoria, Australia). Fetal calf serum (FCS) was a product of Commonwealth Serum Laboratories (Melbourne, Victoria, Australia). All-trans-retinoic acid was purchased from Sigma Chemical Co. (St. Louis, MO). Human recombinant IGF-1 was a generous gift from CibaGeigy, Ltd. (Basel, Switzerland) in collaboration with Chiron Corp. (Emeryville, CA). Recombinant growth hormone (Somatonorm-R) was purchased from Kabi Vitrum, Ltd. (Stockholm, Sweden). TGF-a and TGF-@were generous gifts from Genentech, Inc. (San Francisco, CA). Insulin (Actrapid-R) was purchased from Novo-CSL Pty., Ltd. (Melbourne, Victoria, Australia). [ U - ~ ~ P ] ~ C(specific TP activity 3000 Ci/mmol) was purchased from Amersham-Australia Pty. (Sydney, New South Wales, Australia). Molecular biology-grade agarose was obtained from International Biotechnologies, Inc. (New Haven, CT). All other reagents were of analytic grade obtained from standard suppliers.

Cell culture UMR-201 cells were routinely grown in a-MEM containing 10% (v/v) fetal calf serum (FCS) as described previously.(6)Incubation was carried out at 37°C and equilibrated with 5% CO, in air. For Northern blot analysis, UMR-201 cells were subcultured into 175 cm' flasks in

of 1 x lo9 dpm/pg DNA according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, FRG). The cDNA probe for osteonectin was a plasmid containing a 1.5 kb fragment of the bovine osteonectin cDNA.'~']cDNA for pro-al(I)-collagen was a 1.6 kb insert containing the triple helical region of pro-al(I)-co11agen.(31) The quantity of RNA for each treatment (lane on the gel) was assessed with reference to ethidium bromide staining of the ribosomal bands.

D N A transfection Cells were plated at a density of 3.0 x lo5cells per well in 9.6 cm' six-place multiwell dishes. The next day, cells were transfected with 1 pg plasmid DNA by the calcium phosphate method with removal of DNA after 6 h incubation with cells.(3z1A glycerol shock (1 minute) was performed, and the cells were washed three times in a-MEM and incubated in a-MEM with 2% (v/v) FCS. The next day, cells were treated with various agents for 24 h. Cells were then scraped in phosphate-buffered saline (PBS) and pelleted in an Eppendorf centrifuge. The cell pellet was sonicated in 100 ml of 0.2 M Tris-HCI, pH 8.0, heated at 60°C for 10 minutes, and centrifuged for 4 minutes to remove cell debris. Chloramphenicol acetyltransferase activity was assessed by incubation of cell supernatant with 1 pM acetyl-CoA (Pharmacia LKB Biotechnology Inc.) and 0.125 pCi of [L4C]chloramphenic~l at 37°C overnight followed by thin-layer chromatography on silica gel [thinlayer chromatography (TLC) plates from Sigma Ltd.] in 9 5 5 chloroform-methanol as described previously.(30,33) The data were quantitated by scraping the TLC plates to determine I4C-labeled acetylated chloramphenicol products.

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IGF-1 EFFECTS ON UMR-201CELLS Osteonectin-CAT construct pGSVo ON RP (a gift from M. Young, NIDR, NIH, Bethesda, MD) was constructed by ligating a 514 bp RsuI/Pstl fragment between -504 and - 10 bp of the bovine osteonectin gene into the PstI site of pGSVo-CAT, a fusion plasmid comprising pGEM 4 2 and a promoterless fragment (2830-5001) of pSV2 CAT.'3o' A promoterless construct (pGSVo-CAT) was transfected to control for CAT expression in the absence of a genomic promoter. As a measure of transfection efficiency, a construct comprising the actin promoter 5' to the CAT gene was transfected. Cells were treated with agonist for 24 h after transfection.

Statistical analysis Data are expressed as the mean f SEM. Statistical differences were analyzed using Student's t-test. RESULTS Northern blot analysis of mRNA for osteonectin and pro-a,(I)-collagen indicated that treatment with as little as M IGF-1 increased these species of mRNA. Increases were consistently seen at and M IGF-1 (Fig. 1). A time course of M IGF-1 treatment showed that the increase in both pro-a,(l)-collagen and osteonectin mRNA was apparent after 12 h and was maximal at 18 and 24 h (Fig. 2). In contrast, neither insulin (10-9-10-7 M) nor GH ( 10-9-10-7 M) significantly increased mRNA levels for osteonectin or pro-a,(I)-collagen in repeated experiments (Fig. 3). A time course of GH treatment was performed from 6 to 48 h and did not reveal any significant changes for either mRNA (data not shown). To determine whether the effects of IGF-1 on osteonectin mRNA levels were exerted at a transcriptional level, UMR-201 cells were transfected with the ON-CAT construct. Figure 4 displays a transfection experiment with treatment with IGF-1 at and M. In each of three experiments, the transcriptional activity of the ON-CAT construct was increased by 24 h of treatment with IGF-1 at M. In the experiment shown here, increases and were 235 and 270%, respectively (mean of three observations; p < 0.001 in comparison with control). These data have been normalized with respect to cell number since there was a significant increase in cell number, reaching 16.5% with lo-' M and 23.5% with M IGF-1. The results from transfection experiments correlate with the increase in mRNA as determined by Northern blot analysis and suggest that the stimulatory effect on mRNA occurred at least partially at the transcriptional level. In contrast, the transcriptional activity of the osteonectin promoter-CAT construct was not affected by treatment with GH at 10" and M in comparison to a control with the ON construct alone. Figure 5 summarizes these data and gives a comparison of the quantitative data of both IGF-1 and GH transfection experiments. The marked increase in acetylated chloramphenicol derivatives after treatment with IGF-1 contrasts with the absence of effect of GH. It was of interest to compare the results of IGF-1 experi-

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FIG. 1. Effect of IGF-1 on mRNA for osteonectin and pro-a,(I)-collagen in rat UMR-201 cells. UMR-201 cells were grown to confluence in monolayer cultures and treated for 24 h with doses of IGF-1 ranging from lo-'' to M. Total RNA was extracted from the cells, and each lane was loaded with 20 pg. RNA was transferred to nylon filters hybridized with a nick-translated 3zP-labeled 1.5 kb cDNA of bovine osteonectin (left) and a 1.6 kb cDNA for rat pro-a,(I)-collagen (right), as described in Materials and Methods, and autoradiographed. Each upper panel shows the ribosomal bands stained with ethidium bromide. Dots and arrows indicate the location of the ribosomal transcripts. These results have been confirmed in three similar experiments.

ON w

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CONT ICF] CONT IGF] CONT IGFl

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18h

24h

FIG. 2. Effect of lo-' M IGF-1 (time course study) on mRNA for osteonectin (ON) and pro-a,(I)-collagen in UMR-201 cells. UMR-201 cells were grown to confluence in monolayer cultures and treated with M IGF-1 for 12, 18, or 24 h, as indicated. Total RNA (20 pg) was loaded in each lane. RNA was transferred to nylon filters hybridized with a nick-translated 32P-labeled 1.5 kb cDNA of bovine osteonectin and a 1.6 kb cDNA for rat pro-al(I)collagen, as described in Materials and Methods. Each upper panel shows the ribosomal bands stained with ethidium bromide. Dots and arrows indicate the ribosomal transcripts.

THIEBAUD ET AL.

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FIG. 3. Effect of GH and insulin on mRNA for osteonectin (ON) and pro-a,(I)-collagen from UMR-201 cells. UMR-201 cells were grown to confluence in monolayer cultures and treated for 24 h with doses of insulin ranging from to M and GH from to lo-' M, as indicated. Total RNA (20 pg) was loaded in each lane. RNA was transferred to nylon filters hybridized with a nick-translated '*P-labeled 1.5 kb cDNA of bovine osteonectin and a 1.6 kb cDNA for rat pro-a,(I)-collagen, as described in Materials and Methods. Each upper panel shows the ribosomal bands stained with ethidium bromide. Dots and arrows indicate the ribosomal transcripts.

ACETYLATED DERIVATIVES

CHLORAMPHENIC: o L

ACTIN

Control

ONconstruct alone

ON const*lGFl

IO-'M

ON const*lGF 1 1O-'M

FIG. 4. Transcriptional activity of ON-CAT constructs transfected into UMR-201 cells and treated for 24 h with IGF-1. UMR-201 cells were seeded at a density of 3.0 x lo5 cells per well in six-place multiwells and transfected the next day with 2 pg pGSVo ON RP using the calcium phosphate technique as described in Materials and Methods. Treatments with lo'* and lo-' M IGF-1 were started 24 h after transfection and incubation continued for a further 24 h. CAT activity, measured in terms of production of acetylated derivatives of ['4C]chloramphenicol, is shown in the autoradiograph. The promoterless pGSVo CAT was transfected as a negative control. As a positive control, a CAT construct comprising the actin promoter was transfected (left). This experiment has been conducted three times with similar results.

765

IGF-1 EFFECTS ON UMR-201 CELLS

COUNTS/MIN 6ooo

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Control l GF l lo.nM IGF/w7M

Control GH IOaM

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GH 1W7bl

FIG. 5. Summary of the transcriptional activity of osteonectin-CAT constructs transfected into UMR-201 cells treated with IGF-1 and GH. UMR-201 cells were transfected as in Fig. 4 and treated with IGF-1 (lo-' and M) and GH (low8and M) for 24 h. Quantitation of CAT activity was made by scraping TLC plates to determine the amounts of ''C-labeled acetylated chloramphenicol products. Results are counts per minute f SD for three replicate wells (*p < 0.001 compared with control). The promoterless pGSVo CAT was transfected as a negative control and yielded 468 + 34 counts per minute.

FIG. 6. Effect of TGF-a and TGF-/3 mRNA on osteonectin (ON) on pro-a,(I)-collagen from rat UMR-201 cells. UMR-201 cells were grown to confluence in monolayer cultures and treated for 24 h with doses of TGF-CYranging from 0.1 to 10.0 ng/ml (left), TGF-0 from 0.1 to 2.5 ng/ ml, and retinoic acid (lo+ M), as indicated. Total RNA was extracted from the cells, and each lane was loaded with 20 pg. RNA was transferred to nylon filters and hybridized with 32P-labeled cDNA for osteonectin and proa,(I)-collagen. Each upper panel shows the ribosomal bands stained with ethidium bromide. Dots and arrows indicate the ribosomal transcripts. Numbers on the bottom line designate the amounts of growth factor (ng/ml) added to the cultures for 24 h.

ments with the growth factors TGF-a and TGF-0, both of which have well-documented effects on bone.(24-z7.34)tion of osteoblastic cells by increasing levels of bone matrix TGF-a had no effect on the expression of either species of protein mRNA. This is in good agreement with the general mRNA in UMR-201 cells when given at doses ranging anabolic effect of somatomedins in tissues"0.14.'5)and profrom 0.1 to 10 ng/ml in repeated experiments (Fig. 6). vides further insight into the possible role of IGF-1 in bone Shown in the same figure is the effect of TGF-/3 at doses formation. Recent s t u d i e ~ ( ~ have , ' ~ ) shown that IGF- 1 ranging from 0.1 to 2.5 ng/ml. These concentrations of mRNA is expressed in osteoblasts and that its level is stimTGF-0 clearly increased mRNA for osteonectin and pro- ulated by PTH, estradiol, GH, and µglobulin (an a,(I)-collagen (as did retinoic acid at M, included as a analog of BDGF isolated from bone matrix and calvarial These observations together suggest an positive control) in UMR-201 cells, consistent with their cells).('2~'8~'9~22~36) previously reported activity to increase osteonectin important role of IGF-1 in the bone-remodeling process and in the linkage between bone resorption and formation. mRNA'8,26)in these and other cells. In contrast, neither insulin nor GH showed any stimulatory effect on osteonectin or pro-a,(I)-collagen mRNA DISCUSSION levels or on the transcriptional activity of the ON-CAT construct. Therefore, GH does not appear to act directly This study shows that treatment with IGF-1 increases upon these differentiated functions in the UMR-201 osteoosteonectin and pro-a,(I)-collagen mRNAs in the UMR- blastic cell line, which nevertheless has been shown to pos201 preosteoblastic cell line. A parallel increase in the tran- sess specific high-affinity receptors for GH (unpublished scriptional activity of an ON promoter-CAT construct sug- observations). Lack of a direct effect of GH on the osteogests that IGF-1 may increase osteonectin mRNA levels by blast [with respect to osteonectin and pro-a,(I)-collagen increasing the transcriptional level of the osteonectin gene. expression] is consistent with previous observations in calThese results clearly illustrate an increase in these bone varial ~ e l l s ( l and ~ . ~confirms ~) that most of the anabolic efmatrix protein mRNA species by IGF-1 in UMR-201 cells. fects of GH are indirect, via IGF-1 and perhaps IGF-2,'38) A slight stimulatory effect on cell replication by IGF-1 was according to the original somatomedin hypothesis.('4.'5.39) also observed. Recently, McCarthy et a1.('61reported an The apparent absence of an anabolic effect of insulin in increase in collagen production and pro-a,(I)-collagen UMR-201 cells may reflect the early stage of osteoblastic mRNA after IGF-1 treatment in rat calvarial cells that was phenotype, consistent with previous showing also independent of the increase in cell numbers. There- different patterns of response to insulin in fetal rat bones fore, IGF-1 not only has the initially reported effect on cell depending on the cellular localization. A slight overlap of replication(35)but also promotes the differentiated func- IGF-1 and insulin biologic activities due to the similarity of

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their receptor structures may be expected at nonphysiologic concentration but did not occur in our model, except for a slight, inconsistent increase in pro-a,(I)-collagen with M insulin. TGF-a is known to act through EGF receptors, which are abundant in most osteoblast cells, and was reported to have inhibitory effects on bone formation and collagen We found no significant changes in mRNA for osteonectin or pro-a,(I)-collagen in UMR-201 cells after 24 h treatment at doses ranging from 0.1 t o 10 ng/ml. In confirmation of previous r e p o r t ~ ' ~ ~in. ~other '~ osteoblastic cell lines, TGF-(3 markedly increased mRNA for osteonectin and pro-a,(I)-collagen. This confirms in this preosteoblastic model that TGF-(3 has a clear differentiation-promoting effect by increasing the expression of bone matrix proteins. Since TGF-6 has been shown to be released from bone matrix after exposure of bone to resorptive h o r m ~ n e s , ' ~it ~suggests ~ ~ ~ ' a role of TGF-(3 as a coupling factor (like IGF-1) in the remodeling process. At least in our model the concomitant increase in mRNA for osteonectin and pro-a,(I)-collagen, both of which are major matrix proteins, is consistent with an anabolic effect of TGF-6 on bone formation. Osteonectin is a relatively abundant bone matrix protein secreted by cells possessing osteoblastic features. Osteonectin has a strong affinity for collagen and calcium and may be involved in the processes of mineralization, although it is also present in nonmineralized tissues. f 2 . 5 ) Some parallels in the regulation of osteonectin and pro-a,(I)-collagen mRNA are shown by the similar response to IGF-1 and TGF-(3 in the present study and may suggest a relationship between these two matrix proteins and a role for osteonectin in bone formation, as already In conclusion, the UMR-201 line is a useful model for the study of osteoblast differentiation. The UMR-201 cells express some but not all the phenotypic characteristics generally associated with more mature osteoblasts, such as osteopontin, matrix gla protein, osteonectin, and proa,(I)-collagen, the latter two shown in the present work to be responsive to regulation by extracellular modulators. Alkaline phosphatase is not expressed constitutively but can be induced by treatment with retinoic acid, a well-recognized differentiating agent. A clonal cell line with very similar characteristics has recently been obtained from fetal rat c a 1 ~ a r i a e . I The ~ ~ ) UMR-201 cells should therefore be considered preosteoblasts committed to the osteoblast lineage.

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Address reprint requests to: Prof. T.J. Martin St. Vincent’s Institute of Medical Research 41 Victoria Parade Fitzroy, 3065 Victoria, Australia Received for publication August 29, 1989; in revised form December 8, 1989; accepted January 26, 1990.