Original Contributions

THE TRANSCRIPTIONAL CONTROL OF TGF-f.3 IN HUMAN OSTEOBLAST-LIKE CELLS IS DISTINCT

FROM THAT OF IL-lf3

Karen Merry and Maxine Gowen

Transforming growth factor 6 (TGF-P) and interleukin 1 (IL-l) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-P and IL-lfi was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-P but not IL-lp mRNA. Treatment of the cells with the systemic hormones 1,25dihydroxyvitamin D, [1,25-(OH),D,] (lo-” M) and parathyroid hormone (lo-’ M) induced an increase in TGF-P mRNA but failed to stimulate the production of IL-l-p mRNA. Retinoic acid (lo-* M) had no effect on either mRNA species. The cytokines IL-la (200 pg/ml), tumour necrosis factor cw (TNF-cw) (17 &ml) and bacterial lipopolysaccharide (LPS) (500 rig/ml) stimulated the production of IL-Q3 mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-P mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (lOma M) resulted in the suppression of both TGF-P and IL-lp mRNA. However, when the stimulating agent 1,25-(OH),D, was added in conjunction with hydrocortisone the mRNA expression of TGF-P mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-la to hydrocortisone-treated cells resulted in no increase in IL-lfi mRNA. In-situ hybridization demonstrated both TGF-P and IL-lp mRNA at the cellular level. These data show that human osteoblasts express TGF-P and IL-lp and that they are differentially modulated by a distinct group of agents. This may reflect the contrasting roles of these cytokines in the control of the bone remodelling cycle.

It is characteristic of bone metabolism that remodelling occurs at discrete sites in the skeleton. At these loci a period of osteoclastic resorption is followed by new osteoblast-mediated bone formation. The local control of bone remodelling is probably mediated at least in part via cytokines and growth factors present in the bone microenvironment. Two

From the Bath Institute for Rheumatic Diseases, and University of Bath, Bath, U.K.

Address correspondence to: Dr M. Gowen, Bath Institute for Rheumatic Diseases, Trim Bridge, Bath BAl 1HD.

Received 5 August 1991; revised and accepted for publication 23 January 1992

0 1992 Academic Press Limited 1043-4666/92/030171+09 $05.00/O

KEY WORDS: transforming growth factor P/interleukin lpkranscription

cytokines with potent and often opposing actions in bone are transforming growth factor p (TFG-P) and interleukin 1 (IL-l).

TGF-P is a 25 kDa dimeric polypeptide, first identified by its ability to cause a phenotypic transformation of rat fibroblasts.’ It has many actions in different cell types* and its actions in bone, where it is deposited in the extracellular matrix in a latent form,3 are complex. Activation in vitro can be achieved by limited proteolysis or acidification, and Oreffo et aL4 demonstrated that isolated osteoclasts are able to activate latent TGF-P, presumably in the acidic microenvironment under their ruffled border. TGF-P is a potent stimulator of osteoblast proliferation in vitro, although this seems to be a biphasic effect and dependent on cell density.5 TGF-P also affects the deposition of extracellular matrix: it upregulates transcription and translation of type I

CYTOKINE, Vol. 4, No. 3 (May), 1992: pp 171-179 171

I IL I Merry and Gowen CYTOKINE, Vol. 4, No. 3 (May 1992: 171-179)

collagen, fibronecti# and osteonectin7 which results in an increase in matrix deposition of these proteins. In addition to providing a stimulus for matrix biosynthesis by osteoblasts, it has been suggested that active TGF-l3 is chemotactic for these cells.8 Its role in bone resorption remains controversial since it has been shown to inhibit,9 and promotelO bone resorption in different model systems. Since active TGF-P may be liberated from the matrix by osteoclastic resorption, and has such a profound effect on osteoblast metabolism, it is an ideal candidate as a ‘coupling factor’ between bone resorption and formation. IL-l was one of the earliest cytokines to be identified as having a regulatory role in bone cell function, and remains the most potent bone resorbing agent so far described. l1 Its identification as an osteoclast activating factor12 and its implication in a number of diseases such as myeloma13 and osteoporosis14 cause it to remain an important factor in bone remodelling. Anabolic effects of IL-l in bone metabolism have also been documented. IL-l stimulates osteoblastic proliferation15 and increases DNA and collagen synthesis in organ cultured bones16 although inhibitory effects have also been demonstrated. l7

Preliminary reports have shown that human osteoblast-like cells are capable of producing both TGF-P18 and IL-lp19,20 although little is known about the control of this expression. Studies on cytokine release from osteoblasts of various species show this to be regulated in a species or cell line specific fashion. For example, the production of IL-6 is stimulated by PTH in neonatal mouse calvarial cells*l and ROS 17/2.8 cells,z2 but is unaffected by PTH in human osteoblasts.22 Our results verify the production of TGF-l3 and IL-lp from human osteoblasts and describe how the expression of TGF-P and IL-ll3 mRNA is modulated by a range of osteotropic hormones and cytokines implicated in signalling between cell types during human bone remodelling. We have used human cells derived by outgrowth from trabecular bone obtained at surgery. These cells have been widely used for many years as a model of human osteoblasts, and most recently to study cytokine production.23

RESULTS

We examined the effect of a-variety of cytokines and growth factors on the levels of mRNAs for TGF-l3 and IL-ll3 in human osteoblast-like cells. The cells derived from twelve patients constitutively expressed TGF-l3 mRNA when cultured in the presence of 10% FCS. Both TGF-l3 and 11-l mRNA expression was demonstrated at the cellular level using in-situ hybridization (Fig. lA, B). Sense transcripts were

used as negative controls to assess any non-specific binding (Fig. 1C).

TGF-l3 mRNA was increased c. three-fold after culturing the cells for 8 hours with 1,25-(OH),D, (lo-* M) (Fig. 2). Retinoic acid (1O-8 M) is known to act as a differentiating agent in certain osteoblastic cell lines. However, it had no effect on TGF-P mRNA expression (Fig. 2, lane 2). The increase in TGF-l3 mRNA using 1,25-(OH),D, was detectable after 6 hours of treatment reaching a peak at 8 hours; after 24 hours the level decreased to basal expression (not shown).

Parathyroid hormone was an effective stimulus for TGF-l3 mRNA accumulation. Treatment of cells with 1O-7 M PTH for 6 hours resulted in a nine-fold increase in TGF-P mRNA (Fig. 3). The effect was apparent after 6 hours, but the mRNA levels returned to control levels after 12 hours. PTH was shown to be at least three-fold more effective in stimulating TGF-l3 mRNA expression than 1,25-(OH),D, (lop8 M) using the same donor cells (Fig. 3).

It has been reported that TGF-P is able to autoregulate its own expression in a tumour cell line.24 Cells treated with 100 pM TGF-P in serum free culture showed a small increase in TGF-l3 mRNA expression after 6 hours (Fig. 4). The lower dose of 10 pM, although capable of stimulating proliferation in these cells (results not shown), did not increase TGF-l3 expression.

In contrast, the treatment of human bone cells with IL-lo, LPS and TNF-a (data not shown) resulted in no increase in TGF-P mRNA, which remained at the basal level.

The human osteoblast-like cells showed no constitutive expression of IL-ll3 mRNA. Treatment with the osteotropic agents 1,25-(OH),D, (lOms M), retinoic acid (lo-* M) and parathyroid hormone (1O-7 M) failed to induce any expression of IL-lp mRNA by these cells over a 24-hour time course. In contrast IL-lo (200 pg/ml) and the non-specific inflammatory agent LPS (500 rig/ml) stimulated IL-l/3 mRNA levels by 23 and 5 fold respectively. TNF-o (17 rig/ml) also proved capable of stimulating IL-lp mRNA by these cells. Both IL-la and TNF-CY elicited a response after 6-8 hours (Figs 5, 6). However, TNF-o was far less potent, acting as a stimulus at go-fold greater con- centration than that of IL-l. The increase of IL-lp mRNA after treatment with TNF-o, was followed by a five-fold increase in bioactive IL-l.

Corticosteroids have previously been reported to abrogate cytokine expression.2s The expression of both TGF-l3 and IL-l@ mRNA were completely inhibited by treatment with (1O-7 M) hydrocortisone (Fig. 7, lane 3 and Fig. 8, lane 2). This inhibition was partially alleviated in the case of TGF-l3 mRNA by the addition of 1,25-(OH),D, (10m8 M) (Fig. 7,

TGF-P and IL-l@ expression in osteoblasts / 173

Figure 1. In-situ hybridization of TGF-P and IL-lf3 mRNA in human osteoblast-like cells.

(A,B) Binding of antisense ?S-radiolabelled riboprobes, for TGF-P and IL-lp respectively, which give rise to black silver grains over the cells. (C) Sense probe negative controls for IL-1B.

174 / Merry and Gowen CYTOKINE, Vol. 4, No. 3 (May 1992: 171-179)

Figure 2. Induction of TGF-P mRNA by 1,25dihydroxyvitamin D3.

Ten micrograms of total RNA was loaded on each lane. Lane 1 shows the treatment with 10 -8M 1,25-dihydroxyvitamin D,. for 8 h. Lane 2 shows the treatment with 10 -8M retinoic acid for 8 h. Lane 3, untreated cells. 28s and 18s show the sizes of the ribosomal RNA bands. All lanes were normalized using a p-actin cDNA probe, shown in the panel below.

lane 1). The mRNA level was restored to c. 70% of the stimulated level. Treatment with IL-la in addition to hydrocortisone failed to restore the expression of IL-@ mRNA (Fig. 8, lane 2).

DISCUSSION We have demonstrated that mature human

osteoblast-like cells are capable of expressing both TGF-P and IL-lp mRNA. Various cytokines and osteotropic factors were investigated to determine whether they modulated the expression of TGF-P mRNA. Generally the factors fell into two groups. TGF-p mRNA levels were induced by the systemic

.; 0.8 c 3

0.0

I Control 0 1,25-D?, m PTH

6 12 Time (h)

Figure 3. Induction of TGF-P mRNA with 1,25dihydroxyvitamin D, and parathyroid hormone.

Messenger RNA was quantified with a scanning densitometer. Each point represents a densitometry reading corrected for loading differences with a p-actin cDNA probe. The data shows a direct comparison between treatment with 10 -“M 1,25-dihydroxyvitamin D, and 10 -‘M parathyroid hormone treatment for 6 and 12 h on the same donor’s cells.

Figure 4. Autocrine regulation of TGF-P mRNA.

Ten micrograms of total RNA was serially diluted two-fold to give 8 diluted aliquots of each sample; the vertical dots represent these serial dilutions. Lanes 1, 6, untreated cells at 6 and 12 h respectively, lanes 2, 3, treatment with 10 pM TGF-P for 6 and 12 h respectively; lanes 4, 5, treatment with 100 pM TGF-P for 6 and 12 h respectively. All samples were normalized with p-actin probe as shown in the panel below.

Figure 5. The induction of IL-lp mRNA by IL-k and LPS.

Lane 1, treatment with 200 pg/ml IL-la for 6 h; lane 2, treatment with 500 rig/ml LPS for 6 h; lane 3, untreated cells. All lanes were normalized using a @actin cDNA probe, shown in the panel below.

TGF-l3 and IL-ll3 expression in osteoblasts / 175

hormones 1,25dihydroxyvitamin D, and parathyroid hormone, but remained unaffected by the cytokines rhIL-la and rhTNF-a at concentrations which previously had been shown to stimulate proliferation and prostaglandin release in these cells.15 Retinoic acid, an agent implicated in the maturation of osteoblasts, failed to induce TGF-l3 mRNA at physiological concentrations.

Kim et al.24 have reported that TGF-P is autoregulated in the human adenocarcinoma cell line A-549 and our data also suggest that TGF-l3 may have an autocrine role in human bone cells. In addition the data we have presented show that TGF-l3 mRNA expression is modulated by the systemic hormones 1,25dihydroxyvitamin D, and parathyroid hormone, suggesting that the endocrine status of an individual may regulate TGF-l3 expression by human osteoblasts and act as another regulatory mechanism in the bone remodelling cycle. In contrast the modulation of IL-ll3 mRNA expression was induced by the cytokines IL-la and TNFol but remained unaffected by treatment with the systemic hormones.

In agreement with studies in other cell types, hydrocortisone markedly decreased the expression of both TGF-P mRNA and IL-lp mRNA. It is interesting to note that the addition of 1,25- dihydroxyvitamin D, to hydrocortisone-treated cells results in a partial reduction of the inhibitory effect. Since osteoblasts do not constitutively express IL-ll3 mRNA, it was only possible to demonstrate an inhibition with hydrocortisone on IL-la stimulated cells.

Figure 6. The induction of IL-Q3 mRNA by TNF-(Y.

Lane 1, untreated cells; lanes 2-5, stimulation with 17 @ml TNF-a for 6, 8, 12 and 24 h respectively; lane 6, no treatment at 24 h. All samples were normalized with @actin cDNA probe as shown in the panel below.

176 I Merry and Gowen CYTOKINE, Vol. 4, No. 3 (May 1992: 171-179)

Figure 7. The effect of hydrocortisone on TGF-P mRNA expression.

Lane 1, treatment with both 1OF M hydrocortisone and 1,25- dihydroxyvitamin D, for 6 h; lane 2, treatment with lo-* M 1,25 dihydroxyvitamin D, for 6 h; lane 3, treatment with lo-” M hydrocortisone for 6 h; lane 4, no treatment. All samples were normalized with p-actin cDNA probe as shown in the panel below.

It is interesting to note that human osteoblast-like cells constitutively expressed TGF-P mRNA but not IL-lp mRNA. This may reflect the fact that the former is secreted as an inactive propeptide whilst the latter is secreted in an active form and is a very potent bone resorptive agent. It is likely that such an important event as bone resorption would be regulated by a factor which could be induced by locally produced cytokines, such as TNF-(x and IL-l, and quickly fall to basal level. The instability of IL-l mRNA coupled with the data obtained for IL-lp mRNA expression are consistent with this pattern of modulation, and the induction of TNF-a production by IL-l in these cellsz3 would also allow such a resorptive signal to be further amplified.

It has been suggested that TGF-P could act as a coupling agent between bone resorption and formation in the remodelling cycle. Many biological properties attributed to TGF-P make it an ideal ‘coupling factor’. It is deposited in the bone matrix in large amounts in a latent form, which can be activated by osteoclasts during resorption. It has also been reported to be both chemotactic for osteoblasts and a potent proliferative stimulus. It is conceivable that there are different ‘levels’ of control of bone cell activity. Thus a TGF-P-like factor would be produced and incorporated into the bone matrix throughout the skeleton over a prolonged period under the influence of the endocrine system of the individual. Indeed Finkelman and colleagueP have shown recently that vitamin D deficiency causes a selective reduction in the amount of TGF-6 in rat

Figure 8. The effect of hydrocortisone on IL-Ip mRN

Lane 1, treatment with 200 pg/ml IL-k alone; lane with 1OF M hydrocortisone and 200 pg/ml IL-k treatment. All samples were normalized with p-actin as shown in the panel below.

A exprc ession.

2, tre; itment ; lane 3, no cDNA probe

TGF-p and IL-@ expression in osteoblasts / 177

bone matrix. This stored growth factor may then be activated at a much later time point in a highly localized fashion, such as under an activated osteoclast. Interleukin 1 may represent a more acutely acting family of regulators, being produced in response to locally derived signals and acting briefly over a short range. Other cytokines which may fall into this category include TNF23 and IL-6.22 Further investigation of the complex network of cell-cell communication may lead to improved understanding of defective cell activity in pathological processes.

MATERIALS AND METHODS Recombinant interleukin-la with a specific activity of

6 x 10’ U/mg was a kind gift from Glaxo Group Research UK. Recombinant TNF-(w had a specific activity of 5 X 10’ U/mg and was from Dr R. Adolf, Boehringer Ingelheim. Latent TGF-B was a gift from Dr Edwards at British Biotechnology Ltd, Oxford UK. Activation of TGF-B was achieved by adjusting the protein concentration to 50 pg/ml with BSA fraction V. The TGF-l3 was then heated to 80°C for 8 min and cooled on ice for 5 min. Treatments were carried out in serum-free MEM supplemented with 0.1% BSA. All chemicals were obtained from Sigma UK unless otherwise stated. Radiochemicals were obtained from Amersham International and all molecular biology reagents from Northumbria Biochemicals. Tissue culture media, sera and plastics were obtained from Costar UK and GIBCO UK. The plasmid pghtgfb27 contained the cDNA for TGF /3 in pGEM and was provided by Dr Allen Baxter of Glaxo UK. The plasmid pGEMIL-19 contained the cDNA for IL-1B in pGEM1 and was provided by Dr Alan Shaw, Glaxo Institute for Molecular Biology, Geneva.

Human Bone Cell Culture

The human osteoblast-like cells were obtained from explants of trabecular bone obtained at surgery as previously described by Beresford et al.27,28 After 4-6 weeks in culture the cells were trypsinized and pooled then seeded in 9 cm petri dishes at 1 million per dish. For the serum-free experiments the cells were incubated in serum free MEM + 0.1% BSA for 24 hours prior to the experiment. These cultures have been previously extensively screened for macrophages and monocytes using a panel of monoclonal antibodies and both these cell types have been shown to be absent.29

RNA Preparation

Following culture, the medium was removed from the cells. Denaturing guanidine thiocyanate was added to the petri dishes, which were then scraped to harvest all the cell lysate. The RNA was prepared by a microscale method of Chomininski and Sachi. RNA (10 Fg) was loaded onto a 1% agarose denaturing gel for Northern blot analy- sis according to the method of Goldberg.sl

Slot Blotting

The slot blot method used purified RNA as above, Aliquots (10 bg) were mixed with an equal volume of formamide and denatured at 65°C for 5 min then quenched on ice. The salt concentration was adjusted to 0.25 M with sodium acetate. Two fold serial dilutions were then made and loaded in 100 ~1 volumes onto a GIBCO-BRL slot blot manifold. The samples were washed through with a solution of 50% formamide-0.25 M sodium acetate pH 5.2. The RNA was then fixed to the filter with a solution of 50 mM sodium hydroxide for 5 min. The filter was then washed in 2 X SSC and stained with 2% methylene blue prior to hybridization to check for equal loading.

Radiolubelling Probes

The cDNA probes were radiolabelled using the random hexanucleotide) priming, reaction (Amersham). The specific activity of the probes were routinely in excess of 107cpm/p.g. The hybridization step was as described by Goldberg using 33% formamide and 2 X SSC at 52°C.

Post-hybridization washes were carried out using 2 x SSC at room temperature, followed by 2 x SSC + 0.1% SDS at 65°C. If greater stringency was required, a final wash in 0.1 X SCC at room temperature was performed. Autoradiographs were established by exposing the filter to X-ray film for 24-72 hours at -70°C with an intensifying screen.

The radioactive probe was removed from the Northern blots or slot blots by boiling the filter in a solution of 0.1% SDS. After cooling to room temperature and washing in 2 X SSC the filters were reprobed for the ‘housekeeping gene’ p-actin. The slots and bands were then scanned with a hand held densitometer (Genetic Research Inc.) for quantitation. All loading errors were less than 10% as judged by densitometry readings of p-actin reprobed filters. Three separate readings of density were determined for each dot, the mean of these values were then shown in Fig. 3.

In-situ Hybridization

The cells were plated onto lo-well multi-spot slides at a density of 3000/well. After 24 hours the cells were washed and fixed with 4% paraformaldehyde/PBS for 5 min. After washing in PBS the slides were dehydrated through an ethanol series and stored for up to 1 week. The following pretreatments were performed to decrease the amount of non-specific binding of the RNA probes to the specimen. Proteinase K treatment removed a proportion of the structural proteins, the remaining proteins were acetylated with acetic anhydride. Finally a prehybridization in 2 x SSC 50% formamide was performed.

The RNA probes were synthesized using SP6 and T7 RNA polymerases with [35S-CTP] as the radiolabel according to the Mannheim Boehringer transcription kit. The specific activity of the riboprobes was in excess of lo* cpm/pg. Sense transcripts were synthesized to act as a negative control and assess non-specific binding.

The hybridization was carried out in a solution containing 50% dextran sulphate, 33% formamide, ssDNA,

178 I Merry and Gowen CYTOKINE, Vol. 4, No. 3 (May 1992: 171-179)

tRNA and RNAse inhibitor. The post-hybridization washes were as follows: 4 X 15 min 2 X SSC at room temperature, 4 X 15 min 2 x SSC at 37°C 30 min of RNAse A (200 pg/ml) in 0.1 M Tris, 50 mM EDTA pH 8.0 and a final wash in 0.2 x SSC at 37°C for 30 min. After being washed the slides were dehydrated through an ethanol series, air dried and dipped in LMl (Amersham) photographic emulsion. The slides were exposed at 4°C for up to 1 week prior to developing.

Interleukin-1 Bioassay

The level of expression of IL-l was assessed using the DlON,M bioassay. DlON,M is a murine T-cell line which proliferates in response to IL-l in the presence of IL-1.32 After 72 hours incubation with sample the cells were pulsed with [3H]thymidine (1 pCi per well) for a further 4 hours. The extent of proliferation and hence IL-l bioactivity present in the samples was determined by harvesting the cells, scintillation counting and comparing with a standard curve using rhIL-1. The bioassay is sensitive down to 0.02 pg/ml IL-l.

Acknowledgements

We would like to thank the surgeons and theatre staff at the Royal United Hospital, Bath and the Bristol Infirmary for samples of trabecular bone, and Dr D. Sansom and A. Littlewood for their helpful discussion of the manuscript. This work was supported by Action Research, UK.

REFERENCES 1. Roberts AB, Anzano MA, Lamb LC, Smith JM, Sporn

MB (1981). New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc Nat1 Acad Sci USA 785339-5343.

2. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF (198.5) Type beta transforming growth factor: a bifunctional regulator of cellular growth. Proc Nat1 Acad Sci USA 82:11%123.

3. Gehron Robey P, Young MF, Flander KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB (1987) Osteoblasts synthesize and respond to transforming growth factor-type beta (TGFP) in vitro. J Cell Biol 105:457-463.

4. Oreffo ROC, Mundy GR, Seyedin SM, Bonewald LM (1989) Activation of the bone derived latent TGF beta complex by isolated osteoclasts. Biochem Biophys Res Commun 158:817- 823.

5. Centrella M, McCarthy TL, Canalis E (1987) Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262:286%2874.

6. Ignotz RA, Massague J (1986) Transforming growth factor beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 261: 4337-4345.

7. Thiebaud D, Ng KW, Findlay DM, Harker M, Martin TJ (1990) Insulinlike growth factor 1 regulates mRNA levels of osteonectin and pro-alpha1 (I)-collagen in clonal preosteoblastic calvarial cells. J Bone Min Res 5:761-767.

8. Pfeilschifter J, Wolf 0, Naumann A, Minne HW, Mundy GR, Ziegler R (1990) Chemotactic response of osteoblastlike cells

to transforming growth factor beta. J Bone Min Res 5:825-829. 9. Pfeilschifter JP, Seyechin S, Mundy GR (1988)

Transforming growth factor beta inhibits bone resorption in fetal rat long bones. J Clin Invest 82:680-685.

10. Tashjian AH, Voelkel EF, Lazzaro M (1985) Alpha and beta transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc Nat1 Acad Sci USA 82:4535-4538.

11. Gowen M, Wood DD, Ihrie EJ, McGuire MKB, Russell RGG (1983). An interleukin l-like factor stimulates bone resorption in vitro. Nature 306:378-380.

12. Dewhirst FE, Stashenko PP, Mole JE, Tsurumachi T (1985) Purification and partial sequence of human osteoclast activating factor. J Immunol 135:2562-2568.

13. Kawano M, Yamamoto I, Iwato K, Tanaka H, Asaoku H, Tanabe 0, Ishikawa H, Nobuyoshi M, Ohmoto Y, Hirai Y, Kuramoto A (1989) Interleukin 1 beta rather than lymphotoxin as the major bone resorbing activity in human multiple myeloma. Blood 73:1646-1649.

14. Pacifici R, Rifas L, Teitelbaum S, Slatopolsky E, McCracken R, Bergfeld M, Lee W, Avioli LV, Peck WA1 (1987). Spontaneous release of interleukin 1 from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Nat1 Acad Sci USA 84:46X1-4620.

15. Evans DB, Bunning RAD, Russell RGG (1990) The effects of recombinant human interleukin 1 beta on cellular proliferation and production of prostaglandin E, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone. Biochem Biophys Res Commun 166:20&216.

16. Beresford JN, Gallagher JA, Gowen M, Couch M, Poser J, Wood DD, Russell RGG (1984) The effects of monocyte conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro. Biochim Biophys Acta 80158-65.

17. Stashenko P, Dewhirst FE, Rooney ML, Desjardins LA, Heeley JD (1987) Interleukin-1 beta is a potent inhibitor of bone formation in vitro. J Bone Min Res 2:559-565.

18. Keeting PE, Bonewald LF, Colvard DS, Spelsberg T, Mundy GR, Riggs BL (1989) Estrogen mediated release of TGFP by normal human osteoblast-like cells. J Bone Min Res 4: S655.

19. Hughes DE, Gowen M, Russell RGG (1988) Interleukin 1 as a paracrine factor in bone effects on osteoclast-like cell formation and production by osteoblast-like cells. J Bone Min Res 3:S196 (abstract)

20. Keeting PE, Rifas L, Peck WA, Riggs BL (1989) Production of interleukin 18 by stimulated normal human osteoblast-like cells. J Bone Min Res 4:S152.

21. Feyen JHM, Elford P, Di Padova FE, Trechsel U (1989). Interleukin-6 is produced by bone and modulated by parathyroid hormone. J Bone Min Res 4:633-638.

22. Littlewood AJ, Russell J, Harvey G, Hughes DE, Russell RGG, Gowen M (1991) The modulation of the expression of IL-6 and its receptor in human osteoblasts in vitro. Endocrinology 129:1513-1520.

23. Gowen M, Chapman K, Littlewood A, Hughes DE, Evans DB, Russell RGG (1990) Production of tumor necrosis factor by human osteoblasts is modulated by other cytokines, but not by osteotropic hormones. Endocrinol 126:1250-1255.

24. Kim S, Angel P, Lafyatis R, Hattori K, Kim KY, Sporn MB, Karin M, Roberts AB (1990) Autoinduction of transforming growth factor pl is mediated by the AP-1 complex. Mel Cell Biol 10:1492-1497.

25. Gallagher JA, Beresford JN, McGuire MKB, Ebsworth NM, Meats JE, Gowen M, Elford PR, Wright D, Poser J, Coulton LA, Sharrard M, Imbimbo B, Kanis JA, Russell RGG (1984) Effects of glucocorticoids and anabolic steroids on cells derived from human skeletal and articular tissues in vitro. In Avioli LV,

TGF-8 and IL-1B expression in osteoblasts / 179

Gennarie C, Imbimbo B (eds), Advances in Experimental Medicine and Biological Consequences, Plenum Pub1 Corp., New York, pp 279-292.

26. Finkelman RD, Linkhart TA, Mohan S, Lau KHW, Baylink DJ, Bell NH (1991) Vitamin D deficiency causes a selective reduction in the deposition of transforming growth factor beta in rat bone; possible mechanism for impaired osteoinduction. Proc Nat1 Acad Sci USA 88:3657-3660.

27. Beresford JN, Gallagher JA, Poser JW, Russell RGG (1984) Synthesis of osteocalcin by human bone cells in vitro: Effects of 1,25(OH),D, and PTH and glucocorticoids. Metab Bone Dis Rel Res 5:229-234.

28. Beresford JN, Gallagher JA, Russell RGG (1986) 1,25- dihydroxyvitamin D, and human bone-derived cells in vitro: effects on alkaline phophatase, type I collagen synthesis and proliferation. Endocrinology 119:177&1785.

29. Skjodt H, Moller T, Freiesleben SF (1989) Human osteoblast-like cells expressing MHC class II determinants stimulate allogeneic and autologous peripheral blood mononuclear cells and function as antigen-presenting cells. Immunology 68:416- 420.

30. Chomininski P, Sachi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Analiochem 162:156159.

31. Goldberg ML, Lefton RP, Stark GR, Williams JG (1979) Isolation of specific RNAs using DNA covalently linked to diazobenzyloxymethyl cellulose on paper. Meth Enzymol 68206210.

32. Hopkins SJ, Humphreys M (1989) Simple, sensitive and specific bioassay of interleukin-1. J Immunol Meth 120:271-276.