EP0463004A1 - Metabolic effects of leukaemia inhibitory factor on bone - Google Patents

Abstract

The present invention generally relates to the effect of the leukemia inhibiting factor (IDF) in the bones of animals and more particularly to the use of IDF and / or of polypeptides of the same type to induce, promote and / or reinforce the effects. metabolic on animal bones.

Description

METABOLIC EFFECTS OF LEUKAEMIA INHIBITORY FACTOR ON BONE

The present invention relates generally to the effect of leukaemia inhibitory factor (LIF) on bone in animals. More particularly, the present invention relates to the use of LIF and/or LIF-like polypeptides to induce, promote and/or enhance metabolic effects on bone in animals. The cytokine, leukaemia inhibitory factor (LIF), is a protein that has previously been purified, cloned and produced in large quantities in purified recombinant form from both Escherichia coli and yeast cells

(International Patent Application No. PCT/AU88/00093). LIF was originally isolated on the basis of its capacity to induce differentiation in and suppress the murine myeloid leukaemic cell line. M1. LIF has been shown to have a powerful differentiation suppressing action on normal embryonic stem cells. LIF has no apparent

proliferative effects on normal hemopoietic cells

although LIF receptors were detected on cells of the monocyte-macrophage lineage.

Several cytokines and growth factors have been implicated in bone remodelling and in particular have been shown to promote bone resorption in vitro. The term 'osteoclast activating factor' (OAF) was applied to the bone resorbing activity detected in supernatants from lectin-transformed lymphocytes (1), and was used to explain the excessive bone resorption accompanying certain hematological malignancies. It is now accepted that OAF in fact consists of several cytokines, including interleukin 1 (IL-1) and tumour necrosis factors alpha and beta (TNFα, TNFβ), all of which are potent bone resorbing agents (2). LIF has no significant structural or sequence homology with the aforementioned cytokines. It was surprising, therefore, that following an analysis of the possible physiological and biochemical effects of LIF on bone remodelling in vitro and in vivo, including studies of the cellular targets of LIF action, it was discovered, in accordance with the present invention, that LIF has a metabolic effect on bone by inducing, promoting and/or enhancing bone resorption and/or bone formation.

In accordance with this invention, it has been discovered that LIF is an important paracrine regulator in bone and exerts a number of specific biochemical actions on osteoblasts. These include inhibition of plasminogen activator (PA) activity in osteoblasts, most likely through increased synthesis of PA inhibitor-1 formation. Furthermore, LIF itself is expressed in osteoblastic cells and primary osteoblasts. Thus several lines of evidence establish cells of the osteoblast lineage as targets for LIF action in a paracrine or autocrine manner.

Accordingly, one aspect of the present invention relates to a method of inducing, promoting and/or

enhancing metabolic effects on bone in an animal

comprising administering to said animal an effective amount of leukaemia inhibitory factor (LIF) and/or LIF- like polypeptides alone or in combination with other cytokines for a time and under conditions sufficient to effect bone metabolism.

Another aspect of the present invention is directed to a pharmaceutical composition useful in inducing, promoting and/or enhancing metabolic effects on bone in animals comprising an effective amount of

leukaemia inhibitory factor (LIF) and/or LIF-like

polypeptides in combination with one or more other molecules such as cytokines, inorganic or organic

molecules and which affect bone resorption, calcification and/or formation and one or more pharmaceutically

acceptable carriers and/or diluents.

Still yet another aspect of the present invention contemplates the use of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides in the manufacture of a medicament useful in the induction, promotion and/or enhancement of metabolic effects on bone in animals.

A further aspect of the present invention

contemplates a method for the treatment of injuries and/or diseases which result in bone degeneration or fracture in an animal comprising administering to said animal a bone-forming increasing-effective amount of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides alone or in combination with other active molecules for a time and under conditions sufficient to effect bone calcification.

In a preferred embodiment of the subject

invention, the animal is mammalian or avian and more preferably is human.

The following abbreviations are used herein:

LIF Leukaemia inhibitory factor

TNFα Tumour necrosis factor alpha

TNFβ Tumour necrosis factor beta

TGFβ Transforming growth factor beta

PTH Parathyroid hormone

PA Plasminogen activator

PAI Plasminogen activator inhibitor

CSF Colony stimulating factor

GM-CSF Granulocyte-macrophage colony-stimulating factor PBS Phosphate buffered saline

SDS Sodium dodecyl sulphate

BSA Bovine serum albumin

MEM Minimal essential medium

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

FCS Fetal calf serum

UV Ultra violet

FD cells CSF-dependent continuous hemopoietic cell line

FD/LIF cells FD cells expressing LIF gene

HEPES 4-(2-Hydroxyethyl)-1- piperazinoethanesulfonic acid The present invention arose, in part, from an analysis of the effects of LIF on certain responses in osteoblast-like cells and its effects on bone resorption in calvariae in organ culture. Furthermore, to test the effects of LIF in vivo and to delineate LIF's

physiological functions, mice were generated with

chronically-elevated levels of LIF. With respect to this latter test, use was made of cells of the colony

stimulating factor (CSF)-dependent continuous hemopoietic cell line FDC-P1 ("FD" cells) which, although non-leukaemic, have been found to persist and accumulate in the spleen, lymph nodes and bone marrow of recipient animals. By superinfecting FDC-P1 cells with a

retrovirus containing the gene which encodes LIF, cloned sublines were developed that constitutively produce high levels of LIF and such cells were injected into mice to generate animals containing a continuous source of LIF-producing cells. Upon analysis of these animals,

together with the in vitro data, it was discovered that LIF is an important regulator of bone cell function causing the induction, promotion and/or enhancement of such activities as bone resorption and/or bone formation. The in vivo exemplified aspects of the present invention are performed using a mouse model. This is done, however, with the understanding that the present invention extends to the effect of LIF on bone in all animals. Preferably, however, the animals are mammalian or avian but more preferably mammalian. In its most preferred embodiment, the present invention is directed to humans. Accordingly, one aspect of the present invention relates to a method of inducing, promoting and/or

enhancing metabolic effects on bone in an animal

comprising administering to said animal an effective amount of LIF and/or LIF-like polypeptides alone or in combination with one or more other cytokines or one or more active substances, for a time and under conditions sufficient to effect bone anabolism.

"Metabolic effects", as used in the specification and claims herein, include, although are not necessarily limited to, such processes as bone formation and/or bone resorption, increased protein synthesis in bone tissue and increased thymidine uptake in bone tissue. The term "metabolic effects" also encompasses anabolic and/or catabolic activities of LIF on bone.

By "LIF and/or LIF-like polypeptides" is meant derivatives and homologues of LIF and extends to the entire natural length LIF molecule or derivatives thereof carrying single or multiple amino acid substitutions, deletions and/or additions and includes substitution, deletion and/or addition of any other molecules

associated with LIF and its derivatives such as, inter alia, carbohydrate, lipid and polypeptide moieties, provided said derivatives retain sufficient activity to be useful in the practice of the present invention. LIF-like polypeptides extend to molecules having substantially similar activity as LIF while carrying amino acid rearrangements or alterations. The

preparation of various derivatives of LIF will be

apparent from the disclosure in PCT/AU88/00093 of the recombinant LIF molecule and its corresponding DNA.

Reference herein to LIF encompasses LIF-like polypeptides and vice verca.

Hence, the present invention extends to naturally occurring but substantially pure LIF (i.e. greater than or equal to 70% by weight of LIF relative to other proteins or molecules and preferably greater than or equal to 85% and even more preferably greater than or equal to 90%), to recombinant LIF and to synthetic LIF made, for example, by chemical means.

Depending on the animal to be treated or the disease state to be treated, LIF may be from a range of sources such as, but not limited to, human, mouse, dog, cow, pig, sheep or other ruminant. In some cases it will be preferable to use homologous LIF to the animal being treated, for example, human LIF on humans. But in other circumstances, heterologous LIF may be more convenient and/or more effective. The choice of source of LIF may depend on the exigency of the treatment required or the animal requiring treatment.

In one embodiment of the subject invention, LIF and/or LIF-like polypeptides are used alone. In another embodiment, they are used in a combination with one or more other cytokines, such as, but not limited to, IL-1, TNFα and/or TNFβ, and which affect various aspects of bone resorption, and/or formation. The present invention also extends to the use of LIF and/or LIF-like polypeptides in combination with other active molecules such as agonists, antagonists, calcium or any inorganic or organic molecule which aids in the bone resorbing and/or forming process either directly or by enhancing the effect of LIF or LIF-like polypeptides.

The injection of FD cells into mice produces animals in which a progressively increasing population of FD cells accumulate. Injection of FD cells producing LIF ( "FD/LIF cells") had the same consequence. In agreement with previous studies, this accumulation was accelerated by pre-irradiating the recipients. Northern analyses for LIF mRNA and bioassays for LIF indicated that active production of LIF was achieved in certain organs known to be the site of accumulation of FD cells, i.e. the spleen, lymph nodes and sometimes the marrow and there was a substantial elevation of circulating LIF levels. No elevation of LIF production or circulating LIF levels were seen in recipients of FD cells. The consequence of injecting LIF-producing FD cells was inter alia the development within an interval as short as 10 days of bone overgrowth with consequent extramedullary

haemopoiesis in the spleen and liver.

The absence of any of these effects in recipients of FD cells alone or in cachectic GM-CSF transgenic mice that have been studied extensively in the inventors' laboratory argue that the pathological changes observed are the specific consequence of excess LIF levels. Since such mice often have few or no FD/LIF cells in the marrow, the tissue effects seem likely to be mediated by circulating LIF.

The most dramatic primary change was the pattern of excess bone formation with a histological appearance resembling osteosclerosis. This involved three

simultaneous changes: an accumulation and activation of osteoblasts; a depletion of pre-existing hemopoietic cells (in unirradiated recipients); and, in many bones, activation of osteoclasts. Osteoblasts express receptors for LIF (27) and LIF is known to release calcium in vitro from bone tissue (Reid et al. Personal Communication). It is conceivable, therefore, that the osteoblast changes were due to direct actions of LIF which , if so, would suggest that LIF may be capable of influencing osteoblast precursor migration , osteoblast proliferation and bone formation. A well-documented association exists between osteoblast and osteoclast activity but, since osteoclasts lack LIF receptors (27), their altered behaviour may be secondary to changes in osteoblast activity. Although the perturbation in bone biology resulted in excess bone formation, many mice can be assumed to have had excess calcium levels because of calcium deposits particularly in muscle and liver tissue.

The administration of LIF, therefore, may, under appropriate conditions and requisite time, induce bone formation and may be extremely useful in the repair of fractured bones and to stimulate bone growth and should be particularly useful in the treatment of injuries and/or diseases where bone degeneration or fracture is a symptom, or a result, such as in increasing bone

calcification in osteoporosis, such as in post-menopausal osteoporosis, or in stimulating bone fracture repair.

Administration of LIF will be by standard procedures, for example, intravenous, interperitoneal, intramuscular, or topically to the extent that it is applied to the

fractured bone. Certain modifications may need to be made to LIF in order to increase its serum half life or to protect is from hydrolytic enzymes. All such

modifications to LIF or its pharmaceutical composition comprising LIF or LIF-like polypeptides are encompassed by the present invention. Alternatively, the

modifications may comprise adding protective compounds (e.g. enzyme inhibitors) to pharmaceutical compositions. Suitable carrier vehicles and their formulations,

Inclusive of other human proteins (e.g. human serum albumin), are described, for example, in Remington's Pharmaceutical Sciences 16th ed., 1980, Mach Publishing Company, edited by Osol et al which is herein

incorporated by reference. The effective amount of LIF or LIF-like polypeptides required in pharmaceutical compositions or in vivo to induce metabolic effects will vary according to the animal and condition being treated. For example, an effective amount of from 0.01 to 1 million units may be necessary but in some circumstances a range of 0.1 to 10,000 units may be required. In other circumstances it may be propitious to administer LIF at 0.01 μg to 100,000 mg per kg of body weight of animal. Another preferred range may be 0.1 jug to 10,000 mg of LIF or LIF-like polypeptides per kg of body weight of animal. The aforementioned effective amounts may, depending on the situation, be per hour or per day or possibly per week or month. Administration may be single or multiple application. Accordingly, "effective amount" refers to the amount necessary to give the desired effect.

Pharmaceutical compositions useful in the

induction of bone formation or increasing bone resorption comprising a bone formation-inducing or bone resorption-effective amount of LIF and/or LIF-like polypeptides may be prepared according to standard practices.

Furthermore, the present invention extends to the use of LIF and/or LIF-like polypeptides in the manufacture of a medicament for the induction, promotion and/or

enhancement of metabolic effects on bone in animals and in particular mammals or birds and even more particularly humans. In a preferred embodiment, the present invention contemplates the use of LIF and/or LIF-like polypeptides in the manufacture of a medicament for treating injuries and/or diseases in animals, and in particular humans, where bone degeneration or fracture is a symptom or a result such as in osteoporosis or bone fracture. The present invention extends to parts or derivatives of LIF and/or LIF-like polypeptides defined by their ability to interact with osteoblasts. In particular, the

interaction may comprise the binding of the LIF fragment to a LIF receptor on an osteoblast cell. Such parts or derivatives of LIF and/or LIF-like polypeptides would be isolated by any number of means such as chemically treating the LIF molecule, isolating cleaved or altered sections and testing whether the sectioned or fragmented LIF has activity in bioassays as described in

PCT/AU88/00093. The active fragments would then be further analysed as described herein using in vitro

techniques. Alternatively, selected fragments or

portions of LIF would be synthesized using standard recombinant DNA expression systems using segments or deriviates of the LIF coding region. The polypeptide so produced would then be tested using bioassays and, if active, would be further tested in in vitro osteoblast cell systems. Therefore, use of the term "LIF-like polypeptides" herein extends to fragments or parts of LIF capable of interacting with osteoblasts.

The in vitro results described herein suggest that LIF stimulates bone resorption by a mechanism possibly involving prostaglandin production and involving an increase in the number of osteoclasts (bone resorbing cells) in the calvaria. Its action on thymidine

incorporation probably reflects responses of osteoblasts, and does not appear to involve local prostaglandin production since it is unaffected by the presence of indomethacin. These data further suggest that LIF may be an important regulator of bone cell function.

Furthermore, the effect of LIF on the osteoblast-like cells is largely metabolic, in that there is clear evidence that LIF effects an increase synthesis of some of the important specific components of bone protein. Additionally, one of the clear specific actions of LIF reported herein is upon the PA-plasmin system in

osteoblasts. Several bone-resorbing hormones stimulate PA activity in osteoblast-like cells in culture. In order to localize the proteolytic activity of the PA-plasmin system at sites where it is required, precise regulatory control is needed. This is provided by the stimulating hormones and paracrine factors and also by PA inhibitors (PAIs), which bind to and limit the activity of both tissue-type PA (tPA) and urokinase. The specific inhibitors, PAI-1 and PAI-2, which bind to and inactivate PA, are the products of separate genes.

In the present work, the production of PAI-1 by normal osteoblasts is identified from rat calvaria and from the osteoblast-like osteogenic sarcoma line, UMR 106-01.

Furthermore, LIF is shown to enhance production of PAI-1 protein and mRNA, an effect which is most likely

responsible for the net decrease in PA activity released by the cells.

A further positive action of LIF is the ability to enhance the stimulation of alkaline phosphatase activity by retinoic acid in UMR 201 cells, an effect similar to that obtained previously with recombinant TNF (11).

Although the effects of LIF resemble in some respects those of TGFβ the latter has no such effect on alkaline phosphatase activity. LIF, therefore, exhibits a regulatory effect on the PA-PA inhibitor system, further evidencing the potential of LIF as an important regulator in the microenvironment of bone cells and, therefore, important in bone remodelling. The effect of LIF on PA does not appear to be acting by the generation of TGFβ. The present invention is also described by the following non-limiting Figures and Examples. Although the Examples use mice as the animal model, the techniques used and results obtained will be equally applicable to other animals including avian and mammalian (e.g. human) species, and, hence, the present invention encompasses the use of LIF in inducing, promoting and/or enhancing bone formation and resorption and other metabolic

activites in all animals.

In the Figures:-

Figure 1 is a graphical representation showing survival of mice, with or without exposure to radiation, following injection of FD cells expressing LIF.

Figure 2 is a graphical representation showing serum levels of LIF in irradiated and non-irradiated mice following injection of FD cells and FD cells expressing LIF.

Figure 3 is a pictorial representation of the femur from mouse injected with FD cells (A) and FD/LIF cells (B). Note the excess new bone formation and abnormal trabeculae in FD/LIF recipient.

[Hematoxylin/eosin (H & E; x 54)]. (C) Higher power view of marrow from a recipient of FD/LIF cells showing depletion of hemopoietic cells, excess numbers of

osteoblasts, and new bone formation. (H & E; x 450). (D) Heart of recipient of FD/LIF cells showing calcium deposits (arrows). (H & E; x54). (E and F) Pancreas from recipient of FD cells (E) and from recipient of FD/LIF cells (F). Note acinar degeneration and edema but normal islet (arrow). (H & E; x 54). (G and H) Ovary from a recipient of FD cells (G) (the normal corpora lutea are indicated by arrows) and from a recipient of FD/LIF cells (H). Note the absence of corpora lutea in H. (H & E; x 54). Figures 4 and 5 are graphical representations showing, respectively, murine and human LIF-stimulated ⁴⁵Ca release from pre-labelled mouse calvaria in a dose-dependent manner.

Figure 6 is a graphical representation showing that the increase in bone resorption is associated with an increase in osteoclast numbers in the calvaria.

Figure 7 is a graphical representation showing the ability of 10^-7M indomethacin to block LIF stimulated bone resorption.

Figure 8 is a graphical representation showing that [³H]-thymidine incorporation into the calvaria is stimulated by LIF.

Figure 9 is a graphical representation showing that stimulation by LIF of [³H]-thymidine incorporation into the calvaria is not blocked by the presence of indomethacin.

Figure 10 is a graphical representation showing PA activity in calvarial osteoblasts (A) and UMR 106-01 cells (B). UMR 106-01 cells were incubated in the presence (●) and absence (o) of a submaximal (20ng/ml) dose of PTH. Results are the means ± SEM of triplicate determinations.

Figure 11 is a photographic respresentation showing (A) Time courses of PAI-1 mRNA levels following treatment of UMR 106-01 cells with LIF (1000 U/ml).

Total RNA (10μg) was electrophoresed on an

agarose/formaldehyde gel, transferred to Hybond N and the RNA hybridized with labelled RNA transcripts; (B)

Response of PAI-1 mRNA following 1 h treatment of UMR 106-01 cells with increasing concentrations of LIF.

Conditions were as described in (A).

Figure 12 is a photographic representation of an autoradiograph of a Northern blot showing the effect of LIF on mRNA for α1(I) collagen (A) and osteonectin (B) in UMR201 cells. The 6 lanes on the left in each case are the LIF treatment samples. The cells were incubated with LIF for 24 hours in 2% v/v uv irradiated fetal calf serum. An aliquot of 20μg total RNA was electrophoresed as described in Example 5.

Figure 13 is a photographic representation showing autoradiographs of cells from newborn rat long bones labelled with ¹²⁵I-LIF. Specifically, the photograph shows osteoblasts and an osteoclast in the absence of

unlabelled competitor (x400).

Figure 14 is a diagrammatic representation showing (A) saturation binding isotherm of total (o), specific

(Δ) and non specific (o) binding of ¹²⁵I-LIF to UMR 106-06 cells at 4°C. (B) scatchard analysis of specific binding data illustrated in panel A.

Figure 15 is a photographic representation showing the detection of LIF transcripts in osteoblastic cells. Equivalent amounts of RNA from UMR 106-06, UMR 201 or osteoblast-rich primary cultures (1°Ca) were probed for LIF or GAP-DH transcripts (as a positive control) by PCR amplification as described in Example 5. The negative control track represents a "no cell" sample carried in parallel through RNA extraction, cDNA synthesis and PCR reaction. In the case of UMR 106-06 and UMR 201 cells, the + and - symbols refer to treatment with or without TGF β (2ng/ml) for the number of hours indicated.

EXAMPLE 1

General effect of LIF in mice

Commencing 10 days after the injection of FD/LIF cells, mice that had previously been given 6 Gy whole-body irradiation began to show evidence of weight loss, ruffled hair and restricted movement. Within days, such mice became moribund and by 28 days after injection 80% of these mice had become moribund (Figure 1). Irradiated mice injected with control FD cells remained in good health during this period. Unirradiated mice injected with FD/LIF cells developed an identical clinical syndrome but with a delayed onset. Unirradiated mice first became moribund 30 days after injection but

thereafter the rate of disease development was parallel to that of irradiated mice, 80% becoming moribund by 56 days. Again, unirradiated mice injected with control FD cells remained apparently healthy during this period. All the observations were performed on moribund mice injected with FD/LIF cells, control mice injected with FD cells being killed on each occasion for comparative analysis. The serum levels of LIF in irradiated and non-irradiated mice following injection of FD and FD/LIF cells are shown in Figure 2.

Moribund irradiated and non-irradiated mice injected with FD/LIF cells appeared dehydrated and showed a consistent weight reduction of 21% and 24%,

respectively, compared with control mice. The moribund mice exhibited circulatory collapse and a reduced

circulating blood volume. The hematocrit levels (Table 1) did not exhibit an expected rise due to

homoconcentration, suggesting a reduction in red cell mass. Collection of serum from such mice was difficult because of the reduced volume of blood obtained at killing and a failure of clot retraction in specimens from FD/LIF mice.

TABLE 1

Parameters of Moribund Mice Injected with FD/LIF cells

Parameter OGy 6Gy

FD/LIF FD FD/LIF FD

Number of Mice

Analysed 13 9 12 12

Body Weight g 17.8±2.1 22.4±1.8

Haematocrit % 47±8 30±4 49±4 42±2

Total White

Cells/μl 19,940± 5470± 10,600± 2370±

8000 1990 4550 1030

Polymorphs 10,780± 630±390 9130± 630±310

5580 4100

Lymphocytes 7870± 4600± 850±760 1130±590

2420 1680

Monocytes 1270± 230±40 610±350 3370±200

1610

Eosinophils 30±60 20±30 10±30 10±10

Mean values ± standard daviations

EXAMPLE 2

Hemopoietic changes

White cell levels were consistently elevated in FD/LIF mice due to a selective 4-5 fold elevation of mature neutrophils (Table 1). In unirradiated mice, monocyte levels were also significantly higher in mice injected with FD/LIF cells than in control mice injected with FD cells.

All mice injected with FD/LIF cells exhibited moderate spleen enlargement that was more evident in TABLE 2

Spleen and Marrow Changes in Mice Injected with FD/LIF Cells

Treatment OGy 6Gy

FD FD/LIF FD FD/LIF

SPLEEN

Number of Mice 12 21 15 16

Spleen Weight mg 110±30 230±70 120±40 170±40

PERCENT

Blasts 3.2± 2.0 6.1+ 3.3 2.5± 2.1 7.9± 4.0

Myeloblasts/ 1.3± 2.2 7.7± 6.7 2.5± 2.0 9.8± 5.2 Myelocytes

Neutrophils 3.2± 2.6 15.0± 5.9 5.6± 2.5 26.0+18.2

Lymphocytes 77.6± 8.0 31.5±13.7 36.9±12.0 9.7± 5.0

Monocytes 2.4± 2.1 7.5± 3.1 5.8± 3.1 11.7+ 4.9

Eosinophils 5.4± 4.9 3.1± 3.0 16.6±11.0 2.1± 2.0

Nucleated 6.8± 3.0 29.1±17.9 30.6±20.0 32.7±27.7

Erythroid Cells

BONE MARROW

Number of Mice 10 8/19 14 9/19

Total Cells x 10⁶ 25.2± 5. 9 5.5± 0. 7 24.0± 4. 0 6.0±4.0

PERCENT

Blasts 3.2± 1. 2 3.0± 1.9 3.7± 2. ,3 4.1± 4.4

Myeloblasts/ 5.8± 3. 4 11.1± 3. ,8 8.4± 5.7 10.8± 7.4

Myelocytes

Neutrophils 33.6± 9. ,1 66.9+11.0 29.1+12. 6 68.6±25.3

Lymphocytes 23.7± 5. ,3 7.5± 5.0 21.1+ 8. ,1 2.7+ 3.2

Monocytes 5.2+ 2. .3 6.3± 3.9 5.9+ 3. ,7 7.0± 5.2

Eosinophils 19.2±13.8 3.1+ 1.9 14.3+11.7 2.3+ 2.5

Nucleated 9.3± 4.3 2.1+ 1 .1 17.5+ 7.7 4.5± 7.3

Erythroid Cells

Mean Values ± Standard Deviations unirradiated recipients (Table 2). The spleen was

slightly paler than normal but exhibited no surface nodules and in both types of recipient exhibited a

reduced percentage of lymphoid cells, an elevated

percentage of granulocytic cells and, particularly in unirradiated recipients, an elevated percentage of

nucleated erythroid cells.

A striking feature of the femoral bone marrow in mice injected with FD/LIF cells was the difficulty

experienced in inserting a needle to flush out marrow cells. Marrow cells were not able to be recovered from 10 of 19 irradiated and 11 of 19 unirradiated mice.

Where cells were recovered from mice injected with FD/LIF cells, there was a 4-5-fold reduction in total cell number (Table 2) in marrow from both types of recipient. In these marrows there was a significant rise in the proportion of granulocytic cells and a significant reduction in lymphoid and nucleated erythroid cells.

Interestingly, recipients of FD cells exhibited a major increase in eosinophils, usually myelocytes and

promyelocytes, a change not seen in the marrow of

recipients of FD/LIF cells. EXAMPLE 3

Bone changes

The femur, tibia and sternum in mice injected with FD/LIF cells showed a remarkable overgrowth of bone, seen as thickening of the bone cortex and the formation of

irregular-shaped trabeculae in marrow cavity (Figure 3). The marrow cavity was convoluted and narrowed in

unirradiated and irradiated recipients and was depleted of hemopoietic cells, those remaining being mainly granulocytic cells although in some but not all marrows, megakaryocytes were present. Replacing the hemopoietic tissue,

particularly in the ends of the long bones were elongated cells oriented in parallel bundles. These cells were enlarged close to bone surface and merged with the developed bone. From the calcification evident in the cytoplasm of these cells, they appeared to be osteoblasts but, although the total number seemed well in excess of the number that could have been present in the normal marrow cavity, no obvious migration was seen through bone foramina. In some bones, osteoclasts were prominent and adjacent bone had irregular scalloped edges. Irregular foramina were also present in many bones particularly at the ends of long bones. These could have been the consequence of increased osteoclastic activity. The open channels between the marrow and the tissues outside the bone could have allowed free entry of osteoblast precursors or exit of marrow cells.

The red pulp of the spleen was enlarged and was packed with hemopoietic cells, the lymphoid follicles being severely depleted of cells. In many livers, there were also prominent foci of hemopoietic cells and some livers showed areas of necrosis and/or calcification. Less commonly, cirrhotic changes were present but there was no mitotic activity in parenchymal cells and there was consistent reduction in the volume of individual parenchymal cells. Areas of calcification were seen in striated muscle and the heart in some but not all animals but no foci of cellular infiltration were observed in these locations. In several mice, prominent macroscopic areas of calcification were visible in the liver.

Mice injected with FD cells showed none of the above pathological changes and the only condition evident in some of these mice was the presence of small focal

accumulations of FD cells in the spleen or mesenteric node. EXAMPLE 4

In vitro Effects of LIF in neonatal mouse calvaria

Bone resorption was measured as the release of ⁴⁵Ca from bones which had been pre-labelled in vivo. Hemicalvaria were dissected as previously described (3) and preincubated for 24 hours in minimal essential media plus 1% v/v fetal calf serum. Bones were then changed to media containing the experimental compounds and incubated for a further 48 hours, at the end of which time media and bone were harvested for measurement of ⁴⁵Ca content. In

experiments involving the measurement of [³H]-thymidine and [³H]-phenylalanine, these compounds were added in the last 4 hours of the 48 hour experimental period and their content in the calvaria quantified by the method of Canalis (4).

Both recombinant murine (Figure 4) and human (Figure 5) LIF stimulated ⁴⁵Ca release from pre-labelled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in osteoclast numbers in the calvaria (Figure 6). The ability of LIF to stimulate bone resorption was blocked by the presence of 10^-7M indomethacin, an inhibitor of prostaglandin synthesis (Figure 7; Table 3)

The effect of LIF on [³H]-thymidine incorporation, a measure of cell proliferation, was also studied. LIF was found to stimulate [³H] - thymidine incorporation into calvaria but the dose response relationship was different from that for bone resorption in that it was a more

sensitive response (Figure 8). Furthermore, this effect of LIF was not blocked by the presence of indomethacin (Figure 9). LIF also increased the incorporation of [³H]-phenylalanine into bones (control 21420 ± 1110 cpm; LIF Table 3 Effect of indomethacin on the LIF response to PA in calvarial osteoblasts and UMR 106-01 cells.

PA Activity ( % Tcpm )

+ Indomethacin

1.5 X 10^-5M

Osteoblasts

Control 13.0± 0.9 10.6 ± 0.9

UF 7.0± 0.7 5.4± 0.3

UMR 106-01

Control 12.7±2.2 9.4± 0.1

LIF 4.3± 0.5 4.5 ± 0.4

Calvarial osteoblasts and UMR 106-01 cells were subcultured onto 125-I fibrin coated plates as described in the Methods section. Cells were preincubated for 8 h in serum free conditions as described in the legend in Table 4. The cells were incubated in the presence and absence of LIF (1000 U/ml ) and indomethacin (1.5 x 10^-5M ).

26890 ± 1253 cpm, p < 0.01), indicating stimulation of protein synthesis.

Furthermore, LIF has significant effects on [³H]-thymidine incorporation in neonatal mouse calvaria even at doses as low as 100 units/ml ( Table 4) .

Accordingly, these results indicate that LIF stimulates bone resorption by a mechanism possibly involving prostaglandin production and involving an increase in the number of osteoclasts (bone resorbing cells) in the

calvaria. Its action on thymidine incorporation probably reflects responses of osteoblasts, and does not appear to involve local prostaglandin production since it is

unaffected by the presence of indomethacin. These data further indicate that LIF may be an important regulator of bone cell function.

EXAMPLE 5

Effect of LIF on certain responses in osteoblast-like cells

Experiments were carried out to determine the effect of LIF on the expression of mRNA of the bone-specific proteins, α1(I) collagen and osteonectin, and upon

plasminogen activator (PA) activity and the production of mRNA for plasminogen activator inhibitor - 1(PAI-1) in osteoblast-like cells. 1. MATERIALS AND METHODS

(i) Chemical and biological materials

Synthetic human parathyroid hormone (PTH) (1-34) was from Beckman Pty. Ltd., Palo Alto, CA, U.S.A., while human recombinant TGFβ, TNFα and TNFβ, and full-length rat actin cDNA were gifts from Genentech Inc., San Francisco, CA, U.S.A. 1,25-dihydroxyvitamin D₃ was generously provided by Dr. M. Uskokovich, Hoffman La Roche Inc., New Jersey, and

Table 4 Effect of low concentrations of hLIF on p[³H]-thymidine incorporation and 4⁵Ca release in neonatal mouse calvaria.

[ hLIF] [³H]-Thymidine ⁴⁵Ca Release (units/ml) Incorporation (treatment/control)

(treatment/control)

100 1.31±0.12^a 1.01±0.07

300 1.17±0.07^a 1.04±0.06

1000 1.38±0.08^b 1.05±0.07

Data are mean ± sem and are given as treatment/control ratios for ease of comparison of the two indices. Significant differences from control are shown: a, p<0.05; b, p<0.01. n=3-4 in each group. Similar results were obtained in two other experiments.

prostaglandin E₂ was purchased from Upjohn Company,

Kalamazoo, Michigan. Recombinant human LIF was produced in both yeast and E.coli and purified to homogeneity as

described. The specific biological activity of pure LIF is -10⁸ units/mg. Antiserum against TGFβ was purchased from R and D Systems, Inc. Minneapolis, M.D. 55413 U.S.A.

Rabbit antiserum against PAI-2, prepared form U937 cells, was by courtesy of Professor E.K.O. Kruithof, Centre Hospitaller Universitaire, Vaudois, Lausanne, Switzerland. Rabbit anti-rat hepatoma cell (HTC)PAI-1 (S) and rat PAI-1 cDNA cloned into the EcoRl site of pBluescript SK(-)(6) were kindly provided by Drs. R. Zeheb and T.D. Gelehrter,

University of Michigan, Ann Arbor, MI. The cDNA probe for IGFβ was provided by Genentech, San Francisco. The cDNA probe for murine LIF was the plasmid pLIF7.2b which spans the coding region of the murine LIF mRNA. ¹²⁵I-fibrinogen, [α³²P]UTP, [α₃₂P]CTP and a T7/SP6 paired promoter system were from Amersham International, T₃RNA polymerase was from

International Biotechnologies Inc., New Haven, CT, 06535, U.S.A. and nick-translation kits from Boehringer Mannheim, Mannheim, West Germany. Sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) M_r standards were from Bio-Rad Laboratories, Richmond, CA 94804, U.S.A.

(ii) Osetoblast Systems

The UMR 106-01 and UMR106-06 cells are a subclone of the UMR106 cells which are a clonal line of rat

osteogenic sarcoma cells in which many features of the osteoblast phenotype are preserved (1, 8, 9). The

experiments described were carried out on cells between the 7th and 15th passages and maintained in Eagles Minimal

Essential Medium (MEM) supplemented with non-essential amino acids, 14mM 4-(2-Hydroxyethyl)-1-piperazinoethanesulfonic acid (Hepes), gentamycin (80μg/ml), minocyclinee(1 mg/ml) and 5% (V/V) fetal calf serum (FCS) at 37°C in 5% CO₂ in air. The UMR201 clonal cell line, which was derived from nerborn rat calvaria (10), has properties consistent with those of an osteoblast precursor. Treatment with retinoic acid leads to substantial increase in alkaline phosphatase activity and mRNA, and the cells produce predominantly type I collagen and have other osteoblastic features (10, 11).

Osteoblast-rich calvarial cells were prepared as described previously (7, 8) and were used on the first subculture. Conditions of culture maintenance for UMR201 and osteoblast-rich cells were as described for osteogenic sarcoma cells.

Osteoclasts were isolated from newborn long rat bones as previously described (24), and allowed to settle onto glass coverslips for periods of greater than 30 mins before washing, to ensure significant contamination with osteoblasts (25). (iii) RNA Preparation and Hybridization

(Figures 11 and 12)

Cells were washed twice with phosphate buffered saline (PBS) and preincubated in minimal essential medium (MEM) and 0.1% w/v bovine serum albumin (BSA) for 15 h. At the end of this period the medium was replaced with MEM and 0.1% w/v BSA with and without added LIF. The cells were incubated for varying times and concentrations. Total RNA was prepared as previously described (12). An aliquot of 10 μg of total RNA was electrophoresed on a 1.5% w/v

agarose/formaldehyde gel as previously described (12) and transferred to Hybond N (Amersham International) by Northern blotting. Labelled riboprobes for PAI-1 were prepared from rat PAI-1 cDNA cloned into the EcoRI site of Bluescript SK(-) using T3 RNA polymerase. Hybridization conditions were essentially as described (12) in 50% v/v formamide, 6 x SSC, 5 x Denhardt's, 0.1% w/v SDS at 65ºC. Filters were washed to a stringency of 0.1 x SSC at 65°C and exposed to Kodak XAR-5 X-ray film backed by intensifying screens.

After removing DNA bound to the RNA by boiling for 5 min in 0.1 x SSC, 0.1% w/v SDS filters were probed a second time with a nick-translated full-length rat actin cDNA probe. The filters were washed to a stringency of 0.1 x SSC, 0.1% w/v SDS at 42ºC and exposed as above. Similar hybridization conditions were used in detecting mRNA for osteonectin and type I collagen. In each case the probes were labelled by nick translation.

(IV) Plasminogen Activator (PA) Activity and

Alkaline Phosphatase Cells were subcultured at 20,000/200^1 into ¹²⁵I-fibrin-coated wells of 96 well tissue culture plates (13). After 24 hours in maintenance medium the cells were washed twice with Dulbecco's PBS and the medium was replaced with MEM and 0.1% w/v BSA. After 8 hours the medium was replaced with fresh Eagle's MEM and 0.1% w/v BSA together with human plasminogen (1.56 ug/ml). Plasminogen-independent activity was measured in parallel in the absence of added human plasminogen. After 24 hours an aliquot of medium was removed and counted for solubilized radioactivity.

Plasminogen dependent fibrinolytic activity is expressed as percentage of total radioactivity released from the plate by trypsin.

For studies of regulation of alkaline phosphatase activity, UMR 201 cells were subcultured into 9.6cm² six-plate multiwell dishes in alpha modified MEM (α-MEM)

containing 10% v/v FCS. When the cells were semi-confluent, the medium was changed to α-MEM with 2% v/v vitamin A-depleted FCS containing the indicated concentrations of LIF plus 1μM retinoic acid. Medium was changed after 48 h and fresh treatments added. Specific activity of alkaline phosphatase was measured as described previously (10). (v) LIF Receptor Studies

UMR 106-01 and 106-06 cells were subcultured, centrifuged in Hanks' BSS and kept on ice after which cells were resuspended and layered over 180 μl fetal calf serum (FCS) in tapered flexible plastic tubes. Cell-associated and free ¹²⁵I-LIF were separated by centrifugation of the cells through FCS. The tip of the tube containing the cell pellet was cut off and the pellet and supernatant were counted in aγ-counter (Packard Crystal Multi-Detecter,

Packard, Downers Grove, IL). Saturation isotherms were analyzed by the method of Scatchard (14) using the nonlinear curve fitting programme Ligand (15).

Cells isolated from newborn rat long bones were allowed to adhere to glass coverslips within the wells of a 12-well tissue culture plate (Flow Laboratories, McLean VA), and were covered with 1.0 ml KHF containing 5x10⁸ cpm ¹²⁵I-LIF with or without a 40-fold excess of unlabelled LIF.

Incubation was carried out at 37°C for 30 minutes. The medium was removed and the cells were washed with 4x4 ml aliquots of warm KHF and 2x4 ml aliquots of warm 20mM sodium phosphate, 0.15 ml NaCl at pH 7.4. The cells were fixed in 4 ml of 2.5% u/v glutaraldehyde in PBS and washed with 2x4 ml aliquots of water. The coverslips were attached to gelatin-coated glass slides using DePex mounting medium, allowed to air-dry overnight, dipped in Kodak NTB2

photographic emulsion at 42°C in a dark room and allowed to dry. Slides were then sealed in light proof boxes

containing Drierite and allowed to expose for 4 weeks at 4°C. Prior to development, slides were warmed to room temperature and then developed for 1 min in Kodak D19 developer (40g/500 ml water) and fixed in Agfa G333c X-ray fixer for 3 min. (vi) Fibrin and Reverse Fibrin Autography

Cells were subcultured at 500,000 cells/well (9.6 cm²) in MEM and 5% v/v FCS, After 25 h the medium was aspirated, cells washed twice with PBS and the medium replaced with 1 ml MEM and 0.1% w/v BSA. After 8 h the medium was aspirated and 1 ml of fresh MEM and 0.1% w/v BSA added together with treatment. At 25 h the medium was aspirated and centrifuged to remove any cell debris. The cells were washed twice with PBS and lysed in 1ml. 0.1% v/v triton X 100. Conditioned medium (CM) and cell extracts, prepared as described above, were resolved by SDS-PAGE on 10% w/v slab gels under non-reducing conditions as described (16). PA and PAI activity were visualized by fibrin (17) and reverse fibrin (18) gel autograph, respectively.

(vii) Immunoblottino Conditioned media from confluent cultures of UMR

106-01 cells and calvarial osteoblasts incubated for 24 h in serum-free MEM were concentrated by ultrafiltration with a 30,000 M_r cutoff, while cells were washed twice with PBS, scraped, centrifuged and lysed in 0.1% v/v triton X 100. The proteins were resolved by SDS-PAGE as above and

transferred to nitrocellulose. PAI-1 was detected by incubation of the nitrocellulose with rabbit polyclonal anti-rat HTC PAI-1 (5) followed by anti-rabbit Ig conjugated with alkaline phosphatase and visualization with 5' bromo-4-chloro-3-indolyl phosphate (19).

(viii) RNA Detection Using the Polymerase Chain Reaction

(Figure 15) Total cytoplasmic RNA was isolated from UMR 201 or UMR 106-01 cells or primary osteoblast populations (see above) as described (26) and subjected to first strand cDNA synthesis in a 20 μl reaction containing 50 mM Tris-C1 (pH 8.3 at 42°C), 20 mM KCl, 10mM MgC12, 5 mM dithiothretiol, 1 mM of each dNTP, 20 /ug/ml oligo-DT₁₅ and LIF specific

oligonucleotide primers, and 20 units AMV reverse

transcriptase (Boehringer Mannheim) for 40 min at 42°C.

After completion of first-strand synthesis, the reaction was diluted to 100 μl with distilled water and 5 μl used to each PCR reaction. PCR reactions (in a volume of 50 μl )

contained 200 μM of each dNTP, 1 μm of each specific primer, buffer as supplied in the GeneAmp kit (Cetus Corp., USA) and 1.25 units Taq polymerase. The primers used for PCR

reactions were: LIF, 5' - CCGAATTCGAAAACGGCCTGCATCTAAGG and 3' - CCGAATTCGCCATTGAGCTGTCCAGTTG, defining a 300 bp

fragment of mRNA, and GAPDH, 5' - ACCACCATGGAGAAGGCTGC, GAPDH, 5' - ACCACCATGGAGAAGGCTGC and 3'-

CTCAGTGTAGCCCAGGATGC, defining a 500 bp fragment. The PCR reaction conditions were: 1.5 min at 94°C; 2 min at 60°C; 3 min at 72°C for 25 cycles in a Perkin-Elmer-Cetus DNA

Thermal Cycler. A portion of the PCR reaction was

electrophoresed through a 1.2% w/v agarose gel and

transferred to nitrocellulose. Filters were prehybridized, hybridized and washed in 2 x SSC-containing buffers as described (21). The hybridization probes were the gel-purified cDNA inserts of plasmids pLIF7.2b and hGAP-DH, radiolabeled to a specific activity of approximately 2x10⁸ cpm/ug by nick translation and included in the hybridization at approximately 2x10⁷cpm/ml.

2. RESULTS

(i) Effect of LIF on bone resorption

In the PA response it was found that LIF inhibited PA activity in osteoblast-like cells in a dose dependent manner. The inhibitions was apparent in control cells, or in cells which had been treated with an agent which increases PA activity, e.g. parathyroid hormone (PTH). An example of such an experiment is provided in Figure 10, showing inhibition of PA activity in the UMR106-01 cells. Similar inhibition was demonstrated in the calvarial osteoblasts. These experiments are not possible in the UMR201 cells, which do not survive sufficiently long enough in culture in the absence of serum.

Because the effect of LIF to inhibit PA activity might be due to an increase in the amount of a PA inhibitor, the possible effect of LIF on the formation of PAI-1, the PA inhibitor produced by osteoblasts, was examined. Evidence based on reverse fibrin autography, western blotting with a specific antiserum to PAI-1, and demonstration of PAI-1 mRNA by northern blots of calvarial osteoblasts and UMR106 cells showed this to be the case. When UMR 106 cells were treated with LIF, there was an increase in mRNA for PAI-1,

consistent with the possibility that the inhibition of PA activity of LIF was due to the generation of increased amounts of PAI-1. Regulation of PA activity by inhibitor formation is well recognised as an important factor in determining nett PA activity. Figure 11 shows a time course up to 24 hours of the PAI-1 mRNA with response to LIF. As early as 1 hour after the beginning of LIF treatment there was a clear increase in PAI-1 mRNA. This effect of LIF on inhibition of PA activity and promotion of PAI-1 mRNA is very similar to the effect of transforming growth factor β (TGFβ) in the same cells. To exclude the possibility that LIF might be acting through the generation of TGFβ in the cells, two experiments were carried out. In the first, an antibody against TGFβ was used to determine whether it blocked the action of LIF. No blocking action was found (Table 5). The same antibody was able to block the action of TGFβ. The second experiment was to determine whether LIF influenced mRNA for TGFβ in the osteoblast-like cells. In the UMR106 cells and in calvarial osteoblasts, no effect of LIF on TGFβ mRNA was found. It is concluded that the LIF action of PAI-1 was not mediated in the osteoblast-like cells by any generation of TGFβ as an intermediate in its action.

TABLE 5

Effect of anti human TGF antibody (ab) on PA Activity in

LIF UMR106-01 cells.

Expt. 1 PA Activity (% Tcpm)

Control 39.7 ± 1.4

LIF (100 u/ml) 20.9 ± 0.8 TGFβ ab (30 ug/ml) 43.5 ± 0.4 LIF + TGFβ ab 23.1 ± 0.7

Expt. 2

Control 20.8 ± 0.3

PTH (10 ng/ml) 46.0 ± 1.1

TGFβ (0.5 ng/ml) 3.2 ± 0.1

PTH + TGFβ 12.0 ± 0.7

PTH + TGFβ + ab (30 ug/ml) 51.7 ± 4.7

PTH + TGFβ + TGFβ ab 38.6 ± 2.2

LIF treatment of UMR201 cells resulted in a dose-dependent increase in mRNA for both type I collagen and osteonectin. Northern blots to demonstrate this are provided in Figure 12. Both these effects are also demonstrated by TGFβ in UMR 201 cells. However, not all TGFβ-like effects are

demonstrated by LIF, even in these cells. For example, whereas TGFβ attenuates the rise in alkaline phosphatase activity and mRNA in response to retinoic acid in these cells, the reverse takes place with LIF. LIF actually enhances the alkaline phosphatase response, which is an effect very similar to that of another cytokine, tumour necrosis factor α (TNFα).

The effect of LIF on the osteoblast-like cells is largely anabolic, in that there is clear evidence of a LIF effect to increase synthesis of some of the important specific components of bone protein, and a regulatory effect on the PA-PA inhibitor system, which give LIF the potential of being an important regulator in the microenvironment of bone cells, and therefore important in bone remodelling.

The effect of LIF to increase bone resorption in mouse calvariae is one which is dependent upon the synthesis within those bones in organ culture of prostaglandins. The evidence for this is that blocking prostaglandin synthesis inhibits the LIF stimulation of resorption. On the other hand, the LIF stimulation of ³H-thymidine into acid-precipitable macromolecules was not inhibited by

indomethacin (Example 4). TGFβ also promotes resorption in the mouse calvarial organ culture system, but it is

inhibited when prostaglandin synthesis is inhibited, however, TGFβ is not a promoter of resorption in a system using fetal rat long bones. The mouse calvarial system is one in which the generation of prostaglandins is virtually certain to result in increased resorption. However, TGFβ has its predominant effect on promoting formation of bone, as does LIF. In other types of experimental systems there is good evidence that prostaglandins in fact stimulate bone formation.

(ii) Receptor Studies

Receptor autoradiography with ¹²⁵I-LIF was carried out to determine which cells of newborn rodent bone bound LIF specifically. Osteoclast preparations from newborn rat long bones were prepared so as to ensure that the cultures were heavily contaminated with osteoblasts, macrophages and other cells. This was achieved by allowing a long settling time onto coverslips after isolation of cells (25). In these experiments, specific binding of ¹²⁵I-LIF was

demonstrated on cells with the characteristics of

osteoblasts (Figure 14) and to macrophages as the inventors have shown previously (22), but no evidence of LIF binding to osteoclasts or to mononuclear tartrate-resistant acid phosphatase-positive cells (osteoclast precursors) could be found.

In view of the identification of LIF receptors on primary osteoblasts, biochemical studies of LIF binding were carried out on the osteoblast-like, clonal osteogenic sarcoma cell line, UMR106-06. Specific binding was readily demonstrated, and Scatchard analysis indicated a receptor number of 300 per cell, and K_D of 60 pM (Fig. 14). The receptor number compares favourably with the 400-500 per cell which had been previously demonstrated on the murine myeloid leukemia cell line, M1 (20). Specific binding was also demonstrated on UMR 201 cells, although at lower receptor numbers.

In view of the growing realization that locally produced factors are important in regulating bone metabolism (7), and the demonstration of GM-CSF production by osteoblasts (23) evidence was sought for production of LIF by osteoblast-like cells.

(iii) Production of LIF by osteoblasts Biological assays revealed the presence of very low levels of M1 cell differentiating activity in medium conditioned by primary osteoblast populations and by the osteoblastic cell lines UMR 201 and 106-01. In order to confirm that this activity was due to LIF, these cells were assayed for LIF transcripts. LIF transcripts were not detected in RNA from these sources using conventional Northern blots (not shown), possibly because the amounts of LIF formed were very low. LIF transcripts were, however, demonstrable in these cells by using a sensitive RNA detection approach based on the polymerase chain reaction. It appears from such analyses (eg Figure 15) that LIF is transcribed constitutively in both the UMR 201 and UMR 106-01 cells, as well as in primary osteoblast populations, although in some experiments

modulation of the level in UMR 106-01 was apparent on treatment with TGFβ (Figure 15). However, given the

possible difficulties in quantifying PCR reactions it cannot be dermined with certainty the level of induction in

response to TGFβ. It was consistently found that UMR 201 cells contained higher levels of LIF transcripts than UMR 106-01 cells. (iv) Effect of LIF on alkaline phosphatase activity in UMR201 cells

Treatment of UMR 201 cells with LIF for 3 days resulted in dose-dependent potentiation of the retinoic acid-induced alkaline phosphatase activity (Table 6). This effect is very similar to that we have previously obtained with TNFα in the same cells (11). In the latter work evidence was obtained that the effect was due to stabilization of mRNA for alkaline phosphatase. Thus, although LIF and TNFα resemble each other in this action, they have effects in opposing directions on PA activity in osteoblasts (Table 7).

Effect of LIF on PA Ac tivity in Control and Stimulated UMR-106-01 cells.

PA Activity (% T cpm)

Treatment -LIF +LIF(1000U/ml)

Control 10.5 + 0.3 2.0+0.1

PTH (3.3 x 10^-9M) 42.1 + 1.1 14.7 +1.0

PGE2 (10^-7M) 22.0 + 1.6 3.5 + 0.2

U5 (OH)₂ D3 (10^-8M) 22.3 + 0.2 5.5 + 0.1

TNFα (200 U/ml) 25.3 + 0.3 4.6 + 0.1

TNFβ (5000U/ml) 32.4 + 1.5 5.3 + 0.3

Cells were subcultured on to ¹²⁵-I-fibrin-coated plates as described in the Me thods section. Cells were preincubated for 8 h in MEM plus 0.1% BSA before the addition of fresh medium containing the bone resorbing factors. The incubation was carried out for 24 h.

REFERENCES;

1) Horton, J. E., Raisa, L.G., Simmons, H.A., Oppenheim, J.J., & Meyerhager, S.E. Science, 177:793-795. 1972. 2) Martin, T.J., Ng, K.W., & Nicholson, G.C. Clin. Endocr. Metab. 2:1-29, 1988.

3) Canalis, E. Endocrinol. 118:78-81, 1986. 4) Reid, I.R., Katz, J., Ibbertson, H.K., & Gray, D.H.

Clacid. Tissue Int. 38:38-43, 1986.

5) Zeheb, R., Rafferty, U.M., Rodriguez, M.A., Andreasen, P., & Gelehrter, T.D. Thromb. & Haemost. 58:1017-1022, 1987.

6) Zeheb, R., Gelehrter, T.D. Gene 73:459-468, 1988.

7) Martin, T.M., Ng, K.W., Partridge, N.D. & Livesey, S.A. Methods in Enzvmol. 185: 324-336, 1987.

8) Partridge, N.C., Alcorn, D., Michelangeli, V.P., Ryan, G., & Martin, T.J. Cancer Res., 43:4388-4394. 1983. 9) Forrest, S.M., Ng, K.W., Findlay, D.M., Michelangeli, V.P., Livesey, S.A., Partridge, N.C., Zajac, J.D., & Martin, T.J. Calc. Tiss. Int. 37:51-56, 1985.

10) Ng, K.W., Gummer, R.R., Michelangeli, V.P., Bateman, J.F., Mascara, T., Cole, W.G., & Martin, T.J., J. Bone. Min. Res. 3:53-61, 1988.

11) Ng, K.W., Hudson, P.J., Power, B.E., Manji, S.S.,

Gummer, P.R., & Martin, T.J., J. Mol. Endocrinol., 3: 57-64, 1989. 12) Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular cloning: a Laboratory Manual, Cold spring Harbour

Laboratory, Cold Spring Harbour, N.Y., USA, 1982. 13) Allen, E.H., Hamilton, J.A., Metcalf, D.C., Kabota, M. & Martin, T.J. Biochim. Biophys. Acta 888:199-207. 1986.

14) Scatchard, G. Ann. NY. Acad. Sci. 51:600-672, 1949. 15) Munson, P.J., & Rodbard, D. Anal. Biochem. 107:220-237, 1980.

16) Laemmli (1970). 17) Granelli-Piperino, A., & Reich, E. J. Exp. Med.

148:223-234, 1978.

18) Erickson, L.A., Lawrence, P.A., & Loskutoff, D/J. Anal. Biochem. 137:454-463. 1984.

19) Knecht, D.A., & Dimond, R.L. Anal. Biochem. 136: 180-184, 1984.

20) Hilton, D.J., Nicola, N.A., & Metcalf, D. Proc. Natl. Acad. Sci. (USA) 85:5971-5975, 1988.

21) Gearing, D.P., King, J.A., Gough, N.M. & Nicola, N.A. EMBO J. 8:3667-3676, 1989. 22) Hilton, D.J., Nicola, N.A. & Metcalf, D. J. Cell.

Physiol. (In Press), 1990.

23) Felix, R., Elford, P.R., Stoerile, D., Cecchin, M., Wetterward, A., Trechsel, U., Fleisch, H. & Shadier, B.M. J. Bone Min. Res. 3:27-32, 1988. 24) Nicholson, G.C., Moseley, J.M., Sexton, P.M.,

Mendelsohn, F.A.O. & Martin, T.J. J. Clin. Invest. 78: 355-360, 1986. 25) McSheehy, P.M.J. & Chambers, T.J. Endocrinology 118: 824-828, 1986.

26) Gough, N.M. Anal. Biochem. 173 : 93-95, 1988. 27) Allen, E.H., D.J. Hilton, M.A. Brown et al. J. Cellular Physiology (submitted for publication), 1990.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for inducing, promoting and/or enhancing metabolic effects on bone in an animal comprising

administering to said animal an effective amount of

leukaemia inhibitory factor (LIF) and/or LIF-like

polypeptides for a time and under conditions sufficient to effect bone metabolism.

2. The method according to claim 1 wherein the animal is mammalian or avian.

3. The method according to claim 2 wherein the animal is mammalian.

4. The method according to claim 3 wherein the mammal is a human.

5. The method according to any one of claims 1 to 4 wherein the metabolic effect comprises bone formation and/or bone resorption, increased protein synthesis in bone tissue and/or thymidine uptake in bone tissue.

6. The method according to any one of claims 1 to 5 optionally further comprising the administration of one or more active molecules in combination with LIF and/or LIF-like polypeptides.

7. The method according to claim 6 wherein the active molecule is another cytokine.

8. A pharmaceutical composition useful in inducing, promoting and/or enhancing metabolic effects on bone in an animal comprising an effective amount of leukaemia

inhibitory factor (LIF) and/or LIF-like polypeptides and one or more active molecules and one or more pharmaceutically acceptable carriers and/or diluents.

9. The pharmaceutical composition according to claim 8 wherein the animal is mammalian or avian.

10. The pharmaceutical composition according to claim 9 wherein the animal is mammalian.

11. The composition according to claim 10 wherein the mammal is a human.

12. The composition according to claim 8 wherein the active molecule is a cytokine.

13. The composition according to claim 8 wherein the active molecule is an inorganic or organic molecule.

14. The use of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides in the manufacture of a medicament for the induction, promotion and/or enhancement of metabolic effects on bone in animals.

15. The use according to claim 14 wherein the

animal is avian or mammalian.

16. The use according to claim 15 wherein the

animal is mammalian.

17. The use according to claim 15 wherein the mammal is human.

18. The use according to any one of claims 14 to 17 wherein the metabolic effect comprises bone formation and/or bone resorption, increased protein synthesis in bone tissue and/or increased thymidine uptake in bone tissue.

19. A method for the treatment of injuries and/or diseases which result in bone degeneration or fracture in an animal said method comprising the administration to said animal of a bone forming - increasing - effective amount of leukaemia inhibitory factor (LIF) and/or LIF-like

polypeptides for a time and under conditions sufficient to effect bone formation, and/or bone fracture repair.

20. The method according to claim 19 wherein said animal is a human.

21. The method according to claim 19 or 20 wherein said injury is a bone fracture.

22. The method according to claim 19 or 20 wherein said disease is post-menopausal osteoporosis.

23. The method according to any one of claims 19 to 22 optionally further comprising administering one or more active molecule in combinationn with LIF and/or LIF-like molecules.

24. The method according to claim 23 wherein the active molecule is a cytokine.

25. The method according to claim 23 wherein the active molecule is an inorganic or organic molecule.

26. A method for stimulating bone formation in a mammal comprising administering to said human a bone forming-increasing-effective amount of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides for a time and under conditions sufficient to effect bone formation.

27. The method according to claim 26 wherein said mammal is a human.

28. A method for stimulating bone fracture repair in a mammal comprising administering to said mammal a bone-forming effective amount of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides for a time and under conditions sufficient to effect bone formation.

29. The method according to claim 28 wherein said mammal is human.

30. The use of leukaemia inhibitory factor (LIF) and/or LIF-like polypeptides in the manufacutre of a medicament for treating injuries and/or diseases in animals where bone degeneration or fracture is a symptom or a result.

31. The use according to claim 30 wherein the animal is avian or mammalian.

32. The use according to claim 31 wherein the animal is mammalian.

33. The use according to claim 32 wherein the animal is human.

34. The use according to any one of claim 30 to 33 wherein the injury is bone fracture.

35. The use according to any one of claims 30 to 33 wherein the disease is osteoporosis.

36. The use according to claim 35 wherein the disease is post-menopausal osteoporosis.