JP3055802B2 - Metabolic effect of leukemia inhibitory factor on bone - Google Patents

Description

The present invention relates generally to the effects of leukemia inhibitory factor (LIF) on animal bone. 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 animal bone.

The cytokine leukemia inhibitory factor (LIF) is a protein that was previously purified from both Escherichia coli and yeast cells, cloned, and mass produced in purified recombinant form (International Patent Application No. PCT / AU88 / 00093
issue). LIF is originally a mouse myeloid leukemia cell line (M1)
Was isolated on the basis of its ability to induce and suppress differentiation. LIF has been found to have a potent differentiation inhibitory effect on normal fetal stem cells. Despite the LIF receptor being detected on cells of the monocyte-macrophage cell lineage, LIF has no apparent proliferative effect on normal hematopoietic cells.

Several cytokines and growth factors have been implicated in bone remodeling and have been shown to promote bone resorption, especially in vitro. The term "osteoclast activator" (OAF) applies to bone resorption activity detected in supernatants from lectin-transformed lymphocytes (1) and is associated with certain types of hematopoietic malignancy Used to explain excessive bone resorption. In fact, today OAF is using Interleukin 1 (IL
-1) and tumor necrosis factors α and β (TNFα, TNFβ)
And several other cytokines, all of which have been found to be potent bone resorption factors (2).

LIF has no significant structural or sequence homology to the above cytokines. Therefore, an analysis of the potential physiological and biochemical effects of LIF on bone remodeling in vitro and in vivo, including studies of the cellular targets of LIF action, has shown that LIF has a role in bone resorption and / or bone formation. It has been found that by inducing, promoting and / or enhancing has a metabolic effect on bone,
That is surprising.

According to the present invention, it has been found that LIF is an important regulator of paracrine secretion in bone and has many biochemical effects specific to osteoblasts. These effects include the suppression of plasminogen activator (PA) activity in osteoblasts via increased synthesis to produce the most likely PA inhibitor-1. LIF itself is expressed in osteoblasts, mainly osteoblasts. Thus, various lines of evidence establish that cells of the osteoblast lineage are targets for LIF action in paracrine or autocrine mechanisms.

Therefore, one embodiment of the present invention provides an animal with a leukemia inhibitory factor (LI
F) and / or a method of inducing, promoting and / or enhancing metabolic effects on bone in animals, comprising administering a LIF-like polypeptide alone or in combination with other cytokines.

Another embodiment of the present invention relates to a leukemia inhibitory factor (LI
F) and / or an effective amount of a LIF-like polypeptide is determined by combining cytokines, inorganic or organic molecules, and one or more other molecules that affect bone resorption, calcification and / or bone formation, and pharmaceutically It is an object of the present invention to provide a pharmaceutical composition useful for inducing, promoting and / or enhancing metabolic effects on bone in animals containing one or more acceptable carriers and / or diluents in combination.

In another aspect, the invention relates to a leukemia inhibitory factor (LIF) in the manufacture of a medicament useful for inducing, promoting and / or enhancing metabolic effects on bone in animals.
And / or devises the use of LIF-like polypeptides.

In yet another aspect, the invention provides an effective amount of a leukemia inhibitory factor (LIF) and / or LIF-like polypeptide that increases bone formation, alone or under conditions and conditions sufficient to effect bone calcification. A method of treating damage and / or disease that results in bone degeneration or fracture in an animal, including administering to the animal in combination with other effective molecules, is devised.

In a preferred embodiment of the invention, the animal is a mammal or bird, more preferably a human.

 The following abbreviations are used in this specification.

LIF Leukemia inhibitory factor TNFα Tumor necrosis factor α TNFβ Tumor necrosis factor β TGFβ Tumor growth factor β PTH Parathyroid hormone PA Plasminogen activator PAI Plasminogen activator inhibitor CSF Colony formation stimulator GM-CSF Granulocyte-macrophage Colony formation stimulating factor PBS Phosphate buffered saline SDS Sodium dodecyl sulfate BSA Bovine serum albumin MEM Minimum essential medium DNA Deoxyribonucleic acid RNA ribonucleic acid FCS Fetal bovine serum UV Ultraviolet FD cell CSF-dependent continuous hematopoietic cell line FD / LIF cell FD Cell-expressed LIF gene HEPES 4- (2-hydroxyethyl) -1-piperazinoethanesulfonic acid This invention relates, in part, to the effect of LIF on certain responses in osteoblast-like cells, and to the calvaria in organ culture. Arising from the analysis of the effect of LIF on bone resorption in rats. It was also chronically elevated to test the effect of LIF in vivo and to outline the physiological function of LIF.
Mice were generated at LIF concentrations. In this latter test,
Colony formation stimulating factor (CSF) dependent continuous hematopoietic cell line FDC
Using -P1 ("FD" cells), these cells were found to persist and accumulate in the spleen, lymph nodes and bone marrow of recipient animals, albeit non-leukemic. Superinfection of FDC-P1 cells with a retrovirus containing the gene encoding LIF results in the expression of a cloned subline that produces essentially high concentrations of LIF, and injection of such cells into mice Animal LIF
Animals were produced that contained a continuous source of producer cells. By analyzing these animals, in conjunction with in vitro performance, LIF could be a key regulator of bone cell function causing induction, promotion and / or enhancement of activities such as bone resorption and / or bone formation. It turned out to be.

In the present invention, a state in a living body is exemplified by using a mouse model. However, this is done with the understanding that the invention can be extended to LIF effects on all animal bones. Preferably, however, the animal is a mammal or a bird, more preferably a mammal. In the most preferred embodiment, the invention is for human application.

Therefore, one embodiment of the present invention is to provide LIF and / or LIF under conditions and for a time and under conditions that allow sufficient bone anabolic activity.
, Promoting and promoting metabolic effects on bone in an animal, comprising administering to the animal an effective amount of a polypeptide similar to the above, alone or in combination with one or more other cytokines, or one or more active agents. And / or enhancement methods.

The term “metabolic effect” as used herein and in the claims encompasses processes such as bone formation and / or bone resorption with increased protein synthesis in bone tissue and increased thymidine uptake in bone tissue. However, it is not necessarily limited to this. The term "metabolic effect" also includes the anabolic and / or catabolic effects of LIF on bone.

The term "LIF and / or LIF-like polypeptide" refers to LI
Derivatives and homologs of F means LIF molecule of natural full length, or single or multiple amino acid substitutions, deletions,
And / or derivatives thereof including additions, LIF
Substitutions, deletions, and / or additions, and derivatives of any other molecule associated with the derivative itself, carbohydrates, lipids, and polypeptide moieties, provided that the derivative possesses sufficient activity useful in the practice of this invention. Such derivatives are expanded. In addition, LIF-like polypeptides are expanded to molecules that have amino acid transpositions or changes and have substantially similar activity to LIF. The production of various derivatives of LIF is based on PCT / AU for recombinant LIF molecules and the corresponding DNA.
This is clear from the report of 88/00093. A reference herein to LIF includes a LIF-like polypeptide, and vice versa.

Therefore, this invention relates to naturally occurring substantially pure LIF
(Ie, 70% of LIF compared to other proteins or molecules)
% By weight or more, preferably 85% by weight
, More preferably equal to or greater than 90% by weight), recombinant LIF, and synthetic LIF, for example, produced by chemical means.

Depending on the animal being treated or the disease state being treated, the LI
F can vary across sources such as, but not limited to, humans, mice, dogs, cows, pigs, sheep, or other ruminants. In some cases, it is preferable to use a LIF homologous to the animal to be treated, for example a human LIF if human. However, in other situations, heterologous LIFs may be more convenient and / or more effective. The choice of source of LIF may vary depending on the treatment required or the urgency of the animal requiring the treatment.

In one aspect of the invention, LIF and / or LIF-like polypeptides are used alone. In yet another embodiment, they affect various aspects of bone resorption and / or bone formation, such as, but not limited to, IL-1, TNFα and / or TNFβ1. Or in combination with other cytokines. The invention also relates to agonists, antagonists, calcium, or other such compounds such as agonists, antagonists, calcium, or any inorganic or organic molecules that assist the bone resorption and / or bone formation process, either directly or by enhancing the effects of LIF or LIF-like polypeptides. LIF combined with active molecules of
Or extend to the use of LIF-like polypeptides.

Injection of FD cells into mice results in animals accumulating a growing population of FD cells. FD cell production LIF (FD / LIF
Injection of cells) shows the same result. Consistent with previous studies, this accumulation was accelerated by pre-irradiation of recipient animals. As a result of Northern analysis of LIF mRNA and bioassay for LIF, active production of LIF is achieved in certain organs known as FD cell accumulation sites, namely, the spleen, lymph nodes, and possibly bone marrow, where Circulation LI
A substantial increase in F concentration was observed. There was no increase in LIF production or circulating LIF concentration in FD cell recipient animals. Injection of LIF-producing FD cells resulted in bone hyperproliferation with extramedullary hematopoiesis in the spleen and liver, especially for as short as 10 days.

Despite extensive studies in our lab, recipient animals with FD cells alone or cachectic GM-CS
The absence of any of these effects in F transgenic mice is evidence that the observed pathological changes are a specific consequence of excessive LIF concentrations. Since such mice often have little or no FD / LIF cells in the bone marrow, it is likely that these tissue effects are mediated by circulating LIF.

The most dramatic initial change was a pattern of excessive bone formation with a histological appearance resembling sclerosis. This involves three simultaneous changes including osteoblast accumulation and activation, depletion of pre-existing hematopoietic cells (for non-irradiated recipients), and activation of osteoclasts in many bones. Osteoblasts express the receptor for LIF (2
7), LIF is known to release calcium from bone tissue in vitro (Ride et al., Personal communication). Thus, alterations in osteoblasts are presumed to be due to a direct effect of LIF, and if so, LIF is likely to migrate osteoblast precursors, osteoblast proliferation, and osteogenesis. May be affected. There is a well-documented link between osteoblasts and osteoclasts, but because osteoclasts lack the LIF receptor (27), their altered behavior can be attributed to altered osteoblast activity. It can be secondary. Although perturbations in bone biology lead to excessive bone formation, causium accumulates particularly in muscle and liver tissues, so it can be assumed that many mice possess excessive calcium concentrations.

Therefore, administration of LIF is very useful for inducing bone formation and stimulating bone repair and bone growth for the required time and under suitable conditions, where bone degeneration or fracture occurs after postmenopause. It may be particularly useful for the treatment of injuries and / or diseases that result in increased bone mineralization in osteoporosis, such as osteoporosis, or stimulation during fracture repair. The administration of LIF can be performed by standard methods, for example, intravenously, intraperitoneally, intramuscularly, or locally, to the extent applicable to the fractured bone. Certain modifications may need to be made to LIF to increase serum half-life or to protect against hydrolases. All such modifications to LIF and pharmaceutical compositions containing LIF or LIF-like polypeptides are included within the scope of this invention. Alternatively, the modification can include adding a protective compound (eg, an enzyme inhibitor) to the pharmaceutical composition. Suitable carrier media and their formulations include other human proteins (eg, human serum albumin),
For example, Remington's Pharmaceutical Sciences, 16th edition (1980), edited by Ozole et al. (Match Publishing, Inc.), incorporated herein by reference.
Company). The effective amount of LIF or LIF-like polypeptide required to induce a metabolic effect in a pharmaceutical composition or in vivo may vary depending on the animal and the condition being treated. For example, an effective amount of 1 to 1 million units may be required, but in some circumstances a range of 0.1 to 10000 units may be required. In other situations, LIF / kg of animal weight
It may be convenient to administer from 0.01 μg to 100000 mg. Another preferred range may be from 0.1 μg to 10,000 mg LIF or LIF-like polypeptide per kg body weight of the animal.
The above-mentioned effective amounts may be hourly, or daily, or possibly weekly or monthly depending on the circumstances. Administration can be in one or multiple applications. Thus, "effective amount" refers to the amount necessary to achieve the desired effect.

Pharmaceutical compositions useful for inducing osteogenesis or increasing bone resorption that contain an effective amount of inducing or resorbing LIF and / or a LIF-like polypeptide are produced by standard procedures. The invention further encompasses utilizing LIF and / or LIF-like polypeptides for the manufacture of a medicament for inducing, promoting and / or enhancing metabolic effects on bone in animals, particularly mammals or birds, especially humans. As a preferred embodiment, the present invention relates to the manufacture of a medicament for the treatment of injuries and / or diseases in animals, in particular humans, in which bone degeneration or fracture is such a symptom or result in osteoporosis or fracture, LIF and / or Consider utilizing a LIF-like polypeptide.

The invention extends to LIF and / or LIF-like polypeptide parts or derivatives defined by their ability to interact with osteoblasts. In particular, the interaction depends on the osteoblast LI
It may involve binding of the LIF fragment to the F receptor. like that
Portions or derivatives of LIF and / or LIF-like polypeptides may be chemically treated, isolated of truncated or altered fragments of LIF molecules, and truncated or fragmented as reported in PCT / AU88 / 00093. The isolated LIF can be isolated by any of a number of means, such as testing whether it has activity in a biological assay. Active fragments are then further analyzed using the in vitro procedure reported herein. Alternatively, LI
Selected fragments or portions of LIF can be synthesized using standard recombinant DNA expression systems that use segments or derivatives of the F coding region. The polypeptide so produced is then tested by a bioassay and, if effective, further tested in vitro on an osteoblast cell line. Thus, the use of the term "LIF-like polypeptide" herein includes fragments or portions of LIF that can interact with osteoblasts.

The results of the in vitro studies described here show that LIF stimulates bone resorption by a mechanism that probably involves prostaglandin production and includes an increase in the number of osteoclasts (bone resorbing cells) in the calvaria. Suggest that you Since it is not affected by the presence of indomethacin, its effect on thymidine incorporation probably reflects the osteoblast response and does not imply local prostaglandin production. Furthermore, these results suggest that LIF may be an important regulator of bone cell function. Also, the effect of LIF on osteoblast-like cells is likely to be metabolic, for which there is clear evidence that LIF increases the synthesis of certain key specific components of bone proteins.

One of the apparent specific effects of LIF, which is additionally reported here, is the effect on the PA-plasmin system in osteoblasts. Some bone resorbing hormones stimulate PA-activity of osteoblast-like cells in culture. Precise regulatory control is required to determine the proteolytic activity of the PA-plasmin system at sites that require it. It is provided by stimulating hormone and paracrine elements, and by PA inhibitors (PAIs), which bind to both tissue type PA (tPa) and urokinase and limit their activity. Specific inhibitors, PAI-1 and PAI
-2 binds to PA and inactivates it, but they are the product of another gene.

In this study, the production of PAI-1 by normal osteoblasts was determined in rat calvaria and osteoblast-like osteosarcoma cell line UMR1.
Identify from 06-01. LIF was also found to enhance the production of PAI-1 protein and mRNA, an effect that appears to be the most likely cause of the net decrease in PA activity released by cells.

Another positive effect of LIF is to enhance the stimulation of alkaline phosphatase activity by retinoic acid in UMR201 cells. This effect is similar to that previously obtained with recombinant TNF (11). The effect of LIF is similar in some respects to that of TGFβ, but TGFβ has no effect on such alkaline phosphatase activity.

Thus, LIF has a regulatory effect on the PA-PA inhibitor system, further evidencing the potential of LIF as a key regulator in the bone cell microenvironment, and thus its importance in bone reconstruction. The effect of LIF on PA is T
It does not appear to operate by the production of GFβ.

The present invention will be further described with reference to the drawings and examples below. These are intended to illustrate the invention and not to limit the scope of the invention.
Although the examples use mice as an animal model, the procedures used and the results obtained are equally applicable to other animals, including birds and mammalian (eg, human) species, and thus the invention Include the use of LIF to induce, promote and / or enhance bone formation and bone resorption and other metabolic activities in all animals.

In the drawings, FIG. 1 is a graph showing the survival rate of mice irradiated with or without irradiation after injection of LIF expressing FD cells.

FIG. 2 shows LIF of mice irradiated or not irradiated with FD cells and LIF expressing FD cells.
5 is a graph showing the serum concentration of.

FIG. 3 is a drawing description of a femur from a mouse injected with FD cells (A) and FD / LIF cells (B). Note excessive new bone formation and abnormal trabeculae in FD / LIF recipients [hematoxylin / eosin (H &E; x54)].
(C) High magnification image of bone marrow from FD / LIF cell recipient.
Hematopoietic cell depletion, excessive osteoblast numbers, and new bone formation are seen (H &E; x450). (D) Heart and calcification of FD / LIF cell recipient are seen (arrow) (H)
&E; x 54). (E and F) FD cell recipient (E)
And spleen from FD / LIF cell recipient (F). Note that vesicle degeneration and edema are noted, but the islets are normal (arrow) (H &E; × 54). (G and H) Ovaries from FD cell recipients (G) (normal corpus luteum is indicated by arrows) and ovaries from FD / LIF cell recipients (H).
Note that there is no corpus luteum in H (H &E; × 54).

Figures 4 and 5 are graphs showing the respective dose-dependent ⁴⁵ Ca release from mouse calvaria pre-labeled by stimulation of mouse and human LIF.

FIG. 6 is a graph showing that bone resorption increases with an increase in the number of osteoclasts in the calvaria.

FIG. 7 is a graph showing the inhibitory ability of 10 ⁻⁷ M indomethacin to inhibit LIF-stimulated bone resorption.

FIG. 8 shows that the uptake of [ ³ H] -thymidine into the calvaria was LI.
Graph showing stimulation by F.

FIG. 9 shows [ ³ H] − to the calvaria stimulated by LIF.
Graph showing that thymidine incorporation is not blocked by the presence of indomethacin.

FIG. 10 is a graph showing PA activity in calvarial osteoblasts (A) and UMR106-01 cells (B). UMR106-01 cells were incubated with (存在) and without (○) a submaximal dose of PTH (20 ng / ml). The results are shown as the average of three measurements ± SEM.

FIG. 11 shows (A) UMR106-01 cells in LIF (1000 U / ml)
Shows the time course of PAI-1 mRNA concentration after treatment with. Total RNA (10 μg) was electrophoresed on an agarose / formaldehyde gel, transferred to Hybond N, and the RNA was hybridized with the labeled RNA transcript.
(B) PAI-1 mRNA response 1 hour after UMR106-01 cells treated with increasing concentrations of LIF. Conditions are the same as (A).

FIG. 12 shows LIF for mRNA for α1 (I) collagen (A) and osteonectin (B) in UMR201 cells
Autoradiograph of Northern blot showing the effect of. In each case, the left 6 lanes are LIF treated samples. UV cells
Incubated with LIF in irradiated 2% (v / v) fetal calf serum for 24 hours. An aliquot of 20 μg of total RNA was electrophoresed as described in Example 5.

FIG. 13 is a photograph of an autoradiograph of cells from a long bone of a neonatal rat labeled with ¹³⁵ I-LIF. Specifically, the photograph shows osteoblasts and osteoclasts without unlabeled competitors (x400).

FIG. 14 (A) ¹³⁵ I- bound to UMR106-01 cells at 4 ° C.
FIG. 4 is a diagram showing a saturation binding isotherm of LIF. (●) is the whole,
(△) is specific binding, (○) is non-specific binding. (B) Scatchard analysis of the specific binding data shown in FIG.

FIG. 15 is a photograph explanatory view showing the detection of LIF transcript in osteoblasts. Equivalent RNA from UMR106-01, UMR201, or osteoblast-enriched primary culture (1 ° C. a) was PCR amplified as described in Example 5 for LIF or GAP-DH transcripts (positive control) Probed by The negative control track represents a "cell free" sample in which RNA extraction, cDNA synthesis, and PCR reactions were performed in parallel. In the case of UMR106-01 and UMR201 cells, the + and-symbols indicate that TGFβ (2 ng
/ ml) or not processed.

Example 1 General Effects of LIF in Mice Ten days after injection of FD / LIF cells, mice that had been subjected to 6 Gy whole body irradiation in advance began to show signs of weight loss, piloerection, and movement restriction. Such mice became moribund within a few days, and by 28 days after injection, 80% of these mice were moribund (FIG. 1). During this period, irradiated mice injected with control FD cells remained in good health. Mice injected with FD / LIF cells and not irradiated exhibited the same clinical symptoms,
The time to onset was delayed. Non-irradiated mice became moribund for the first time 30 days after injection, but the rate of disease progression paralleled that of irradiated mice, with 80% dying by 56 days. During this period, non-irradiated mice injected with control FD cells were again apparently healthy. FD / LIF
The moribund mice injected with cells and the control mice injected with FD cells were sacrificed in each case for comparative analysis and all observations were made. Serum from irradiated and unirradiated mice after injection of FD and FD / LIF cells
The LIF concentration is shown in FIG.

Dying irradiated and non-irradiated mice injected with FD / LIF cells developed dehydration and showed constant weight loss of 21% and 24%, respectively, compared to control mice. The moribund mice fell into circulation collapse and showed a decrease in circulating blood volume. Hematocrit values (Table 1) did not show the expected increase due to the same concentration, suggesting a decrease in red blood cell count. With samples from FD / LIF mice, it was difficult to collect serum from such mice due to reduced blood volume obtained at sacrifice and impaired clot retraction.

Example 2 Changes in hematopoietic system In FD / LIF mice, mature neutrophils were selectively increased 4 to 5-fold, so that the leukocyte count was constantly increased (Table 1). In non-irradiated mice, monocyte counts 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 showed moderate splenic hypertrophy, more pronounced in non-irradiated recipients (Table 2). The spleen was slightly paler than normal but showed no surface nodules, and both types of recipients showed a decrease in the percentage of lymphocytes and an increase in the percentage of granulocytes, especially when irradiated. None of the recipients showed an increase in the percentage of nucleated red blood cells.

A notable feature of femoral bone marrow in mice injected with FD / LIF cells was the difficulty experienced during insertion of a needle to harvest bone marrow cells. Bone marrow cells were 10 out of 19 irradiated mice
No mice and 11 out of 19 unirradiated mice could not be recovered. When cells could be recovered from mice injected with FD / LIF cells, the total number of bone marrow cells decreased by a factor of 4 to 5 for both types of recipients (Table 2). In these bone marrow, there was a significant increase in granulocyte ratio and a significant decrease in lymphocyte and nucleated red blood cell ratio. Interestingly, recipients of FD cells showed a marked increase in eosinophils (usually myeloid and promyelocytes), with changes not seen in the bone marrow of recipients injected with FD / LIF cells .

Example 3 Bone Changes The femur, tibia, and sternum of mice injected with FD / LIF cells had marked bone hyperplasia with thickening of the cortex and irregular trabecular formation in the medullary canal. (FIG. 3). In non-irradiated and irradiated recipients, the medullary cavity was swirled and narrowed, and hematopoietic cells were depleted. What remained was mainly granulocyte cells, and not all bone marrow, but occasionally megakaryocytes. Instead of hematopoietic tissue, long elongated cells, especially at the ends of long bones, were arranged in bundles in the same direction. These cells adhered to the surface of the bone and became giant and fused with the developed bone. The apparent calcification in the cytoplasm of these cells seemed to indicate that they were osteoblasts, but their total number appeared to be well above the number that could be present in the normal bone marrow cavity. Nevertheless, there was no apparent migration through the bone stoma. In some bones, osteoclasts were prominent, and the surrounding bones had irregular shell-like edges. Irregular stomas were also present in many bones, especially at the ends of long bones. It is believed that these may be the result of increased osteoclast activity. Open channels between the bone marrow and the outside of the tissue allowed osteoblast precursors to enter and bone marrow cells to enter and exit freely.

The red medulla of the spleen is enlarged, packed with hematopoietic cells,
Lymph follicles were markedly depleted of cells. In many livers, again, distinct hematopoietic cell foci were present, and in some livers areas of necrosis and / or calcification were noted.
There was less common cirrhosis, but parenchymal cells had no mitotic activity and individual parenchymal cell volumes were uniformly reduced.

In some animals (but not all), calcified areas were found in striated muscle and heart, but no cellular infiltrates were found at these locations. In some mice, macroscopically significant calcified areas were found in the liver.

Mice injected with FD cells do not show any of the above pathological changes, and the only apparent condition in some of these mice is the presence of nested FD cell accumulation in the spleen or mesenteric node. there were.

Example 4 In Vitro Effect of LIF on Neonatal Mouse Calvaria Bone resorption was measured by the release of ⁴³ Ca pre-labeled in vivo from bone. As previously reported (3) the hemi-calvaria was transected and pre-incubated for 24 hours in minimal essential medium supplemented with 1% (v / v) fetal calf serum. The bone was then transferred to a medium containing the experimental compound and incubated for a further 48 hours.
^The medium and bone were collected to determine ⁴⁵ Ca content.
^[3 H] - thymidine and ^[3 H] - In experiments involving measurement of phenylalanine, the compound is added to the end of the 4 hour experimental period 48 hours, Canalis way their content in the calvaria ( Quantified by 4).

Mouse (Fig. 4) and human (Fig. 5) recombinant LI
F stimulated the release of ⁴⁵ Ca from pre-labeled calvaria, both in a dose-dependent manner. Increased bone resorption was accompanied by an increase in the number of osteoclasts in the calvaria (FIG. 6). LI
The ability of F to stimulate bone resorption was blocked by the presence of 10 ^-7 M indomethacin, a prostaglandin synthesis inhibitor (FIG. 7, Table 3).

Calvarial osteoblasts and UMR106-01 cells were subcultured on ¹²⁵ I-fibrin coated plates. Cells were pre-incubated for 8 hours under serum-free conditions as indicated in the description in Table 4. Cells were harvested with LIF (100 U / ml) and indomethacin (1.5 × 10 ^−5).
M) Incubated with or without presence.

The effect of LIF on [ ³ H] -thymidine incorporation, which measures cell proliferation, was examined simultaneously. LIF ^[3 H] into calvaria - was found to stimulate thymidine incorporation, the dose - (FIG. 8 which was different from the case of bone resorption is a response relationships more sensitive reaction ). Also this LI
The effect of F was not blocked by the presence of indomethacin (FIG. 9).

LIF also increased [ ³ H] -phenylalanine uptake into bone (control: 21420 ± 1110 cpm, LIF: 26890 ± 1253 cp)
m, p <0.01) and was found to stimulate protein synthesis.

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

Thus, these results indicate that LIF probably stimulates bone resorption by a mechanism of action involving the production of prostaglandins and an increase in the number of osteoclasts (bone resorbing cells) in the calvaria. Since its effect on thymidine incorporation is not affected by the presence of indomethacin, this probably reflects the osteoblast response and does not imply local prostaglandin production. Furthermore, these results indicate that LIF may be an important regulator of bone cell function.

Values are mean ± standard error and are shown as treatment / control ratios for each of the two index comparisons. Significant difference from control is a: p
<0.05, b: p <0.01 (n = 3 to 4 in each group). The other two
Similar results were obtained in one experiment.

Example 5 Effect of LIF on some responses in osteoblast-like cells Bone-specific protein α1 (I) in osteoblast-like cells
Experiments were performed to determine the effects of LIF on mRNA expression of collagen and osteonectin, and mRNA production for plasminogen activator (PA) activity and plasminogen activator inhibitor-1 (PAI-1). Was.

(1) Materials and Methods (i) Chemical and Biological Materials Synthetic human parathyroid hormone (PTH) (1-34) was obtained from Beckman Pty., Inc. (Palo Alto, Calif., USA) and obtained from human recombinant TGFβ. , TNFα and TNFβ, and full-length rat actin cDNA were provided by Genentech (San Francisco, CA, USA). 1,25-dihydroxyvitamin D ₃ is M. Yusukokobitchi Dr. (Hoffman
La Roche, is provided by courtesy of New Jersey), prostaglandin E ₂ was purchased from Upjohn Company (Kalamazoo, Michigan). Recombinant human LIF
Was produced in both yeast and Escherichia coli and purified to homogeneity as previously reported. Pure LIF
The specific biological activity of a -10 ⁸ units / mg. Antiserum against TGFβ is available from R & D Systems, Inc. (Minneapolis, M .;
D., 54413, USA).

Rabbit antiserum against PAI-2 prepared from U937 cells was
Courtesy of Professor EKO Kruitov (Center Hospitalité Universitaire, Baudaz, Lausanne, Switzerland). Rabbit anti-rat hepatoma cell (HTC) P
AI-1 (S) and pBluescript SK (-) (6)
Rat PAI-1 cDNA cloned into the EcoR1 site was kindly provided by Professors R. Zeheb and TD Gellertel (University of Michigan, Ann Arbor, MI). A cDNA probe for TGFβ was provided by Genentech (San Francisco). Mouse LI
The cDNA probe for F is plasmid pLIF7.2b,
It spans the entire coding region of mouse LIF mRNA.
¹²⁵ I-fibrinogen, [α ³² P] UTP, [α ³² P] CTP
And T7 / SP6- from combinatorial promoter system Amersham International, T ₃ RNA polymerase International Biotechnologies Inc. (New Haven, CT, 06535, USA), nick translation kit Boehringer Mannheim (Mannheim, West Germany ). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Mr standard is from Bio-Rad Laboratories (Richmond, CA, 94
804, USA).

(Ii) Osteoblast cell lines UMR106-01 and UMR106-06 cells are subclones of UMR106 cells, a clonal line of rat osteogenic sarcoma cells that possess many features of the osteoblast phenotype ( 1,
8, 9). The experiments reported reported that cells from passage 7 to 14 were converted to non-essential amino acids, 14 mM 4- (2-hydroxyethyl)-.
Eagle's Minimum Essential Medium (MEM) supplemented with 1-piperazinoethanesulfonic acid (Hepes), gentamicin (80 μg / ml), minocycline (1 mg / ml) and 5% (v / v) fetal calf serum (FCS) Performed on cells maintained at 37 ° C. with 5% CO ₂ in air.

The UMR201 clonal cell line is derived from neonatal rat calvaria (10) and has properties consistent with those of osteoblast precursors. Retinoic acid treatment leads to a substantial increase in alkaline phosphatase activity and mRNA, with cells producing significant type I collagen and possessing other osteoblast characteristics (10,11).

Osteoblast-enriched calvarial cells were generated by previously reported methods (7,8) and used in primary subcultures. UM
Culture maintenance conditions for R201 cells and osteoblast-rich cells are similar to those reported for osteogenic sarcoma cells.

Osteoclasts were isolated from long bones of neonatal rats as previously reported (24), left on coverslips for at least 30 minutes, then washed and isolated from significant contamination by osteoblasts (25). .

(Iii) RNA preparation and hybridization (FIGS. 11 and 12) The cells were washed twice with phosphate buffered saline (PBS),
Pre-incubation for 15 hours with minimal essential medium (MEM) and 0.1% (w / v) bovine serum albumin (BSA). At the end of this period, the medium was supplemented with and without MEM and LIF at 0.1% (w /
v) Replaced with BSA. The cells were incubated at different times and concentrations. (12) Whole RNA as previously reported
It was created. As previously reported (12), one of the total RNA
A 0 μg aliquot was electrophoresed on a 1.5% (w / v) agarose / formaldehyde gel and transferred to Hybond N (Amersham International) by Northern blotting. Bluescript using T3 RNA polymerase
Rat PAI-1 cD cloned into EcoR1 site of SK (-)
A riboprobe labeled for PAI-1 was prepared from NA. Hybridization conditions were mainly 6 × SSC, 5 × 5% in 50% (v / v) formamide as reported in (12).
Den Hearts, 0.1% (w / v) SDS, 65 ° C. The filters were washed at 65 ° C. to a tightness of 0.1 × SSC and exposed to Kodak XAR-5 X-ray film with intensifying screens as a back. 0.
After removing the DNA bound to the RNA by boiling for 5 minutes in 1 × SSC, the nick-translated full-length rat actin cDNA probe was used again for 0.1% (w /
v) Probed SDS filter. 0.1 × S filter
Washed to SC stringency and exposed as above at 0.1% (w / v) SDS, 42 ° C. Similar hybridization conditions were used to detect mRNA for osteonectin and type I collagen. In each case, the probes were labeled by nick translation.

(IV) Plasminogen activator (PA) activity and alkaline phosphatase Cells were subcultured at 20000/200 μl into ¹²⁵ I-fibrin coated wells (13) of a 96-well tissue culture plate.
After 24 hours in maintenance medium, cells were washed twice with Dulbecco's PBS and the medium was replaced with MEM and 0.1% (w / v) BSA.
Eight hours later, the medium was replaced with fresh Eagle's MEM and 0.1%
(W / v) Human plasminogen (1.56 μg / ml) was added and replaced with BSA. In parallel, plasminogen-independent activity without human plasminogen was measured. After 24 hours, an aliquot of the medium was taken and the solubilized radioactivity was counted. Plasminogen-dependent fibrinolytic activity was expressed as a percentage of the total radioactivity released from the plate by trypsin.

To examine the regulation of alkaline phosphatase activity, 10% of UMR201 cells were placed in six 9.6 cm ² multiwell dishes.
(V / v) Subcultured in α-modified MEM containing FCS (α-MEM). Once the cells are semi-confluent, add the medium to each concentration of LIF
And 2% (v / v) vitamin A deficient FCS containing 1 μM retinoic acid was added to α-MEM. After 48 hours, the medium was changed and a new treatment was added. Previously reported methods (1
In 0), the specific activity of alkaline phosphatase was measured.

(V) LIF receptor study UMR106-01 and 106-06 cells were subcultured, centrifuged in Hank's BSS, left on ice, and resuspended in 180 μl bovine fetus in a tapered soft plastic tube. Layered on serum (FCS). By centrifugation of the cells with FCS, they were separated and ¹²⁵ I-LIF of ¹²⁵ I-LIF and cell-free with cells. The test tube containing the cell pellet is cut off, and the pellet and the supernatant are subjected to a γ-counter (Packard Crystal Multidetector, Packard,
(Downers Grove, IL). Saturation isotherms were analyzed by the Scatchard method (14) using a non-linear curve fitted to the program ligand (15).

Cells isolated from the long bones of neonatal rats were plated in 12-well tissue culture plates (Flow Laboratories, McLean, V
5) Adhesion to cover slips in wells of A), 5 × 10 ⁸ cpm ¹²⁵ with or without 40-fold excess of unlabeled LIF
Coated with 1.0 ml of KHF containing I-LIF. Incubation was performed at 37 ° C for 30 minutes. Remove medium and place cells in warm KH
An aliquot of 4 × 4 ml of F and warm 20 mM sodium phosphate,
Washed with 2 x 4 ml aliquots of 0.15 ml NaCl (pH 7.4).
Cells were fixed in 4 ml of 2.5% (u / v) glutaraldehyde in PBS and washed with 2 x 4 ml aliquots of water. A cover glass was stuck to a gelatin-coated slide glass using a Depex-attached medium, air-dried overnight, immersed in a Kodak NTB2 photographic emulsion at 42 ° C. in a dark room, and air-dried. The slide was then sealed in a light-tight box containing drierite and exposed at 4 ° C. for 4 weeks. Prior to development, the slides were warmed to room temperature, then developed with Kodak D19 developer (40 g / 500 ml water) for 1 minute and fixed with Agfa G333c X-ray fixer for 3 minutes.

(Vi) Fibrin autography and reverse fibrin autography Cells were subcultured in MEM and 5% (v / v) FCS at 500,000 cells / well (9.6 cm ² ). After 25 hours, the medium was aspirated, the cells were washed twice with PBS, and the medium was washed with 1 ml MEM and 0.1 ml MEM.
% (W / v) BSA. After 8 hours, aspirate the medium,
1 ml of fresh MEM and 0.1% (w / v) BSA were added along with the treatment. After 25 hours, the medium was aspirated and centrifuged to remove any cell debris. Wash cells twice with PBS and add 1 ml 0.1%
(V / v) Dissolved in Triton × 100. The conditioned medium (CM) and cell extract prepared as described above were subjected to SDS-PAGE on a 10% (w / v) slab gel under non-reducing conditions as reported in (16).
Decomposed. PA and PAI activity in fibrin (17)
And by reverse fibrin (18) gel autography.

(Vii) Immunoblot conditioned medium from confluent cultures of UMR106-01 cells and calvarial osteoblasts incubated for 24 hours in serum-free MEM was concentrated by ultracentrifugation at a 30,000 Mr cut-off,
Washed twice with BS, scraped, centrifuged and dissolved with 0.1% (v / v) Triton × 100. The protein was converted to SDS-
Degraded by PAGE and transferred to nitrocellulose. Nitrocellulose for rabbit polyclonal anti-rat HTC PAI-
1 (5), then bind anti-rabbit Ig with alkaline phosphatase and add 5'-bromo-phosphate
PAI-1 was detected by visualization with 4-chloro-3-indolyl (19).

(Viii) RNA detection using polymerase chain reaction (Fig. 15) Total cytoplasmic RNA was converted to UMR201 or UMR201 as described in (26).
Isolated from MR106-01 cells or primary osteoblast populations (see above), 50 mM Tris-Cl (pH 8.3 at 42 ° C.), 20 mM KC
l, 10 mM MgCl ₂ , 5 mM dithiothreitol, 1 mM each dNT
P, 20 μg / ml oligo-DT ₁₅ , and LIF-specific oligonucleotide primers, AMV reverse transcript (Boehringer
Mannheim) in a 20 μl reaction containing 20 units at 42 ° C.
First strand cDNA synthesis was performed for 40 minutes. After the first strand synthesis was completed, the reaction was diluted with 100 μl of distilled water and 5 μl was used for each PCR reaction. PCR reaction (50 μl volume)
TP contained 200 μM, 1 μl of each specific primer, buffer supplied with Gene Amp kit (Cetus, USA), and 1.25 units of Taq polymerase. P
Primers used in the CR reaction were LIF, 5'-CCGAATTCGAAAACGGCCTGCATCTAAGG and 3'-CCGAATTCGCCATTGAGCTGTCCAGTTG, which define a 300 bp fragment of mRNA, and GAPD
H, 5'-ACCACCATGGAGAAGGCTGC and 3'-CTCAGTGTAGCCCAGGATGC defining a 500 bp fragment. The conditions for the PCR reaction were Perkin Elmer Cetus DNA Thermal
25 cycles in a cycler, 1.5 minutes at 94 ° C, 2 minutes at 60 ° C, 3 minutes at 72 ° C. One part of the PCR reaction was 1.2% (w /
v) Electrophoresed on agarose gel and transferred to nitrocellulose. Filters were prehybridized, hybridized and washed with 2xSSC containing buffer as reported in (21). The hybridization probe is a cDNA insert of the gel-purified plasmids pLIF7.2b and hGAP-DH, approximately 2 × 10 ⁸ cpm / hr by nick translation.
Radiolabeled to a specific activity of μg and added to the hybridization at a concentration of about 2 × 10 ⁷ cpm / ml.

(2) Results (i) Effect of LIF on bone resorption In the PA response, LIF was expressed in a dose-dependent manner in osteoblast-like cells.
It was found to suppress A activity. This suppression was evident in control cells or cells treated with drugs that increase PA activity, such as parathyroid hormone (PTH). An example of such an experiment is provided in FIG. 10, which shows suppression of PA activity in UMR106-01 cells. Similar suppression was demonstrated in calvarial osteoblasts. These experiments were not possible in UMR201 cells that did not survive for long enough in serum-free culture.

Since the effect of LIF that suppresses PA activity may be due to an increase in the amount of PA inhibitor, the possibility of LIF on the production of PA inhibitor PAI-1 produced by osteoblasts was examined. Reverse fibrin autoradiography, Western blot with antiserum specific for PAI-1, and calvarial osteoblasts and
Evidence based on evidence of PAI-1 mRNA by Northern blotting of UM106 cells proved to be the case. Treatment of UMR106 cells with LIF yielded mR for PAI-1.
PAI- with increased NA and increased suppression of LIF PA activity
This was in good agreement with the possibility of one quantity production. Regulation of PA activity by inhibitor formation is well recognized as an important factor in determining net PA activity. No.
FIG. 11 shows the time course of PAI-1 mRNA up to 24 hours in response to LIF. After the LIF treatment was started, a clear increase of PAI-1 mRNA was observed in about 1 hour.

L for suppression of this PA activity and promotion of PAI-1 mRNA
The effect of IF is on tumor growth factor β in the same cells.
Very similar to the effect of (TGFβ). To exclude the possibility that LIF acts via TGFβ production in cells, two experiments were performed. The first experiment used an antibody to TGFβ to determine if it blocked the effect of LIF. No blocking effect was observed (No. 5).
table). This same antibody was able to block the action of TGFβ.

In a second experiment, m for TGFβ in osteoblast-like cells
We examined whether LIF affects RNA. LIF had no effect on TGFβ mRNA in UMR106 cells and calvarial osteoblasts. It was concluded that the effect of LIF of PAI-1 was not mediated by producing TGFβ as an intermediate in its action in osteoblast-like cells.

Treatment of UMR201 cells with LIF resulted in a dose-dependent increase in mRNA for both type I collagen and osteonectin. A Northern blot to prove this is shown in FIG. These effects are also enhanced by TGFβ
Both were proven in MR201 cells. However, even in these cells, LIF did not necessarily show all TGFβ-like effects. For example, TGFβ reduces alkaline phosphatase activity and mRNA elevation in these cells in response to retinoic acid, whereas the opposite occurs in LIF. In fact, LIF enhances the alkaline phosphatase response, an effect very similar to that of another cytokine, tumor necrosis factor alpha (TNFα).

The effects of LIF on osteoblast-like cells are probably anabolic, i.e. clear evidence of the effect of LIF on increasing the synthesis of some key specific components of bone proteins and its regulatory effect on the PA-PA inhibitor system Indicate that LIF is an important regulator in the bone cell microenvironment and therefore may be important in bone reconstruction.

The effects of LIF on increasing bone resorption in mouse calvaria are dependent on their internal synthesis in prostaglandin organ cultures. Evidence suggests that blocking prostaglandin synthesis suppresses LIF reabsorption stimulatory effects. On the other hand, the stimulatory effect of LIF on incorporation of ³ H-thymidine into acid-precipitating macromolecules is not suppressed by indomethacin (Example 4). TGFβ also promotes reabsorption in mouse calvarial organ cultures, but is inhibited when prostaglandin synthesis is inhibited. However, TGFβ is not a promoter of resorption in the system using long bones of fetal rats. The mouse calvarial system is one of the systems in which prostaglandin production is substantially certain to result in increased resorption.
One. However, TGFβ, like LIF, has a remarkable effect on promoting bone formation. In another type of experimental system, there is good evidence that prostaglandins actually promote bone formation.

(Ii) Receptor studies Receptor autoradiography with ¹²⁵ I-LIF was performed to determine which cells of the rodent newborn bone specifically bind LIF. To prevent severe contamination of the culture with osteoblasts, macrophages and other cells, osteoclast preparations were prepared from the long bones of neonatal rats. This was achieved by isolating the cells and placing them on coverslips for an extended period of time (2
Five). In these experiments, we previously demonstrated specific binding of ¹²⁵ I-LIF in cells with osteoblast characteristics as shown by macrophages (22) (FIG. 14). No evidence was found for LIF binding to cells or mononuclear tartrate-resistant acid phosphatase-positive cells (osteoclast precursors).

For the identification of LIF receptors on primary osteoblasts,
Osteoblast-like cell cloned osteogenic sarcoma cell line UMR106-
At 06, a biochemical study of LIF binding was performed. Specific binding is readily demonstrated, receptor number per cell by Scatchard analysis is 300, was found to be K ₀ of 60 pM (Figure 14). Receptor counts were determined to be between 400 and 500 (2 per cell) previously demonstrated in the mouse myeloid leukemia cell line M1.
0) and conveniently compared. UMR201 cells also demonstrated lower specific but specific receptor binding.

It has been progressively elucidated that locally produced factors are important in the regulation of bone metabolism (7), and GM-CS by osteoblasts
In view of the demonstrated F production (23), we explored evidence for LIF production by osteoblast-like cells.

(Iii) LIF production by osteoblasts As a result of the biological assay, extremely low concentrations of M1 cell differentiation activity were found in the primary osteoblast population and the medium conditioned by the osteoblast-like cell lines UMR201 and UMR106-01. It was revealed. To confirm that this activity was due to LIF, these cells were assayed for LIF transcripts. Perhaps the amount of LIF produced is very low, so RNA from these sources using regular Northern blots
No transcript was detected (data not shown).

But a sensitive RN based on the polymerase chain reaction
By using the A detection technique, LIF transcripts were verifiable in these cells. From such an analysis (eg, FIG. 15), in one experiment, treatment with TGFβ
At −01, the modulation of the concentration was evident (FIG. 15).
Was essentially transcribed in both UMR201 and UMR106-01 cells, and in the primary osteoblast population. However, given the possible difficulties in quantifying the PCR reaction, the level of induction in response to TGFβ could not be determined reliably. UMR201 cells have higher concentrations of LIF than UMR106-01 cells.
It was consistently found to contain transcripts.

(Iv) Effect of LIF on alkaline phosphatase activity in UMR201 cells Treatment of UMR201 cells with LIF for 3 days resulted in a dose-dependent enhancement of retinoic acid-induced alkaline phosphatase activity (Table 6). This effect is similar to the effect previously obtained by TNFα in the same cells by the inventors (1
Very similar to 1). The above study provided evidence that this effect was due to mRNA stabilization for alkaline phosphatase. That is, although LIF and TNFα are similar to each other in this reaction, they have a reaction direction effect on PA activity in osteoblasts (Table 7).

Cells were subcultured on ¹²⁵ I-fibrin coated plates as described in the methods section. Transfer cells to MEM + 0.1% BSA
After pre-incubation for 8 hours, fresh medium containing bone resorption factor was added. Incubation was performed for 24 hours.

[References] (1) Houghton, JE, Liza, LG, Simmons, H.
A., Oppenheim, JJ, Meyerhager, SE,
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(2) Martin, TJ, Gu, KW, Nicholson, GC,
Clinical End Clinology and Metabolism, Vol. 2, pp. 1-29 (1988).

(3) Canalis, E., End Clinology, 118, 78
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(4) Ride, IR, Katz, J., Ibertson, H.
K., Gray, DH, Calculated Tissue International, 38, 38-43 (1986).

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Thrombosis and Hemostasis, Vol. 58, 1017
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(8) Partridge, NC, Alcorn, D., Michelangeli, VP, Ryan, G., Martin, TJ, Cancer Research, 43, 4388-4439 (1983).

(9) Forrest, SM, Gu, KW, Findlay, D.
M., Michelangeli, VP, Ribessy, SA, Partridge, NC, The Jacques, JD, Martin, TJ, Calcified Tissue International, Volume 37,
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(11) Gu, KW, Hudson, PJ, Power, BE, Manji, SS, Gamma, PR, Martin, TJ, Journal of Molecular End Clinology, Volume 3,
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(12) Maniatis, T., Flitch, EF, Sunbrook, J., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
(1982).

(13) Allen, EH, Hamilton, JA, Metcalfe,
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(14) Scatchard, G., Anals of the New York Academy of Sciences, 51 volumes,
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(15) Manson, PJ, Rodvard, D., Analytical Biochemistry, 107, 220-237 (1980)
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(19) Knecht, DA, Dimond, RL, Analytical Biochemistry, 136, 180-184 (1984)
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(20) Hilton, DJ, Nikola, NA, Metcalfe,
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(21) Gearing, DP, King, JA, Gaff, NM,
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(22) Hilton, DJ, Nikola, NA, Metcalfe,
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(23) Felix, R., Elford, PR, Storaryl, D., Checkin, M., Wetterward, A.,
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────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Martin, Thomas John 3101 Victoria, Kew, Stock Avenue No. 6 (56) Reference J. Bone. Miner. Res. , Vol. 3, No. 6, (1988) p. 635-645 (58) Fields studied (Int. Cl. ⁷ , DB name) A61K 38/00 A61P 3/14 A61P 19/10 CA (STN) MEDLINE (STN)

Claims (11)

(57) [Claims] 1. A leukemia inhibitory factor (LIF) and / or LIF
Inducing, promoting and / or enhancing metabolic effects on animal bones, comprising the like-like polypeptides as active ingredients. 2. The agent according to claim 1, wherein the animal is a mammal or a bird. 3. The agent according to claim 2, wherein the animal is a mammal. 4. The agent according to claim 3, wherein the mammal is a human. 5. The metabolic effect is an increase in bone formation and / or bone resorption, protein synthesis in bone tissue and / or thymidine uptake in bone tissue.
The agent according to any one of the above. 6. The agent according to claim 1, further comprising one or more active molecules. 7. The agent according to claim 6, wherein the active molecule is a cytokine. 8. The agent according to claim 6, wherein the active molecule is an inorganic or organic molecule. 9. Injuries resulting in bone degeneration or fractures and / or
Or any one of claims 1 to 8 for treating a disease.
The agent according to item. 10. The agent according to claim 9, wherein the injury is a fracture. 11. The agent according to claim 9, wherein the disease is postmenopausal osteoporosis.