Background of the Invention

Osteogenic protein-1 (OP-1) is a member of the transforming growth factor (TGF) -β family which induces de novo bone formation by stimulating collagen synthesis and proliferation and differentiation of osteoblasts. (Sampath, T.K., et al. , J. Biol. Chem. , 267:20352-20362 (1992)) . The OP-1 protein was initially isolated in demineralized bone matrix. (Sampath, T.K., J. Biol . Chem. , 265:13198-13205 (1990)) . The murine OP-1 gene encoding the homolog of human OP-1 (Ozkaynak, E., et a_l. EMBO J. 9:2085-2093 (1990)) was isolated from cDNA and genomic libraries using human OP-1 cDNA as a probe. Northern blot analysis with murine OP-1 cDNA showed two major species of 2.4 kb and 4.0 kb, which may represent alternative splices. Tissue specific expression studies revealed maximal levels of OP-1 in the kidney (Ozkaynak, E., et, al. , Bioche . Biophys. Res. Comm. , 30:116-123 (1991)) , a unique feature among the so called bone morphogenetic proteins (BMPs) (Ozkaynak, E., et. al _. . , J. Biol . Chem. , 267:25220-25227 (1992)) .

The expression of OP-1 in the kidney suggests its potential role as a link between this organ and cellular elements in the skeleton, and may explain an earlier observation (Huggins, Arch. Surer. , 22:377-408 (1931)) regarding the capability of the urinary tract epithelium to induce new bone formation.

However, the specific localization of OP-1 mRNA and protein species along the nephron, as well as the role of OP-1 in the cell, and molecular biology of the kidney remain unknown.

Moreover, the study of OP-1 expression in the kidney has been limited by the unavailability of an acceptable model system. To understand the function of different nephron segments, it is necessary to isolate and culture cells representative of the many different cell types that comprise the nephron. A major limitation in this regard is that cells isolated from microdissected kidney tubules have a limited life span in culture. Furthermore, expression studies of osteogenic proteins have been difficult to reproduce in primary cell cultures due to minimal levels of OP-1 mRNA expression under physiological conditions. In vitro transfection of cells with agents such as viruses or oncogenes produce immortalized cell lines, but often these cells lose their differentiated functional characteristics. (Gullans, S.R., et al. , Am. J. Physiol. , 265 (1993)) . Thus, the need exists to determine the specific nephron location and cell type responsible for OP-1 mRNA expression, and to devise a model assay system for the rapid screening and identification of substances which induce new bone formation by regulating OP-1 mRNA expression and OP-1 protein synthesis.

Summary of the Invention

The present invention relates to Applicants' characterization of mRNA expression and protein synthesis of osteogenic protein-1 (OP-1) in kidney sections. Additionally, by screening a variety of cultured renal epithelial cell lines, Applicants have found a cell line with markedly high baseline OP-1 mRNA expression. As a result of Applicants' work, presented herein, it has been determined, for the first time, that OP-1 mRNA transcripts and proteins are present in the renal medulla and that inner medullary collecting duct (IMCD) cells synthesize abundant OP-1 protein.

As a further result of Applicants' work, a model assay system is now available to rapidly identify substances capable of inducing new bone formation by the evaluation of the effect of test substances on the expression of OP-1 mRNA expression and OP-1 protein synthesis in inner medullary collecting duct cells.

In one embodiment of the present invention, identification of a substance capable of inducing new bone formation is determined by evaluating the effect the substance has on inducing OP-1 mRNA expression in mammalian cells . Mammalian cells are grown in culture and the substance to be tested (i.e., the test substance) is introduced into the cell culture medium, or into the cell. The test substance is introduced in an amount which would produce a detectable increase in the expression of OP-1 mRNA if the substance possesses OP-1 mRNA inducing properties. The cells are maintained in culture under conditions which support cell growth and RNA transcription, for a period of time sufficient for RNA transcription to occur. The cellular RNA is then isolated, and the amount of OP-1 mRNA expressed by the cultured cells is determined. The amount of OP-1 mRNA expressed by the cultured cells in the presence of test substance is compared to the amount of OP-1 mRNA expressed by cultured cells under similar conditions, but without the presence of the test substance, to determine the effectiveness of the test substance to induce OP-1 mRNA.

In another embodiment, identification of a substance capable of inducing new bone formation is determined by evaluating the effect the substance has on inducing the synthesis OP-1 protein in mammalian cells. Mammalian cells are grown in culture and the test substance is introduced into the cell culture medium, or into the cell. The test substance is introduced in an amount which would produce a significant increase in the synthesis of OP-1

protein if the substance possesses OP-1 protein inducing properties. The cells are maintained in culture under conditions which support cell growth and protein synthesis and the amount of OP-1 protein synthesized by the cultured cells is determined. The amount of OP-1 protein synthesized by the cultured cells in the presence of test substance is compared to the amount of OP-1 protein synthesized by cultured cells under similar conditions, but without the presence of the test substance, to determine the effectiveness of the test substance to induce OP-1 protein synthesis.

In another embodiment, the present invention provides a method of therapeutically treating a patient with bone disease, such as osteoporosis, to induce bone growth (bone morphogenesis) . This method comprises administering to the patient an effective amount of a substance capable of inducing bone growth by stimulating OP-1 mRNA expression, or OP-1 protein synthesis. Conversely, in another embodiment, the present invention provides a method of therapeutically treating a patient with a bone disease, characterized by overproduction of bone (i.e., osteopetrosis) to suppress bone formation. This method comprises administering to the patient an effective amount of a substance capable of suppressing bone growth by suppressing OP-1 mRNA expression, or OP-1 protein synthesis.

Thus, as described herein, the present invention provides a rapid and sensitive method for screening candidate substances (test substances) to determine their effect on OP-1 mRNA expression, or OP-1 protein synthesis and secretion, and thus, determine the effect of the substance on bone formation. It also provides a model system to study the interaction of OP-1 protein with other known regulators of bone homeostasis, such as vitamin D, calcitonin, parathyroid hormone or growth factors such as

TGF-β, and other substances like mineralocorticoids, and the response of OP-1 production to changes in pH, calcium, phosphorous concentration. Finally, the present invention provides a therapeutic treatment for patients suffering from bone disease.

Brief Description of the Figures

Figure IA and IB depicts the murine OP-1 cDNA sequence (SEQ ID NO: 1) and encoded amino acid sequence (SEQ ID NO: 2) . Figure 2 is a photograph of a Northern blot showing the results of Poly A ^* RNA from kidney (cortex, medulla and papilla) , lung, liver, brain, ovary and small intestine probed with OP-1 cDNA.

Figure 3A and 3B depicts two graphs showing the ratio OP-l//3-actin signal calculated for the 2.4 and the 4.0 kb OP-1 transcripts for each tissue (n=6) .

Figure 4 is a photograph of an immunoblot showing the results of Western blot analysis showing that 12G3 recognizes OP-1 protein. The arrows denote the positions of molecular weight standards expressed as molecular mass X 10 ³ .

Figure 5 is a photograph of an immunoblot showing that mouse monoclonal antibody 12G3 identifies multiple OP-1 protein bands in homogenates from rat kidney and cultured mouse inner medullary collecting duct (IMCD) cells. The small arrowheads point to the top (T) and bottom (B) of the gel.

Figure 6A-6D is a series of micrographs showing that mouse monoclonal 12G3 localizes OP-1 staining to cells in kidney glomeruli, tubules and vessels in rat kidney. Figure 7A-H is a series of micrographs showing localization of OP-1 expression in mouse kidney by in si tu hybridization.

Figures 8A-B are photographs of a Northern blot showing the results of northern analysis of MDCK and IMCD cells probed with OP-1 cDNA.

Detailed Description of the Invention Applicants have determined, by expression studies in dissected kidney sections and immunohistochemistry analysis, that the renal medulla contains high levels of the OP-1 protein. Specifically, Applicants have demonstrated that OP-1 mRNA is highly expressed in inner medullary collecting duct cells. Based on Applicants results, a rapid and sensitive method for screening candidate substances to determine their effect on OP-1 mRNA expression, or OP-1 protein synthesis, and thus, determining the effect of the substance on bone formation in now provided.

As described in detail in Example 1, Poly A ⁺ RNA extracted from kidney sections (cortex, medulla and papilla) were analyzed by Northern blot analysis using a ³² P-labeled OP-1 cDNA probe (SEQ ID NO: 1) . The probe, shown in Figure 1, b.p. 370-1050, is a 680 b.p. fragment that covers two thirds of the pro-domain and one third of the mature murine OP-1 cDNA, immediately upstream to the highly conserved 7-cysteine domain present in all members of the TGF-jS superfamily (Ozkaynak, E., et. al. , Biochem. Biophvs. Res. Comrnun., 30:116-123 (1991) the teachings of which are incorporated herein by reference) . To compare kidney expression of OP-1 mRNA with extrarenal expression, lung, liver, brain, ovary and small intestine mRNA were also isolated and subjected to hybridization with the OP-1 cDNA probe. After hybridization with the OP-1 cDNA probe, the same membranes were stripped and reprobed with mouse β-actin (Figure 2, lower row) to control for mRNA content and quality. As illustrated in Figure 2, Northern blot analysis identified the OP-1 transcript (2.4 and 4.0 kb)

in all tissues of the normal rats surveyed. However, the signal was markedly higher in kidney sections, and OP-1 mRNA expression was greatest in the renal medulla, followed by cortex and papilla. Figure 3A and 3B shows the percentage of OP-1 mRNA expression (mean ± SEM, n=6 animals) for the rat tissues studied. The highest value was taken as 100%, and the percentages for the other tissues were derived by relating their ratios to the highest one. The calculated means ± SEM of the mRNA percentages in the tissues analyzed is shown in Figure 3. OP-1 mRNA expression for the 2.4 and 4.0 kb transcripts, respectively, was highest in the renal medulla followed by cortex and papilla. The relative mRNA expression for the 2.4 and 4.0 kb transcripts was respectively higher in the renal medulla (77.6 ± 11.2% and 65.0 ± 8.3% ) followed by cortex (72.7 ± 12.2% and 41.6 ± 7.8% ) and papilla (62.7 ± 8.3% and 53.9 ± 11.9%) . Relative mRNA expression in kidney sections was significantly greater than that of extrarenal tissues (p=0.0002 for the 2.4 kb band and p=0,0001 for the 4.0 kb band) . Brain showed the lowest signal (9.0 ± 3.3 and 8.3 ± 3.0) whereas ovary (18.8 ± 5.2 and 15.1 ± 3.1) elicited the highest extrarenal expression. Results for liver (15.2 ± 5.2 and 8.2 + 2.0) , intestine (12.4 ± 3.7 and 8.1 + 2.3) and lung (10.8 + 3.0 and 8.2 ± 2.0) were more variable.

Although the putative amino acid sequence of the OP-1 cDNA has been deduced and OP-1 expressed in transfected mammalian cells in culture (Ozkaynak, E., et _. al.. , EMBO J. , 9:2085-2093 (1990) ; Sampath, T.K., et al., J. Biol. Chem. , 267:20352-20362 (1992) ; Jones, W.K., et al. , Bone and Mineral Research, S348. (1993)), little is known with regard to endogenous processing of OP-1 by various tissues. The OP-1 cDNA predicts a primary translation polypeptide of 49 kD that includes a signal secretory sequence (29 A.A or -3.5 kD) , a pro domain (-270 A.A. or

31 kD) containing two polybasic proteolytic processing sites and a TGF-/3 domain (138 A.A. or 16 kD) . OP-1 is a glycoprotein (Sampath, T.K., et _. al _. . , J. Biol . Chem. , 265:13198-13205 (1990) ; Sampath, T.K., et al. , J. Biol. Chem. , 267:20352-20362 (1992)) possessing 4 distinct potential glycosylation sites that would increase the apparent molecular mass of any of these OP-1 polypeptides.

The processing of human OP-1 has been partially characterized after its expression in either cultured CHO or BCC-1 cells (Sampath, T.K., e_t al. , J. Biol . Chem.. 267:20352-20362 (1992) ; Jones, W.K., et al. , Bone and Mineral Research, S348. (1993)) . These data suggest the major intracellular form of OP-1 in CHO cells is polypeptide of approximately 50 kD that is dimmerized upon its secretion. This OP-1 complex undergoes further cleavage and processing is an acidic intracellular vesicular compartment and is finally secreted as a complex of mature OP-1 homodimers.

A monoclonal antibody, antibody 12G3, was produced as described in detail in Example 2. To verify the specificity of mouse monoclonal antibody 12G3 for OP-1 proteins, 0.5 ugs of spent media derived from CHO cells expressing recombinant human OP-1 was probed. (Sampath, T.K., et al., J. Biol. Chem. , 267:20352-20362 (1992) ; Jones, W.K., et _. al _. . , Bone and Mineral Research, S348.

(1993)) As shown in Figure 4, monoclonal 12G3 recognized 3 bands of 65 kD, 44 kD and broad band of 27-19 kD. As reported previously (Jones, W.K., et _. al. , Bone and Mineral Research, S348. (1993)) . These bands correspond to the glycosylated primary translation OP-1 polypeptide (65 kD) , pro-OP-1 (44 kD) and mature OP-1 (27-19 kD) proteins. These data demonstrate that 12G3 recognizes an epitope present on both primary and mature OP-1 proteins.

To obtain evidence for the presence of OP-1 species, as well as mRNA, in the kidney, homogenates of kidney

tissue were probed with monoclonal 12G3. As shown in Figure 5, whole kidney (lane 1) as well as renal cortex (lane 2) , medulla (lane 3) and papilla (lane 4) with monoclonal antibody 12G3 specifically reacted with 12G3. Either omission of 12G3, or substitution of another irrelevant monoclonal resulted in the complete absence of bands. Monoclonal 12G3 recognized 6-7 major OP-1 bands including species of 53 kD, 45 kD, 36 kD, 34 kD, 2 closely spaced bands of 19-17 kD and a 15 kD band of variable intensity. The overall pattern of OP-1 bands was similar in homogenates of renal cortex and medulla (lanes 2 and 3, of Figure 5) . In contrast, the 53 kD was less intense in the renal papilla (lane 4, of Figure 5) as compared to renal cortical and medullary homogenates. Several of the bands identified in kidney homogenates by 12G3 possess molecular masses identical to that described for OP-1 species harvested from OP-1 producing CHO cells shown in Figure 4. The major OP-1 bands of 45 kD, 19 kD and 17 kD are identical to those displayed in Figure 3 and likely correspond to pro-OP-1 protein as well as the 2 mature glycosylated OP-1 products. Previous data (Sampath, T.K., et _. al.. , J. Biol. Chem., 267:20352-20362 (1992); Jones, W.K., et al . , Bone and Mineral Research, S348. (1993)) have identified a 15 kD OP-1 band as possessing multiple amino termini and thus is likely the result of additional proteolytic degradation. The 53 kD band is similar to the molecular mass of the primary translation OP-1 polypeptide and less intense bands in the 65 kD region of Figure 4 may correspond to glycosylated forms of this initial OP-1 polypeptide shown in Figure 4. The bands of 36 and 34 kD identified by 12G3 have not been reported as major bands in immunoblotting analyses of CHO cell homogenates containing processed OP-1 polypeptides (Jones, W.K., et. al. , Bone and Mineral Research, S348. (1993)) . Several possibilities could

account for the apparent molecular masses on SDS-PAGE. These include either differences in the carbohydrate content between endogenous and CHO cells OP-1 proteins, incomplete reduction of 17 kD and 19 kD dimers or additional OP-1 processing steps that are not present in CHO cells. For example, at least 2 polybasic proteolytic cleavage sites are present in the prodomain of OP-1 (Ozkaynak, E., e_t al. , Biochem. and Biophysical Research Comm. , 179:116-123 (1991)) that may be utilized by kidney cells expressing OP-1.

As shown in Figure 6A-D, immunohistochemistry staining using monoclonal 12G3 localizes OP-1 to glomeruli and adjacent large vessels (Panel B) as well as epithelial cells of renal tubules (Panel C) and small vessels located in medullary portions of the kidney (Panel D) . Glomerular OP-1 staining appears to be localized in both epithelial cells in Bowman's capsule as well as other cells within the glomerular tuft. In addition, endothelial and smooth muscle cells of vessels including the arterioles of the juxtaglomerular apparatus display prominent OP-1 staining. Mouse monoclonal 12G3 is unsuitable for use as a probe for mouse tissues. However, in si tu hybridization analysis of serial sections of mouse kidney was performed to determine whether OP-1 mRNA expression displayed a pattern similar to that observed for OP-1 protein in rat kidney. As shown in Figure 7A-H, paraffin sections of whole kidney were hybridized with either antisense (Panels B and F) or sense (Panels D and H) RNA synthesized from the same cDNA probe used in Figure 2. In a f shion similar to that demonstrated by northern analysis of rat kidney shown in Figure 2, specific hybridization by the OP-1 antisense probe was observed in the renal cortex, medulla and papilla. Specific labeling of tubular and/or vascular elements in the papilla and inner medulla of the kidney were readily discernable as shown in Panel F of

Figure 7. There was also specific labeling of both medullary and cortical structures including areas corresponding to glomeruli as shown by the large open arrows in Panels A and B. Specific OP-1 hybridization also appeared to be distributed in a nonhomogeneous manner that was especially prominent in the inner medulla and papilla (see Panel F) . This patter of OP-1 mRNA expression is similar to that observed in rat kidney using immunohistochemistry as shown in Figure 6. To obtain a kidney derived cell line to further study OP-1 expression, confluent cells from 3 types of cultured epithelial cells were screened by northern and immunoblot analyses using the cDNA and 12G3 antibody OP-1 probes described above. Opossum kidney (OK) cells, canine (MDCK cells and an SV-40 transformed mouse IMCD cell line each form polarized epithelia. Both OP and MDCK cells have been utilized extensively in studies of epithelial cell polarity and transport. The IMCD cell line is derived from the cells of the IMCD and it retains many characteristics of this nephron segment including increased transepithelial resistance in response to dexamethasone, inhibition of sodium flux by amiloride and atrial natriuretic peptide and the ability to adapt to hypertonicity (Rauchman, M.I., Am. J. Physiol . , 265-271 (1993) ) .

In contrast to OK cells where no OP-1 expression was detected, both MDCK and IMCD cell poly A ⁺ RNA contained both 4.0 and 2.4 kb OP-1 transcripts as shown in Figure 8. OP-1 expression by MDCK cells confirms previous reports using a canine OP-1 cDNA probe (Ishibashi, K. , et, al . , Biochem. and Biophysical Research Comm. , 193:235-239 (1993)) . However, OP-1 expression by IMCD cells (Right Panel) was consistently larger when compared to MDCK cells (Left Panel) even under conditions of reduced stringency to account for species differences.

Immunoblot analysis using 12G3 also detected the presence of 4 OP-1 species in IMCD cell homogenates (lane 5, Figure 5) that were identical to the pattern exhibited by rat kidney homogenates (lanes 1-4, Figure 5) . The major IMCD OP-1 bands included bands of 36 kD, 19 kD, 17 kD and 15 kD. Although IMCD cells did display 12G3 reactive bands of 53 kD and 45 kD, both were less intense as compared to cortical (lane 2) and medullary (lane 3) homogenates. The abundance of OP-1 protein species in IMCD cells is reflected in the fact that lane 5 contains approximately one half the total protein content of lanes 1-4 of Figure 5.

The observation that most of the antibody localized to tubular structures in medullary rays, plus the high baseline mRNA expression found in IMCD cells, indicates that tubular epithelial cells in the renal medulla are an important source of the OP-1 protein. Thus, Applicants have determined that renal epithelial cells, in particular, tubular epithelial cells in renal medulla express high levels of OP-1 mRNA and OP-1 protein. As a result of this work, , a method is now available to identify substances capable of inducing bone formation by determining the effect a substance has on OP-1 mRNA expression, or OP-1 protein synthesis. As used herein, the term "effect on OP-1 mRNA expression" means effect on the transcription of genetic information from DNA to mRNA. As used herein, the term "effect on OP-1 protein synthesis" means the effect on the translation of mRNA to protein molecules, as well as effects on the biological activity of the protein molecule, or secretion of protein molecules from the cell. OP-1 protein is a potent bone morphogen member of the transforming growth factor beta (TGF-β) superfamily and induces de novo bone formation by stimulating collagen synthesis and the proliferation and differentiation of

osteoblasts. Thus, upregulation of the expression of OP-1 mRNA, or an increase of OP-1 protein synthesis, provides a means to induce new bone formation. Alternatively, downregulation of OP-1 mRNA expression, or a decrease in OP-1 protein synthesis, provides a means to suppress new bone formation. Thus, as used herein, substances that affect OP-1 mRNA expression, or OP-1 protein synthesis also affect bone formation.

The regulation of expression of OP-1 mRNA can be accomplished by any method that regulates the transcription of OP-1 DNA to mRNA. Upregulation of the expression of OP-1 mRNA can be accomplished, for example, by a transcriptional activator factor, such as an ancillary protein necessary to initiate the transcriptional process. Upregulation can also be accomplished, for example, by removal of a transcriptional factor which prevents initiation of the transcriptional process. For example, the upregulation of OP-1 mRNA expression can be accomplished by introducing into the cell an antisense oligonucleotide which binds to a gene encoding a regulatory protein, that, when present, binds to OP-1 DNA and prevents initiation of transcription of OP-1 DNA to OP-1 mRNA. Removal of this regulatory protein thus upregulates the expression of OP-1 mRNA expression. Alternatively, removal of the regulatory protein can be accomplished by introducing into the cell and an antibody, or antibodies, that bind to the regulatory protein and, thus, block, or neutralize, its activity. Removal of this regulatory protein initiates transcription of OP-1 DNA to mRNA, and, thus, induces new bone formation.

The expression of OP-1 mRNA can also be downregulated by any method that which regulates the transcription of OP-1 DNA to mRNA. Downregulation of mRNA expression can be accomplished, for example, by sense, or antisense, DNA complementary to a portion of OP-1 DNA, which hybridizes

with all, or a portion, of the OP-1 gene, thus, preventing transcription.

The regulation of synthesis of OP-1 protein can be accomplished by any method that regulates protein synthesis, or regulates the biological activity of the protein, or the secretion of protein from a cell. For example, the synthesis of OP-1 protein can be regulated by substances that affect the translation of mRNA to protein, such as the binding of an antisense oligonucleotide to OP- 1 mRNA, thus preventing translation. Alternatively, the biological activity of OP-1 protein can be altered by a substance that interferes with the maturation (e.g., biological activity) of the protein, such as cleavage of the pro region, or glycosylation of the protein. Moreover, regulation can occur by substances that stimulate, or inhibit secretion of OP-1 protein from the cell.

The method for the identification of substances capable of inducing new bone formation is performed as follows.

Renal medullary cells are grown under standard laboratory conditions at pH 7.4, at 37°C in 5% C0 ₂ , in Dulbecco's Modified Eagle's Medium (DMEM) as described in Example 1. Renal medullary cells can be obtained from a variety of sources. These include, for example, dissecting mammalian kidneys and establishing primary renal medullary cell cultures, or using a cell line such as the Madin-Darby canine kidney (MDCK) cell line. Applicants have demonstrated that inner medullary collecting duct (IMDC) cells exhibit significantly higher expression levels of OP-1 mRNA and OP-1 protein than other kidney cells. A convenient source of IMDC cells, suitable for use in the present invention, is the mIMCD-3 cell line derived from a transgenic SV-40 mouse as described in Gullans, S.R., et al. , Am. J. Phvsiol . , 265 (1993) , the

teachings of which are incorporated herein by reference. Alternatively, other culture media can be used to practice this assay. Selection of suitable culture medium will depend on the ability of the medium to support growth of renal cells and to provide an environment which permits the test substance to detectably affect OP-1 mRNA expression or OP-1 protein synthesis, and thus, induce bone formation.

Substances identified by the method described herein, capable of inducing new bone growth formation, are substances which posses properties which affect the expression of OP-1 mRNA or synthesis of OP-1 protein. Candidate substances (test substances) can be oligonucleotides, peptides, proteins, small molecules or organic molecules, including molecules that alter the pH, or ionic strength, of the cellular environment. For example, a test substance can be a peptide hormone (e.g., parathyroid hormone, anti-diuretic hormone, or atrial naturetic peptide) or a steroid hormone such as vitamin D, or one of its active metabolites. Mineralocorticoids can also be tested in IMCD cells. Minerals such as calcium and phosphorous, as well as substances that affect calcium and phosphorous concentration in the cellular environment, such as chelating agents, can also be tested. Test substances can also be antisense oligonucleotides or antibodies. Accordingly, a test substance can be any substance reasonably believed to affect bone formation. However, the candidate substance must be sufficiently soluble in an aqueous medium to allow a sufficient amount to dissolve so that the effect of the substance on OP-1 mRNA expression, or OP-1 protein synthesis, can be determined.

The test substance is then introduced into the culture medium, in sufficient amount and in a form that allows the test substance to affect the expression of OP-1

mRNA or synthesis of OP-1 protein. A sufficient amount of the test substance, (e.g., an effective amount) is determined for each individual substance to be tested. For example, it is known that 10 ^~7 M atrial natriuretic peptide (ANP) inhibits luminal-to-basal transport of Na in IMCD cells. (Gullans, S.R., Am. J. Phvsiol . , 265 (1993) . Thus, 10 ^"7 M ANP would be a reasonable amount of ANP to be tested in the method of the present invention. This substance would also be tested in a greater or lesser amount than 10 ^"7 M to determine the optimum dose of ANP effective to induce bone formation.

The test substance must be in a form so as to allow the substance to affect the OP-1 mRNA expression or OP-1 protein synthesis. Preferably, the test substance is sufficiently soluble to allow an effective amount of substance to dissolve in the aqueous culture medium and subsequently to enter into the IMCD cell. However, the test substance can be introduced into the culture medium, or the cell via other routes, such as encapsulation by a liposome, or by other standard methods used to introduce substances into culture media and cultured cells.

Once the test substance is introduced into the culture medium or cell, the test culture is incubated under conditions (e.g., time, temperature, C0 ₂ content) suitable for growth of the cells. Incubation conditions and duration will depend on the nature of the individual substance being tested. A control culture is run in tandem with the test culture, under similar conditions, but without the presence of the test substance. In one embodiment of the present invention, after appropriate incubation, total RNA is isolated from the cultured cells and prepared for Northern blot analysis as described in Example 1. After fractionation of mRNA on a 1% agarose formaldehyde gel using standard techniques, the mRNA is transferred to a membrane suitable for Northern

blot analysis and hybridized with a ³² P-labeled OP-1 cDNA probe under conditions of high stringency. For example, the probe can be all or a portion of the murine OP-1 cDNA sequences described in Ozkaynak, E., et al. , Biochem. Biophys . Res. Commun. , 30:116-123 (1991) . Alternatively, the OP-1 cDNA probe can be all or a portion of the human OP-1 cDNA sequence (Ozkaynak, E., et al. , EMBO J. , 9:2085- 2093 (1990) , deposited in the EMBL data library under accession number X51801) . Other OP-1 oligonucleotide probes can also be used in the Northern blot analysis. For example, a labeled OP-1 RNA oligonucleotide probe can be used. The probes can be of a length different from the cDNA probe used in Example 1. However, the probe must be of a length sufficient to specifically hybridize with OP-1 mRNA under conditions of high stringency. The probes can also vary in nucleotide sequence from the probe used in Example 1. However, the probe sequence must contain sufficient number of similar nucleotides to permit specific hybridization of the probe to OP-1 mRNA under conditions of high stringency.

The oligonucleotide probes used in this embodiment of the present invention can be labeled with radioactive molecules, such as ³² P. mRNA on the membrane which hybridized with the ³² P-labeled OP-1 oligonucleotide probe is then visualized by autoradiography, and quantified, for example, by laser scanning densitometry as described in Example 1. Alternatively, the probes can be labeled with non-radioactive molecules, such as peroxidase, biotin or digoxigenin. mRNA hybridized with non-radioactive OP-1 oligonucleotide probes is then visualized by color development by enzyme assay, binding to labeled strep- avidin, or chemiluminescence.

After quantification, the amount of OP-1 mRNA expressed in the test culture (i.e., cells cultured in the presence of test substance) is compared to the amount of

mRNA expressed in the control culture (i.e., cells cultured without the presence of the test culture) . If the amount of OP-1 mRNA expressed in the test culture is higher than the amount of OP-1 mRNA expressed in the control culture, the presence of the test substance induced OP-1 mRNA expression, and thus, is capable of inducing bone formation. If the amount of OP-1 mRNA expressed in the test culture is less than the amount of mRNA expressed in the control culture, the presence of the test substance suppressed OP-1 mRNA expression, and thus, is capable of suppressing bone formation.

In another embodiment of the present invention, the synthesis of OP-1 protein is determined. Cultured cells can be prepared, denatured by SDS and heat treatment, and subjected to SDS polyacrylamide (PAGE) electrophoresis as described in Example 2. After separation on the basis of size, the separated proteins are transferred to a membrane suitable for Western blot analysis, and incubated with antibody specific for OP-1 protein (e.g., an OP-1 specific monoclonal, polyclonal antibody, or an antibody mix) .

Detection and quantification of OP-1 protein analyzed by Western blot analysis is performed as described in Example 2. Alternatively, OP-1 protein secreted into the culture medium can be measured by standard laboratory techniques (e.g., ELISA or RIA) , also using antibodies specific for OP-1.

After quantification, the amount of OP-1 protein synthesized in the test culture (i.e., cells cultured in the presence of test substance) is compared to the amount of OP-1 protein synthesized in the control culture (i.e., cells cultured without the presence of the test culture) . If the amount of OP-1 protein synthesized in the test culture is higher than the amount of OP-1 protein synthesized in the control culture, the presence of the test substance induced OP-l protein synthesis, and thus,

is capable of inducing bone formation. If the amount of OP-1 protein synthesized in the test- culture is less than the amount of OP-1 protein synthesized in the control culture, the presence of the test substance suppressed OP-1 protein synthesis, and thus, is capable of suppressing bone formation.

The biological activity of the OP-1 protein synthesized, or secreted, can be measured using, for example, the in vivo bone matrix implant bioassay described in Rueger, D.C., et. al. , J. Biol . Chem. , 265:13198-13205 (1990) , the teachings of which are incorporated herein by reference. This assay measures the bone-inducing activity of OP-1 protein on bone matrix implanted in rats. Thus, if a test substance affects the biological activity of the OP-1 protein (e.g., by affecting maturation of the protein, or glycosylation of the protein) the increase, or decrease in biological activity can be determined.

Abnormalities in mineral and bone metabolism begin in early stages of loss of kidney function, and bone disease represents a major contributor to the morbidity of patients with end stage renal disease. Decreased renal synthesis of 1,25 dihydroxyvitamin D is clearly a major factor in the production of hyperparathyroid bone disease. However a subset of patients with renal osteodystrophy develop a low turnover bone disease not associated with aluminum or iron deposition (Salusky, I., et, al _. . , Miner. Electrolyte Metab.. 17:237-280 (1991) ; Mathias, R. , et al.. J. Am. Soc. Nephrol . , 3:1938-1946 (1993)) . The etiology of this aplastic bone disease remains unknown. Thus, other factors produced by the kidney aside from activated vitamin D, may be necessary for adequate bone homeostasis.

Peptide hormones classically involved in bone metabolism could modulate OP-1 expression and production

by the kidney via activation of the protein kinase A pathway, to stimulate the level of OP-1 mRNA expression. However, based on the finding that IMCD cells expressed OP-1 at such high levels, it is reasonable to believe that renal OP-1 production may be an integral part of a mechanism distinct from parathyroid hormone (PTH) and vitamin D by which the kidney influences skeletal maturation and growth. The mIMCD-3 cell line, together with the assay described herein, are particularly useful as a model system to study the pathway of bone morphogenesis regulated by OP-1. The IMCD cell line was developed from the terminal inner medullary collecting duct of a large T antigen SV40 mouse and it retains many characteristics of this nephron segment, including increased transepithelial resistance in response to dexamethasone, inhibition of sodium flux by amiloride and atrial natriuretic peptide, and the ability to adapt to hypertonicity. Receptors to calcitriol or PTH have not been found in inner medulla. (Karashima, H., _______ al. , Kidney Int . , 29:98 (1986) ; Alexander, E. and Schwartz, J. , American J. of Kid. Pis., 18 (5) :612-618 (1991); Yoshitaka, M., et al., Am. J. Phvsiol.. 263 (32) :F319-F327 (1992)) . Therefore, it is reasonable to believe that alterations in acid-base status and/or mineralocorticoid effect might modulate expression of OP-1 in this segment of the nephron. Thus, OP-1 protein may serve as a link between ion sensing cells in the inner medulla and cellular elements of the skeleton.

Substances identified in the method of the present invention, found to affect OP-1 mRNA expression, or OP-1 protein synthesis, can be used to therapeutically treat a patient with bone disease, such as osteoporosis, to induce bone growth (bone morphogenesis) . This method comprises administering to the patient an effective amount of a

substance capable of inducing bone growth by inducing OP-1 mRNA expression, or OP-1 protein synthesis.

Administration of such bone-inducing substances can be by medically accepted techniques, including intravenous, subcutaneous, or oral administration.

Appropriate amounts, or effective dosages will, of course, vary from individual to individual and by type of disease and/or severity of the disease. Appropriate dosages can be calculated by those skilled in the art taking such factors into account.

Conversely, the present invention provides a method of therapeutically treating a patient with bone disease, such as osteopetrosis, to suppress an increase in bone formation. This method comprises administering to the patient an effective amount of a substance capable of suppressing bone growth by suppressing OP-1 mRNA expression, or OP-1 protein synthesis.

Of the extrarenal tissues studied, as described herein, ovary tissue had the highest OP-1 mRNA expression. Thus, a possible role of the OP-1 synthesized by the ovary in the maintenance of bone homeostasis is also reasonable, especially in the relationship of age related OP-1 mRNA expression and OP-1 protein synthesis (e.g., post- menopausal osteoporosis) . The method of the present invention can also be used to identify substances capable of inducing bone formation by their effect on OP-1 mRNA expression, or OP-1 protein synthesis in ovary cells.

Additionally, IMCD cells of the kidney may make a unique form of OP-1 that does not cause calcification immediately but would have to be processed to become active in calcification processes. Alternatively, IMCD OP-1 may have other functions unique to the kidney itself. Since IMCD cells secrete OP-1, these cells can be used in the described methods to further elucidate these functions. Unique properties of IMCD-derived OP-1 would

have considerable value. For example, because these cells are epithelial cells, it is reasonable to predict that OP-

1 is only secreted on one side of the layer of cells, either the apical side (urine facing) or basolateral side (blood facing) of epithelial plasma membranes. Thus, the different forms of OP-1 could reflect one form of OP-1 going to one surface and the targeting of another form to the opposite surface.

It is also reasonable to predict that OP-1 secreted by renal epithelial cells is also present in the urine. Assay systems using IMCD cell supernatants can be designed to measure the urine and hence body stores, of OP-1 using OP-1 specific monoclonal antibodies such as 12G3. If renal IMCD cells make a specialized form of OP-1 this form could be distinguished from OP-1 made by other regions of the kidney as well as that filtered by glomeruli from the serum. Such an assay could distinguish between the various forms of OP-1. This information would be of value, for example, to distinguish which patients might benefit from treatment with OP-1 and/or stimulation of synthesis of OP-1 exogenous as opposed to patients requiring other therapy.

In addition to the role of OP-1 as a mediator of bone metabolism, OP-1 may also function as a "repair" molecule in mammalian kidney tissue. As discussed above, is has been demonstrated that OP-1 mRNA and protein expression is

2 fold higher in rat kidney as compared to 5 other tissues. Comparison of the OP-1 expression in the same 6 tissues in young (4 week old) versus old (2 year old) rats demonstrated a significant decline in renal medullary OP-1 expression as a function of age. In addition, both in situ hybridization and immunocytochemistry studies showed that OP-1 is expressed by glomerular tufts and adjacent vessels in the renal cortex as well as tubules and vasculature in the kidney medulla. (J. Amer. Soc.

Nephrol . , 4:700 Abstracts 83P and 23P (1993) ; Paredes, A., et al., J. Amer. Soc. Nephrol . , 5:855 Abstract 68P (1994) ) .

A similar pattern of OP-1 has now been demonstrated by staining human kidney biopsy samples using identical antibody probes (the monoclonal antibody, 12G3) . In tissue biopsy sections of both rat and human kidneys, the distribution of OP-1 among renal tubules is not homogenous. In selected renal tubules, there is abundant OP-1 protein, whereas tubules immediately adjacent have little, or no detectable OP-1 expression. These data suggest that the observed heterogeneity in detectable OP-1 protein may be due to the fact that some tubules are undergoing remodeling or repair, whereas others are quiescent.

Moreover, described herein for the first time, examination of a limited number of (n=4) of kidney biopsy sections from children with renal dysplasia revealed abundant OP-1 staining with the OP-1 specific antibody, 12G3. The overall pattern of OP-1 staining also included primarily the tubular epithelial cells and the adjacent interstitium. These data reasonably suggest that the amount of OP-1 protein detected is indicative of the amount of remodeling of diseased kidney tissue thus, supporting the paracrine role of OP-1 protein as a repair molecule.

Other reports provide support for the role of OP-1 as a modulator of tissue remodeling. Studies have shown that OP-1 appears to act as a potent renal morphogen in the developing kidney. When assayed in an i-n vitro organ culture system consisting of explants of mouse metanephric blastema cultured in a serum free media, OP-1 dramatically altered tubular development and gave rise to a new cell type that possesses many characteristics of chondrocytes (Avener, E.D., J. Amer. Soc. Nephrol., 3:461 Abstract 82P

(1993)) . OP-1 is also highly expressed in embryonic tissues (Ozkaynak, E., et al., EMBO J. , 9:2085-2093 (1992)) and in neuronal cells in tissue culture alters the patterns of aggregation and development of these cells in a highly specific fashion (Perides, G. et _. al . , Proc. Natl. Acad. Sci., 89:10326-10330 (1992)) .

In addition, studies of unrelated proteins have provided examples of proteins synthesized by the kidney that have both autocrine (acting at a distance) and paracrine (acting locally) effects. Several proteins derived from the kidney have been demonstrated to possess effects on other organs as well as serving to modulate cellular growth within the kidney. Renin/angiotensin proteins alter systemic blood pressure and acts as a factor for kidney growth (Ichikawa, I. and Harris, R.C., Kidney International, 40:583-596 (1991)) . Endothelin, produced by kidney endothelial cells and exerts both local and systemic vasoconstrictive effects and effects proliferation of glomerular endothelial cells (Kon, V. and K. Bodi, Kidney International, 40:1-12 (1991)) . Epidermal growth factor (EGF) is produced largely by epithelial cells of the thick ascending limb of Henle, but EGF receptors are present in all epithelial cells of the kidney where it stimulates tubular proliferation (Fine, L.J., Amer. Soc. of Nephrol. , 2:1163-1170 (1992)) .

Most renal biopsies are performed in an effort to prognosticate the outcome, as well as determine the cause of a patient's renal disease. Examination of OP-1 expression using antisera in these renal biopsies can be used to assess the kidney's response or lack thereof to a specific renal injury. Such a test can also be useful to determine specific diseases where OP-1 might play a significant fold in its pathogenesis. Lastly, since epithelial cells are shed into the urine and, as discussed above, it has been demonstrated the OP-1 is secreted from

renal epithelial cells, OP-1 antisera can be useful to determine the level of OP-1 expression in urine samples from patients with renal disease.

Thus, as a result of Applicants' work, a model assay system is now available to identify substances capable of inducing new bone formation by the evaluation of the effect of test substances on the expression of OP-1 mRNA expression and OP-1 protein synthesis in inner medullary collecting duct cells. Significant levels of OP-1 mRNA transcripts and proteins are present in the renal medulla. Therefore, renal OP-1 production in the inner medulla may act as an alternative pathway to the classic PTH and vitamin D mechanisms by which kidney modulates bone homeostasis. Moreover, based on additional data presented herein, OP-1 may also act in the repair of normal and abnormal kidney tissue.

The invention is further illustrated by the following specific examples.

Example 1: Northern Blot Analysis to Detect OP-1 mRNA Expression

Animals : Young adult female Sprague-Dawley rats approximately 300 gms (2-4 months old) were purchased from Charles River Laboratories (Wilmington, MA) . After euthanasia under general anesthesia, organs were flushed with ice-cold saline via intracardiac puncture. Renal cortex, medulla and papilla were immediately dissected. Equal amounts of lung, liver, brain, small intestine and ovary were also extracted.

Cultured Cells: The following cell lines were surveyed: opossum kidney (OK) cells, a gift from Dr. M.H. Montrose, Johns Hopkins University, MD; Madin Darby Conni Kidney (MDCK) cells obtained from American Type Culture

Collection (Rockville, MD) ; and inner medullary collecting duct (IMCD) cells derived from a transgenic SV-40 mouse (Rauchman, M.I., et al. , Am. J. Phvsiol . , 265-271 (1993)) were obtained from Dr. M. Ziedel, Brigham and Women's Hospital, Boston, MA. The cells were grown to confluency on tissue culture dishes (Nunclon Delta; Nunc, Inc; Naperville, IL) at pH 7.4 in incubators at 37°C with a 5% C0 ₂ 95% air atmosphere. OK and IMCD cells were fed on alternate days in Dulbecco's Modified Eagle's Medium (DMEM) -F12 (GIBCO, Gaithersburg, MD) supplemented with 29 mM bicarbonate, 2mM glutamate, 100 IU/cc penicillin, 100 pg/cc of streptomycin and 10% (OK cells) or 5% (IMCD cells) fetal calf serum. MDCK cells were fed on alternate days with a standard MEM culture medium (GIBCO, Gaithersburg, MD) supplemented with Earles Salt, 2 mM glutamate, 10% fetal calf serum, 100 IU/cc penicillin and 100 pg/cc of streptomycin.

Probes: A 680 bp fragment (SEQ ID NO: 1) containing two thirds of the pro domain and one third of the mature murine OP-1 cDNA (nucleotides 370-1050 immediately upstream to the highly conserved 7-cysteine domain present in all members of the TGF-/3 superfamily) was used as a probe for Northern analysis. (Ozkaynak, E., et al. , Biochem. Biophvs. Res. Commun., 30:116-123 (1991)) . USB Sequenase Random Prime DNA labeling Kit (United States Biochemical, Cleveland, OH) and c_ ³² P dCTP (New England Nuclear, Boston, MA) were used for standard labeling reactions. A 40 bp oligonucleotide of mouse β- act in (Oncogene Science, Uniondale, NY) was used as control gene.

RNA Preparation: Total RNA was isolated from animal tissues (100 mg/assay) and cultured cells (90-100% confluency, 100 x 20 mm plate/assay) using the guanidine

isothyocyanate/phenol-chloroform method (Chomczynski, P. and Sacchi, N. , Anal . Bioche . , 162:156-159 (1987)) . Poly A ⁺ was selected by oligo dT cellulose using the mRNA MicroFastTrack Kit from Invitrogen, San Diego, CA.

Northern Blot Analysis : Equal amounts of mRNA were fractionated on a 1% agarose formaldehyde gel using standard techniques. RNA was transferred to Duralon -UV membranes (Stratagene, La Jolla, CA) by capillary blotting with lOx standard sodium citrate (SSC) . The membranes were then baked at 80oC under vacuum for 2 hours. After this treatment, membranes were prehybridized with 50% deionized Formamide (Sigma) , 10% Dextran Sulfate, 1% sodium dodecyl sulfate (SDS) , 1 M NaCI and 100 pg/ml of denatured sonicated salmon sperm DNA (Sigma) for 2 hours at 42°C in a hybridization oven. Using the same solution, the membranes were then hybridized for 20 hours at 42©c with 1 x 10 ⁶ cpm/ml of the ³² P-labeled OP-1 cDNA probe. Membranes were washed for 30 minutes with 0.lx SSC-O.lx SDS at 65°C and then subjected to autoradiography at -80oc for 24 hours using Kodak XAR-5 film and an intensifying screen (Lightning Plus; Eastman Kodak, Rochester, NY) . To control for mRNA content and quality, the blots were stripped and subsequently hybridized with mouse /--actin. The intensity of the signal was quantified by laser scanning densitometer (model SL-504-XL; Biomed Instruments,

Fullerton, CA) . The ratio OP-1 mouse -actin signal was calculated for each tissue and the highest value within an animal was assignated as 100%. The percentages of mRNA expression for the rest of the tissues were figured out by comparison of their ratios to the highest one.

Statistical analysis: Data are expressed as means ± SEM. Since the variables were not normally distributed, OP-1 mRNA expression among tissues was statistically analyzed

by Man Whitney non parametric tests. Statistical significance was defined as P < 0.05.

Example 2: Western Blot Analysis of OP-1 Protein Synthesis

Generation of the Anti-OP-1 Antibody 12G3 The selection of the IgG _j monoclonal anti-OP-1 antibody 12G3 was accomplished by injecting recombinant human OP-01 and fusing the spleen cells with nurine myeloma cells, followed by screening using Western blots of oxidized and reduced OP-1 (Vukicevic, S., et, al. , Biochem. Biophys. Res. Commun. 198, 693-700 (1994)) The injected recombinant human OP-1 used in the generation of 12G3 was composed of amino acids 293-431 where amino terminal methionine of pre-pro OP-1 is defined as residue #1 (Ozkaynak, E., et al. , EMBO J. 9, 2085-2093 (1993) ) .

Gel Electrophoresis and Immunoblottincf: Tissue samples were minced and denatured by rapid addition of 10% SDS in a 1:1 dilution and heating to 100° C for 5 minutes. The samples were then stored at -80° C after determination of their protein content using the BCA protein assay (Pierce

Chemical Co., Rockford, IL) . Forty micrograms of protein from each sample were separated by SDS-PAGE using a Mighty Small apparatus, (Hoefer Scientific Instruments, San Francisco, CA) (Laemmli, U.K., Nature London, 277:680-685 (1980) ) . After SDS-PAGE, the proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) using a solution of 25 mM Tris, 200 mM glycine, and 20% methanol . These membranes were then removed from the transfer apparatus and either stained with 0.1% Coomassie blue or immunoblotted with monoclonal antibody 12G3 after blocking with 5% bovine serum albumin (Sigma Chem. Co., St. Louis, MO) dissolved in water. The blots were incubated

overnight using 12 μg/ml of 12G3 diluted in 50 mM NaP0 ₄ , pH 7.4, 100 mM NaCl phosphate buffered saline (PBS) with 0.01% Tween-20 (PBS-Tween) . After incubation with monoclonal 12G3, the blots were rinsed repeatedly with 5 Tween-PBS and bound immunoglobulin was detected using affinity purified peroxidase conjugated goat antimouse antisera (Organon Teknika, West Chester, PA) and chemiluminescence (ECL Amersham Life Sci., Arlington, IL) .

Example 3 : Immunocvtochemical Analysis to Detect the ° Presence of OP-1 Protein

Immunocytochemistry (IC) : Following an overnight incubation in 3% normal goat serum at 4° C, rat kidney sections were incubated in primary monoclonal antibody 12G3, that was diluted in PBS, 1% fetal calf serum and 5 0.1% sodium azide for 60 minutes at room temperature. Subsequently sections were exposed to a peroxidase- conjugated goat anti-mouse immunoglobulin (Jackson Immunoresearch, Westgrove, PA) in the same diluent, at a dilution of 1:40 for an additional 60 minutes. The slides 0 received sequential washes of 5 minutes duration in PBS then PBS containing 0.2% gelatin after each incubation. After the above series of incubations, the specimens were exposed to 5 minute intervals of 0.1 M sodium acetate buffer (pH 5.2) and 0.25 mg/ml 3-amino 9-ethylcarbazole 5 (AEC) (Sigma Chem. Co., St. Louis, MO) in 2% N-N dimethylformamide (Sigma Chem. Co., St. Louis, MO) followed by 0.1 M sodium acetate buffer (pH 5.2) with 0.03% hydrogen peroxide. The specimens were finally washed in tap water for 5 minutes, counterstained with 0 hematoxylin, and mounted in glycerol gelatin (Sigma Chem. Co. , St. Louis, MO) .

In Situ Hybridization: A ³ P-labelled antisense RNA probe was prepared by linearizing Bluescript (Stratagene, La Jolla, CA) containing the 680 bp OP-1 cDNA with Hind III followed by transcription with T7 polymerase (Promega Inc., Madison, WI) in the presence of [ ³² P] CTP (800

Ci/mmol.) . As a control, ³² P-labelled sense RNA probe was prepared by linearizing the same vector with BAM HI followed by transcription with T3 polymerase under identical conditions. Paraffin sections of adult C57BL/6 mouse kidney were subjected to in si tu hybridization as described in Arceci, R.J., et al., Proc. Natl. Acad. Sci. USA, 86:8818-8822 (1989) using [ ³ P] CTP-labeled sense (Control) or antisense (Experimental) RNA probes transcribed form Bluescript-OP-1 vector as described above. After autoradiography, the slides were stained with hematoxylin/eosin prior to photographic analysis.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Children's Medical Center Corporation

300 Long ood Avenue Boston, Massachusetts 02115

(ii) TITLE OF INVENTION: METHOD OF IDENTIFYING A SUBSTANCE CAPABLE OF INDUCING BONE FORMATION

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P.C.

(B) STREET: Two Militia Drive

(C) CITY: Lexington

(D) STATE: Massachusetts

(E) COUNTRY: USA

(F) ZIP: 02173-4799

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/153,183

(B) FILING DATE: 16-N0V-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Brook, David E.

(B) REGISTRATION NUMBER: 22,592

(C) REFERENCE/DOCKET NUMBER: CMCC-349

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-861-6240

(B) TELEFAX: 617-861-9540

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1872 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 104..1390

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115

Met His Val Arg

1

TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163 Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro 5 10 15 20

CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211 Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 25 30 35

GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 259 Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser Gin Glu Arg Arg 40 45 50

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307 Glu Met Gin Arg Glu lie Leu Ser lie Leu Gly Leu Pro His Arg Pro 55 60 65

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 355 Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 70 75 80

GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 403 Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 85 90 95 100

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 451 Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 105 110 115

TTA GCC AGC CTG CAG GAC AGC CAC TTC CTC ACT GAC GCC GAC ATG GTC 499 Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp Met Val 120 125 130

ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 547 Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 135 140 145

CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG 595 Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys lie Pro Glu 150 155 160

GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 643 Gly Glu Arg Val Thr Ala Ala Glu Phe Arg lie Tyr Lys Asp Tyr lie 165 170 175 180

CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG 691 Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr Val Tyr Gin Val 185 190 195

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739 Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 200 205 210

CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 Arg Thr lie Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp lie Thr 215 220 225

GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 835 Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 230 235 240

CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC AAC CCC AAG TTG 883 Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser lie Asn Pro Lys Leu 245 250 255 260

GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AAC AAG CAA CCC TTC ATG 931 Ala Gly Leu lie Gly Arg His Gly Pro Gin Asn Lys Gin Pro Phe Met 265 270 275

GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CTC CGT AGT ATC CGG TCC 979 Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser lie Arg Ser 280 285 290

ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TCC AAG ACG CCA AAG AAC 1027 Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn 295 300 305

CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 310 315 320

CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123 Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 325 330 335 340

CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GAA GGC TAT GCT GCC TAC 1171 Leu Gly Trp Gin Asp Trp lie lie .Ala Pro Glu Gly Tyr Ala Ala Tyr 345 350 355

TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCC TAC ATG AAC GCC 1219 Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 360 365 370

ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 Thr Asn His Ala lie Val Gin Thr Leu Val His Phe lie Asn Pro Asp 375 380 385

ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala lie Ser 390 395 400

GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC ATC CTG AAG AAG TAC AGA 1363 Val Leu Tyr Phe Asp Asp Ser Ser Asn Val lie Leu Lys Lys Tyr Arg 405 410 415 420

AAC ATG GTG GTC CGG GCC TGT GGC TGC CACTAGCTCT TCCTGAGACC 1410

Asn Met Val Val Arg Ala Cys Gly Cys 425

CTGACCTTTG CGGGGCCACA CCTTTCCAAA TCTTCGATGT CTCACCATCT AAGTCTCTCA 1470

CTGCCCACCT TGGCGAGGAG CCAACAGACC AACCTCTCCT GAGCCTTCCC CTCACCTCCC 1530

CAACCGGAAG CATGTAAGGG TTCCAGAAAC CTGAGCGTGC AGGCAGCTGA TGAGCGCCCT 1590

TTCCTTCTGG CACGTGACGG ACAAGATCCT ACCAGCTACC ACAGCAAACG CCTAAGAGCA 1650

GGAAAAATGT CTGCCAGGAA AGTGTCCATT GGCCACATGG CCCCTGGCGC TCTGAGTCTT 1710

TGAGGAGTAA TCGCAAGCCT CGTTCAGCTG CAGCAGAAGG AAGGGCTTAG CCAGGGTGGG 1770

CGCTGGCGTC TGTGTTGAAG GGAAACCAAG CAGAAGCCAC TGTAATGATA TGTCACAATA 1830

AAACCCATGA ATGAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 1872

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 429 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30

Leu Asp Asn Glu Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser 35 40 45

Gin Glu Arg Arg Glu Met Gin Arg Glu lie Leu Ser lie Leu Gly Leu 50 55 60

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 65 70 75 80

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 85 90 95

Pro Asp Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr 100 105 110

Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 115 120 125

Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 130 135 140

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 145 150 155 160

Lys lie Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg lie Tyr 165 170 175

Lys Asp Tyr lie Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr 180 185 190

Val Tyr Gin Val Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 195 200 205

Leu Leu Asp Ser Arg Thr lie Trp Ala Ser Glu Glu Gly Trp Leu Val 210 215 220

Phe Asp lie Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 225 230 235 240

Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser lie 245 250 255

Asn Pro Lys Leu Ala Gly Leu lie Gly Arg His Gly Pro Gin Asn Lys 260 265 270

Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 275 280 285

Ser lie Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 290 295 300

Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 305 310 315 320

Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val 325 330 335

Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp lie lie Ala Pro Glu Gly 340 345 350

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 355 360 365

Tyr Met Asn Ala Thr Asn His Ala lie Val Gin Thr Leu Val His Phe 370 375 380 lie Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 385 390 395 400

Asn Ala lie Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val lie Leu 405 410 415

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 420 425