Polypeptide cartilage-inducing factors found in bone

Abstract

Two proteins that are found in bone and that have in vivo chondrogenic/osteogenic activity in combination with a co-factor are described. Both proteins also were active in combination with EGF in the in vitro TGF-β assay. Each has a molecular weight of approximately 26,000 daltons by SDS-PAGE. Each is reduced to a single polypeptide indicating that the proteins are probably homodimers. One has an N-terminal sequence identical to that of human placenta-derived TGF-β whereas the other has an N-terminal sequence that is different from that of TGF-β derived from human placenta. The two proteins may be purified to homogeneity using RP-HPLC or acetic acid-urea gel electrophoresis.

We claim: . .1. A method of promoting proliferation of cells in an animal comprising 
     
       Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp-..!.. .5.  A method for treating osteoporosis or osteopetrosis, which comprises: systemically administering an effective amount of a composition comprising a TGF-beta activating agent, a pharmaceutically acceptable excipient, and a polypeptide that (a) is found in mammalian bone, (b) is active in the TGF-beta assay, (c) is a co-factor for inducing cartilage formation in vivo, (d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE, and (e) is substantially pure, wherein said polypeptide and activating agent are present in amounts sufficient to promote bone growth..!.. .6. The method of claim 5 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp-..!.. .7. The method of claim 3 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp..!.. .8. The method of claim 5 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pr   
     
     
       o-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp..!..Iadd.9.  A method for inducing cartilage and bone formation for repairing, replacing, and/or augmenting cartilage and bone tissue in animals, which method comprises administering to an animal in need of bone repair, replacement, or augmentation a composition comprising a TGF-beta activating agent, a pharmaceutically acceptable excipient, and a polypeptide that (a) is found in mammalian bone, (b) is active in the TGF-beta assay, (c) is a co-factor for inducing cartilage formation in vivo, (d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE, and (e) is substantially pure, wherein said polypeptide and activating agent are present in amounts sufficient to promote bone growth..Iaddend..Iadd.10. The method of claim 9 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp (TGF-β1)..Iaddend..Iadd.11. The method of claim 9 wherein each chain of said dimer has the following N-terminal sequence:   Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp   
     
     
        (TGFβ-2)..Iaddend..Iadd.  .  A method for treating osteoporosis or osteopetrosis, which comprises systemically administering to an individual in need of such treatment an effective amount of a composition comprising a TGF-beta activating agent, a pharmaceutically acceptable excipient, and a polypeptide that (a) is found in mammalian bone, (b) is active in the TGF-beta assay, (c) is a co-factor for inducing cartilage formation in vivo, (d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE, and (e) is substantially pure, wherein said polypeptide and activating agent are present in amounts sufficient to promote bone growth..Iaddend..Iadd.13. The method of claim 12 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp (TGFβ-1)..Iaddend..Iadd.14. The method of claim 12 wherein each chain of said dimer has the following N-terminal sequence:   Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp (TGFβ-2)..Iaddend..Iadd.15. A method for inducing cartilage and bone formation for repairing, replacing, and/or augmenting cartilage and bone tissue in animals, which method comprises administering to an animal in need of bone repair, replacement, or augmentation a composition comprising a pharmaceutically acceptable excipient and a polypeptide that (a) is found in mammalian bone, (b) is active in the TGF-β assay, (c) is a co-factor for inducing cartilage formation in vivo, (d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE, and (e) is substantially pure, wherein said polypeptide is   
     
     
        present in an amount sufficient to promote bone growth..Iaddend..Iadd.16. The method of claim 15 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp (TGFβ-1)..Iaddend..Iadd.17. The method of claim 15 wherein each chain of said dimer has the following N-terminal sequence:   Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp (TGFβ-2)..Iaddend..Iadd.18. A method for treating osteoporosis or osteopetrosis, which comprises systemically administering to an individual in need of such treatment an effective amount of a composition comprising a pharmaceutically acceptable excipient and a polypeptide that (a) is found in mammalian bone, (b) is active in the TGF-β assay, (c) is a co-factor for inducing cartilage formation in vivo, (d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE, and (e) is substantially pure, wherein said polypeptide is present in an amount sufficient to promote bone growth..Iaddend..Iadd.19. The method of claim 18 wherein each chain of said dimer has the following N-terminal sequence:   Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp   
     
     
        (TGFβ-1)..Iaddend..Iadd.0.  The method of claim 18 wherein each chain of said dimer has the following N-terminal sequence: Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp (TGFβ-2)..Iaddend.