Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)

Abstract

Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies. Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies. IGFs 1The abbreviations used are: IGFinsulin-like growth factorIGFBP-5IGF-binding protein 5α-X-gal5-bromo-4-chloro-3-indolyl β-d-galactopyranosideADactivation domainBDbinding domainPBSphosphate-buffered salinerFHL2recombinant FHL2bpbase pair(s)SELDIsurface-enhanced laser desorption ionizationkbkilobase are growth-promoting peptides that play an important role in growth, remodeling, and repair of skeletal tissues as well as in modulating development, growth, and cell survival in many other tissues (1.Rosen C.J. Donahue L.R. Proc. Soc. Exp. Biol. Med. 1998; 219: 1-7Crossref PubMed Scopus (112) Google Scholar, 2.Collett-Solberg P.F. Cohen P. Endocrine. 2000; 12: 121-136Crossref PubMed Google Scholar, 3.Butt A.J. Firth S.M. Baxter R.C. Immunol. Cell Biol. 1999; 77: 256-262Crossref PubMed Scopus (163) Google Scholar, 4.Bayes-Genis A. Conover C.A. Schwartz R.S. Circ. Res. 2000; 86: 125-130Crossref PubMed Scopus (385) Google Scholar). IGFs are the most abundant growth factors stored in bone, and they are the most abundant growth factors produced by osteoblast cells in culture (5.Mohan S. Baylink D.J. Horm. Res. (Basel). 1996; 45: 59-62Crossref PubMed Scopus (76) Google Scholar). IGF-I serum levels correlate with serum levels of bone formation markers (osteocalcin and alkaline phosphatase) as bone formation increases during puberty and after growth hormone therapy or decreases with growth hormone deficiency, menopause, and aging (1.Rosen C.J. Donahue L.R. Proc. Soc. Exp. Biol. Med. 1998; 219: 1-7Crossref PubMed Scopus (112) Google Scholar,6.Rajaram S. Baylink D.J. Mohan S. Endocr. Rev. 1997; 18: 801-831Crossref PubMed Scopus (971) Google Scholar, 7.Mohan S. Baylink D.J. Endocrine. 1997; 7: 87-91Crossref PubMed Google Scholar, 8.Boonen S. Mohan S. Dequeker J. Aerssens J. Vanderschueren D. Verbeke G. Broos P. Bouillon R. Baylink D.J. J. Bone Miner. Res. 1999; 14: 2150-2158Crossref PubMed Scopus (103) Google Scholar). Bone formation is severely compromised in mice lacking a functional IGF-I gene (9.Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2597) Google Scholar). insulin-like growth factor IGF-binding protein 5 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside activation domain binding domain phosphate-buffered saline recombinant FHL2 base pair(s) surface-enhanced laser desorption ionization kilobase IGFs stimulate bone formation by regulating proliferation, differentiation, and apoptosis of osteoblasts (1.Rosen C.J. Donahue L.R. Proc. Soc. Exp. Biol. Med. 1998; 219: 1-7Crossref PubMed Scopus (112) Google Scholar, 10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar). Actions of IGFs on osteoblasts depend not only on the amounts of IGFs but also on the other components of the IGF system including type-I and -II IGF receptors, IGF binding proteins (IGFBPs), and IGFBP proteases (10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar, 11.Mohan S. Baylink D.J. J. Clin. Endocrinol. Metab. 1996; 81: 3817-3820Crossref PubMed Scopus (75) Google Scholar). IGFBPs either stimulate (e.g. IGFBP-3 and -5) or inhibit (e.g. IGFBP-4 and -6) IGF effects on target cells, and hence, they are an important regulatory part of the IGF system in bone (1.Rosen C.J. Donahue L.R. Proc. Soc. Exp. Biol. Med. 1998; 219: 1-7Crossref PubMed Scopus (112) Google Scholar, 10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar, 12.Ferry Jr., R.J. Katz L.E. Grimberg A. Cohen P. Weinzimer S.A. Horm. Metabol. Res. 1999; 31: 192-202Crossref PubMed Scopus (139) Google Scholar, 13.Clemmons D.R. Mol. Cell. Endocrinol. 1998; 140: 19-24Crossref PubMed Scopus (337) Google Scholar). Of the various IGF system components, IGFBP-5 has several features that suggest it is a key component of the IGF system. IGFBP-5 is the most abundant IGFBP stored in bone, because it is also the only IGFBP that binds avidly to hydroxyapatite (10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar). Decreased skeletal content of IGFBP-5 has been shown to correlate with decreased skeletal content of IGF-I that may contribute to the impairment in coupling of bone formation to resorption (14.Nicolas V. Mohan S. Honda Y. Prewett A. Finkelman R.D. Baylink D.J. Farley J.R. Calcif. Tissue Int. 1995; 57: 206-212Crossref PubMed Scopus (58) Google Scholar). Among the IGFBPs known, IGFBP-5 has been shown to stimulate both osteoblast cell proliferation and activity in vitro (15.Andress D.L. Loop S.M. Zapf J. Kiefer M.C. Biochem. Biophys. Res. Commun. 1993; 195: 25-30Crossref PubMed Scopus (117) Google Scholar, 16.Kling L. Dony C. Leser U. Popp F. Bauss F. Lang K. J. Bone Miner. Res. 1996; 11 (Abstr. P249): 153Google Scholar, 17.Richman S. Baylink D.J. Lang K. Dony C. Mohan S. Endocrinology. 1999; 140: 4699-4705Crossref PubMed Google Scholar). Recent findings demonstrate that IGFBP-5 itself is a growth factor with cellular effects that are not dependent on IGFs (18.Miyakoshi N. Richman C. Kasukawa Y. Linkhart T.A. Baylink D.J. Mohan S. J. Clin. Invest. 2001; 107: 73-81Crossref PubMed Scopus (188) Google Scholar). In this regard, IGFBP-5 treatment increased bone formation parameters in vitro and in vivo in osteoblasts derived from IGF-I knockout mice. IGFBP-5 binds to a putative receptor on the osteoblast cell surface, which may induce downstream signaling pathways (10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar, 19.Mohan S. Nakao Y. Honda Y. Landale E. Leser U. Dony C. Lang K. Baylink D.J. J. Biol. Chem. 1995; 270: 20424-20431Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar,20.Andress D.L. Am. J. Physiol. 1998; 274: E744-E750PubMed Google Scholar). IGFBP-5 also contains a nuclear localization sequence that mediates transport of IGFBP-5 to the cell nucleus (21.Schedlich L.J. Le Page S.L. Firth S.M. Briggs L.J. Jans D.A. Baxter R.C. J. Biol. Chem. 2000; 275: 23462-23470Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 22.Schedlich L.J. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google it may affect gene on we the that IGFBP-5 interacts with transcription factors to stimulate transcription of genes that to increased osteoblast The that IGFBPs may affect cells by is not to IGFBP-3 has been shown to effects on a of cell in part an Y. G. Rosenfeld R.G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, R. Cohen P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Firth S.M. A.J. Baxter R.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. V. Roberts Jr., C.T. F. Rosenfeld R.G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins been using the yeast two-hybrid F. L.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Weinzimer S.A. D.R. Cohen P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). it has been shown that IGFBP-3 interacts with and this interaction in the of the activity of which is IGFBP-3 effects on apoptosis Weinzimer S.A. D.R. Cohen P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). apoptosis was in knockout cells, and IGFBP-3 and receptor apoptosis in cells. the of IGFBP-5 bone formation by an it is to identify cellular proteins that with proteins be IGFBP-5 as well as nuclear proteins that We therefore a yeast two-hybrid S. PubMed Scopus Google screen to identify proteins that bind to IGFBP-5 using human IGFBP-5 as bait a human osteosarcoma U2 cDNA library. We clones the or sequence of the protein FHL2 P. A. Cell Biol. 1997; PubMed Scopus Google Scholar, S.M. 1998; PubMed Scopus Google and shown that FHL2 binds IGFBP-5 but not IGFBP-3, or FHL2 was a from of U. E. A. A. C. R. R. J. 2000; PubMed Scopus Google Scholar). human IGFBP-4 and expressed in and purified as S. Baylink D.J. Lang K. Dony C. Mohan S. Endocrinology. 1999; 140: 4699-4705Crossref PubMed Google Scholar, N. Richman C. Kasukawa Y. Linkhart T.A. Baylink D.J. Mohan S. J. Clin. Invest. 2001; 107: 73-81Crossref PubMed Scopus (188) Google Scholar). was from human IGFBP-3 was a from A. The yeast two-hybrid system was from and the cDNA the IGFBP-5 from the the cDNA was and into the and confirmed that the cDNA the IGFBP-5 was with The bait was into the yeast and protein from yeast cell lysates using or as by the of the two-hybrid system The of the bait protein was by Western blot analysis using a is to of gene the two-hybrid the yeast was with the using the as in the two-hybrid system The contains and with and and are expressed from and which strongly to cells on and as well as on contains a as a growth was to the to screen of The in the yeast from human osteosarcoma cell was from Because a screen to of on the yeast and the the in was to the We of from E. cells using the The contains the gene in E. and a in the yeast The the was with the of using the as in the The cells and and The after of and a to and and and growth was and on that gene that of the genes in the The the protein was from yeast cells using a from The was used to E. and with The and into 5 of and with was using a from the screen used to the the to activation of cells on clones that confirmed by with an analysis system. that to in the screen not human osteoblasts as T.A. Linkhart D.L. J. Bone Miner. Res. PubMed Scopus Google from and bone from the Tissue which is by the of the cells from and from the and used cells an Mohan S. Baylink D. Linkhart T.A. J. Endocrinol. 1996; PubMed Scopus (58) Google Scholar). and human cells from the human osteosarcoma cells are a alkaline by Farley J.R. S.L. S. Full Text PDF PubMed Scopus Google Scholar). in with of serum and from human osteoblasts derived from and and human osteosarcoma cell and was using the of was on a and the was using J. Molecular after FHL2 cDNA was using a Northern cDNA was used as a in the of using Nuclear and from human and osteoblasts and from U2 cells as Y. Linkhart T.A. Baylink D.J. Biophys. 2001; PubMed Scopus Google Scholar). The nuclear used Western blot analysis using the FHL2 by of cells in in with The the was with and cells an human recombinant IGFBP-5 was of in the or of recombinant cells with and with IGFBP-5 protein localization was using IGFBP-5 and cells with stored in and using an cDNA to the FHL2 was into the with the The FHL2 sequence was by from the We used a to and and The was sequence to and the sequence to the of and the was sequence to the and the sequence to the The was purified using with and and to the was confirmed by to be with the The was into E. and was with The was purified to in the the FHL2 and IGFBP-5 in bone cells, we a We the FHL2 and IGFBP-5 cDNA of and in of the gene in a Baylink D.J. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The FHL2 cDNA was by using a of the was and the was the IGFBP-5 cDNA was by using the and the in The was and to The transcription of the FHL2 and IGFBP-5 in and was the of the cells with the or and a using the J. D.J. J. 1999; PubMed Google Scholar). The and the of the was by and by cells as Baylink D.J. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). U2 cells in of the cells with of the of or as Baylink D.J. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of was on of after the cells and The was by the cells was to be after U2 cells with used to and nuclear Western blot analysis using the FHL2 U2 cells with and used to whole cell U2 cells with and used to whole cell lysates using a by of cell lysates in of of protein to FHL2 or to only of protein on a The protein four with the and in of The was from protein by and was and by in The IGFBP-5 in the was by IGFBP-5 in Western blot analysis to E. D. Scholar). and as S. Linkhart T.A. Baylink D.J. Biophys. PubMed Scopus Google Scholar). The of FHL2 to bind to IGFBP-5 was by The FHL2 protein was with FHL2 and in of serum and Jr., C. D.R. Endocrinology. 2000; PubMed Scopus Google Scholar). binding IGFBP-5 was in to of protein was and the with The with and in of The was from protein by and and by in by FHL2 was by The was used to binding of FHL2 and -6. interaction using proteins was also with IGFBP-5 to and the to FHL2 using the as FHL2 in the was by Western blot to E. D. Scholar). used the analysis of FHL2 with The in with that binds to the Inc., of FHL2 was to on an and with to the with of or of IGFBP was to and on an The are with and the of to the The and using the system as Mohan S. N. Baylink D.J. Biophys. 2000; PubMed Scopus Google Scholar). using laser of and with protein Mohan S. N. Baylink D.J. Biophys. 2000; PubMed Scopus Google Scholar). We used the to identify proteins that with protein of The IGFBP-5 cDNA sequence was to the binding domain sequence of in the The yeast was with this and cells on the of the protein in cells was confirmed by Western blot analysis using an to the binding domain of the protein cells the bait but not cells expressed protein of the the of bait in yeast cells was we by the the IGFBP-5 on lacking the and In the as a the of the gene that The that the IGFBP-5 not the genes on the We the with an U2 human osteosarcoma cDNA to the in the We several clones from screen of the human osteosarcoma cDNA which to a clones as The that of the genes in the and used to the E. We the clones and the cDNA using the Five clones a gene sequence in the of we a and a cDNA the sequence of we it a protein of The sequence as a protein that contains four and a half LIM S.M. 1998; PubMed Scopus Google Scholar, U. E. A. A. C. R. R. J. 2000; PubMed Scopus Google Scholar). The cDNA of the FHL2 protein and strongly interacted with IGFBP-5 as by the two-hybrid clones using the yeast two-hybrid system to screen the human osteosarcoma cDNA clones of cDNA sequence to human FHL2 of the yeast with with the and of and human human and is a that a of and that is to bind that the on that the not on in a The clones of cDNA sequence to human FHL2 of the yeast with with the and of and human human and is a that a of and that is to bind that the on that the not on the FHL2 clones by we yeast cells with the and to the I the screen and The that both the and the cDNA clones of FHL2 strongly with IGFBP-5 as by growth of the on growth was either the or the with and to the the interaction was FHL2 is expressed in other bone cell U2 human osteosarcoma cells, from and human osteosarcoma cells was with the FHL2 cDNA Northern analysis that the FHL2 is expressed in of the human osteoblast cell The FHL2 cDNA a the of which is to that in other cell Western blot analysis of nuclear and from and cells using the FHL2 The FHL2 protein was in the nuclear and of both human osteoblasts and U2 osteosarcoma cells and In to the that to in the nuclear may of FHL2 or of FHL2 with or other proteins that with the FHL2 used in this was was used Western not of IGFBP-5 in human osteoblast was in osteosarcoma cells that IGFBP-5 protein to the cells to to the nucleus of cells as by IGFBP-5 and was of nuclear localization in the of recombinant IGFBP-5 in cells, which not levels of IGFBP-5 (10.Mohan S. Baylink D.J. Rosenfeld R.G. Roberts C.T. The IGF System, Molecular Biology, Physiology, and Clinical Applications. Humana Press Inc., Totowa, NJ1999: 457-496Crossref Google Scholar). The of IGFBP-5 in of IGFBP-5 in human osteoblasts derived from which IGFBP-5 Mohan S. N. Baylink D.J. Biophys. 2000; PubMed Scopus Google IGFBP-5 in the nucleus in the of The of IGFBP-5 increased of IGFBP-5 in the nucleus are not FHL2 sequence was with the in the The protein was expressed in the E. and was purified using of FHL2 was confirmed by Western blot analysis using a and the FHL2 We studies by the FHL2 protein with IGFBP-5 in the of FHL2 in that FHL2 interacts with IGFBP-5 in an in vitro to FHL2 was with FHL2 binding IGFBP-5 was and proteins in the FHL2 not with IGFBP-4 or that FHL2 binding to IGFBP-5 is We in vitro the interaction by in the of protein IGFBP-5 The was by Western using IGFBP-5 and the and The that FHL2 was by IGFBP-5 but not by the interaction between FHL2 and IGFBP-5 occurs in whole cells, cell from U2 cells overexpressing FHL2 and IGFBP-5 was with FHL2 and with by immunoblot that was by FHL2 in whole U2 cell with the yeast two-hybrid that the interaction between FHL2 and IGFBP-5 occurs in whole cells after and of both FHL2 and The of interaction was verified using an Mohan S. N. Baylink D.J. Biophys. 2000; PubMed Scopus Google Scholar). The system proteins on and with FHL2 was on the of the and used to the IGFBP-5 be on the The IGFBP by laser was and the was by a of that IGFBP-5 was by FHL2 to the of the other IGFBPs -4, or -6) was by FHL2 to with the that the binding of FHL2 to IGFBP-5 is the yeast two-hybrid system and IGFBP-5 as we several FHL2 from a human osteosarcoma cDNA library. interaction was by in vitro and by localization of both FHL2 and IGFBP-5 in the the functional of the between IGFBP-5 and FHL2 has not been the findings that IGFBP-5 itself is a growth factor that to nucleus (21.Schedlich L.J. Le Page S.L. Firth S.M. Briggs L.J. Jans D.A. Baxter R.C. J. Biol. Chem. 2000; 275: 23462-23470Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 22.Schedlich L.J. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and that FHL2 as a of the receptor U. E. A. A. C. R. R. J. 2000; PubMed Scopus Google the that interaction may play a role in the of osteoblast cell proliferation this is the to of an IGFBP-5 binding in cell using the yeast two-hybrid system. We to screen proteins in osteoblasts on the of IGFBP-5 in bone (15.Andress D.L. Loop S.M. Zapf J. Kiefer M.C. Biochem. Biophys. Res. Commun. 1993; 195: 25-30Crossref PubMed Scopus (117) Google Scholar, 16.Kling L. Dony C. Leser U. Popp F. Bauss F. Lang K. J. Bone Miner. Res. 1996; 11 (Abstr. P249): 153Google Scholar, 17.Richman S. Baylink D.J. Lang K. Dony C. Mohan S. Endocrinology. 1999; 140: 4699-4705Crossref PubMed Google Scholar, N. Richman C. Kasukawa Y. Linkhart T.A. Baylink D.J. Mohan S. J. Clin. Invest. 2001; 107: 73-81Crossref PubMed Scopus (188) Google and on the that IGFBP-5 has effects on cells and to the nucleus (18.Miyakoshi N. Richman C. Kasukawa Y. Linkhart T.A. Baylink D.J. Mohan S. J. Clin. Invest. 2001; 107: 73-81Crossref PubMed Scopus (188) Google Scholar, D.L. Am. J. Physiol. 1998; 274: E744-E750PubMed Google Scholar, L.J. Le Page S.L. Firth S.M. Briggs L.J. Jans D.A. Baxter R.C. J. Biol. Chem. 2000; 275: 23462-23470Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 22.Schedlich L.J. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We that IGFBP-5 interacts with other cellular nuclear with this clones corresponded to a four and a half LIM domain gene which is also as P. A. Cell Biol. 1997; PubMed Scopus Google Scholar, S.M. 1998; PubMed Scopus Google Scholar). the of of the other four The that the in the screen only LIM and that the of FHL2 the LIM is FHL2 interaction with We confirmed the interaction between FHL2 and IGFBP-5 in yeast with in vitro studies using purified recombinant human purified recombinant human and FHL2 or was also in whole cell from U2 cells overexpressing FHL2 and IGFBP-5 binding to FHL2 was verified using a in which IGFBP-5 was using FHL2 to We that the interaction between IGFBP-5 and FHL2 is on the FHL2 interacted with IGFBP-5 but not with either IGFBP-3, -4, or which are to be produced by human osteoblasts R. Baylink D.J. Linkhart T.A. Mohan S. J. Bone Miner. Res. 1997; 12: PubMed Scopus Google Scholar). The of FHL2 binding to IGFBP-3, which is to IGFBP-5 in the and that of IGFBP-5 may be FHL2 IGFBP-3 may bind to FHL2 but with a with IGFBP-5 and that this binding not be by the used in this osteoblasts in culture been shown to LIM Y. D. L. Endocrinology. 1998; PubMed Scopus Google we not identify of in yeast two-hybrid In this in lysates of U2 cells overexpressing both FHL2 and that the interaction between FHL2 and IGFBP-5 in whole cells that are to both of they not that proteins bind interaction studies using human osteoblasts of either FHL2 or IGFBP-5 are to that the interaction between FHL2 and IGFBP-5 occurs that the FHL2 interaction with IGFBP-5 in other osteoblast cell U2 human osteosarcoma cells, we FHL2 in human osteoblasts as well as in other human osteosarcoma cell Northern analysis showed that the FHL2 is strongly expressed in human osteoblasts derived from and and in U2 cells. Of the various human osteoblast cell FHL2 was in human osteosarcoma cells with other human osteoblast cell on the findings that cells functional and that FHL2 is in cell functional and in cell P. E. C. J. Cell Biol. 2000; PubMed Scopus Google it is that is an important of FHL2 In that FHL2 is strongly expressed in human osteoblasts the that FHL2 is to the system Y. D. L. Endocrinology. 1998; PubMed Scopus Google Scholar, P. E. C. J. Cell Biol. 2000; PubMed Scopus Google Scholar, S. S.M. J. R. Cell 2001; PubMed Scopus Google Scholar, P. C. J. 2000; PubMed Scopus Google Scholar). FHL2 has been shown to with other In this regard, it is that a of the LIM in by S.M. J. Cell. Biochem. 2001; PubMed Scopus Google or R. 1998; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). using the yeast two-hybrid FHL2 has also been shown to with S.M. S.M. J. Cell. Biochem. 2000; PubMed Scopus Google and V. D. E. D. N. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google the factors Exp. Cell Res. 2001; PubMed Scopus Google and Mol. 2000; PubMed Scopus Google Scholar). Because FHL2 contains four and a half LIM of which contains it is that LIM may be involved in FHL2 interaction with the various binding V. D. E. D. N. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, Exp. Cell Res. 2001; PubMed Scopus Google Scholar). In this regard, on the of a FHL2 cDNA which in the yeast two-hybrid that the LIM may be interaction with The of a interaction between FHL2 and IGFBP-5 in osteoblasts only be this In this regard, it has been shown by U. E. A. A. C. R. R. J. 2000; PubMed Scopus Google that FHL2 binds and the activity of in an and Y. D. L. Endocrinology. 1998; PubMed Scopus Google that LIM protein is an intracellular of osteoblast differentiation, which to effects on bone formation in the that FHL2 as an intracellular mediator of IGFBP-5 that the interaction between IGFBP-5 and FHL2 occurs and that proteins are in the nucleus of human osteoblasts the the that IGFBP-5 may bind to a transcription to stimulate transcription of putative IGFBP-5 target genes that may be involved in of osteoblast cell proliferation and We the by and We Farley cells.