Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m110872200
ISSN1083-351X
AutoresYousef G. Amaar, Garrett R. Thompson, Thomas A. Linkhart, Shin‐Tai Chen, David J. Baylink, Subburaman Mohan,
Tópico(s)Muscle Physiology and Disorders
ResumoRecent 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. 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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. Young T.F. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; 273: 18347-18352Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), where it may affect gene transcription. Based on these exciting findings, we have proposed the concept that IGFBP-5 interacts with transcription factors to stimulate transcription of genes that lead to increased osteoblast proliferation. The idea that IGFBPs may affect cells by IGF-independent mechanisms is not restricted to IGFBP-5. For instance, IGFBP-3 has been shown to mediate its effects on a variety of cell types in part via an IGF-independent mechanism (23.Oh Y. Muller H.L. Lamson G. Rosenfeld R.G. J. Biol. Chem. 1993; 268: 14964-14971Abstract Full Text PDF PubMed Google Scholar, 24.Rajah R. Valentinis B. Cohen P. J. Biol. 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For example, it has been shown that IGFBP-3 interacts with retinoid X receptor-α, and this interaction results in the modulation of the transcriptional activity of retinoid X receptor-α, which is essential for mediating IGFBP-3 effects on apoptosis (28.Liu B. Lee H.Y. Weinzimer S.A. Powell D.R. Clifford J.L. Kurie J.M. Cohen P. J. Biol. Chem. 2000; 275: 33607-33613Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). IGFBP-3-induced apoptosis was abolished in retinoid X receptor-α knockout cells, and IGFBP-3 and retinoid X receptor ligands enhanced apoptosis in prostate cancer cells. To understand the molecular mechanism of how IGFBP-5 stimulates bone formation by an IGF-independent pathway, it is essential to identify cellular proteins that interact with IGFBP-5. These proteins could be IGFBP-5 receptors as well as nuclear proteins that regulate transcription. We therefore utilized a yeast two-hybrid assay (29.Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar) screen to identify proteins that bind to IGFBP-5 using human IGFBP-5 as bait for screening a human osteosarcoma U2 cDNA library. We have identified clones encoding the full or partial coding sequence of the LIM-only protein FHL2 (30.Genini M. Schwalbe P. Scholl F.A. Remppis A. Mattei M.G. Schafer B.W. DNA Cell Biol. 1997; 16: 433-442Crossref PubMed Scopus (114) Google Scholar, 31.Chan K.K. Tsui S.K. Lee S.M. Luk S.C. Liew C.C. Fung K.P. Waye M.M. Lee C.Y. Gene. 1998; 210: 345-350Crossref PubMed Scopus (124) Google Scholar) and have shown that FHL2 binds IGFBP-5 but not IGFBP-3, IGFBP-4, or IGFBP-6. FHL2 monoclonal antibody was a kind gift from Dr. Muller, University of Freiberg, Germany (32.Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Crossref PubMed Scopus (289) Google Scholar). Recombinant human IGFBP-4 and -5 were expressed in Escherichia coli and purified as previously described (17.Richman S. Baylink D.J. Lang K. Dony C. Mohan S. Endocrinology. 1999; 140: 4699-4705Crossref PubMed Google Scholar, 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). IGFBP-6 was purchased from Upstate Biotechnology, Lake Placid, NY. Recombinant human IGFBP-3 was a gift from Dr. A. Sommer (Celtrix Corp., Palo Alto, CA). The yeast Matchmaker two-hybrid system 3 was purchased from CLONTECH, Palo Alto, CA. BamHI and SalI double-digestion excised the cDNA fragment encoding the entire IGFBP-5 from the plasmid pGEM-T. Then the cDNA fragment was gel-purified and cloned into the BamHI/SalI-digested pGBKT7 vector, and DNA sequencing confirmed that the cDNA encoding the IGFBP-5 (bait) was in-frame with GAL4 BD (amino acids 1–147). The bait vector was transformed into the yeast reporter strain AH109, and protein samples were prepared from selected yeast cell lysates using standard protocols or as recommended by the supplier of the Matchmaker two-hybrid system 3 kit (CLONTECH). The expression level of the bait IGFBP-5/GAL4 BD fusion protein was determined by Western blot analysis using a GAL4 BD monoclonal antibody (CLONTECH). It is crucial to test each BD:bait plasmid construct for auto-activation of reporter gene promoters before performing the two-hybrid screen. To check for auto-activation, the yeast reporter strain AH109 was transformed with the BD:IGFBP-5 plasmid using the Lithium acetate method as described in the Matchmaker two-hybrid system 3 user manual. The AH109 reporter contains three reporter genes, ADE2, HIS3, and MEL1, with GAL4-responsive upstream-activating sequences (AUS) and TATA boxes. ADE2, HIS3, and MEL1 are expressed from GAL1 and GAL2 promoters, which strongly respond to GAL4. Transformed cells were plated on synthetic complete medium (CM) minus adenine and histidine as well as on CM minus tryptophan (bait plasmid contains a tryptophan as a selection marker for growth control). α-X-Gal was added to the medium at 20 mg/ml to screen for auto-activation of MEL1. The GAL4 AD:cDNA library constructed in the yeast plasmid pACT2 from U-2Os (U2) human osteosarcoma cell (ATCC HTB-96) mRNA was purchased from CLONTECH. Because a library screen requires up to 500 μg of AD:cDNA plasmid DNA, depending on the yeast strain and the BD:bait plasmid, the AD:cDNA plasmid library in pACT2 was amplified according to the supplier's instructions. We prepared 6 mg of plasmid DNA from amplified E. coli cells using the Nucleobond Mega plasmid prep kit (CLONTECH). The plasmid contains the ampicillin gene for selection in E. coli and a leucine marker for selection in the yeast host. The reporter strain AH109 containing the BD:IGFBP-5 plasmid was transformed with the 0.5 mg of AD:cDNA plasmid library using the lithium acetate method as outlined in the THS3 manual. The transformed cells were plated onto low stringency minimal selection medium (lacking histidine, leucine, and tryptophan) and incubated for 4–21 days at 30 °C. The plates were checked for colonies after 4 days of incubation at 30 °C, and positive colonies were picked over a 3-week time period. Positive colonies were transferred to high stringency minimal selection medium plates (lacking Ade, His, Leu, and Trp and containing α-X-gal) plates and incubated at 30 °C until sufficient growth was achieved and colonies turned blue. Positive colonies were maintained on high stringency selection media that select for reporter gene activation. Colonies that activated all of the reporter genes in the AH109 stain were further analyzed. The AD:cDNA plasmid encoding the interacting protein was isolated from yeast cells using a modified Qiagen (Valencia, CA) plasmid prep method obtained from Qiagen technical support. The plasmid DNA prepared was then used to transform E. coli (HB101), and colonies containing AD:cDNA plasmid were selected with ampicillin (50 μg/ml). The AmpR colonies were picked and inoculated into 5 ml of LB-Amp medium and grown overnight at 37 °C with shaking. Plasmid DNA was isolated using a Qiagen kit. AD:cDNA plasmids isolated from the primary screen were used to transform the AH109 strain containing the BD:IGFBP-5 plasmid to test for activation of reporter genes. Transformed cells were plated on high stringency selection medium containing α-X-gal. AD:cDNA clones that confirmed positive were further characterized by DNA sequencing with an automated Applied Biosystems 373A genetic analysis system. Clones that failed to grow in the reconfirmation screen were not pursued for any further analysis. Normal human osteoblasts were isolated as described (33.Linkhart T.A. Linkhart S.G. MacCharles D.C. Long D.L. Strong D.D. J. Bone Miner. Res. 1991; 6: 1285-1294Crossref PubMed Scopus (100) Google Scholar) from calvaria and rib bone specimens obtained from the Cooperative Human Tissue Network, which is supported by the National Cancer Institute, National Institutes of Health. For the present study, cells isolated from calvaria and rib were grown from frozen stocks made at the second passage and were used at passage 3–4. These cells maintain an osteoblastic phenotype for more than six passages (34.Chevalley T. Strong D.D. Mohan S. Baylink D. Linkhart T.A. Eur. J. Endocrinol. 1996; 134: 591-601Crossref PubMed Scopus (58) Google Scholar). U-2 Os (HTB-96) and MG63 (CRL-1427) human osteogenic sarcoma cells were from the American Type Culture Collection (Manassas, VA). SaOs human osteosarcoma cells are a low alkaline phosphatase subline developed by Farley et al. (35.Farley J.R. Hall S.L. Herring S. Tarbaux N.M. Matsuyama T. Wergedal J.E. Metabolism. 1991; 40: 664-671Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Cells were grown at 37 °C in humidified incubator with 5% CO2. Growth medium consisted of Dulbecco's modified Eagle's medium (Invitrogen), 10% iron-supplemented newborn calf serum (Hyclone, Logan, UT), and 1% antibiotics (Cellgro). Total RNA from untransformed normal human osteoblasts derived from calvaria and rib and human osteosarcoma cell lines (SaOs-2 and U2) was isolated using the Trizol reagent (Invitrogen). 20 μg of total RNA was loaded on a 1.2% agarose gel, and the gel was blotted using standard techniques (36.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) after electrophoresis. FHL2 full-length cDNA was randomly32P-labeled using a commercial kit (New England BioLabs) for Northern hybridization. A glyceraldehyde-3-phosphate dehydrogenase cDNA was used as a control probe. Probes were hybridized at 42 °C in the presence of 50% formamide using standard protocols. Nuclear and cytoplasmic extracts from normal human calvaria and rib osteoblasts and from U2 cells were prepared as described (37.Dailly Y.P. Zhou Y. Linkhart T.A. Baylink D.J. Strong D.D. Biochim. Biophys. Acta. 2001; 1518: 145-151Crossref PubMed Scopus (18) Google Scholar). The nuclear extracts were then used for Western blot analysis using the FHL2 monoclonal antibody kindly provided by Dr. Muller, University of Freiburg, Freiburg, Germany. MG63 cells were plated at 2000 cells/well in 96-well plates in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. The next day the medium was replaced with serum-free medium, and cells were incubated for an additional 24 h before 10 nm human recombinant IGFBP-5 was added. After 48 h of incubation in the presence or absence of recombinant IGFBP-5, cells were fixed, rendered permeable with ethanol, and rinsed with PBS. IGFBP-5 protein localization was detected using IGFBP-5 guinea pig polyclonal antibody and fluorescent anti guinea pig secondary antibody. After staining, cells were rinsed three times with PBS, stored in 50% glycerol, and visualized using an Olympus IX70 epifluorescence microscope. A full-length cDNA clone corresponding to the FHL2 was cloned into the expression vector pQE32 (Qiagen) in-frame with the 6-His tag. The FHL2 coding sequence was amplified by PCR from the selected AD:cDNA plasmid template. We used a primer pair to create BamHI and SalI restriction enzyme sites at 5′ and 3′ ends, respectively. The forward primer was 5′-CGCGGATCC TGACTGAGCGCTT-3′ (the bold sequence corresponds to BamHI site, and the underlined sequence correspond to the N terminus of FHL2), and the reverse primer was 5′-ACGCGTCGAC AAGTGAACTTGCGGGGTTTTCAGTATCTACG-3′ (the bold sequence corresponds to the SalI restriction site, and the underlined sequence corresponds to the pACT2 vector 3′ sequence). The PCR product was subsequently purified using QIAXII (Qiagen), digested with BamHI and SalI, and ligated to the vector pQE32. A selected clone was confirmed by DNA sequencing to be in-frame with the 6-His tag. The pQE32 expression vector was transformed into E. coli host M15, and expression was induced with isopropyl-β-d-thiogalactopyranoside. The rFHL2 was purified according to instructions in the Qiagen Expressionist handbook. To overexpress the FHL2 and IGFBP-5 cDNAs in bone cells, we constructed a retroviral expression vector. We cloned the FHL2 and IGFBP-5 cDNA coding sequences of 850 and 750 bp, respectively, in place of the β-galactosidase gene in a murine leukemia virus-based retroviral vector plasmid, pCLSAβ-gal (38.Peng H. Chen S-T. Wergedal J.E. Polo J.M. Yee J-K. Lau W.K.-H. Baylink D.J. Mol. Ther. 2001; 4: 95-104Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The FHL2 850-bp cDNA fragment was generated by PCR using a pair of oligonucleotides; the forward primer was 5′-ACGCGTCGACATGACTGAGCGCTTT-3′, and the reverse primer was 5′-GCGCGGATCCAATTCAGATGTCTTTCCCAC-3′; the IGFBP-5 cDNA fragment was generated by PCR using the forward primer 5′-ACGCGTCGACATGGGCTCCTTCGTGCAC-3′ and the reverse primer 5′-CGCGGATCCATCACTCAACGTTGCTGCTG-3′ (restriction sites noted in bold). The PCR product was digested SalI/BamHI, purified, and ligated to SalI/BamHI-digested retroviral vector plasmid, pCLSAβ-gal. The transcription of the FHL2 and IGFBP-5 cDNAs in pCLSA-FHL2 and pCLSA-IGFBP-5 was under the control of the murine leukemia virus long terminal repeat promoter. To produce retroviral expression vector, 293T cells were co-transfected with the pCLSA-FHL2 or pCLSA-IGFBP-5 plasmid and a viral envelope expression plasmid pCMV-G using the calcium phosphate method (39.Gasmi M. Glynn J. Jin M.J. Jolly D.J. Yee J.K. Chen S.T. J. Virol. 1999; 73: 1828-1834Crossref PubMed Google Scholar). The viral vectors were harvested 36 h post-transfection, and the titer of the viral vector was determined by end point dilution and by transducing HT1080 cells as described (38.Peng H. Chen S-T. Wergedal J.E. Polo J.M. Yee J-K. Lau W.K.-H. Baylink D.J. Mol. Ther. 2001; 4: 95-104Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). U2 cells were seeded at 1 × 105 cells/well in 6-well plates. After 24 h of incubation, the cells were transduced with 100 μl each of the viral stocks of pCLSA-FHL2 plus pCLSAβ-gal, pCLSA-IGFBP-5 plus pCLSAβ-gal, or pCLSA-FHL2 plus pCLSA-IGFBP-5 vectors as described (38.Peng H. Chen S-T. Wergedal J.E. Polo J.M. Yee J-K. Lau W.K.-H. Baylink D.J. Mol. Ther. 2001; 4: 95-104Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Multiplicity of infection was 10 based on viral vector titer of ∼1 × 107 transforming units/ml. Twenty-four hours after transduction, the cells were rinsed and passaged twice for expansion. The transduction efficiency was determined by staining the cells for β-galactosidase expression was estimated to be ∼90% after two passages. U2 cells transduced with pCLSA-FHL2 were used to prepare cytoplasmic and nuclear extracts for Western blot analysis using the FHL2 monoclonal antibody. U2 cells transduced with pCLSA-FHL2 and pCLSA-IGFBP-5 were used to prepare whole cell lysates. U2 cells transduced with pCLSA-FHL2 and pCLSA-IGFBP-5 were used to prepare whole cell lysates using a procedure recommended by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 250 μl of cell lysates were incubated in presence of 25 μl of protein A-Sepharose conjugated to FHL2 monoclonal antibody or to only 25 μl of protein A-Sepharose at 4 °C for 14 h on a rotary shaker. After incubation, samples were centrifuged for 1 min at 14,000 rpm. The protein A-Sepharose pellets were washed four times with the lysis buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 1.0% Triton X-100) and resuspended in 50 μl of SDS sample buffer. The immune complex was dissociated from protein A by boiling, and 20 μl was removed and analyzed by SDS-PAGE in 12% acrylamide gel. The IGFBP-5 in the immune precipitate was detected by IGFBP-5 antibody in Western blot analysis according to standard protocols (42.Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 269-309Google Scholar). IGFBP-5, IGFBP-4, and IGFBP-6 were 125I-labeled as described previously (40.Mohan S. Jennings J.C. Linkhart T.A. Baylink D.J. Biochim. Biophys. Acta. 1988; 966: 44-55Crossref PubMed Scopus (182) Google Scholar). The ability of FHL2 to bind to IGFBP-5 was analyzed by immunoprecipitation. The FHL2 protein (100 ng/ml) was incubated with FHL2 monoclonal antibody (1:500 dilution) and 125I-IGFBP-5 (100,000 cpm/ml) for 14 h at 4 °C in 250 μl of incubation buffer (30 mm sodium phosphate, pH 7.4, 10 mmEDTA, 0.1% bovine serum albumin, and 0.5% Tween 80) (41.Nam T.J. Busby Jr., W.H. Rees C. Clemmons D.R. Endocrinology. 2000; 141: 1100-1106Crossref PubMed Scopus (94) Google Scholar). For competitive binding experiments, 1 μg/ml unlabeled IGFBP-5 was included in addition to 125I-IGFBP-5. After incubation, 10 μl of protein A-Sepharose (Upstate Biotechnology) was added, and the samples were incubated at room temperature for 1 h with mixing every 10 min, then were centrifuged for 10 min at 12,000 ×g. The pellets were washed three times with incubation buffer and re-suspended in 50 μl of SDS sample buffer. The immune complex was dissociated from protein A by boiling, and 20 μl were removed and analyzed by SDS-PAGE in 12% acrylamide gel. The125I-IGFBP-5 precipitated by FHL2 anti-body was detected by autoradiography. The same procedure was used to test binding of FHL2 to125I-IGFBP-4 and -6. IGFBP-5/FHL2 interaction using unlabeled proteins was also performed with IGFBP-5 polyclonal antibodies to immune precipitate and the 6-His-tag monoclonal antibody to detect FHL2 using the same procedure as mentioned above. FHL2 in the immune precipitate was detected by Western blot according to standard protocols (42.Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 269-309Google Scholar). PS1 ProteinChip arrays were used for the analysis of FHL2 interactions with IGFBPs. The spots in PS1 ProteinChip were pre-activated with iminodiacetate chemistry that covalently binds to the free primary amine groups (Ciphergen Biosystems, Inc., Fremont, CA). Briefly, 200 ng of FHL2 (50 μl) was added to each spot, incubated for 2 h at room temperature on an orbital shaker, and blocked with 0.1 m Tris, pH 8.0, for 30 min to remove nonspecific binding. After rinsing the spots with PBS, 50 μl of PBS or PBS containing 200 ng of IGFBP was added to each spot and incubated for 1 h at room temperature on an orbital shaker. The spots were are then rinsed with 50 mm Tris, pH 8.0, containing 150 mm NaCl and 1% Triton X-100 before the addition of energy absorbing molecule (α-cyano-4-hydroxy cinnamic acid) according to the manufacturer's instructions. The spots were air-dried and analyzed using the SELDI ProteinChip system (PBS-1, Ciphergen) as described previously (43.Li X. Mohan S. Gu W. Miyakoshi N. Baylink D.J. Biochim. Biophys. Acta. 2000; 1524: 102-109Crossref PubMed Scopus (32) Google Scholar). Data were collected using laser intensity of 80% and mass calibrated with protein standards (43.Li X. Mohan S. Gu W. Miyakoshi N. Baylink D.J. Biochim. Biophys. Acta. 2000; 1524: 102-109Crossref PubMed Scopus (32) Google Scholar). We used the THS3 to identify candidate proteins that interact with our protein of interest, IGFBP-5. The IGFBP-5 cDNA sequence was fused to the DNA binding domain sequence of GAL4 in the expression vector pGBDT7. The yeast reporter strain AH109 was transformed with this plasmid, and cells were plated on the appropriate selection medium. Expression of the GAL4-IGFBP-5 fusion protein in AH109 cells was subsequently confirmed by Western blot analysis using an antibody (CLONTECH) to the GAL4 DNA binding domain of the fusion protein (Fig. 1). AH109 cells containing the bait but not control AH109 cells expressed BD
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