title: Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-(beta) in mesenchymal chondroprogenitor cell growth.
abstract: Cartilage formation is driven by mesenchymal chondroprogenitor cells (MCCs) that proliferate and differentiate into chondrocytes. The molecular mechanisms by which growth factors regulate MCC fate are not well defined. Insulin-like growth factor binding protein-3 (IGFBP-3) has intrinsic bioactivity that is independent of IGF binding. We previously reported that IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs, and requires STAT-1 activation to mediate its apoptotic effect. Transforming growth factor-beta (TGF-beta) is a key chondroinductive growth factor. The objective of the study is to define the interactions between IGFBP-3 and TGF-beta in MCC growth and their intracellular signaling pathways. We used the RCJ3*1C5*18 mesenchymal chondrogenic cells that without biochemical or oncogenic transformation progress in culture from MCCs to differentiated chondrocytes. Cell proliferation was assessed in MCCs treated with IGFBP-3 or transfected with IGFBP-3, in the presence or absence of TGF-beta. To demonstrate that IGFBP-3 effects were IGF-independent an IGFBP-3 analog that lacks IGF binding was used (GGG-IGFBP-3). To determine the functional roles of the TGF-beta-mediated signaling and the STAT-1 pathway, cells were either stably transfected with a dominant negative TGF-beta type II receptor (MCC-DNTbetaRII) or treated with a STAT-1 morpholino antisense oligonucleotide. We found that in MCCs, TGF-beta antagonized the antiproliferative effect of IGFBP-3. IGFBP-3 increased the cyclin-dependent kinase inhibitor p21 expression and this effect was abolished by TGF-beta. Furthermore, TGF-beta inhibited STAT-1 phosphorylation induced by IGFBP-3. Similarly to TGF-beta, STAT-1 antisense oligonucleotide inhibited the IGFBP-3 antiproliferative action. Although TGF-beta in MCC-DNTbetaRII lacked Smad-mediated signaling, it persistently antagonized the IGFBP-3 antiproliferative action. However, TGF-beta even in MCC-DNTbetaRII cells induced ERK1/2 phosphorylation, and treatment with MEK inhibitor, UO126, inhibited the antagonistic effects of TGF-beta on IGFBP-3. Furthermore, UO126 blocked the TGF-beta inhibition of STAT-1 phosphorylation induced by IGFBP-3. Collectively, these results demonstrate cross-talk between the IGFBP-3-dependent STAT-1 signaling and the TGF-beta-dependent ERK pathway that regulates MCC proliferation.
authors.affiliation: Vanderbilt University School of Medicine, Department of Pediatrics, T-0107 Medical Center North, Nashville, Tennessee, USA.
authors.forename: L
authors.initials: L
authors.lastname: O'Rear
authors.forename: L
authors.initials: L
authors.lastname: Longobardi
authors.forename: M
authors.initials: M
authors.lastname: Torello
authors.forename: B K
authors.initials: BK
authors.lastname: Law
authors.forename: H L
authors.initials: HL
authors.lastname: Moses
authors.forename: F
authors.initials: F
authors.lastname: Chiarelli
authors.forename: A
authors.initials: A
authors.lastname: Spagnoli
mesh_terms: D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: C493958:Cdkn1a protein, rat; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D050794:STAT1 Transcription Factor; C494081:Stat1 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta