Identification of STAT-1 as a molecular target of IGFBP-3 in the process of chondrogenesis.
The chondrogenesis process requires the ordered proliferation and differentiation of chondrocytes. Insulin-like growth factor-binding protein (IGFBP)-3, well characterized as the carrier of insulin-like growth factor (IGF), has been reported to have intrinsic bioactivity that is independent of IGF binding. The mechanisms involved in this IGF-independent action are still unclear. Using the RCJ3.1C5.18 chondrogenic cells, which in culture progresses from undifferentiated to terminally differentiated chondrocytes, we have shown previously that IGFBP-3 has an IGF-independent, antiproliferative effect in undifferentiated and early differentiated but not in terminally differentiated chondrocytes. In the present study, cDNA microarray analysis was used to screen for genes: 1) that were regulated by IGFBP-3 in early but not in terminally differentiated chondrocytes; 2) that were regulated specifically by IGFBP-3, but not by IGF-I; and 3) whose regulation was abolished by coincubation of IGFBP-3 with IGF-I. Signal transducer and activator of transcription (STAT)-1 was the gene that, fulfilling the screening criteria, exhibited the greatest up-regulation by IGFBP-3 (>40-fold). STAT-1 gene up-regulation was confirmed by Northern analysis of cells treated with IGFBP-3 or transfected with an IGFBP-3 expression vector. Remarkably, similar results were obtained when cells were transfected with an IGFBP-3 mutant unable to bind IGFs, definitively demonstrating the IGF-independent action of IGFBP-3. Consistent with the up-regulation of STAT-1 mRNA, IGFBP-3 also increased STAT-1 protein expression. Furthermore, both IGFBP-3 and the IGFBP-3 mutant induced STAT-1 phosphorylation and its nuclear localization. An antisense STAT-1 oligonucleotide abolished the IGF-independent cell apoptosis induced by IGFBP-3. We have demonstrated that STAT-1 is a major intracellular signaling and transcriptional target of the IGF-independent apoptotic effect of IGFBP-3 in chondrogenesis.
Spagnoli
Anna
A
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2579, USA. anna.spagnoli@mcmail.vanderbilt.edu
Torello
Monica
M
Nagalla
Srivinasa R
SR
Horton
William A
WA
Pattee
Patrick
P
Hwa
Vivian
V
Chiarelli
Francesco
F
Roberts
Charles T
CT
Rosenfeld
Ron G
RG
D017209:Apoptosis / Q000502:physiology; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D020219:Chondrogenesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators