Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation.
Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.
Song
Jason J
JJ
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Aswad
Rulla
R
Kanaan
Reem A
RA
Rico
Mario C
MC
Owen
Thomas A
TA
Barbe
Mary F
MF
Safadi
Fayez F
FF
Popoff
Steven N
SN
D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D005353:Fibronectins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006609:High Mobility Group Proteins / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D020319:Oligodeoxyribonucleotides, Antisense; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013131:Spine / Q000196:embryology / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism* / Q000494:pharmacology
C526318:CCN2 protein, mouse; D005353:Fibronectins; D006609:High Mobility Group Proteins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor