Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication.
Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18alpha-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 microM 18alpha-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18alpha-GCA for only the first 24-48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18alpha-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18alpha-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication.
Zhang
Wei
W
Department of Surgery, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand.
Green
Colin
C
Stott
N Susan
NS
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D002356:Cartilage / Q000166:cytology* / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002452:Cell Count; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002642:Chick Embryo; D020219:Chondrogenesis / Q000187:drug effects / Q000502:physiology*; D018031:Connexin 43 / Q000378:metabolism; D004247:DNA / Q000032:analysis; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D018878:Limb Buds / Q000166:cytology / Q000196:embryology; D008648:Mesoderm / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D016212:Transforming Growth Factor beta; D015854:Up-Regulation
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D018031:Connexin 43; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; C119129:18alpha-glycyrrhetinic acid; D004247:DNA; D006034:Glycyrrhetinic Acid