Gap-junctional communication is required for the maturation process of osteoblastic cells in culture

• Published in: Bone (New York, N.Y.) Vol. 28; no. 4; pp. 362 - 369
• Main Authors: Schiller, P.C, D’Ippolito, G, Balkan, W, Roos, B.A, Howard, G.A
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01-04-2001; Elsevier Science
• Subjects: 18-α-glycyrrhetinic acid; 3T3 Cells; Alkaline Phosphatase - metabolism; Animals; Base Sequence; Biological and medical sciences; Biomineralization; Cell Communication - drug effects; Cell coupling; Differentiation; DNA Primers; Fundamental and applied biological sciences. Psychology; Gap junctions; Gap Junctions - drug effects; Gap Junctions - physiology; Glycyrrhetinic Acid - pharmacology; Mice; Osteoblasts; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - enzymology; Osteoblasts - metabolism; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; Skeleton and joints; Vertebrates: osteoarticular system, musculoskeletal system; Biomineralization; Cell coupling; Differentiation; Osteoblasts; Gap junctions; 18-α-glycyrrhetinic acid; Cell culture; Biochemical analysis; Cell function; Rodentia; Exploration; Cell differentiation; Experimental study; Gene expression; In vitro; Gap junction; Vertebrata; Mammalia; Mouse; Mineralization; Animal; Established cell line; Osteoblast; Molecular biology
• ISSN: 8756-3282; 1873-2763
• DOI: 10.1016/S8756-3282(00)00458-0

Osteoblastic cells in long-term culture undergo a phenotypic maturation process leading to extracellular matrix (ECM) production and bone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detected between osteoblastic cells within 24 h of plating. We evaluated, in long-term cultures of osteoblastic cells, the effect of inhibiting GJC on the phenotypic maturation process and the expression of specific genes associated with this process. MC3T3-E1 cells were plated, and, after 24 h (day 0), cells were exposed to 18-α-glycyrrhetinic acid (AGA), a nontoxic reversible inhibitor of GJC. GJC, alkaline phosphatase (AP) activity, BN formation, and the relative level of transcripts encoding osteocalcin (OC), bone sialoprotein (bSP), osteopontin (OP), collagen α1 type I (α1ICol), and elongation factor-1a (EF1a) were evaluated on day 0 and every 4–7 days thereafter through day 30. GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse transcription-polymerase chain reaction. GJC was detectable at day 0 and increased with time in culture. AGA (100 μmol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed cells showed a dose-dependent decrease in AP activity and a delay in the appearance of BN. This delayed phenotypic expression coincided with an inhibitory effect on the expression of the osteoblast-specific genes OC and bSP. Expression of α1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affected. Similar results were obtained with oleamide, an additional reversible inhibitor of GJC. In contrast, cells exposed to either vehicle or 100 μmol/L glycyrrhizic acid (a noninhibitory glycoside of 18-β-glycyrrhetinic acid) were indistinguishable from untreated cells for all parameters evaluated. We conclude that GJC inhibition interferes with the maturation process of osteoblastic cells in culture, possibly by affecting signals regulating the expression of genes involved in the maturation/differentiation of the osteoblastic phenotype.