Gap-junctional communication is required for the maturation process of osteoblastic cells in culture.
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-alpha-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 alpha1 type I (alpha1ICol), 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 micromol/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 alpha1ICol 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 micromol/L glycyrrhizic acid (a noninhibitory glycoside of 18-beta-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.
Schiller
P C
PC
Geriatric Research, Education, and Clinical Center, and Research Service, Veterans Affairs Medical Center, University of Miami School of Medicine, Miami, FL 33125, USA. pschille@med.miami.edu
D'Ippolito
G
G
Balkan
W
W
Roos
B A
BA
Howard
G A
GA
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002450:Cell Communication / Q000187:drug effects; D017931:DNA Primers; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D000469:Alkaline Phosphatase; D006034:Glycyrrhetinic Acid