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

Osteoblastic cells in long-term culture undergo a phenotypic maturation pro cess leading to extracellular matrix (ECM) production and hone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detect ed between osteoblastic cells within 24 h of plating. We evaluated, in long -term cultures of osteoblastic cells, the effect of inhibiting GJC on the p henotypic: maturation process and the expression of specific genes associat ed with this process, MC3T3-E1 cells were plated, and, after 24 h (day 0) c ells were exposed to 18-alpha -glycyrrhetinic acid (AGA), a nontoxic revers ible inhibitor of GJC, GJC, alkaline phosphatase (AP) activity, BN formatio n, and the relative level of transcripts encoding osteocalcin (OC), bone si aloprotein (bSP), osteopontin (OP). collagen alpha1 type I(alpha 1ICol), an d elongation factor-1a (EF1a) were evaluated on day 0 and every 4-7 days th ereafter through day 30, GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse tran scription-polymerase chain reaction. GJC was detectable at day 0 and increa sed with time in culture, AGA (100 mu mol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed tells showed a dose-dependent decr ease in AP activity and a delay in the appearance of BN. This delayed pheno typic expression coincided with an inhibitory effect on the expression of t he osteoblast-specific genes OC and bSP, Expression of alpha 1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affect ed. Similar results were obtained with oleamide, an additional reversible i nhibitor of GJC, In contrast, cells exposed to either vehicle or 100 mu mol /L glycyrrhizic acid (a noninhibitory glycoside of 18-beta -glycyrrhetinic acid) were indistinguishable from untreated cells for ail parameters evalua ted. 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, (Bone 28:362-369; 2001) (C) 2001 by Elsevier Scienc e Inc. All rights reserved.