Isolation and characterization of gap junctions in the osteoblastic MC3T3-E1 cell line.

Gap junctions are channels connecting cells that function in cell-to-cell communication. Gap junctions are abundant in osteoblastic cells. Membranes enriched for gap junction plaques were obtained by differential centrifugation, followed by treatment of the membranes with potassium iodide and sarkosyl before sucrose density gradient centrifugation. Electron microscopy showed that the preparation was enriched for electron-dense membranes consistent with gap junctions. Coomassie Blue staining of SDS-PAGE preparations revealed a prominent band at approximately 41 kD. Western analysis with a site-directed antibody, CT-360 (D. Laird, California Institute of Technology, Pasadena, CA), to the C-terminal portion of the rat heart connexin 43 molecule was positive in the MC3T3-E1 cell line, a phenotypic osteoblastic cell line derived from normal neonatal mouse calvariae. Western analysis using a monoclonal antibody, R5.21C, to rat liver connexin 32 was negative. Additionally, a prominent band at 59 kD was detected by CT-360 in both gap junction-enriched preparations and cell lysates. Treatment of diluted samples of gap junction-enriched preparations with sulfhydryl reducing agents in combination with detergents resulted in the enhancement and diminution of the 41 and 59 kD bands, respectively. Immunoprecipitation following [35S]methionine/[35S]cysteine labeling revealed a significant band detected at 122 kD in addition to the 41 kD band. To demonstrate functional gap junctions, transfer of lucifer yellow dye to surrounding cells was monitored after microinjection of a target cell. Between passages 10 and 25 in culture, functional cell coupling was found in approximately 70% of injected cells. Coupling was detected within 1-2 minutes after injection. Simultaneous microinjection of the CT-360 antibody with lucifer yellow resulted in the decoupling of cells. In conclusion, (1) MC3T3-E1 cells possess a 41 kD protein that is recognized by connexin 43 antibody to rat heart gap junction; (2) multimers of the MC3T3-E1 gap junctions occur in the preparation; and (3) functional coupling demonstrated by dye transfer may be regulated by region(s) in the C terminus of the connexin molecule.