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

Abstract

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.

Attenuated Response to In Vivo Mechanical Loading in Mice With Conditional Osteoblast Ablation of the Connexin43 Gene (Gja1)

“…Strains were recorded through a signal conditioning amplifier system (SCX1-1001; National Instruments). Peak forces of 4,8,12,16,20, and 24 N were applied with a 160-s recovery interval between each set of eight cycles. Peak strains were determined for cycles 4-8, and the average was computed.…”

“…When two connexons on adjacent cells dock to each other, they form a transcellular gap junction channel. (1,2) The most abundant connexin found in bone is connexin43 (Cx43), (3)(4)(5) although connexin45 (Cx45) and connexin46 are also present. (3,6) The importance of Cx43 in the skeletal system was shown by the skeletal developmental abnormalities and osteoblast dysfunction in mice with a null mutation of the Cx43 gene (Gja1), (7) and it is further underscored by the skeletal malformations featuring oculodentodigital dysplasia, a human disease caused by loss-of-function mutations of Gja1.…”

Role of Cx43-Based Gap Junction in Murine Osteoblast-Like Mc3t3-E1Cells Exposed to 17-Βestradiol

“…Connexins have been identified in bone tissues by morphological, structural, and functional analyses [4][5][6][7]. Among the members of the connexin family, Connexin43 (Cx43) is the most ubiquitously expressed in virtually all types of bone cells [8][9][10][11]. Genetic studies have demonstrated that both gap junction and Cx43 play essential roles in the bone development and turnover of human and animal models in vivo.…”

Gap junctions and hemichannels in signal transmission, function and development of bone

“…In the skeleton system, gap junctions are present in all the cells, but are particularly abundant among the osteoblasts and osteocytes. Though three different connexins, Cx43, Cx45 and Cx46 are expressed in the bone cells, Cx43 is the most ubiquitously expressed in virtually all types of bone cells including human bone marrow stromal cells [6], mice, rat, bovine chondrocytes [44,45], mandibular bone and periodontal ligament cells of teeth [46,47], newborn rat calvarial osteoblasts [48], osteoblast-like MC3T3-E1 cells [49,50], primary chick calvarial osteocytes [51], and osteocyte-like MLO-Y4 cells [52]. The ubiquitous expression of Cx43 in all the bone cells makes it the most preferred candidate for the formation of gap junctions, allowing the passage of molecules less than 1.2 kDa to go across the channels and facilitate networking among the different cells especially osteocytes and osteoblasts.…”