Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant‐negative connexin 43

Abstract

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins

“…These data confirm and extend the results of other studies, suggesting that Cx43 mutants with deletions of this region act as dominant-negative inhibitors of Cx43 based on inhibition of Lucifer yellow or Alexa 568 transfer and desynchronization of calcium transients [14][15][16]. Moreover, our data suggest a mechanism for this inhibition: the formation of heteromeric connexons between Cx43del 130-136 and wild-type Cx43.…”

“…In light of the results presented here, it is likely that the previous conclusion was based on the effect of Cx43del 130-136 upon endogenous Cx43. It has also been suggested that Cx43del [130][131][132][133][134][135][136] formed functional channels with reduced permeability [16]. However, we have excluded that possibility by showing that infection of cells with Ad-Cx43del 130-136 not only did not allow transfer of either Lucifer yellow or neurobiotin, but it also did not produce any electrical conductance detectable by double whole-cell patch-clamp.…”

“…Several publications have suggested that deletion of portions of the cytoplasmic loop in Cx43 (including amino acids 130-136) produces a dominant negative connexin (Cx43del [130][131][132][133][134][135][136] ) which can inhibit the function of co-expressed wild-type Cx43 [14][15][16], but they do not agree on the mechanisms for its effects nor have they evaluated its effects on other connexins besides Cx43. We prepared a recombinant adenovirus (Ad-Cx43del [130][131][132][133][134][135][136] ) containing this mutant and characterized its cellular fate and physiological behavior when expressed alone, together with wild-type Cx43, or with several other connexins.…”

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

“…Likewise, cells from Cx43 ϩ/Ϫ mice have deficient calcium mobilization in response to endothelin-1. Additionally, several in vitro studies have upregulated (Steinberg et al, 1994;Gramsch et al, 2001) or down-regulated Cx43 abundance or function (Van der Lecanda et al, 1998;Li et al, 1999Li et al, , 2006Upham et al, 2003;Plotkin et al, 2008) with profound effects on osteoblast function.…”

“…Likewise, cells from Cx43 ϩ/Ϫ mice have deficient calcium mobilization in response to endothelin-1. Additionally, several in vitro studies have upregulated (Steinberg et al, 1994;Gramsch et al, 2001) or down-regulated Cx43 abundance or function (Van der Molen et al, 1996;Lecanda et al, 1998;Li et al, 1999Li et al, , 2006Upham et al, 2003;Plotkin et al, 2008) with profound effects on osteoblast function.Our ultimate goals are to gain insight into how osteoblasts utilize intercellular communication in order to regulate function and to identify biologically relevant molecules being passed through Cx43 gap junction channels in osteoblasts. Accordingly, our approach is to work our way from a defined transcriptional response affected by gap junctions back through the signaling cascade-systematically examining a path from the nucleus to membrane.…”