A connexin 43 mutant lacking the carboxyl cytoplasmic domain inhibits both growth and motility of mouse 3T3 fibroblasts

Abstract

Connexins have been shown to inhibit the growth of a wide number of communication-deficient cells both in vivo and in vitro, but the molecular mechanism remains largely unknown. In previous work we have shown that stable transfectants of 3T3 A31 fibroblasts, which express a Connexin 43 (Cx43) mutant (Cx43-256M) consisting of amino acids 1-256 of rat Cx43 fused to a c-myc tag, exhibit a decreased basal growth rate and weakened mitogenic response to platelet derived growth factor compared with either the parent cell line or cells transfected with an expression vector that did not encode a functional protein. Here we have investigated further the growth characteristics of these cells in order to establish the mechanism by which this protein suppresses cell growth. Analysis of DNA synthesis in individual cells by immunofluorescence staining of bromodeoxyuridine incorporation demonstrated that the slow growth of Cx43-256M cells was due to a decrease in the number of cells that undergo DNA synthesis following growth factor stimulation. This was associated with an increased proportion of the cell population in the G2/M phases of the cell cycle suggesting growth may be arrested during G2 or metaphase. In addition to effects on cell growth, Cx43-256M expression inhibited cell motility as assayed both in a wounding assay and in a Boyden chamber assay. These results now raise the question as to whether the primary effect of the Cx43-256M protein is on cell growth or cell motility.

Connexin43 enhances glioma invasion by a mechanism involving the carboxy terminus

“…The CT truncation of Cx43 alters GJ channel properties and hence GJIC yet neither our study nor that of Moorby et al (2000) were able to detect alterations in motility as a result of this truncation. Many proteins have been identified to interact with the cytoplasmic tail of Cx43, providing a context for Cx43 to be involved in signalling events leading to cytoskeletal rearrangement.…”

“…Previous studies have identified both junction‐dependent (Oliveira et al,2005) and ‐independent (Moorby,2000; Wei et al,2005) mechanisms for Cx43‐mediated motility. Therefore, the observed attenuation in cell motility may not be a direct result of reduced GJIC.…”

“…In murine neuroblastoma (N2a) cells, single channel analysis indicated that truncation of the Cx43 CT (Cx43M257 mutant) did not significantly modify the magnitude of the main unitary conductance of Cx43 channels, while the mean open time of Cx43M257 channels was considerably prolonged, along with disappearance of the residual state, compared with full length channels (Moreno et al,2002). Moorby et al (2000) have also directly examined the consequence of Cx43 CT truncation on motility, albeit using different constructs in different cells (Cx43M256 in 3T3 A31 versus Cx43Δ244‐382GFP in C6 cells). In wound healing and Boyden chamber assays, they observed decreased motility in cells expressing truncated Cx43 compared with untransfected control cells, although they reported no differences when they employed transwell assays, a result which could be due to differences in experimental conditions.…”

The carboxyl tail of Cx43 augments p38 mediated cell migration in a gap junction-independent manner

“…The mechanisms by which Cx43 can modulate cell migration are still unclear. However, in C6-glioma cells, increased cell motility was only seen when a full length Cx43 was expressed while cells expressing a C-terminal truncated Cx43 did not show it, implying an important role for the carboxyl tail of Cx43 in the regulation of glia cell migration (Bates et al, 2007;Moorby, 2000). Similar results were obtained in migrating neurons during brain development (Cina et al, 2009).…”

Connexins in the control of vasomotor function

“…Connexins have been shown to be involved in the control of migration of endothelial cells 105,111,112 and many other cell types, among them glioma cells, 106 astrocytes 113 and fibroblasts. 114 We and others have shown that connexin-dependent migration control can occur in the absence of functional gap junction channels. The expression of the C-terminus alone in HeLa cells, 105 endothelial precursor cells 105 and glioma cells 115 was sufficient to enhance migration.…”