Submitted on June 19, 2007
Accepted on November 13, 2007
Connexin Expression and Functional Analysis of Gap Junctional Communication in Mouse Embryonic Stem Cells
Philipp Wörsdörfer 1,
Stephan Maxeiner 1,
Christian Markopoulos 1,
Gregor Kirfel 2,
Volker Wulf 1,
Tanja Auth 1,
Stephanie Urschel 1,
Julia von Maltzahn 1,
Klaus Willecke 2*
1 Institute of Genetics, Division of Molecular Genetics, University of Bonn, 53117 Bonn, Germany
2 Institute of Genetics, Division of Molecular Genetics, University of Bonn, 53117 Bonn, Germany, Institute of Cell Biology, University of Bonn, 53121 Bonn, Germany
* To whom correspondence should be addressed. E-mail: genetik{at}uni-bonn.de.
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Abstract |
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Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, i.e. the subunit proteins of gap junction channels. For this purpose we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43 and Cx45 to be expressed as proteins. We have demonstrated by co-immunoprecipitation that Cx31 and Cx43 as well as Cx43 and Cx45 probably form heteromeric gap junction channels while Cx31 and Cx45 do not.
The pharmacological inhibitors reduced GJIC between ES cells to about 3% and initiated apoptosis, suggesting an anti-apoptotic effect of GJIC. In contrast to these results, reduction of GJIC to about 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43 deficient ES cells did not lead to apoptosis. Further studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, in order to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.
Key Words.
gap junctions, Cx31, Cx43, Cx45, apoptosis, ES cells
