Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions

Abstract

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK-kcdc and Mβ16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mβ16tsA-wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mβ16tsA-kcdc cells with Mβ16tsA-wt, N2A or NRK-wt cells had no effect on pol β levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions. © The Physiological Society 2005.