An Alternatively Translated Connexin 43 Isoform, GJA1-11k, Localizes to the Nucleus and Can Inhibit Cell Cycle Progression

Abstract

Connexin 43 (Cx43) is a gap junction protein that assembles at the cell border to form intercellular gap junction (GJ) channels which allow for cell-cell communication by facilitating the rapid transmission of ions and other small molecules between adjacent cells. Non-canonical roles of Cx43, and specifically its C-terminal domain, have been identified in the regulation of Cx43 trafficking, mitochondrial preconditioning, cell proliferation, and tumor formation, yet the mechanisms are still being explored. It was recently identified that up to six truncated isoforms of Cx43 are endogenously produced via alternative translation from internal start codons in addition to full length Cx43, all from the same mRNA produced by the gene GJA1. GJA1-11k, the 11kDa alternatively translated isoform of Cx43, does not have a known role in the formation of gap junction channels, and little is known about its function. Here, we report that over expressed GJA1-11k, unlike the other five truncated isoforms, preferentially localizes to the nucleus in HEK293FT cells and suppresses cell growth by limiting cell cycle progression from the G₀/G₁ phase to the S phase. Furthermore, these functions are independent of the channel-forming full-length Cx43 isoform. Understanding the apparently unique role of GJA1-11k and its generation in cell cycle regulation may uncover a new target for affecting cell growth in multiple disease models.

Keywords: cell cycle; connexin43; internal translation; trafficking.

Figure 1

Localization of alternatively translated Cx43 isoforms expressed in HEK293FT cells. (A). The fixed-cell immunofluorescence of HEK293FT cells expressing short isoforms of Cx43 was detected by monoclonal anti-V5 (green) and polyclonal anti-Cx43 (red) antibodies, raised against a 17-residue peptide of C-terminal Cx43 (Sigma) with Hoechst staining of the nuclei (blue). The arrow heads show the gap junction (plaque) formation at the cell–cell border of cells with wild type (GJA1-WT) plasmid. Scale bar: 10μm. The results are representative of four independent experiments. (B). The ratio of fluorescent intensity in the nucleus versus cytoplasm in transfected HEK293FT cells. The data are presented as mean ± SEM, n = 20, **** p < 0.0001, by two-way ANOVA followed by Tukey’s multiple comparison test. The intensities were measured and normalized to background using Image J.

Figure 1

Localization of alternatively translated Cx43 isoforms expressed in HEK293FT cells. (A). The fixed-cell immunofluorescence of HEK293FT cells expressing short isoforms of Cx43 was detected by monoclonal anti-V5 (green) and polyclonal anti-Cx43 (red) antibodies, raised against a 17-residue peptide of C-terminal Cx43 (Sigma) with Hoechst staining of the nuclei (blue). The arrow heads show the gap junction (plaque) formation at the cell–cell border of cells with wild type (GJA1-WT) plasmid. Scale bar: 10μm. The results are representative of four independent experiments. (B). The ratio of fluorescent intensity in the nucleus versus cytoplasm in transfected HEK293FT cells. The data are presented as mean ± SEM, n = 20, **** p < 0.0001, by two-way ANOVA followed by Tukey’s multiple comparison test. The intensities were measured and normalized to background using Image J.

Figure 2

(A). Biochemical isolation of GJA1-11k in the nucleus. Western Blot of Cx43 isoforms in subcellular fractions (cytosolic and nuclear fractions) of HEK293FT cells over-expressing exogenous GJA1-11k, GJA1-43k, and GFP-V5 tagged plasmid cDNA, probed to monoclonal Cx43-CT (Millipore) antibodies. To demonstrate enriched biochemical isolation, the same blot was probed using antibodies to different subcellular fraction markers, including cytoplasmic (GAPDH), membrane (Na⁺/K⁺-ATPase), Golgi (GM-130), and nuclear markers (Histone H3, Lamin B1). The results are representative of three independent experiments. (B). The densitometry of CT-43k (11k) subcellular fractions assessed by Western Blot. Uncut immunoblots are provided in Figure S3.

Figure 3

Effect of Cx43 on cell proliferation. The knockdown of endogenous Cx43 increases the cell proliferation of HEK293FT cells. For each day, data are presented as mean ± SEM, n = 9, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by an unpaired two-tail Student’s t test. Western Blot analysis confirmed the knockdown of Cx43 and is representative of three independent experiments.

Figure 4

Effect of Cx43 short isoforms on cell proliferation. The overexpression of GJA1-11k inhibits the cell growth of the HEK293FT cell line. The growth curve is counts of transiently transfected cells each with vectors expressing GJA1-11k, GJA1-43k, GJA1-WT, and the negative control plasmid GFP with V5-tag. For each day data are presented as mean ± SEM, n = 12, ** p < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test. Cell growth analysis revealed that cells expressing GJA1-11k grew significantly slower, than cells overexpressing other isoforms. Western Blot confirmed transient expression of Cx43 isoforms. Uncut immunoblots are provided in Figure S6.

Figure 5

GJA1-11k with nuclear export signal rescues the cell proliferation of the HEK293FT cell line. (A). The immunofluorescence of HEK293FT cells expressing GJA1-11k or GJA1-11k-NES probed with V5 and Cx43 antibodies (Sigma) and the corresponding ratio of fluorescence density in the nucleus versus cytoplasm is shown on the graph. Scale bar: 10μm. The graph results are representative of four independent experiments. The data are presented as mean ± SEM, n = 26, **** p < 0.0001 by an unpaired two-tail Student’s t test. The intensities in the bar graph were measured and normalized to background using Image J. (B). GJA1-11k-NES rescues cell proliferation, suggesting that the effect of GJA1-11k is linked to the nucleus. The number of cells transiently transfected with vectors expressing GJA1-11k, GJA1-11k-NES, GJA1-43k, GJA1-WT, and the negative control plasmid GFP were counted 72 h after transfection. Cell growth analysis has shown that cells expressing GJA1-11k-NES grew significantly faster than cells expressing GJA1-11k. The Western Blot confirmed the transient expression of Cx43 isoforms. The data are presented as mean ± SEM, n = 12, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 6

Overexpression of wild type Cx43 and short isoforms inhibits cell cycle progression in HEK293FT cells. (A). Non-transfected cells were serum-deprived for 48 h to synchronize cells in the G₀/G₁ phase. A representative image showing cell cycle phases in the HEK293FT cell line based on BrdU incorporation versus total DNA staining by 7-AAD. The cell cycle phases were analyzed by FlowJo. (B). The cell cycle distribution of GJA1-WT, GJA1-43k, GJA1-11k, and GJA1-11k NES V5-tagged isoforms were analyzed 72 h post-transfection. The bar graph shows the reduction in the number of cells in the S-phase and accumulation of cells in G₀/G₁ expressing GJA1-11k compare to GJA1-WT or GJA1-43k. GJA1-11k-NES rescues cell cycle progression from G₀/G₁ to S-phase. The data are presented as mean ± SEM, n = 12, * p < 0.05 and ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test. (C). The Proliferation index (using following PI = ((S+G₂/M) / (G₀/G₁+S+G₂/M)) formula) confirmed the reduction in proliferation rate for GJA1-11k. The data are presented as mean ± SEM, n = 12, * p < 0.05 and ** p < 0.01, by one-way ANOVA followed by Tukey’s post-hoc test.