HepaCAM associates with connexin 43 and enhances its localization in cellular junctions

Abstract

HepaCAM (GlialCAM) is frequently deleted in carcinomas, and reintroduction of hepaCAM into transformed cell lines reduces cellular growth and induces senescence. Mutations in HEPACAM give rise to the neurodegenerative disease megalencephalic leukoencephalopathy with subcortical cysts (MLC) since mutated hepaCAM prevents shuttling of MLC1 protein to astrocytic junctions in the plasma membrane. Here we identify that hepaCAM associates with connexin 43, a main component of gap junctions, and enhances connexin 43 localization to the plasma membrane at cellular junctions. HepaCAM also increases the levels of connexin 43, not by enhancing its transcription but by stabilizing connexin 43 protein. In the absence of hepaCAM, connexin 43 undergoes a faster degradation via the lysosomal pathway while proteasomal degradation seems not to be involved. Mutations in hepaCAM that cause MLC, or neutralization of hepaCAM by antibodies disrupt its association with connexin 43 at cellular junctions. By discovering the requirement of hepaCAM for localizing connexin 43, a well-established tumor suppressor, to cellular junctions and stabilizing it there, this study suggests a mechanism by which deletion of hepaCAM may support tumor progression.

Figure 1. HepaCAM associates with connexin 43.

(A) U373 MG cells were stably transfected with pcDNA3.1 vector, wild-type hepaCAM, hepaCAM-R92Q and hepaCAM-R92W. Immunofluorescent staining was performed with antibodies against the hepaCAM cytoplasmic domain (green) and connexin 43 (red). Co-localization of hepaCAM and connexin 43 is indicated by yellow fluorescence. Nuclei were stained with DAPI (blue). Insets show a higher magnification of sites of cell-cell contacts. Cells were visualized by confocal microscopy under a 60× objective. Scale bar: 10 μm. (B) Co-immunoprecipitatation of connexin 43 and hepaCAM. Cell lysates were prepared from U373 MG cells stably transfected with pcDNA3.1 vector and wild-type hepaCAM, and immunoprecipitated with antibody against the hepaCAM extracellular domain (IP hepaCAM). Immunoprecipitation with mouse IgG1 (IP IgG) was included as a negative control. Western blot analysis was performed on the immunoprecipitates and input (3%) using connexin 43 antibody. The efficiency of hepaCAM immunoprecipitation was evaluated with an HRP-conjugated FLAG antibody. The IgG heavy chain detected with an HRP-conjugated anti-mouse antibody is shown as a loading control. (C) Co-immunoprecipitation of wild-type and mutant hepaCAM with connexin 43. Cell lysates were immunoprecipitated with antibody against the hepaCAM extracellular domain (IP hepaCAM). Immunoprecipitation with mouse IgG1 (IP IgG) was included as a negative control. Western blot analysis was performed on the immunoprecipitates and input (2%) using connexin 43 antibody. (D) Expression of wild-type hepaCAM increases connexin 43 protein levels in U373 MG cells. 20 μg of cell lysates were subjected to Western blot analysis. GAPDH was used as a loading control. The result presented is a representative experiment of four independent experiments with similar results. The full view blots are shown in Supplementary Figure 1. (E) Quantification of connexin 43 protein levels in D and in three additional independent Western blot analyses. Using ImageJ the densities of the connexin 43 bands were normalized to the densities of the respective GAPDH bands for each sample, and the mean relative density over the four experiments was calculated. The data presented are the means ± SE (n = 4), **p < 0.01 as assessed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 2. HepaCAM enhances connexin 43 expression.

(A) Treatment of hepaCAM-expressing U373 MG cells with antibodies against the hepaCAM extracellular domain prevents the association of hepaCAM with connexin 43 at cell-cell contacts. Wild-type hepaCAM-expressing U373 MG cells were treated overnight with antibody against the hepaCAM extracellular domain in soluble form (10 μg/ml). Cells were also treated with the isotype mouse IgG1 as a control. The next day, cells were fixed and immunofluorescent staining was performed with antibodies against the hepaCAM extracellular domain (green) and connexin 43 (red). Co-localization of hepaCAM and connexin 43 is indicated by yellow fluorescence. Nuclei were stained with DAPI (blue). Cells were visualized by confocal microscopy under a 60× objective. Scale bar: 10 μm. (B) Treatment of hepaCAM-expressing U373 MG cells with antibodies against the hepaCAM extracellular domain causes a downregulation of connexin 43 expression. Wild-type hepaCAM-expressing U373 MG cells were treated overnight with antibody against the hepaCAM extracellular domain in soluble form (10 μg/ml). Cells were also treated with the isotype mouse IgG1 as a control. The next day, cells were lysed and 20 μg of cell lysates were subjected to Western blot analysis using connexin 43 antibody. GAPDH was used as a loading control. The full view blots are shown in Supplementary Figure 2.

Figure 3. HepaCAM regulates connexin 43 stability.

(A) Evaluation of connexin 43 mRNA expression. Total RNA was analyzed by RT-PCR. GAPDH and no template controls (NTC) were included as housekeeping gene and negative controls, respectively. (B) Evaluation of connexin 43 protein stability by a cycloheximide (CHX) chase assay. Cells treated with CHX (50 μg/ml) for the times indicated were lysed and 30 μg of cell lysates were subjected to Western blot analysis. The result presented is a representative experiment of three independent experiments with similar results. (C) Quantification of all three CHX chase experiments using ImageJ. The densities of the connexin 43 bands were normalized to the densities of the respective GAPDH bands at each time-point. The level of connexin 43 remaining at each time-point was calculated as a percentage of the initial connexin 43 level (time 0 of CHX treatment). The data presented are the means ± SE (n = 3). (D) Expression of hepaCAM in HEK293T cells increases connexin 43 protein levels. HEK293T cells were transiently transfected with pcDNA3.1 vector or wild-type hepaCAM. Two days after transfection, cells were lysed and 60 μg of cell lysates were subjected to Western blot analysis using antibodies against connexin 43 and the hepaCAM extracellular domain. The result presented is a representative experiment of three independent experiments with similar results. (E) Quantification of all three experiments using ImageJ. The densities of the connexin 43 bands were normalized to the densities of the respective GAPDH bands for each sample, and the mean relative density over the three experiments was calculated. The data presented are the means ± SE (n = 3), ***p < 0.0001 as assessed by t-test. (F) HepaCAM slows down connexin 43 turnover by the lysosomal pathway. U373 MG cells stably transfected with pcDNA3.1 vector or wild-type hepaCAM were treated with chloroquine (50 μM) and 30 μg of cell lysates were subjected to Western blot analysis for connexin 43. The result presented is representative of two independent experiments with similar results. The full view blots for (B,D,F) are shown in Supplementary Figures 3,4 and 5, respectively.

Figure 4. Schematic depiction of hepaCAM activities.

(A) HepaCAM associates with connexin 43 at the cell-cell contacts of U373 MG glioblastoma cells. (B) Treatment of cells with antibody against the HepaCAM extracellular domain prevents association of hepaCAM with connexin 43 at cell-cell contacts and downregulates cell surface expression of connexin 43. (C) The R92Q and R92W mutations in the hepaCAM extracellular domain prevent association of hepaCAM with connexin 43 at cell-cell contacts.