title: hDlg controls the trafficking of gap junction protein Cx43 in normal keratinocytes and human papillomavirus-positive tumour cells
Creators: name: Dong, Li
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: Connexin 43, hDlg, keratinocyte, wound healing.

description: Gap junction intercellular channels allow the exchange of ions and small molecules between neighbouring cells. Our laboratory showed previously that tumour suppressor protein and cell polarity regulator hDlg (human homologue of Drosophila Discs Large) binds to the wide-spread gap junction protein Connexin 43 (Cx43) in human papillomavirus (HPV) - positive cervical tumour cells. Cx43 relocated together with hDlg to the cytoplasm of these cells, and hDlg knockdown resulted in some Cx43 reappearing on the plasma membrane. This led to our hypothesis that hDlg could control Cx43 trafficking. High-risk HPV oncoprotein E6 via its PDZ-binding domain targets hDlg for proteasome degradation. Previously, co-immunoprecipitation indicated Cx43 binds to hDlg in vitro in cervical tumour cell lines and proximity ligation assays indicated their interaction in vivo in cervical epithelial tissue from high-grade cervical lesions. Moreover, our protein-protein interaction data indicated that HPV E6 oncoprotein took part in the hDlg/Cx43 interaction. Cx43 and hDlg were observed on the plasma membrane in non-transformed cervical tumour cells (W12G, low level of HPVE6) but appeared in the cytoplasm in fully transformed cervical tumour cells W12GPXY (high level of HPVE6). C33a cells (HPV-negative cervical tumour cells with membranous Cx43) transfected with mutated HPVE6 (loss of ability to bind to hDlg) showed membranous Cx43 while re-location of Cx43 to the cytoplasm was observed in C33a cells transfected with wild-type HPVE6. This indicates that HPVE6 negatively regulates the trafficking of Cx43 through interaction with hDlg. It may be that the ability of hDlg to suppress cell growth relies on correct GJIC communication, and HPVE6 interference with Cx43/hDlg functionality may provide a growth advantage to cells, which would increase viral production. To determine if the Cx43/hDlg interaction requires HPVE6, Cx43/hDlg interaction was investigated in HPV-negative cervical cancer cell lines (C33a and C33aE6 (C33a cells stably expressing HPV16E6). Co-immunoprecipitation indicates Cx43 binds hDlg in these cells. Immunofluorescence confocal microscopy shows the co-localisation of Cx43/hDlg on the plasma membrane in C33a cells and in the cytoplasm in C33aE6 cells. To determine if the Cx43/hDlg interaction is dependent on the cancer environment, similar experiments were carried out in non-tumour epithelial cell lines that do not express E6 (HEK293, HaCaT, NIKS) and NIKS16 cells (NIKS cells stably transfected with HPV16 genomes). Co-immunoprecipitation shows that Cx43 forms a complex with hDlg in HEK293, HaCaT, NIKS cells and the presence of HPVE6 at low levels in NIKS16 cells do not affect their interaction. Co-localisation of Cx43 and hDlg was observed on the plasma membrane in these cells. This indicates the Cx43-hDlg interaction is neither HPV-E6-dependent nor cancer-cell-specific. The role of hDlg in controlling the trafficking of Cx43 has been investigated by siRNA depletion of hDlg in HaCaT, HEK293, NIKS and NIKS16 cells. siRNA depletion of hDlg in these cells led to a reduction in levels and cytoplasmic location of Cx43. HaCaT cells with stable depletion of hDlg (HaCaT shDlg) also displayed a similar pattern of Cx43 rearrangement. Endolysosomal/lysosomal inhibition by ammonium chloride (NH4Cl) and chloroquine (CQ) led to increased levels of Cx43 in HaCaT and HEK293 cells and HaCaT cells with siRNA depletion of hDlg. These data indicate the role of hDlg in maintaining a cytoplasmic pool of Cx43 and suggest that hDlg may play roles in the delivery of Cx43 to the membrane. To investigate if changes in cell signalling can alter the Cx43/hDlg complex formation, a scrape wound healing model was used since the wound healing process shares many similarities with tumour progression. The changes in subcellular location and expression levels of the two proteins during scrape wound closure were investigated. The wound closure rates in the non-tumour epithelial cells were also been checked. At the start of wound closure in HaCaT keratinocytes, Cx43 and hDlg colocalised on the plasma membrane but both proteins moved into the cytoplasm 4-16 hours post-wound. After 24 hours, most of the proteins regained a membrane location. A similar phenomenon was also observed in NIKS cells during wound healing. There were significant reductions in Cx43 and hDlg levels during the early stages of wound closure and changes in Cx43 phosphorylation were also observed during the wound closure process. hDlg is one of the regulators of cell polarity, which is important in cell migration especially in direct migration. In wound closure experiments, hDlg depletion led to cell death and a significantly delayed wound closure rate in HaCaT and NIKS16 cells. The cell monolayer was not repaired after 24 hours (less than 50% closure). Sudden loss of hDlg by siRNA might lead to loss of regulation of cell polarity and affect the wound healing process. Surprisingly, in HaCaT shDlg cells (HaCaT cells stably depleted of hDlg), wound closure was completed at 16h post-wound (faster than control HaCaT cells with 70% closure at 24h) and no cell death was observed. hScrib, a tumour suppressor protein, is involved in the regulation of cell polarity (Dow et al., 2003). Faster wound closure might be due to the role of hScrib in HaCaT shDlg cells to complement the stable loss of hDlg, while the sudden loss of hDlg through siRNA treatment might lead to lower tolerance of cell stresses such as wound healing. Interestingly, the presence of the HPV16 genome drives faster closure of the wound in NIKS16 cells compared to NIKS cells. Wound closure was complete at 16h post-wound in NIKS16 cells while after 24h post-wound a small gap remained in NIKS cell monolayers. This might be due to the effect of beta-catenin. Beta-catenin transits to the nucleus and induces gene expression of proteins involved in cell migration, which enhance wound healing. HPVE6 causes beta-catenin to accumulate in the nucleus. However, none of the cervical cancer cell lines (C33a, C33aE6 and HeLa43) were able to close the scrape wound properly (closure of 17%, 33% and 28% respectively). This might be due to a combination of the effect of membranous Cx43 and HPVE6 on the regulation of nuclear beta-catenin. Nuclear transmission of beta-catenin could be prevented by interaction with membranous Cx43, which could explain the loss of membranous Cx43 at the early stage of wound repair. C33a cells (membranous Cx43, no HPVE6) close the wound at the slowest rate (17%) while C33aE6 (cytoplasmic Cx43, HPVE6) and HeLa43 (membranous Cx43, HPVE6) show faster wound closure (33% and 28%). However, whilst no evidence is presented, it is known that HPVE6 induces accumulation of nuclear beta-catenin via its PDZ-binding domain and thus is a good candidate for future studies. Lastly, through Cx43 C-terminal mutagenesis and co-immunoprecipitation experiments, the Cx43-hDlg binding region has been limited to amino acids 348-382 of the Cx43 C-terminus. Six different serine residues were chosen for the mutation to alanine to mimic un-phosphorylated serine. Four of these were MAPK kinase phosphorylation sites, one was an Akt and one a PKC phosphorylation site. However, these Cx43 C-terminal nonphospho-mutants could still interact with hDlg, suggesting that phosphorylation does not affect Cx43 binding to hDlg. In conclusion, hDlg binds to regions of amino acids 348 -382 of the Cx43 CT. This Cx43-hDlg interaction is not cancer cell-specific and not HPVE6-dependent. The data in this thesis add to accumulating evidence of a role for hDlg in maintaining a cytoplasmic pool of Cx43 and delivery of Cx43 to the plasma membrane.