Role Of CLC-3 In Migration Of Epidermal Stem Cells

Background and Objective:The repair of wounds is one of the most complex biological processes that occur during human life. Wound healing is a process involving several overlapping stages including hemostasis and inflammation, cell proliferation and maturation, and finally, remodeling. Moments after an injury occurs, various intracellular and intercellular pathways must be activated and coordinated if tissue integrity and homeostasis are to be restored(1). It has been reported that the epidermal stem cells(ESCs) could contribute to epidermal repair through the migration of epidermal or infundibulum, which is the portion of the hair follicle between the epidermis and the sebaceous gland(1-5). Nevertheless, not much is known about the factors responsible for the complex biological processes in wound healing.Because of lack of further research of the factors responsible for controlling ESCs volume and migration during wound healing, new knowledge is extremely limited.Studies have shown membrane ion channels play significant roles in tumor development and metastasis, and are essential for some cancer cell behaviors, including proliferation, migration, and apoptosis(6). Voltage-gated sodium(Na+), potassium(K+) and calcium(Ca2+) channels play a role in tumor metastasis and cell migration in breast cancer(7), melanoma(8) and fibrosarcoma(9).Depending on their gating mechanisms, Cl- channels have been classified into five subtypes, one of which is volume-activated Cl- channels(VACC)(10). VACCs respond to changes in volume-activated Cl- currents(ICl,vol) stimulated by osmotic cell swelling. Outflow of Cl- and K+ through VACC and K+ channels leads to a decrease in cell volume termed regulatory volume decrease(RVD)(11,12). Alteration of VACCs has been identified to play an important role in migration of glioma cells(13,14).Although the channel protein responsible for providing ICl,vol currents is still unidentified, CLC-3, a member of the CLC family of voltage-gated Cl- channels, is a strong molecular candidate(15-17). CLC-3 is established as the molecular component involved in activation or regulation of ICl,vol(18,19). Its protein expression has been identified in a variety cancer cell types, including prostate cancer epithelial cells(20), glioma cells(21), PC12 cells(22)and CNE-2Z cells(23).Much attention has been given to expression and involvement of Cl- channels in migration and metastasis of cells. The question immediately arose as to whether CLC-3 expression in ESCs regulates cell volume by altering ICl,vol or RVD. The role of endogenous CLC-3 in ESC migration during wound healing has not been investigated utilizing an excisional wound model. Hence, this study was performed to evaluate expression and functional activities of VACCs in ESCs. Suppression of CLC-3 expression by small interfering RNA(siRNA) was used to evaluate the role of CLC-3 in ESC migration in vitro.Our study will be about the role of CLC-3 in migration of epidermal stem cells, and will be helpful to find a key therapeutic target for intervention of ESCs migration and tailored therapies for advanced skin wound healing.Methods:Study of role of CLC-3 in migration of epidermal stem cells1 Isolation of total RNA in epidermal stem cells and RT-PCR to detect CLC-3 mRNA expression.2 Western Blotting to detect CLC-3 protein expression.3 Immunofluorescence staining to detect CLC-3 protein expression in epidermal stem cells in vitro and in vivo.4 Immunofluorescence staining to study the interaction between CLC-3 and epidermal stem cells in different phase of wounding healing.5 Production, concentration and transduction of recombinant lentiviral vectors.6 Electrophysiological studies by Whole-Cell Patch-Clamp.7 Scratch wound assay and transwell migration assay.8 In vitro live cell imaging-measurement of cell movement velocity and track length.Results:Study of role of CLC-3 in migration of epidermal stem cells1 Isolation of total RNA in epidermal stem cells and RT-PCR to detect CLC-3 mRNA expression. RT-PCR was performed to measure expression of CLC-3 m RNA levels, It have been confirmed the expression of CLC-3 mRNA in epidermal stem cells.2 Western Blotting to detect CLC-3 protein expression. CLC-3 protein levels were detected by western blot, we detected that the expression of CLC-3 protein in epidermal stem cells.3 Immunofluorescence staining to detect CLC-3 protein expression in epidermal stem cells in vitro and in vivo. Immunofluorescence staining indicated CLC-3 proteins were present in ESCs. In adult animals, CLC-3 expression was detected in normal skin.4 Immunofluorescence staining to study the interaction between CLC-3 and epidermal stem cells in different phase of wounding healing. In adult animals, CLC-3 expression was detected in skin undergoing wound healing. Notably, expression of CLC-3 in skin was significantly higher during wound healing compared with normal skin. These results suggest CLC-3 is highly expressed in ESCs and may contribute to wound and epidermal repair by promoting ESC migration from neighboring uninjured epidermis.5 Production, concentration and transduction of recombinant lentiviral vectors.To investigate the physiological function of CLC-3 in ESCs, we used lentiviralmediated RNAi and overexpression technology, a powerful method to knockdown or overexpress endogenous genes. Two days after lentiviral infection, fluorescent microscopy detected a high percentage of cells successfully transduced by lentivirus as indicated by fluorescence of expressed GFP fusion proteins. These results demonstrate lentiviralmediated RNAi was successfully taken up by most ESCs. Moreover, q RT-PCR indicated that CLC-3 m RNA levels within the Lv-CLC-3-overexpression group were significantly increased compared with the Lv-CLC-3-mock-vehicle control group; whereas, Lv-CLC-3-sh groups(1, 2, and 3) were significantly reduced compared with Lv-CLC-3- mockvehicle controls. Western blotting confirmed appropriate overexpression and silencing of CLC-3, as strong bands were only detected in the Lv-CLC- 3- overexpression group, while weak bands were detected in Lv-CLC-3-sh2 and Lv-CLC- 3-sh3 groups. Thus, Lv-CLC-3-mock-vehicle, Lv-CLC-3-overexpression and Lv-CLC-3-sh were efficiently transduced into ESCs. As protein levels of CLC-3 were significantly increased in Lv-CLC-3-overexpression group-infected cells and decreased in Lv-CLC-3- sh2-infected cells, our results suggest constructed Lv-CLC-3-overexpression and Lv-CLC- 3-sh2 can efficiently and specifically overexpress and knockdown CLC-3 within ESCs.6 Electrophysiological studies by Whole-Cell Patch-Clamp.Changes in whole cell Cl- currents were examined by patch clamp electrophysiology. For controls, background Cl- currents from the Lv-CLC-3-mock-vehicle group were recorded under isotonic conditions. ICl,Vol was induced by a 47% hypotonic bath solution. Compared with Lv-CLC-3-mock-vehicle controls, treatment with Lv-CLC-3-overexpression and Lv-CLC-3-sh had a significant impact on ICl,Vol induced by 47% hypotonic bath solution. Specifically, ICl,Vol was significantly amplified in cells treated with Lv-CLC-3-overexpression and attenuated in cells treated with Lv-CLC-3-sh. Within the LvCLC-3-mock-vehicle group, mean density of peak currents induced by hypotonic solution(at +100-mV steps) was 722.77 ± 48.24 p A. In the presence of specific Lv-CLC-3-overexpression, these currents increased to 1257.39 ± 92.98 p A in ESCs; whereas, in the presence of Lv-CLC-3-sh, currents decreased to 367.43± 41.17 pA. Thus, whole-cell recordings indicated hypotonicity-activated Cl- currents in ESCs were promoted by LvCLC-3-overexpression and inhibited by Lv-CLC-3-sh.7 Scratch wound assay and transwell migration assay.We demonstrated CLC-3 protein expression is involved in regulation of ESC migration. To investigate these effects, an in vitro scratch wound assay and transwell cell migration assay were employed.8 In vitro live cell imaging-measurement of cell movement velocity and track length.The effects of CLC-3 on ESCs(e.g., the velocity of migration) were further assessed by examining migration of these cells using an in vitro live cell imaging system(IVIS). The velocity of ESC migration was significantly affected by treatment with either Lv-CLC3-overexpression or Lv-CLC3-sh for 48 h. The Lv-CLC3-overexpression group showed high migratory level ratios, while Lv-CLC3-sh group cells were lower than the NC group. No significant difference in track lengths was observed between Lv- CLC3- overexpression group cells and NC cells. P= 0.128. However, Lv-CLC3-sh reduced cell track length.Conclusion:CLC-3 expression levels increased in skin during wound healing and transfection of ESCs with Lv-CLC-3-overexpression and Lv-CLC-3-sh successfully regulates expression of CLC-3 and its subsequent cell processes. suggesting this protein may be involved in repairing wounds and/or contributing to epidermal repair through a role in ESC migration.