When the skin is injured, the keratinocytes that cover the skin's surface are scraped away, exposing the sensitive tissue, which causes pain and bleeding. It is known that when wound healing, keratinocytes remaining around the wound proliferate and migrate to re-cover the wound area. It has been known that TRPV3, one of the thermosensitive TRP channels, is involved in proliferation and migration of keratinocytes. However, it was not well understood how TRPV3 regulates keratinocytes functions. We had previously found that calcium influx through TRPV4 activates a ANO1, a calcium-activated chloride channel, which is thought to lead to the secretion of cerebrospinal fluid in choroid plexus epithelial cells. Other epithelial cells were also reported to have both TRPV4 and ANO1 channels. For instance, these ion channels are co-expressed by both salivary and lacrimal gland acinar cells, and saliva and tear secretions could be accelerated by functional interaction between TRPV4 and ANO1. Thus, TRP channel/ ANO1 complexes have distinct functions even though each TRP channel works independently.
TRPV3 is a warmth-sensitive TRP channel, and calcium permeability is approximately ten times higher than that of sodium. Although TRPV3 is reportedly expressed throughout the body, its physiological significance is not well understood. Previous studies showed that TRPV3 contributed to itch, warmth sensation and wound healing in keratinocytes. ANO1 is reportedly a carcinoma-related gene, and a recent study revealed that ANO1 is involved in the proliferation of prostate epithelial cells in benign prostatic hyperplasia as well as in HaCaT cells, a special cell line of keratinocytes. In addition, ANO1 inhibition reduced cell migration in some cancer cells. However, the physiological function of ANO1 in normal skin keratinocytes is not clear even though many epithelial cells express it. Therefore, we investigated interaction of TRPV3 and ANO1, and involvement of these channels in wound healing. We performed whole-cell patch-clamp experiments using normal human epidermal keratinocytes (NHEK), and TRPV3 activator-induced chloride currents were observed. In addition, the chloride currents were inhibited by ANO1 inhibitors. These results indicate that TRPV3-ANO1 interaction in NHEKs.
Furthermore, to investigate the involvement of TRPV3 and ANO1 in wound healing, we analyzed the effects of TRPV3 or ANO1 inhibitor on in vitro wound healing model called culture insert assay. Cell migration and/or proliferation were significantly inhibited by of a TRPV3 inhibitor and ANO1 inhibitors (Figure 1), but not by a TRPV4 inhibitor. The fact that cell migration and/or proliferation were reduced by both TRPV3 and ANO1 inhibitors, strongly indicated that TRPV3-ANO1 interaction is involved in the migration and/or proliferation of NHEKs. In addition, these results suggested the importance of chloride ions for cell migration and proliferation. Therefore, we performed culture insert assay in a low chloride medium. Cell migration and/or proliferation were drastically reduced in the low chloride-containing medium, an effect that was lost after the change back to the control medium. These results indicated that chloride flux through ANO1 plays critical roles in cell migration and/or proliferation.
Although the previous results suggested the importance of chloride ions for cell migration and/or proliferation, the actual roles of chloride ions in keratinocytes are largely unknown. To address this question, we attempted to determine the direction of chloride movement. Chloride permeation through chloride channels depends on intracellular chloride concentrations and membrane potentials. Therefore, we performed chloride-imaging experiments using the chloride indicator. The calculated intracellular chloride concentrations of NHEKs were relatively low (6.8 ± 1.3 mM). Because the resting membrane potentials of skin keratinocytes are reported to range from −24 to −40 mV, ANO1 opening should induce chloride influx and its inhibition likely decreases intracellular chloride ions within keratinocytes.
All of our data suggest that the chloride influx induced by TPRV3 activation is important mechanism for wound healing. The importance of chloride ions in a therapeutic setting has not been emphasized. Our analysis of TRPV3-ANO1 interaction represents a promising starting point for future investigations into the detailed mechanisms underlying intracellular chloride concentrations. Furthermore, ANO1 activators could be candidates for wound healing agent. Thus, our study could shed light on the importance of chloride ions in skin homeostasis.
This work was supported by grants to M.T. from a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (#21H02667 and #20H05768; Scientific Research on Innovative Areas “Thermal Biology”).
Please read our original article published in Communications Biology to learn more.