The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

BACKGROUND AND PURPOSE: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. EXPERIMENTAL APPROACH: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. KEY RESULTS: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. CONCLUSION AND IMPLICATIONS: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.

Random/aligned electrospun PCL fibrous matrices with modified surface textures: Characterization and interactions with dermal fibroblasts and keratinocytes.

Micro- or nano-surface topography of a biomaterial can improve various cellular activities for obtaining functional tissues. Electrospun fibers can gain further functionality when introduced topographic details to their surfaces. In this regard, we produced random and aligned polycaprolactone (PCL) micron/submicron fibers by the electrospinning method. Simultaneously, the surface structure of the fibers was altered by applying phase separation processes including non-solvent-induced phase separation (NIPS) and vapor-induced phase separation (VIPS) mechanisms. As a result, PCL fibers with porous, wrinkled, grooved, and crater-like morphology were obtained. Human dermal fibroblasts (BJ cells) and human keratinocytes (HS2) were cultured onto the fiber surfaces and the data were evaluated in terms of cell-material interactions. Results showed that not only the orientation of fibers but also fiber topography affected both cell-fiber and cell-cell interactions in different manners. It was observed that the wrinkled topography is the most suitable for both dermal fibroblasts and keratinocytes in terms of cell attachment and proliferation. We also concluded that cellular behavior was varied according to the morphology of the cells used. Morphological observations showed that HS2 cells proliferated more intensively on all surfaces compared to BJ cells. All these findings can be evaluated in terms of the design of tissue scaffolds, especially in skin tissue engineering.