Introduction

Human body can sense a wide array of mechanical stimuli and our sense of touch can distinguish between a soft breeze floating over skin and a painful pinch, or detect the stretch of a muscle or even blood pressure. The ability to sense these different stimuli is due to their specific recognition by mechanoreceptors present on cell membrane that convert them in electrochemical signals exciting cell membranes or triggering calcium signaling. Two related proteins Piezo1 and Piezo2, evolutionally conserved in animals, plants, and protozoa, are essential components of mechanically activated ion channel. In particular, in animals Piezo2 was recently identified in Merkel cells, the skin cells specialized in mechano-sensation whereas Piezo1 is expressed in non-neuronal cells, like endothelial ones that respond to forces such as shear and osmotic force. In plants Piezo1, an ortholog of mammalian Piezo, is required for root penetration and the mutants not expressing Piezo1 have impaired primary root growth into harder media.

Aim of the paper

Human skin is a multifaceted mechanoresponsive interface and considering the central role of Piezo1 and Piezo2 in this process, the aim of this work was the development of innovative and reliable systems based on skin and plant 3D models to identify natural products able to regulate Piezo1 and Piezo2 receptors.
Methods: Gene expression analysis in human cell lines; Calcium assay in human cell lines expressing endogenously Piezo receptors or transfected ones; cloning of PIEZOs in expression vectors for mammalian cell lines and plants; Atomic Force Microscopy; differentiation of SH-SY5Y cells and co-culture with skin explants; Immunofluorescence; plant transformation; phenotypic analysis of root growth.

Results

To determine the activation of Piezo1 and Piezo2 in mammalian skin cell lines we assessed the expression of the two genes in cell lines representing the main skin cell populations. Consistent with the literature, our data show that Piezo1 was expressed in HUVEC, HDF and HaCaT, whereas Piezo2 was mainly expressed in Merkel cells. We showed that the treatment of Piezo1-expressing cells with Yoda1, a known activator of Piezo1, led to an increase in calcium influx into the cells and this effect was completely abolished in the presence of GsMTX4, which inhibits mechanoreceptors. In order to have a screening platform to find natural regulators of Piezo 1 and Piezo2 we cloned their cDNA sequences in a mammalian expression vector and introduced them in HEK293 cells, lacking endogenous Piezo receptors. We demonstrated that the cells transfected with Piezo1 robustly respond to Yoda stimulation, indicating the ability to modulate the receptor. Again, this effect was abolished in the presence of GsMTx4. On the contrary, cells transfected with Piezo2 expressing vector did not respond to Yoda treatment and were mechanically stimulated with Atomic Force Microscopy.
To set up a model resembling the real human skin, we generated reinnervated skin in vitro using a skin biopsy punch co-cultured with neuronal cells. To this aim, we differentiated the neuroblastoma cell line SH-SY5Y into mature neuron-like cells by using retinoic acid and confirmed the phenotype through quantification of beta-III tubulin, a neuronal marker. We then co-cultured these cells with skin explants. To monitor the activation of Piezo2 receptor, we used neuronal cells transfected with a calcium reporter construct and we demonstrated that when the skin explant was mechanically stimulated with AFM cantilever, the calcium reporter was activated, suggesting that the model of reinnervated skin was functional. In order to monitor Piezo1 regulation in plant system we employed Piezo1 Knock out Arabidopsis plants further transformed with human Piezo1 and studied for its root penetrating properties. We revealed that treating these transformed plants with Yoda1 completely reversed their impaired root phenotype. With this new system we were able to monitor the regulation of human piezo 1 in plant following an easily measurable phenotype such as root penetration.

Discussion and conclusions

This study describes comprehensive and innovative 3D models to monitor the regulation of Piezo1 and Piezo2 and their output in skin system and plant roots. We generated a model of reinnervated skin that functions as a biosensor for Piezo2 activation and established an innovative plant system that, following a simple phenotype like root elongation, can provide new insights into Piezo regulation.