We previously demonstrated that a series of neural receptors, those play a crucial role in nerve system, also expressed in epidermal keratinocytes. We also demonstrated that topical application of glycine on hairless mice skin after the barrier disruption also accelerated the barrier recovery and it was blocked by the strychnine. Glycine is known as one of the most important inhibitory neurotransmitters in the mammalian central nervous system. Thus, we hypothesized that glycine receptor also functionally expressed in the epidermal keratinocytes. In the present study, we first studied the expression of glycine receptor message in cultured human keratinocytes. Then we demonstrate for the first time the existence of a functional receptor with electrophysiological properties of glycine receptors in cultured human epidermal keratinocytes. Finally, we demonstrated immune‐histochemical study against anti‐glycine receptor subunits in human skin. Results of the present study might indicate new target of the clinical dermatology.

Background Epidermal keratinocytes express multiple receptors known to play crucial roles in the nervous system 1. Neuronal excitation and inhibition are associated with influx of calcium and chloride ions, respectively, into neurons. Activation of excitation receptors in cultured human keratinocytes similarly induces calcium ion influx, and topical application of receptor agonists on barrier‐disrupted hairless mouse skin delays barrier recovery 1. Cultured human keratinocytes also express GABA(A) as an inhibitory receptor, and its activation induces chloride ion influx. Topical application of GABA(A) agonists on barrier‐disrupted hairless mouse skin accelerates barrier recovery 1. We previously found that topical application of glycine on barrier‐disrupted hairless mouse skin also accelerated barrier recovery, and this effect was blocked by strychnine 2. Glycine is one of the most important inhibitory neurotransmitters in the mammalian central nervous system 3. These results suggested that glycine receptor (GlyR) might also be expressed in epidermal keratinocytes and play a role in epidermal permeability barrier homeostasis. GlyR is an anion‐permeable channel protein, which is composed of four different α‐subunits, α1‐4, and one β‐subunit (s1). The inhibitory actions of glycine are chloride dependent and are competitively antagonized by the alkaloid strychnine (s2).

Question addressed Is GlyR functionally expressed in epidermal keratinocytes?

Experimental design To test the hypothesis, we first evaluated the expression of GlyR subunit mRNAs in cultured human keratinocytes, using RT‐PCR. We then confirmed functional activity of the receptor by examining the electrophysiological properties of cultured human epidermal keratinocytes. Finally, immunohistochemical staining with anti‐GlyR subunit antibodies was carried out to confirm the presence of the receptor proteins in human skin.

Results First, using RT‐PCR, we detected a subset of GlyR subunits, α1, α2, α3, α4 and β, in undifferentiated and differentiated cultured human keratinocytes (Fig. 1a). α2, α4 and β mRNAs were clearly expressed in both types of keratinocytes, while α1 and α3 mRNAs were weakly expressed in both. Next, a patch‐clamp study showed that glycine (10 μm–1 m) dose dependently evoked inward currents in human epidermal keratinocytes (Fig. 1b). The reversal potential for glycine current was −20 mV. Normalized dose–response curves indicated that the concentration of glycine required for generating a half‐maximal response was 20 mm (data not shown). β‐Alanine and taurine, endogenous GlyR agonists 4, also evoked inward currents, although millimolar concentrations were required (Fig. 1c,d). The response to glycine (10 mm) was inhibited by strychnine (100 nm, 1 μm and 10 μm; 80.0 ± 24.9%, 74.0 ±30.3%, 37.8 ± 8.0%; n = 3–9, Fig. 1e). This concentration of 10 μm strychnine is extremely high compared to the IC 50 value reported for strychnine‐sensitive GlyR 4. However, it should be noted that GlyR α1 subunit but not α2 subunit is highly expressed in adult spinal cord and brainstem, while the α2 subunit is highly expressed in foetal brain and spinal cord, and then its expression decreases postnatally 5. We used neonatal keratinocytes, in which GlyR α2 has a lower sensitivity to strychnine 6. We also examined the effects of picrotoxinin, a chloride channel blocker. Glycine‐evoked current was inhibited by a high concentration of picrotoxinin (1 mm; 36.0 ± 27.0%; n = 5, Fig. 1e). The low sensitivity to the inhibitory effect of picrotoxinin indicates that keratinocyte GlyR is probably composed of heteromeric α/β subunits (7, s3, s4). Figure 1 Open in figure viewer PowerPoint (a) Results of RT‐PCR study. Expression of GlyR subunit α1, α2, α3, α4 and β mRNAs was detected in undifferentiated and differentiated cultured human keratinocytes (a). ‘Diff.’ meant differentiated keratinocytes, and ‘Undiff.’ meant undifferentiated keratinocytes. Responses of human epidermal keratinocytes to GlyR agonists and antagonists in patch‐clamp experiments are shown in b–e. (b), Representative responses evoked by glycine (1 m m and 100 m m ) at a holding potential of −30 mV. Bar graphs show dose–response relationships for glycine‐evoked current (b), β‐alanine‐evoked current (c) and taurine‐evoked current (d). (e), Concentration dependency of inhibitory effect of strychnine (100 n m –10 μ m ) or picrotoxinin (100 μ m –1 m m ) on glycine‐evoked current. The cells were stimulated with 10 m m glycine for 20 s. Antagonists were applied 30 s before glycine stimulation. Peak amplitude of glycine‐evoked current in the presence of antagonists was normalized to the current before antagonist application. Asterisks indicate statistically significant differences from the control (10 m m glycine, alone). Columns and bars represent mean ± SD of 3–9 cells. The immunoreactivity of human epidermis to anti‐GlyR α2 subunit and anti‐GlyR β subunit antibodies is shown in Fig. 2a–h. Immunoreactivity to anti‐α2 antibody was observed in the uppermost layer of human epidermis (antibody: Fig. 2a, merged image with Dapi: Fig. 2b, and Nomarski image: Fig. 2c). The immunoreactivity was blocked by a blocking peptide (Fig. 2d). Immunoreactivity to anti‐β antibody was observed throughout human epidermis (antibody: Fig. 2e, merged image with Dapi: Fig. 2f, and Nomarski image: Fig. 2g). The immunoreactivity was blocked by a blocking peptide (Fig. 2h). Immunoreactivity against anti‐α1 and anti‐α3 antibodies could not be clearly observed (data not shown). We did not conduct immunohistochemical study with anti‐GlyR α4, because GlyR α4 gene is reported to be a pseudogene in humans (s5), and there is little evidence for its functional expression (s6). Figure 2 Open in figure viewer PowerPoint Immunostaining of GlyR subunits in human epidermis. Immunoreactivity to anti‐GlyR α2 subunit antibody was observed in the uppermost layer of human epidermis (a–c), while immunoreactivity to anti‐GlyR β subunit antibody was observed throughout human epidermis (e–g). Antibody staining: a, e; merged images with Dapi: b, f; and Nomarski images: c, g. Immunoreactivity was blocked by appropriate blocking peptides (d, h, respectively). Bars show 50 μm.

Conclusion We detected expression of α1, α2, α3, α4 and β GlyR subunit mRNAs in cultured human keratinocytes, and patch‐clump experiments showed that inward current was induced in undifferentiated keratinocytes in response to three GlyR agonists. Glycine‐induced current was partially blocked in the presence of GlyR antagonists. These results suggested that GlyR is functionally active in human epidermal keratinocytes. Immunohistochemical study of human epidermis suggested that GlyR α2 subunit was localized in the stratum granulosum of epidermis. However, differentiated keratinocytes in the stratum granulosum form a cornified envelope around their surface, so that it is difficult to carry out patch‐clump experiments, and we could not establish whether GlyR is functionally active at the uppermost layer of the epidermis. RT‐PCR study of cultured keratinocytes indicated that α2 mRNA was similarly expressed in undifferentiated and differentiated keratinocytes. However, various factor(s) might influence expression of α2 at the protein level in human epidermis. Cornified envelope precursors, S100 proteins, play an important role in keratinocytes (s7), and calmodulin‐like protein is increased in atopic dermatitis (s8). It would be interesting to examine whether there is any relationship between GlyR and these proteins. We previously demonstrated that topical application of glycine on barrier‐disrupted hairless mouse skin accelerated barrier recovery, and this effect was blocked by co‐application of strychnine 2. Those results, together with the present findings that GlyR is expressed and functionally active in keratinocytes, suggest that GlyR might have an important role in epidermal permeability barrier homeostasis. Glycine, alanine and serine are major free amino acids in the stratum corneum 8, and we showed that the amount of free amino acids in stratum corneum is decreased in artificially induced dry skin 9. Thus, these free amino acids in healthy stratum corneum might be endogenous ligands of GlyR, modulating barrier homeostasis. We consider that GlyR might be available as a target for new drug development in the field of clinical dermatology.

Acknowledgement We appreciate the valuable advice of Dr. Thosii Iida. KI and KT carried out RT‐PCR, KI carried out patch‐clamp, and MD carried out immunohistochemical study. KI and MD wrote the paper.

Conflict of interest The authors have declared no conflicting interests.

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