Vitamin B6 derivative improves skin hydration

It is well-known that VB6 is essential to maintain homeostasis due to its role in protein synthesis, amino acid and lipid metabolism. Focusing on the skin, many reports have shown that defects of VB6 initiate several diseases such as dermatitis seborrheica,1 epidermal thickening, and skin and mucosa lesions. In addition, topical application of VB6 to skin with those diseases actually improves their symptoms.2 Clinical knowledge strongly supports the possibility that VB6 plays an important role in maintaining epidermal function. In the cosmetic field, VB6 is usually used as an agent to treat skin roughness by physicians.

In our study, we found that VB6 up-regulates profilaggrin mRNA levels, and improves skin dryness. Those results support the efficacy of VB6 as skin roughness treatment. In addition, we found that a lipophilic ester of VB6, Pyridoxine Tris-Hexyldecanoate (VB6-IP), has improved stability against oxidation and heat and shows stronger effect in comparison with pure VB6.

Regarding the mechanism of action of VB6, some reports have been published as follows. In 1994, Trezise et al showed that pyridoxal-5-phosphate, a chemical analogue of VB6, acts as an antagonist of P2X receptors in the rat vagus nerve and vas deferea.3 P2X receptors, which are ATPgated ion channels, are expressed in the suprabasal, spinosum and granular layers of the epidermis. Furthermore, it has been reported that P2X receptors contribute to the regulation of epidermal differentiation by regulating intracellular calcium concentration.4 Those studies suggest the possibility that VB6 has an effect on profilaggrin mRNA levels via P2X receptors.

However, at the present time, the specific mechanism of VB6 has not been clarified. In this study, we characterised the effects of VB6 on levels of profilaggrin mRNA and filaggrin protein using human epidermal keratinocytes. Using reconstructed skin equivalents and human skin (in vivo) helped to identify the efficacy of VB6-IP. In addition, the results suggest that the P2X receptor contributes to filaggrin up-regulation by VB6.

Materials and methods

RT-PCR analysis of keratinocytespecific markers
Keratinocytes were seeded in 35 mm dishes with KG2 medium (KURABO) and were cultured for 4 days. After that, cells were cultured in KG2(-BPE) medium (KURABO) containing various concentrations of samples and were then further cultured for 48 hours. Total RNAs were extracted from keratinocytes using Trizol reagent following the manufacturer’s instructions. cDNA was synthesised using Superscript II (Invitrogen) following the manufacturer’s instructions. PCR was performed using Taq polymerase (Roche) and primer sets as shown in Table 1. PCR was performed according to the manufacturer’s protocol for each gene in an automated thermal cycler (Trio Thermoblock 48, Biometra).

Dot-blot (immunoblot) analysis of filaggrin
Keratinocytes were seeded in 96-well microplates with KG2 medium (KURABO) and cells were cultured for 24 hours. After that, cells were cultured in KG2(-BPE) medium (KURABO) containing various concentrations of samples and were then further cultured for 72 hours. Total protein was extracted by lysis in 0.5% Triton X-100 (Wako), then was blotted on Nitrocellulose membranes (BIO-RAD). Membranes were incubated with anti-human filaggrin specific antibody (ARGENE), and then were incubated with HISTOFINE simple stain PO(M) (Nichirei). Finally, Lumi-Light Western Blotting Substrate (Roche) was applied on the membranes and filaggrin was visualised using chemiluminescence.

Immunohistochemical staining for filaggrin
VB6 and VB6-IP (1.5 mM) were applied topically to reconstructed skin equivalents (TOYOBO). After 1 week of cultivation, frozen cross-sections of reconstructed skin equivalents were prepared. Tissue sections were incubated with anti-human filaggrin specific antibody (ARGENE), then with FITC-labelled mouse anti-IgG (ZYMED Laboratories). Finally, filaggrin was visualised using fluorescence microscopy.

Human use test (skin moisturisation)
The skin hydration effect of a gel containing 3% VB6-IP and a placebo gel were evaluated by the split face method. Both gels were applied to human volunteers (n=10) twice daily. After 7, 14 and 28 days of application, water content was evaluated by measuring skin-surface conductance (SKICON).

Results

Effect of VB6 on the expression of keratinocyte-specific markers of terminal differentiation
The effect of VB6 on the expression of keratinocyte-specific markers related to terminal differentiation was examined by RT-PCR. VB6 drastically increased profilaggrin mRNA expression whereas it had no effect on the expression of SPT, involucrin or K10 (Fig. 1).

Effect of VB6 on filaggrin protein levels
As shown in Figure 2, VB6 significantly increased filaggrin protein levels in keratinocytes (dot-blot analysis).

Effect of P2X antagonists on filaggrin protein levels
We evaluated the effect of several P2X antagonists, pyridoxal-phosphate-6- azophenyl-20,40-disulfonic acid (PPADS), 20,30-O-(2,4,6-trinitrophenyl)adenosine 50 –triphosphate (TNP-ATP) and suramin, on filaggrin protein levels using dot-blot analysis. As shown in Figure 3, all P2X antagonists increased filaggrin in dosedependent manners.

Oil-soluble VB6 derivative: Pyridoxine Tris-Hexyldecanoate (VB6-IP)
The effect of VB6-IP on the expression/ production of filaggrin was evaluated. As shown in Figures 4 and 5, VB6-IP significantly increased filaggrin expression/ production.

Immunohistochemical staining of filaggrin
To examine the potential use on human skin, we examined the effect of VB6-IP on filaggrin levels in reconstructed skin equivalents, as an alternative for actual human skin.

Topical application of VB6-IP on the three-dimensional epidermis model induced filaggrin expression more dramatically than pure VB6 (Fig. 6; staining represents filaggrin content).

In vivo test (skin moisturisation)
In general, it is well known that filaggrin is the precursor of natural moisturising factor (NMF). Thus, it is expected that skin water content should be enhanced by VB6-IP application. Effect of VB6-IP on actual skin moisturisation was evaluated in vivo. As shown in Figure 7, water content in skin treated with the VB6-IP gel formulation showed initial increase after 7 days and further significantly increased after 14 days. Therefore, we can suggest that increase in skin hydration is explained by higher NMF synthesis due to increase in fillagrin content.

Discussion

By examining the effects of VB6 on epidermal function, we found that VB6 significantly accelerates filaggrin expression/production. In general, it is known that filaggrin expression/production is closely associated with epidermal differentiation. However, VB6 did not affect the expression of other keratinocytespecific markers related to terminal differentiation. Therefore, it can be suggested that filaggrin expression enhanced by VB6 is independent of epidermal differentiation.

The P2X receptor might be involved in the effects of VB6 in the epidermis. Therefore, we examined the effects of three P2X receptor antagonists on filaggrin production and found that all of them accelerated filaggrin production. These results suggest that a novel pathway is associated with the function of the P2X receptor in VB6-specific filaggrin production.

In intact skin, filaggrin is converted into amino acids, a NMF, by aminopeptidase5,6 and carboxypeptidase.7 Since NMF is important to maintain skin water content, this suggests the possibility that the up-regulation of filaggrin levels leads to improved skin conditions.

However, VB6 is unstable against heat and oxidation, and has low skin penetration potential due to its hydrophilic nature. A new lipophilic derivative of VB6 (VB6-IP) also up-regulated filaggrin protein levels in cultured keratinocytes similar to VB6.

Furthermore, when applied on reconstructed skin equivalent, lipophilic VB6-IP accelerated filaggrin production in the cornified layer better than VB6. This result suggests that the lipophilic VB6-IP has superior skin penetration compared with VB6. In addition, the in vivo tests showed that VB6-IP effectively improved skin dryness by increasing NMF content in skin.

References

1 Schereiner A.W., Slinger W., Hawkins V.R., and Vilter R.W. Eborrheic dermatitis; a local metabolic defect involving VB6, J. Lab. Clin. Med., 40 (1952) 121-130.

2 Effersoe H. The effect of topical application of VB6 ointment on the rate of sebaceous secretion in patients with seborrheic dermatitis, Acta. Derm. Venereol., 34 (1954) 272-278.

3 Trezise D.J., Bell N.J., Khakh B.S., Michel A.D., and Humphrey P.A. P2 purinoceptor antagonist properties of pyridoxal-5-phosphate, Eur. J. Pharmacol., 259 (1994) 295-300.

4 Greig A.V.H., Linge C., Terenghi G., McGrouther A., and Burnstock G. Purinergic receptors are part of a functional signaling system for proliferation and differentiation of human epidermal keratinocytes, J. Invest. Dermatol., 120 (2003) 1007-1015.

5 Ito Y., Fukuyama K., Yabe K., and Epstein W.L. Purification and properties of aminoendopeptidase from rat epidermis, J. Invest. Dematol., 83 (1984) 265-269.

6 Boderke P., Merkle H.P., Cullander C., Ponec M., and Bodde H.E. Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy, J. Invest. Dermatol., 108 (1997) 83-86.

7 Kikuchi M., Fukuyama K., Hirayama K., and Epstein W.L. Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells, Biochim. Biophys. Acta., 991 (1989) 19-24.

ABSTRACT
In general, vitamin B6 (VB6) is used to treat skin roughness and acne. It is known that VB6 influences the terminal differentiation process of the epidermis. However, the precise mechanism of VB6 to improve skin roughness is still unclear. The study described in this article was designed to achieve two purposes: to identify the mechanism of VB6 activity in skin and to evaluate the potential of a new VB6 derivative, Pyridoxine Tris-Hexyldecanoate (VB6-IP).