Effects of low molecular weight soybean peptide on mRNA and protein expression levels of differentiation markers in normal human epidermal keratinocytes

Abstract

Low molecular weight soybean peptide (LSP) was applied to normal human epidermal keratinocytes, and the results showed a significant increase in the gene expression levels of involucrin, transglutaminase, and profilaggrin. Filaggrin protein levels were also significantly higher. It is possible that LSP has an epidermal cell differentiation-promoting effect and may be able to regulate metabolism of the epidermis.

Key words:

• low molecular weight soybean peptide
• keratinocyte
• differentiation
• skin
• epidermis

The skin is composed of the epidermis and dermis, with epidermis being subdivided into the stratum corneum, the granular layer, the spinous layer, and the basal layer, in the order from the surface. Basal layer keratinocytes proliferate and differentiate by changing their morphology into cells that form each of these layers, finally becoming the enucleated corneocytes of the stratum corneum; the outermost layer of the stratum corneum then becomes dead skin and falls off. This manner of basal cell differentiation is how the skin is metabolized and renewed. The undifferentiated keratinocytes found in the basal layer produce keratin 5 (K5), which has a molecular weight of 58 kDa, and keratin 14 (K14), which has a molecular weight of 50 kDa. The stratum spinosum layer is composed of 5–10 layers of spinous cells. Like the stratum basal layer, keratin filament expression can be observed, but with pairing of keratin 1 (K1), which has a molecular weight of 67 kDa, and keratin 10 (K10), which has a molecular weight of 56.5 kDa.¹⁾ In addition, there is expression of involucrin, an envelope protein rich in glutamine and lysine.^2,3) The stratum granular layer is composed of two or three layers of granule cells that undergoes the flattening in contrast to the beneath layers of the spinous cells remaining in an intact shape. Loricrin, which is produced by the granule cells,⁴⁻⁶⁾ is cross-linked with involucrin by enzymes such as transglutaminase to form the marginal zone of the stratum corneum.²⁾ Inside the granule cells, there are keratohyalin granules, which include large amounts of profilaggrin, a precursor of filaggrin.⁶⁻⁸⁾ Filaggrin is broken down in the stratum corneum into amino acids; it is also known as the natural moisturizing factor (NMF), as it has a moisture retention function.⁹⁾ The stratum corneum is composed of about 15 layers of corneocytes, which are dead cells (corneocytes) that have been enucleated. The marginal zone described above is stable with respect to physical and chemical stimuli, and is largely responsible for the skin’s barrier function.¹⁰⁻¹²⁾ Because of this release of specific proteins during keratinocyte differentiation, measuring these proteins makes it possible to estimate the extent of cell differentiation.

Soybean peptide (SP) is a broken-down soybean protein. The average molecular weight of SP is about 3–10 kDa, and it is characterized to have a high glutamic acid content. Reported actions of SP include a cholesterol-inhibitory effect,¹³⁾ a weight loss effect,¹⁴⁾ an anti-fatigue effect,¹⁵⁾ and an antihypertensive effect.¹⁶⁾ Low molecular weight SP (LSP), which is a SP that has been further broken down, has an average molecular weight of about 500–1,000 Da; like SP, it has a high glutamic acid content.¹⁷⁾ About 60% of LSP are dipeptides and tripeptides, and it has been reported that di- and tripeptides are absorbed in the small intestine and elsewhere without being broken down.¹⁸⁾ We have previously reported that LSP promoted collagen production in fibroblast cells. In addition, LSP had a moisturizing effect on stratum corneum, transepidermal water loss, and viscoelasticity of the skin of hairless mice exposed to UVA irradiation. This prompted us to explore the potential of LSP as a candidate component of the cosmetic products.¹⁹⁾ In this manuscript, to establish how LSP affects keratinocyte differentiation, an important function in skin physiology, we applied LSP to keratinocytes and examined the gene expression levels of differentiation markers over time by quantitative real-time PCR, and examined protein levels by Western blotting.

LSP was provided by Fuji Oil Co., Ltd (HI-NUTE AM; Osaka, Japan). The LSP used contains large amounts of aspartic acid and glutamic acid, and numerous substances with a molecular weight of about 500–1,000 Da.¹⁷⁾ Normal human epidermal keratinocytes (NHEK) were purchased from Biopredic (Rennes, France). NHEK were cultured in HuMedia-KG2 obtained from Kurabo (Osaka, Japan), and were seeded (2.0 × 10⁵cells/dish) into 60-mm dishes. After culture for 1 day, cells were treated with a final concentration of 0.1 μg/mL LSP for 1, 3, or 9 days. mRNA expression was assayed by real-time PCR. Total RNA was isolated using RNAiso Plus (Takara Bio, Shiga, Japan) and purified with a PrimeScript^® RT reagent Kit (Takara Bio). cDNA was generated using a SYBR^® Premix Ex Taq^TM (Takara Bio). Primer sequences were as follows: GAPDH forward, 5ʹ-GAAGGTGAAGGTCGGAGT-3ʹ, and reverse, 5ʹ-GAAGATGGTGATGGGATTTC-3ʹ; K5 forward, 5ʹ-GAGCTGAGAAACATGCAGGA-3ʹ, and reverse, 5ʹ-TCTCAGCAGTGGTACGCTTG-3ʹ; K10 forward, 5ʹ-CCATCGATGACCTTAAAAATCAG-3ʹ, and reverse, 5ʹ-GCAGAGCTACCTCATTCTCATACTT-3ʹ; involucrin forward, 5ʹ-TGCCTGAGCAAGAATGTGAG-3ʹ, and reverse, 5ʹ-TTCCTCATGCTGTTCCCAGT-3ʹ; profilaggrin forward, 5ʹ-CCATCATGGATCTGCGTGG-3ʹ, and reverse, 5ʹ-CACGAGAGGAAGTCTCTGCGT-3ʹ; TGase forward, 5ʹ-TCTTCAAGAACCCCCTTCCC-3ʹ, and reverse, 5ʹ-TCTGTAACCCAGAGCCTTCGA-3ʹ. Real-time PCR was performed using the ABI PRISM^® 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). Protein levels were assayed by Western blotting. NHEK were washed and subsequently dissolved in lysis buffer solution (62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Samples were then run on 10% SDS-PAGE gels, blotted on polyvinylidene difluoride membranes, and immunodetected with antibodies against K10, involucrin, filaggrin, and β-actin, and with secondary anti-rabbit IgG-HRP-labeled antibodies. Detection was performed using a Lumino image analyzer LAS-1000 plus (Fujifilm Corp., Tokyo, Japan). Analysis was carried out with a MultiGauge (Fujifilm Corp., Tokyo, Japan).

The effects of LSP on viable cell number count were assessed among keratinocytes. To keratinocytes, 0.1 μg/mL LSP was added and viable cell number counts after 24 and 72 h of culture were obtained by trypan blue staining. The results showed that adding LSP did not change cell proliferation or viable cell number (data not shown). To keratinocytes, 0.1 μg/mL LSP was added and gene expression levels for K5, K10, involucrin, profilaggrin, and transglutaminase were measured by real-time PCR on the first, third, and ninth days. The gene expression level was analyzed by a Tukey’s multiple comparison test. The results of relative quantification where gene expression levels with no SP on the first day (first day control) are “1” showed no changes in gene expression levels for K5 and K10 on the first, third, and ninth day after LSP was added (Fig. 1(A) and (B)). Gene expression levels for involucrin on the ninth day after LSP exposure was significantly increased by 1.29-fold (p < 0.05) when compared to that of the control (Fig. 1(C)). Gene expression levels for profilaggrin and for transglutaminase showed significant increases by about 1.73-fold (p < 0.01) and about 1.61-fold (p < 0.01) that of the control on the ninth day, respectively (Fig. 1(D) and (E)). On the other hand, there were no changes in gene expression levels for involucrin, profilaggrin, and transglutaminase on the first and third day after the addition of LSP. To keratinocytes, 0.1 μg/mL LSP was added and protein levels for filaggrin after the ninth day were measured by Western blotting. The protein level of filaggrin was analyzed by a Student’s t-test. The results of relative quantification where gene expression levels with no SP were “1” showed that protein levels for filaggrin on the 9th day after LSP was added had increased by 2.3-fold (p < 0.01).

Fig. 1. Effects of LSP on mRNA Expression Level of Differentiating Gene in Normal Human Keratinocytes.

Note: NHEK were incubated with 0.1 μg/mL of LSP or medium (vehicle) and gene expression levels for K5 (A), K10 (B), Involucrin (C), Profilaggrin (D), and TGase (E) genes expression were measured by real-time PCR on the first, third and ninth day. The results of relative quantification where gene expression levels with no SP of 1st day (control on the 1st day) are “1”. The mRNA levels were normalized by dividing the quantity of GAPDH in each sample. Values are means ± SD of three experiments. *p < 0.05, **p < 0.01: compared to the control on the ninth day group. The statistical analysis was conducted using Tukey’s multiple statistical tests.

This manuscript examined the effects of LSP on differentiation marker expression in epidermal cells. We examined the effects on cell differentiation among keratinocytes. LSP (0.1 μg/mL) was added to epidermal cells and gene expression levels for differentiation markers after the first, third, and ninth day of culture were measured. In this manuscript, we applied and examined 0.1 μg/mL LCP, which increased a gene expression of COL1A1 for skin cells in a previous study.¹⁷⁾ The results showed that gene expression levels for all differentiation markers remained unchanged on the first and third day after LSP was added (Fig. 1). Nine days after LSP was added, however, the gene expression levels for involucrin, a late differentiation marker, showed an increase (Fig. 1(C)), while gene expression levels for profilaggrin and TGase showed significant increases (Fig. 1(D) and (E)). Protein levels for filaggrin also increased significantly (Fig. 2). No changes in cell number caused by LSP were observed (data not shown), for which LSP is believed to have an epidermal differentiation-promoting effect. It is possible that LSP maintains the balance between ceramide, NMF, and the like by activating turnover of the epidermis. As LSP did not have an effect on cell proliferation, it was assumed to affect only cell differentiation. Calcium, retinoic acid, vitamin D, and other substances have been reported to promote differentiation, but LSP contains none of these. Therefore, LSP itself was assumed to have induced differentiation.

Fig. 2. Effects of LSP on Filaggrin Protein Expression Level in Normal Human Keratinocytes.

Note: NHEK were incubated with 0.1 μg/mL of LSP or medium (vehicle) and the gene expression levels for filaggrin protein expression were measured by Western blotting on the ninth day. The results of relative quantification where protein expression levels with no SP of ninth day (control on the ninth day) are “1.” Values are means ± SD of three experiments. *p < 0.05: compared to the control on the ninth day, Student’s t-test.

At present, glutamine receptors, which are the transporters of the amino acids glutamine and glutamic acid, are understood to affect differentiation.²⁰⁾ One of the characteristics of LSP is its high glutamic acid content. It is thus possible that the glutamic acid present in LSP affects differentiation. In the reports given above, however, differentiation was inhibited by glutamic acid, in contrast to our results here. It is not known whether LSP acts as an amino acid or as a peptide. In addition, the mechanisms by which epidermal cell differentiation is promoted must be elucidated, and because 0.1 μg/mL LSP was added in this experiment, which has previously exhibited a type-I collagen production-promoting effect,¹⁷⁾ it is possible that this effect was not observed. In future, its usefulness as a cosmetic ingredient should be elucidated by examining additive concentrations or examining the use of amino acid mixtures formulated with high molecular weight SP or LSP amino acid compositions.

In conclusion, LSP has been shown to have an epidermal cell differentiation-promoting effect. Taking into account the type-I collagen production improvement as well as the repairing effect on the skin of hairless mice irradiated with UVA, one may conclude that LSP has the potential to regulate skin metabolism, and thus may become a material of great interest as a cosmetic additive.

Notes

Abbreviations: K1, keratin 1; K5, keratin 5; K10, keratin 10; K14, keratin 14; LSP, low molecular weight soybean peptide; NHEK, normal human epidermal keratinocytes; NMF, natural moisturizing factor; SP, soybean peptide; TEWL, transepidermal water loss; TGase, transglutaminase.