Laminin peptide YIGSR enhances epidermal development of skin equivalents

https://doi.org/10.1016/j.jtv.2018.02.001Get rights and content

Highlights

  • YIGSR decreased TGF-β1 levels in CCD25-Sk cells.

  • YIGSR decreased the expression of involucrin and loricrin in HaCaT cells.

  • YIGSR increased levels of PCNA, p63, and integrin α6 in skin equivalent models.

  • YIGSR promotes the reconstruction of skin equivalents.

Abstract

Since the use of animal experimentation is restricted with regard to cosmetic materials, alternative in vitro models such as skin equivalents (SEs) are needed. Laminin is one of the major non-collagenous glycoproteins. The pentapeptide YIGSR (Tyr-Ile-Gly-Ser-Arg) is a functional motif of laminin that binds to the laminin receptor. In the present study, we examined whether YIGSR could improve the reconstruction of SEs. YIGSR has no effects on monolayer cell proliferation of CCD25-Sk fibroblasts or HaCaT keratinocytes. Interestingly, YIGSR decreased TGF-β1 levels, although it promoted type Ι collagen synthesis in CCD25-Sk cells. In HaCaT cells, YIGSR decreased the expression of involucrin and loricrin, which are differentiation markers. Furthermore, YIGSR increased levels of proliferating cell nuclear antigen (PCNA), p63, and integrin α6, and decreased involucrin in SE models. In addition, two models containing YIGSR (mixed with dermal equivalents or added into media) did not show any differences in expression levels of PCNA, p63, integrin α6, and involucrin. Therefore, YIGSR is a useful agent for reconstruction of SEs, independent of its method of application. These results indicate that YIGSR stimulates epidermal proliferation and basement membrane formation while inhibiting keratinocyte differentiation of SEs. Taken together, these results indicate that YIGSR promotes the reconstruction of SEs, potentially via decreased TGF-β1 levels and consequent inhibition of epidermal differentiation.

Introduction

As the cosmetic market has grown, interest in novel cosmetic materials has also increased. Previously, safety testing using animals was considered essential before releasing a product. However, the European Union banned animal testing of cosmetics because of animal-rights concerns. Consequently, alternative in vitro methods are needed. Various in vitro tests have been employed, including microbiological tests and cell culture tests using human keratinocytes or fibroblasts seeded in monolayers [1]. Recently, a skin equivalent (SE) containing a dermal equivalent (DE) has emerged as a useful alternative testing model. The DE is an artificial dermis of skin constructed by mixing fibroblasts and collagen gel. The SE is a form of artificial skin containing epidermal layers and dermis constructed by seeding human keratinocytes onto a DE to produce a fully differentiated epidermis [2]. The reconstruction of SEs also facilitates research in dermatology and skin pharmacology.

In human skin, the extracellular matrix (ECM) plays a pivotal role in regulating cell proliferation, migration, and adhesion in tissues. There are many different ECM proteins, such as collagen, fibronectin, elastin, and laminin, among others. Collagen type I accounts for approximately 25–30% of all proteins in the body, is the main component of dermal skin, and is the major protein comprising the ECM [3]. Collagen is synthesized in fibroblasts and is regulated by various factors [4]. Laminin is a major non-collagenous glycoprotein that acts as an important peptide component of base membranes, and promotes cell growth, migration, adhesion, and differentiation [[5], [6], [7]]. Laminin forms a cruciform structure composed of three chains, α, β1, and β2, which are linked covalently by disulfide bonds [8,9]. The pentapeptide YIGSR (Tyr-Ile-Gly-Ser-Arg) in the laminin β1 chain is a functional motif that binds to the laminin receptor [10]. YIGSR has been reported to facilitate cellular attachment and help bear mechanical stress in silicone membranes [11]. In a previous study, YIGSR was found to have effects on collagen synthesis in human dermal fibroblasts [12]. In the skin, the composition of the dermis can affect the epidermis. Therefore, in the present study, we examined whether YIGSR influences SEs.

Studies in many cell types have shown that proliferation and differentiation are inversely correlated processes [13,14]. Recent work has made it clear that cell proliferation and differentiation are regulated simultaneously but independently, that cells often start differentiating long before they stop dividing, and that activation of differentiation is not limited to any particular segment of the cell cycle [13]. Thus, we investigated the relationship between proliferation and differentiation in an SE model.

Section snippets

Materials

YIGSR peptides were obtained from ANYGEN Co. (Gwangju, Korea). Powdered Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 nutrient mixture (F-12) powder, l-ascorbic acid, formaldehyde, sodium bicarbonate, HEPES, hydrocortisone, isoproterenol, involucrin antibody (l9018), and insulin were obtained from Sigma (St. Louis, MO, USA). Recombinant human epidermal growth factor (EGF) and Fetal Bovine Serum (FBS) were purchased from Invitrogen Co. (Gibco, Camarillo, CA, USA). DMEM/Nutrient Mixture

YIGSR has no effect on monolayer cell proliferation

To examine the effects of YIGSR on cell proliferation, CCD25-Sk fibroblasts and HaCaT keratinocytes were treated with YIGSR at concentrations of 0–20 μM for 3 days. As shown in Fig. 1, YIGSR has no effect on monolayer cell proliferation of CCD25-Sk or HaCaT cells.

YIGSR increases collagen production in CCD25-Sk fibroblasts

YIGSR increases collagen synthesis [12]. To confirm that YIGSR is functional in CCD25-Sk cells, we checked whether YIGSR affects collagen production in these cells. As shown in Fig. 2A, YIGSR increased type Ι collagen levels.

Discussion

In this study, we observed that YIGSR promotes the reconstruction of SEs. However, YIGSR has no effect on the proliferation of human fibroblasts or human keratinocytes. Although YIGSR did not induce cellular proliferation in monolayer cultures, it did affect the differentiation of monolayer keratinocytes.

A recent paper reported that YIGSR enhances collagen synthesis in human dermal fibroblasts [12]. We also showed that the expression of type 1 collagen was increased by YIGSR treatment,

Conflicts of interest

The authors have no conflicts of interest to declare.

Acknowledgments

This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (Grant No. A103017).

References (20)

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