日本香粧品学会誌–5（2013）of HAS and COL1A1 mRNA by HA4 in NHDFs
Vol. 37, No. 1, pp. 1Expression

1〈Regular Article〉

Expression of Hyaluronan Synthase and Collagen Type I mRNA byHyaluronan Tetrasaccharides in Normal Human Dermal Fibroblasts
Madoka KAGE1, Yoshihiro TOKUDOME1, Yukiko MATSUNAGA2,Takeshi HARIYA2, Fumie HASHIMOTO *, 1
(Accepted: December 3, 2012)

Abstract
The major compounds in the dermis are hyaluronan and type I collagen, which decrease with aging, UV and various
other factors. The loss of hyaluronan and collagen with aging is associated with increased dehydration and wrinkling of theskin. We aimed to investigate the inﬂuence of hyaluronan tetrasaccharide (HA4) on regulation of high molecular weight hyaluronan (HA) and collagen synthesis in normal human dermal ﬁbroblasts (NHDFs). Expression of hyaluronan synthase(HAS) 1–3 and collagen (COL) 1A1 mRNA were evaluated by quantitative real-time PCR. HAS1 mRNA expression was
found to be increased in NHDFs treated with HA and HA4. In addition, it was observed that NHDFs co-cultured with normal human epidermal keratinocytes (NHEKs) showed up-regulation of HAS1 mRNA expression, as compared with NHDFs
single cell culture treated with HA4. Treatment of NHDFs with HA4 ＋ derivatized vitamin C (VC-PMg) signiﬁcantly increased COL1A1 mRNA expression. In this study, we conﬁrmed that HA4 affected HAS1 and COL1A1 mRNA expression;
thus, HA4 application may show various action in skin.
Key words:

1.

hyaluronan tetrasaccharide, hyaluronan synthase, collagen, mRNA.

Introduction

Hyaluronan, which is composed of repeated β-1,4-glucuronic acid-β-1,3-N-acetylglucosamine disaccharide units, is a
non-sulfated glycosaminoglycan with a molecular weight ofover 1,000 kDa. Hyaluronan is abundant in the extracellularmatrix (ECM) of the skin, and is involved in many biologicalprocesses such as tissue homeostasis, cell proliferation, cellmigration, cell differentiation, angiogenesis, tumor biology
and repair processes by interacting with various proteins1). In
addition, because of physical properties such as viscoelasticity, hydrophilicity and extensibility, hyaluronan seems to play
an important role as a space ﬁller, osmotic buffer, plasma protein sieve and lubricant2). In the skin, hyaluronan synthases(HAS) are synthesized by dermal ﬁbroblasts and epidermal
keratinocytes. Synthesis of hyaluronan is accomplished bythree HAS (HAS1, HAS2 and HAS3), which produce different sizes of polysaccharide chains with average molecularweights of about 3×105 to 2×106 Da for HAS1 and HAS2,
and about 2×105 to 3×105 Da for HAS33).
Collagen is a major component of ECM, and type I collagen accounts for approximately 80％ of the total collagen in
adult human dermis4). Type I collagen is a product of twogenes, α1 (I) (COL1A1) and α2 (I) (COL1A2), which are coordinately regulated5). The major structural protein in the dermis is type I collagen, which comprises 90％ of the dry weight–of skin and diminishes with normal aging6) 8). Collagen plays
a role in cell adhesion, and is important for maintaining nor-mal tissue architecture and function.
Stimulation of living tissue by hyaluronan appears to depend on various factors, including hyaluronan chain length.
For example, high molecular weight hyaluronan was demonstrated to suppress angiogenesis9) and inﬂammation10), whereas low molecular weight hyaluronan stimulated induction ofangiogenesis11), inﬂammation12) and expression heat shockprotein 7213). In addition, injection of cross-linked hyaluronan
stimulates collagen synthesis, partially restoring dermal matrix components that are lost in photo-damaged skin14). In thisway, hyaluronan possesses numerous functions, but little is
known about the effects of hyaluronan oligosaccharides onskin.
In this study, we aimed to investigating the inﬂuence ofhigh molecular weight hyaluronan (HA) and hyaluronan tetrasaccharide (HA4) on regulation of HASs and COL1A1mRNA expression in normal human dermal ﬁbroblasts(NHDFs).
2.

Materials and Methods

2–1. Materials
HA4 (99.14％) (776.3 Da) was provided by Glycoscience
Laboratories Inc. (Tokyo, Japan). HA (＞1,200 kDa) produced by a Streptococcus zooepidemicus was used. All otherchemicals and solvents were of analytical grade. Abdominal
skin origin normal human dermal ﬁbroblasts (NHDFs) werepurchased from Kurabo (Osaka, Japan). Abdominal skin origin normal human epidermal keratinocytes (NHEKs) were*, 1 Faculty of Pharmaceutical Sciences, Josai University (1–1 Keyaki- 2 SHISEIDO Co., Ltd., Research Center
dai, Sakado, Saitama 350–0295, Japan)
連絡先 E-mail: hasimoto@josai.ac.jp