Effects of an Amino acid Deficiency on Hyaluronan Synthesis in Human Dermal Fibroblasts

Abstract

Molecules involved in skin function are greatly affected by nutritional conditions. However, the mechanism linking amino acid (AA)s with these alterations is not well understood. We examined the effects of an AA deficiency on hyaluronan synthesis in human dermal fibroblasts. A deficiency of all AAs or of valine, leucine, isoleucine, threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, or glutamine significantly decreased hyaluronan levels in the culture supernatant. The deficiencies of all AAs, valine, leucine, cysteine or glutamine tended to decrease hyaluronan synthase 2 mRNA levels in human dermal fibroblasts. The present study would contribute to the elucidation of the mechanisms underlying the effects of AAs on skin function.

Introduction

Hyaluronan plays an important role in the formation of scaffolds to promote tissue repair and in cellular proliferation and migration in skin (Saavalainen et al., 2005). In mammals, hyaluronan is synthesized by isoforms of hyaluronan synthase (HAS), namely, HAS1, HAS2, and HAS3; these synthases are products of distinct genes that differ in tissue distribution. For example, HAS2 is the major producer of hyaluronan in the dermis, whereas HAS3 is the major hyaluronan producer in the epidermis. Hyaluronan synthesis along with HAS2 transcripts is greatly affected by nutritional conditions.

We previously demonstrated that hyaluronan synthesis was down-regulated by a low quality of dietary protein, such as gluten (Oishi et al., 2003). The quality of dietary protein can be estimated from the amino acid (AA) composition (van den Borne et al., 2012). AAs have been traditionally classified as nutritionally essential or nonessential for animals and humans. Essential AAs [valine (Val), leucine (Leu), isoleucine (Ile), threonine (Thr), methionine (Met), phenylalanine (Phe), tryptophan (Trp), lysine (Lys), arginine (Arg), histidine (His), and proline (Pro)] are not synthesized by animal cells and, therefore, need to be provided from the diet. In contrast, nonessential AAs [glycine (Gly), alanine (Ala), serine (Ser), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamate (Glu), glutamine (Gln), and aspartic acid (Asp)] are synthesized de novo. Some researchers have shown that AAs in the diet influenced the homeostasis of skin components. Murakami et al. (2013) have reported that the combination of branched-chain AAs and Gln is a key factor for the enhancement of skin collagen synthesis in protein-malnourished rats. Bellon et al. (1995) demonstrated that Gln increases procollagen mRNA levels in human fibroblasts. However, few studies have focused on the effects of AAs on hyaluronan synthesis. In the present study, we examined the effects of an AA deficiency on hyaluronan synthesis in human dermal fibroblasts (HDF).

Materials and Methods

Cell Culture and Treatment    Human dermal fibroblastic cell line, WS1, was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in modified Eagle's medium (MEM) containing 10% (v/v) fetal bovine serum (JRH Biosciences, Lenexa, KS), 1% (v/v) MEM non-essential AA solution, and 1% (v/v) sodium pyruvate in a humidified atmosphere of 5% CO₂ at 37°C.

Quantification of Hyaluronan    WS1 cells were seeded in 24-well plates in the culture medium at a density of 4.0 × 10⁴ cells/well and were incubated for 24 h, followed by serum starvation for 48 h. The culture supernatant fraction was collected after 24 h of incubation in an AA-deficient medium. We prepared the test media by adding each AA on the basis of the concentration of each AA in MEM containing 1% (v/v) MEM non-essential AA solution and 1% (v/v) sodium pyruvate. The level of hyaluronan in the culture supernatant fraction was measured with the QnE hyaluronic acid ELISA assay (Biotech Trading Partners, Encinitas, CA) according to the manufacturer's instructions.

RNA Extraction and Quantitative PCR    WS1 cells were cultured in an AA-deficient medium for 4 h. Following the treatment, total RNA was extracted according to the method reported by Chomczynski and Sacchi (1987) and cDNA was prepared with the ReverTra Ace^® qPCR RT Master Mix (Toyobo, Co., Ltd., Japan) according to the manufacturer's instructions. The amplification products of HAS2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected using THUNDERBIRD™ Probe qPCR Mix (Toyobo Co., Ltd., Japan) and TaqMan Gene Expression Assays kits. HAS2 mRNA levels were measured by quantitative PCR using an ABI Prism 7300 apparatus (Applied Biosystems, Foster City, CA) and expressed as values relative to that of GAPDH. The sequences of primers were as follows: HAS2 forward, 5′-GCTTGACCCAGCCTCATCTG-3′, HAS2 reverse, 5′-GAGGAATGAGATCCCAGGAATCGT-3′ and HAS2 probe, FAM-GGAGGTGTTGGGGGAGATGTCCAGA-TAMRA (GenBank accession no.: NM_005328). Amplifications were performed under the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C.

Statistical Analysis    Results are expressed as the mean ± SD. The mean of multiple groups were compared using a one-way ANOVA, followed by the post hoc Dunnett test. A value of p < 0.05 was considered statistically significant.

Results

Effects of each AA deficiency on the level of hyaluronan and HAS2 mRNA expression in WS1 cells    Fig. 1A shows the effect of each AA deficiency on hyaluronan levels in the culture supernatant. The deficiency of all AAs, or of Ile, Leu, Lys, Met, Cys, Phe, Tyr, Thr, Trp, Val, His, Arg, or Gln significantly decreased hyaluronan levels. Fig. 1B shows the effect of each AA deficiency on HAS2 mRNA expression in WS1 cells. The deficiency of all AAs or of Leu, Cys, Val, or Gln tended to decrease HAS2 mRNA levels.

Fig. 1.

Effects of AA deficiency on the level of hyaluronan and HAS2 mRNA expression in WS1 cells. The amount of hyaluronan in the cell culture supernatant (A) was measured using the QnE hyaluronic acid ELISA assay. The levels of HAS2 mRNA in WS1 cells (B) was measured by quantitative PCR and expressed relative to GAPDH. Bars are shown as the mean ± SD (n = 3, 4). *p < 0.05 indicates values that are significantly different from the control.

Discussion

Hyaluronan retains moisture in the skin, facilitates the transport of ion solutes and nutrients, and promotes wound healing (Saavalainen et al., 2005). HAS2 is the major producer of hyaluronan in the dermis (Saavalainen et al., 2005). Some researchers have reported that AAs might have multiple effects on skin functions (Murakami et al., 2012, 2013). However, the mechanism linking AAs with hyaluronan synthesis is not well understood.

Ile, Leu, Lys, Met, Cys, Phe, Thr, Trp, Val, His, or Gln deficiencies resulted in a pronounced decrease in hyaluronan production in WS1 cells. Our results showed that the deficiency of all AAs, or of Leu, Cys, Val, or Gln tended to decrease hyaluronan synthesis along with HAS2 transcription. Corsetti et al. (2010) reported that AA mixtures that included Leu, Pro, Lys, and Gly improved wound healing associated with the modulation of nitric oxide synthase and transforming growth factor-β1. No significant differences in mRNA levels of transforming growth factor-β1, which enhances HAS2 mRNA expression, were observed (data not shown). Therefore, it is likely that cooperative effects of AAs on skin function also play an important role in hyaluronan production in HDF. The detailed mechanism for the down-regulation of HAS2 gene expression by these AA deficiencies will be clarified by future studies. In WS1 cells, the deficiency of Ile, Lys, Met, Phe, Tyr, Thr, Trp, His, or Arg significantly decreased hyaluronan levels, whereas HAS2 mRNA expression was unaffected. The deficiency of Ile, Lys, Met, Phe, Tyr, Thr, Trp, His, or Arg had no effect on HAS2 mRNA levels in dermal fibroblasts. These AAs may be potent regulators of hyaluronan-degrading enzymes.

In summary, this study provides the first evidence of negative effects of an AA deficiency on hyaluronan synthesis in HDF. Each essential AA deficiency resulted in a pronounced decrease in hyaluron production in WS1 cells. In our previous study, we revealed that the expression of the HAS2 gene was highly sensitive to the quantity and quality of the dietary protein (Oishi et al., 2003). Taken together, our results suggest that such dietary patterns may lead to skin dysfunction and various cutaneous diseases. We believe that our findings lead to a new hypothesis whereby functional AAs regulate metabolic pathways to improve skin health.

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