Effects of GABA on the expression of type I collagen gene in normal human dermal fibroblasts

Abstract

We examined the effects of GABA on type I collagen gene expression in normal human dermal fibroblasts. Real-time PCR analysis indicated GABA increased the level of type I collagen transcripts, and suppressed the expression of matrix metalloproteinase-1, which is a collagen-degrading enzyme. These results suggest GABA improves the skin elasticity by regulating type I collagen expression.

Key words:

• GABA
• collagen
• MMP-1
• normal human dermal fibroblasts

Shochu is a traditional Japanese alcoholic beverage that is produced by fermentation from barley, rice, sweet potato and other crops. During the shochu distillation process, a large amount of shochu distillery byproducts (SDB) is generated as a distillery wastewater. SDB contains large amounts of organic matter, such as protein, peptide, amino acids and organic acids. SDB has a growth—stimulative effect on lactic acid bacteria and bifidobacteria.¹⁾ Therefore SDB increases the productivity of several functional substance by bacterial or fungal fermentation, such as protease,²⁾ amino acids,³⁾ polyphenols,⁴⁾ polyunsaturated fatty acids,⁵⁾ and bacteriocin.⁶⁾ Among them, γ-aminobutyric acid (GABA) is one kind of amino acid synthesized by decarboxylation of glutamate that is widely distributed in plants, micro-organism and animals. In mammals, GABA acts as the major inhibitory neurotransmitter in the central nervous system,⁷⁾ and the oral administration of GABA improves the psychiatric conditions such as, stress,⁸⁾ depress⁹⁾ and sleeplessness.¹⁰⁾ Moreover, GABA has the multiple physiological functions, for example, hypotensive effects,¹¹⁾ prevention of obesity and diabetes,¹²⁾ and enhancement of immunity.¹³⁾

We previously reported that the administration of GABA improved AD-like skin lesions in NC/Nga mice by adjusting the Th1/Th2 balance.¹⁴⁾ Furthermore, a placebo-controlled, double-blind study indicated that oral administration of GABA significantly increased the skin elasticity¹⁵⁾ however, its regulatory mechanism is not understood. In this study, we therefore investigate the effects of GABA on the expression of type I collagen (COL1A1 and COL1A2) and matrix metalloproteinase-1 (MMP-1) in normal human dermal fibroblasts (NHDFs).

GABA used in this study was prepared from the fermented barley extract as described previously with some modifications.¹⁴⁾ The nutrition composition of GABA was as follows: protein, 95.5%; fat, 0.1%; carbohydrate, 2.8%; ash, 0.7%; water, 0.9%. The percentage of GABA used in this study was 92.0% of total products. GABA was dissolved in phosphate-buffered saline (PBS). NHDFs were purchased from Kurabo Industries Ltd. (Osaka, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in humidified 5% CO₂ and 95% air.

NHDFs were plated into 24-well plates at a density of 4 × 10⁴ cells/dish for RNA analysis, or 96-well plates at a density of 5 × 10³ cells/well for cell proliferation assay. The cells were cultured in DMEM with 0.5% FBS 24 h before treatment with GABA. The cells were further cultured with various concentrations of GABA for 24 h, and then the cells were collected for RNA analysis or cell proliferation assay. Cell viability was determined by using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instruction. The absorbance of each well was measured at 450 nm. Total RNA was isolated from NHDFs using TRIzol Plus RNA Purification kit (Life Technologies, NY) according to the manufacturer’s instruction. Reverse transcriptions were carried out using SuperScriptIII First-strand Synthesis System (Life Technologies, NY) with Random primers, and the resulting single-stranded cDNA molecules were PCR amplified using gene-specific primers. For a quantification of mRNA, real-time PCR was performed using LightCycler Nano (Roche, Indianapolis, IN). The thermal cycler conditions included 1 cycle at 95 °C for 10 min, 45 cycles at 95 °C for 10 s, 60 °C for 10 s, 72 °C for 15 s. The relative mRNA expression levels were normalized against that of the GAPDH gene using a comparative threshold cycle method.¹⁶⁾ The sets of primers for COL1A1 and COL1A2, MMP-1 and the GAPDH are described in Table 1. Results are presented as means ± SD of three independent experiments, and significant differences (p < 0.05) were evaluated by Tukey’s multiple comparison test.¹⁷⁾

Table 1. Primer sequences.

Primer	Direction	Sequence
GAPDH	Forward	5′-CGG AGT CAA CGG ATT TGG TCG TAT- 3′
	Reverse	5′-AGC CTT CTC CAT GGT GGT GAA GAC- 3′
COL1A1	Forward	5′-AGG ACA AGA GGC ATG TCT GGT T- 3′
	Reverse	5′-TTG CAG TGG TAG GTG ATG TTC TG- 3′
COL1A2	Forward	5′-GTT GCT GCT TGC AGT AAC CTT- 3′
	Reverse	5′-AGG GCC AAG TCC AAC TCC TT- 3′
MMP-1	Forward	5′-ATT CTA CTG ATA TCG GGG CTT TGA- 3′
	Reverse	5′-ATG TCC TTG GGG TAT CCG TGT AG- 3′

The skin is the largest organ in human body, which provides a protective barrier against the environment such as heat, ultraviolet irradiation, bacterial infection, mechanical, and/or chemical stress. The skin is composed of two primary layers, epidermis and dermis, and each layer exhibits physiological functions. The barrier function of skin is exclusively achieved by epidermis, which directly faces the external environment. In contrast, dermis is located below the epidermis, and maintains the strength, elasticity, and moisture of skin. Type I collagen is the most abundant structural protein in the skin, and is mainly produced by dermal fibroblasts. In dermis, the regulation of type I collagen expression is important to the improvement of the skin elasticity.¹⁸⁾

In this study, first of all, we evaluated the cytotoxic effects of GABA on NHDFs by cell proliferation assay. After incubation for 24 h, GABA at concentrations of 0.1, 1, and 10 μg/mL had no cytotoxic effects on the proliferation of NHDFs, while 100 μg/mL of GABA was slightly decrease of cell viability (data not shown). Thus, 0.1–10 μg/mL of GABA was used for further experiments. Type I collagen is composed of two identical a1(I) chains and one a2(I) chain, which are encoded by two different genes. Therefore, we next investigated the effects of GABA on COL1A1 and COL1A2 mRNA expression in NHDFs. As shown in Fig. 1 10 μg/mL GABA significantly increased the expression level of COL1A1 mRNA in NHDFs. Furthermore, the expression of COL1A2 transcript is also increased in NHDFs treated with GABA in a dose-dependent manner, but not significance. These results suggest that GABA upregulates, at least in part, the expression of type I collagen genes in NHDFs. Because the skin strength and elasticity are associated with a balance between collagen synthesis and decomposition, the effects of GABA on MMP-1 expression, which is a degradation enzyme, was further examined. Fig. 2 shows that 1 and 10 μg/mL of GABA significantly suppressed the expression of MMP-1 transcript in NHDFs.

Fig. 1. Effects of GABA on type I collagen mRNA expression in NHDFs.

Notes: Cells were cultured for 24 h with various concentrations of GABA. The expression level of COL1A1 and COL1A2 transcript was analyzed by real-time PCR. Each datum represents the mean ± SD of three independent experiments. Results are compared to untreated group, and significant difference is indicated by an asterisk (*p < 0.05).

Fig. 2. Effects of GABA on MMP-1 mRNA expression in NHDFs.

Notes: Cells were cultured for 24 h with various concentrations of GABA. The expression level of MMP-1 transcript was analyzed by real-time PCR. Each datum represents the mean ± SD of three independent experiments. Results are compared to untreated group, and significant difference is indicated by an asterisk (*p < 0.05).

The dermal fibroblasts are responsible for the synthesis of extracellular matrix, especially type I collagen. The skin aging is mainly associated with the reduction in the level of type I collagen. For example, the excess UV irradiation dramatically decreased dermal collagen content, resulting in wrinkle formation and loss of elasticity in the skin.¹⁹⁾ Thus, type I collagen is a critical structural component of skin connective tissue and provides the strength and elasticity of skin. On the other hand, functional micronutrients and food components exert to prevent skin aging.²⁰⁾ For example, soybean peptide and vegetable peptones upregulate the type I collagen expression.^21,22) GABA is widely distributed in many vegetables and fruits, and has many physiological functions. In human healthy volunteers’ experiments, GABA was rapidly absorbed and reached the peak concentration in the plasma after 1 h of oral administration, resulting in significantly increased circulating insulin level to improve diabetes.²³⁾ We also reported that oral administration of GABA significantly increased the skin elasticity in human study.¹⁵⁾ In addition, GABA stimulated the synthesis of hyaluronic acid and enhanced the survival rate against an oxidative stress in dermal fibroblasts.²⁴⁾ Furthermore, in the wound healing animal model, GABA treatment was effective to accelerate the healing process.²⁵⁾ In this study, we also indicated that GABA upregulated the expression of type I collagen and downregulated the MMP-1 expression on NHDFs in vitro. Taken together, GABA may exhibit the improvement of skin elasticity by the regulating the homeostasis of dermal fibroblasts.

GABA exerts as inhibitory neurotransmitter in central nervous system, while the function in peripheral tissues is not fully understood.⁷⁾ On the other hand, three types of GABA receptor have been identified, GABAA and GABAC receptors are the ligand of transmitter-gated ion channels, while GABAB receptor is a G-protein-coupled receptor.²⁶⁾ If GABAA and/or GABAC receptors are expressed in NHDFs, GABA induces the activation of Cl⁻ and/or K⁺ cannels via its receptors, and subsequently affects several gene expression.^27–29) If GABAB receptor is expressed in NHDFs, it activates the second messenger systems such as phospholipase C, adenylate cyclase, and/or Ca²⁺ cannel via G-coupled proteins.³⁰⁾ In this study, we did not identify which type of GABA receptors were expressed in NHDFs. To understand the mechanism of the regulations of type I collagen and/or MMP-1 gene expressions, therefore, it is necessary to identify the major GABA receptors expressed in human dermal fibroblasts.

In conclusion, we revealed that GABA increased the level of COL1A1 and COL1A2 expression, and suppressed the expression of MMP-1, which is degrading enzyme of collagens in dermal fibroblasts. Therefore, our results suggest that the administration of GABA improves the skin elasticity in human dermal fibroblasts by controlling the balance between the synthesis and degradation of the type I collagen.

Author contributions

Eriko Uehara, Hideki Hokazono, and Noritaka Matsuo conceived and designed the experiments. Eriko Uehara performed the experiments. Eriko Uehara and Noritaka Matsuo wrote the manuscript. All authors discussed the results and commented on the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.