Phytosphigosine-1-phosphate increases sensitivity of EGF-dependent cell proliferation

  • Authors:
    • Hwa Jun Cha
    • Jeong Pyo Lee
    • Kwang Sik Lee
    • Kun Kook Lee
    • Myoung Jun Choi
    • Dong Kyu Lee
    • Kwang Nyeon Kim
    • Sungkwan An
  • View Affiliations

  • Published online on: January 7, 2014     https://doi.org/10.3892/ijmm.2014.1617
  • Pages: 649-653
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Abstract

The dermis is composed of dermal fibroblasts and various synthesized extracellular matrices. Proliferation of these cells is important to skin structure homeostasis. Therefore, human dermal fibroblasts (HDFs) growth factors have been previously evaluated. In the present study, we examined whether phytosphingosine‑1-phosphate (PhS1P) regulates gene expression, particularly cell cycle-related genes. In addition, we investigated whether there was a synergistic effect of proliferation induced by PhS1P and epidermal growth factor (EGF) through PhS1P-regulated genes. A microarray approach was utilized to identify gene expression changes in PhS1P-treated HDFs and data were analyzed using gene ontology (GO). In addition, proliferative synergistic effects were measured using an MTT assay. The results showed that PhS1P regulates various genes, particularly cell cycle-related genes. Microarray data, followed by GO, indicated that PhS1P affected the biological processes involved in the cell cycle (cyclins A2, B1 and B2). Furthermore, these genes synergistically affected EGF-dependent proliferation. The results obtained in this study demonstrated that PhS1P altered gene expression profiles, inducing EGF-dependent cell proliferation. Therefore, PhS1p acts as a synergistic effector for EGF.

Introduction

Human dermal fibroblasts (HDFs) are mesenchymal cells specialized in extracellular matrix synthesis, including collagen, elastin and hyaluronic acid, in the dermis (1,2). Dermal fibroblast proliferation is important in wound healing and skin structure homeostasis (3). Therefore, a growth factor was used for proliferating HDFs in cosmetics (4).

Epidermal growth factor (EGF), one of various growth factors, is a small polypeptide first purified by Cohen from the submaxillary gland of adult male mice (5). EGF is important in cell proliferation, migration and differentiation (5). These roles are mediated via activation of the EGF receptor (EGFR), a transmembrane glycoprotein with tyrosine kinase activity (6). Activated EGFR regulates proliferation and migration via activation of intrinsic signaling molecules (7,8). EGF is added to serum-free media in in vitro cell culture systems as EGF is essential to cell growth (9). Furthermore, EGF exerts cytoprotective effects from cell damage, such as senescence (10). Therefore, EGF is used as an inducer of dermal fibroblast proliferation in cosmetics and medicine (4).

Phytosphingosine-1-phosphate (PhS1P) is derived from fungi and plants, and is structurally similar to sphingosine-1-phosphate (S1P), an endogenous signal lipid in mammalian cells (11,12). PhS1P is an agonist for S1P receptors, with a particularly high affinity for S1P4 (13). S1P receptors include the isomers, S1P1, S1P2, S1P3 and S1P4, in mammalian cells (13). Since each receptor activates different downstream signals, the effects of these S1Ps are slightly different (14,15). Therefore, we aimed to evaluate the PhS1P function in HDFs. The results showed that PhS1P altered gene expression and induced EGF-dependent proliferation as a synergistic effector.

Materials and methods

Cell culture and materials

HDFs were purchased from Lonza (Basel, Switzerland) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated at 37°C in a humidified incubator containing 5% CO2. PhS1P was obtained from Phytos Co., Ltd. (Suwon, Korea).

RNA extraction and microarray

Total RNA was isolated using RiboEX (GeneAll, Seoul, Korea) and quantified based on the optical density ratio (280/260 nm) using a Bioanalyzer 2100 (28S RNA/18S RNA ratio; Agilent Technologies, Santa Clara, CA, USA). Equal amounts of RNA were used to synthesize cDNA and label it with biotin using an RNA amplification kit (Ambion, Austin, TX, USA). After labeling, the microarray was hybridized with biotin-labeled RNA and streptavidin-Cy3 (Invitrogen Life Technologies, Carlsbad, CA, USA). Following hybridization, the microarray was washed using wash E1BC buffer and scanned using the iScan system (both from Illumina, Hayward, CA, USA).

Microarray analyses

Microarray data were analyzed using Genespring GX software version 11 (Agilent Technologies). mRNAs flagged ‘present’ in at least one sample were analyzed using fold-change. The threshold cut-off was 1.3-fold for fold-change between non-treated HDFs and PhS1P-treated HDFs. Significantly altered mRNAs were sorted using the gene ontology (GO) tool.

Quantitative polymerase chain reaction (qPCR)

cDNA was synthesized using MMLV-reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer’s instructions. Synthesized cDNA was used for qPCR (Line gene K; Bioer Technology, Co., Ltd., China) using specific primers for cyclin A1, B1 and B2. Primers were designed by primer 3 (http://frodo.wi.mit.edu) (Table I). Expression was normalized to β-actin.

Table I

Quantitative PCR primer sequences.

Table I

Quantitative PCR primer sequences.

GenePrimer sequences
Cyclin A2Forward 5′-TTATTGCTGGAGCTGCCTTT-3′
Reverse 5′-CTCTGGTGGGTTGAGGAGAG-3′
Cyclin B1Forward 5′-CGGGAAGTCACTGGAAACAT-3′
Reverse 5′-AAACATGGCAGTGACACCAA-3′
Cyclin B2Forward 5′-TTGCAGTCCATAAACCCACA-3′
Reverse 5′-GAAGCCAAGAGCAGAGCAGT-3′
MTT assay

Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HDFs were cultured for 24 h in 96-well plates with PhS1P and EGF. MTT tetrazolium salt (0.5 mg/ml; Sigma) was added to cells for 4 h. After incubation, the medium was replaced with dimethyl sulfoxide in each well. The absorbance of each sample was measured at 595 nm using a plate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Results

PhS1P cytotoxicity in HDFs

To assess the effect of PhS1P on cell viability, HDFs were treated with various concentrations of PhS1P (0, 0.25, 0.5 and 1 μM) for 24 h (Fig. 1). We determined that PhS1P at concentrations of ≤1 μM had no cytotoxicity.

PhS1P alters the mRNA expression profile

To identify genes that may play a PhS1P-dependent role, we compared the expression profiles of non-treated and 1 μM PhS1P-treated HDFs using the Illumina bead chip HumanHT-12. In 47,207 whole genes, we first filtered 35,973 genes that were flagged ‘present’ with a frequency higher than the sensitivity of detection in a minimum of one array. Fold-change was then analyzed in these flag-filtered genes. Genes that changed by 1.3-fold between non-treated and PhS1P-treated HDFs were presented on the heat map (Fig. 2A). The analyses identified 193 upregulated genes and 495 downregulated genes (Fig. 2B).

Using the GO analytical tool, the genes were sorted based on their roles in biological processes (Fig. 3). Genes upregulated in PhS1P-treated HDFs appeared to be involved in cell processes such as cell cycle, cell division, microtubule-based processes, chromosome segregation, cell communication, cellular responses to stimuli, and cellular component organization or biogenesis. In particular, cell cycle-related ontology (cell cycle, cell division and chromosome segregation) was significantly enriched by PhS1P.

Cyclins A2, B1 and B2 were regulated by PhS1P. Cyclins are well-known regulators in cells (16). Therefore, the analyses revealed that PhS1P affected cell proliferation by altering specific mRNAs. In addition, we confirmed the mRNA expression change of cyclin A2, B1 and B2 using qPCR (Fig. 4).

We also examined the synergistic effect of PhS1P and EGF on HDF viability. Compared with PhS1P-treated HDFs in the absence of EGF, the viability of PhS1P-treated HDFs with EGF was markedly increased (Fig. 4D). These data indicate that PhS1P is a co-effector in the induction of EGF-dependent cell proliferation.

Discussion

The aim of the study was to evaluate the effect of PhS1P on EGF-induced proliferation in HDFs. PhS1P is phytochemically derived from fungi, plants, and even mammalian cells (12). In a previous study, it was shown that PhS1P protects against hydrogen peroxide-dependent growth arrest (17). Recent data from Lee et al (17) suggested that PhS1P had no significant effect on the proliferation at concentrations <1 μM in HDFs (Fig. 1). However, PhS1P (1 μM) regulates various cell cycle-related genes in HDFs (Fig. 2). In particular, the cyclins (cyclin A2, B1 and B2), master regulators of the cell cycle, were upregulated by PhS1P. Cyclin A2 regulates S-phase progression and entry into mitosis (18,19). During S phase, cyclin A2 initiates DNA synthesis (20). During the G/M phase, cyclin A2 triggers entry into mitosis by activating cyclin B1-Cdk1 (21).

In the progressing cell cycle, a sustained high expression of cyclins is essential (16). However, overexpression of cyclins is insufficient to induce cell cycle progression (22,23). As shown in Fig. 2, PhS1P upregulated mRNA expression of cyclin A2, B1 and B2 although there was no change in cell viability (Figs. 1 and 2).

Combining the present data with our previous data (17), we determined that <1 μM PhS1P increases cyclin expression, but does not affect viability. Moreover, EGF triggered proliferation in PhS1P-treated HDFs (Fig. 4D). Therefore, together with the results from Ikezawa et al (22), our data suggest that a cyclin-enriched condition results in synergistic growth following treatment with growth factors, such as EGF (24).

In summary, results of the present study have shown that PhS1P regulates cell cycle-related genes. In addition, the changes in gene expression synergistically trigger EGF-induced proliferation.

Acknowledgements

We would like to thank all other members of Damy Chemical Co., Ltd. for their support. This study was supported by the Konkuk University in 2012.

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2014-March
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Spandidos Publications style
Cha HJ, Lee JP, Lee KS, Lee KK, Choi MJ, Lee DK, Kim KN and An S: Phytosphigosine-1-phosphate increases sensitivity of EGF-dependent cell proliferation. Int J Mol Med 33: 649-653, 2014
APA
Cha, H.J., Lee, J.P., Lee, K.S., Lee, K.K., Choi, M.J., Lee, D.K. ... An, S. (2014). Phytosphigosine-1-phosphate increases sensitivity of EGF-dependent cell proliferation. International Journal of Molecular Medicine, 33, 649-653. https://doi.org/10.3892/ijmm.2014.1617
MLA
Cha, H. J., Lee, J. P., Lee, K. S., Lee, K. K., Choi, M. J., Lee, D. K., Kim, K. N., An, S."Phytosphigosine-1-phosphate increases sensitivity of EGF-dependent cell proliferation". International Journal of Molecular Medicine 33.3 (2014): 649-653.
Chicago
Cha, H. J., Lee, J. P., Lee, K. S., Lee, K. K., Choi, M. J., Lee, D. K., Kim, K. N., An, S."Phytosphigosine-1-phosphate increases sensitivity of EGF-dependent cell proliferation". International Journal of Molecular Medicine 33, no. 3 (2014): 649-653. https://doi.org/10.3892/ijmm.2014.1617