Polydeoxyribonucleotide exerts opposing effects on ERK activity in human skin keratinocytes and fibroblasts

Abstract

Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti‑inflammatory and tissue‑regenerating agent. The mitogen‑activated protein kinase pathway modulates cell growth and collagen accumulation. It also regulates inflammation by suppressing the expression of proinflammatory cytokines. In the present study, it was attempted to elucidate the molecular mechanism of PDRN in skin healing by confirming the effects of PDRN treatment on skin keratinocytes and fibroblasts, and by assessing the levels of collagen and inflammatory cytokines regulated by the extracellular signal‑regulated kinase (ERK) pathway. The potential effects of PDRN on skin regeneration were investigated. Fibroblast and keratinocyte proliferation and migration were analyzed using the water‑soluble tetrazolium‑8 and wound healing assays. The upregulation of collagen synthesis by PDRN‑induced ERK activation was analyzed in fibroblasts with or without an ERK inhibitor. Inflammatory cytokine expression levels in keratinocytes were determined using reverse transcription‑quantitative polymerase chain reaction. PDRN promoted the proliferation and migration of keratinocytes and fibroblasts. However, PDRN‑induced ERK phosphorylation differed between keratinocytes and fibroblasts; PDRN increased ERK phosphorylation and collagen accumulation in fibroblasts, while it inhibited matrix metalloproteinase expression. By contrast, PDRN inhibited ERK phosphorylation in keratinocytes, and it decreased inflammatory cytokine expression levels. PDRN affects skin cell proliferation and migration, and collagen and inflammatory cytokine expression levels via ERK signaling. Overall, PDRN exerts a positive effect on skin regeneration, but the mechanism by which it promotes skin regeneration varies among different skin cell types.

Keywords: extracellular signal‑regulated kinase; fibroblasts; keratinocytes; polydeoxyribonucleotide; skin regeneration.

Figure 1.

Cytotoxicity and proliferation induced by PDRN treatment. (A) PDRN cytotoxicity on HDFs and HEKs was determined using a WST-8 assay 24 h after addition of PDRN to culture medium. (B) HDF and HEK proliferation rates were determined using the WST-8 assay at 24, 48 and 72 h after addition of PDRN to the culture medium. The results were normalized and expressed as fold change ± SEM compared with the control without PDRN. *P<0.05, **P<0.01 and ***P<0.005 vs. 0 µg/ml. PDRN, polydeoxyribonucleotide; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; WST-8, water-soluble tetrazolium-8.

Figure 2.

Increased cell migration by PDRN-treated human skin cells. Migration of (A) HDFs and (B) HEKs 24 h after PDRN treatment was observed under a phase-contrast microscope. Original magnification, ×40. The number of migrated (C) PDRN-treated HDFs and (D) HEKs at 24 h were counted. ***P<0.005 vs. 0 µg/ml. PDRN, polydeoxyribonucleotide; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes.

Figure 3.

Effects of PDRN on mitogen-activated protein kinase pathway activation in human skin cells. Cells were cultured without serum for 16 h, followed by addition of PDRN to culture medium for 0, 5, 15, 30, 50 and 120 min. Cell lysates were analyzed by western blotting using targeted protein antibodies in (A) HDFs and (B) HEKs. Phosphorylated ERK was quantified with Image J software by comparing the density of phospho-ERK and ERK in (C) HDFs and (D) HEKs. Similar results were obtained in three different experiments. *P<0.05 and **P<0.01 vs. 0 min. PDRN, polydeoxyribonucleotide; HDF, human dermal fibroblasts; HEK, human epidermal keratinocytes; ERK, extracellular signal-regulated kinase; JNK, c jun N terminal kinase.

Figure 4.

Effect of ERK inhibition on PDRN-induced collagen accumulation around HDFs. To explore the underlying mechanism of PDRN-induced ERK activation, the proliferation and migration of HDFs stimulated with PDRN in the presence of PD98059 were measured using (A) WST-8 and (B) wound healing assays. Original magnification, ×40. Cells were cultured without serum for 16 h, followed by addition of PDRN and PD98059 to the culture medium for 24 h. (C) hPro-Collagen I α 1 and hCollagen type III expression levels were determined using ELISA in HDFs. (D) MMP-1, 2 and 3 expression levels were examined by reverse transcription-quantitative PCR. Each treatment was performed in triplicate at least, and the data are presented as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.005 vs. PDRN or indicated control. PDRN, polydeoxyribonucleotide; HDF, human dermal fibroblasts; ERK, extracellular signal-regulated kinase; WST-8, water-soluble tetrazolium-8; p-, phosphorylated; h, human; MMP, matrix metalloproteinases.

Figure 5.

Decreased expression levels of pro-inflammatory cytokines in PDRN-treated HEKs. HEKs were cultured without serum for 16 h, followed by PDRN addition to the culture medium for 24 h. Inflammatory cytokine expression levels were detected by reverse transcription-quantitative PCR. Each treatment was performed in at least 5 times, and data presented as means ± SEM. *P<0.05, **P<0.01 and ***P<0.005 vs. 0 µg/ml. PDRN, polydeoxyribonucleotide; TNF, tumor necrosis factor; IL, interleucin; MCP1, monocyte chemotactic protein 1; iNOS, inducible nitric oxide synthase; HEKs, human epidermal keratinocytes.