Ann Dermatol.  2016 Aug;28(4):433-437. 10.5021/ad.2016.28.4.433.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

• Hahn HJ
• Youn HJ
• Cha HJ
• Kim K
• An S
• Ahn KJ

• KMID: 2344812
• DOI: http://doi.org/10.5021/ad.2016.28.4.433

Abstract

BACKGROUND
We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model.
OBJECTIVE
We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation.
METHODS
To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation.
RESULTS
Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation.
CONCLUSION
These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation.

Keyword

Cell differentiation; Keratinocytes; Low-dose radiation

MeSH Terms

Blotting, Western
Cell Differentiation
Keratinocytes*
Molecular Biology
Nuclear Energy
Polymerase Chain Reaction
Radiation, Ionizing
RNA, Messenger
RNA, Messenger

Figure

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  Fig. 1 Low-dose radiation stimulates involucrin accumulation in calcium-induced differentiating keratinocytes. (A) Western blot analysis of involucrin protein levels in control HaCaT cells (no exposure to ionizing radiation) and HaCaT cells exposed to 0.1 Gy gamma irradiation. In each experiment, equal loading was demonstrated by immunostaining for β-actin. (B) The band densities of interest were further analyzed by ImageJ. Involucrin levels were normalized against β-actin.

• 

  Fig. 2 Low-dose radiation elevates involucrin messenger RNA (mRNA) expression in calcium-induced differentiating keratinocytes. HaCaT cells were exposed to gamma radiation and control cells were untreated. After 4 h, keratinocyte differentiation was induced in HaCaT cells using 2 mM calcium for 4 and 8 days. Total RNA, isolated using TRIzol, was used to synthesize cDNA by reverse transcription, and was amplified by polymerase chain reaction. β-actin was amplified as a normalization control. Results are expressed as the mean percentage±standard error of the experiments performed in triplicate. *p<0.01.

• 

  Fig. 3 Low-dose radiation reduced cell viability and growth capacity in HaCaT cells. (A) HaCaT cells (1×104 cells) were plated in 96-well plates and cultured for 24, 48, and 72 h after the exposure to the indicated dose of gamma radiation. Cell viability was measured by WST-1 as described in Materials and Methods. Results are expressed as the mean optical density±standard error, as a percentage of the control for the experiments performed in triplicate. *p<0.01. (B) Growth capacity was calculated by calculating the dRV/dT as derivative relative viability (%)/derivative time (h). IR: gamma irradiation.

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  Fig. 4 Low-dose radiation induced p21 (Cip1/Waf1) expression in HaCaT cells. (A) Western blot analysis of p21 (Cip1/Waf1) protein levels in control HaCaT cells and HaCaT cells exposed to 0.1 Gy gamma irradiation. Equal loading in each experiment was demonstrated by immunostaining for β-actin. (B) Band densities of interest were further analyzed by ImageJ; p21 (Cip1/Waf1) levels were normalized against β-actin. IR: gamma irradiation.

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