Effect of cytokines and ultraviolet B radiation on the promoter activity of the metallothionein gene in keratinocytes

doi:10.4236/abb.2013.49117

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Advances in Bioscience and Biotechnology, 2013, 4, 896-899 ABB

http://dx.doi.org/10.4236/abb.2013.49117 Published Online September 2013 (http://www.scirp.org/journal/abb/)

Effect of cytokines and ultraviolet B radiation on the

promoter activity of the metallothionein gene in

keratinocytes

Hiromi Narumi*, Hajime Nakano, Takahide Kaneko, Koji Nakajima, Yasushi Matsuzaki,

Takayuki Aizu, Daisuke Sawamura, Katsumi Hanada

Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

Email: *[email protected]

Received 18 April 2013; revised 19 May 2013; accepted 10 June 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Metallothionein (MT) has many functions that are

modulated by several factors, including ultraviolet

(UV) radiation and cytokines. We thought that these

diverse functions of MT might reflect the specific

regulatory mechanisms of its expression. To under-

stand some of the molecular mechanisms underlying

MT expression, we examined the effects of several

cytokines and UVB on the promoter activity of the

MT gene. First, we introduced the MT promoter con-

struct into the HaCaT keratinocytes and treated them

with various concentrations of interleukin-1α (IL-1α)

and IL-6. The addition of IL-1α and IL-6 led to an

increase in the promoter activity of the MT gene.

UVB is known to induce MT expression in epidermal

keratinocytes, and IL-6 is a possible mediator of MT

induction by UV radiation. Therefore, we investi-

gated whether UVB could induce MT promoter ac-

tivity. Our results showed, interestingly, that UVB

radiation has no or little effect on the promoter activ-

ity. This suggested a complex molecular regulation of

the MT gen e .

Keywords: Sunburn; Cytokine; Epidermis; Ultraviolet

Light; Promoter

1. INTRODUCTION

Metallothionein (MT) is a ubiquitously distributed, cys-

teine-rich, low molecular weight protein having a high

binding capacity for metals such as zinc, copper, and

cadmium. It plays a role in zinc homeostasis and detoxi-

fication of heavy metals [1]. Several studies have shown

that MT acts as a reactive oxygen species scavenger [2]

and MT induction has protective effects against oxidative

stresses such as anticancer drugs and ultraviolet (UV)

radiation [3]. MT gene expression is induced not only by

heavy metals but also by various stress-inducing agents

such as UV [4] and X-ray radiation [5]. We found that

β-thujaplicin induced MT expression in keratinocytes,

both in vitro and in vivo, and thereby reduced UVB irra-

diation-induced apoptosis [6]. Furthermore, a number of

cytokines, including interferon-α and β, tumor necrosis

factor-α, interleukin-1 (IL-1), and IL-6, increase MT ex-

pression, suggesting that MT expression also involves

additional functions, including immunomodulation, cell

growth, and cell differentiation [7].

We thought that these diverse functions of MT might

reflect specific regulatory mechanisms of its expression.

We have recently cloned the promoter region of the MT

gene and performed a functional assay [8]. Thus, to un-

derstand some of the molecular mechanism underlying

MT expression, we focused on the effects of several cy-

tokines and UVB radiation on the promoter activity of

the MT gene.

2. MATERIALS AND METHODS

2.1. Cell Culture

The spontaneously transformed human epidermal kerati-

nocyte cell line, HaCaT, (kindly provided by Dr. Husenig)

was cultured in Dulbecco’s modified minimal essential

medium supplemented with 10% fetal calf serum, 1%

l-glutamine, and 1% antibiotic/antimycotic solution. The

cells were maintained at 37˚C in a humidified atmos-

phere containing 5% CO2. The viability of the cells

treated with cytokines and UVB radiation for 24 h was

approximately 95%, as determined by trypan blue exclu-

sion staining.

*Corresponding author.

OPEN ACCESS

H. Narumi et al. / Advances in Bioscience and Biotechnology 4 (2013) 896-899 897

2.2. Plasmid Constructs

The vector p5’MT-CAT, containing the 5’-flanking re-

gion of the MT-IIA gene, was generated by ligating a

HindIII/BamHI fragment of the MT-IIA promoter, span-

ning from −764 to +79, (American Type Culture Collec-

tion, Rockville, MD, USA) to the pBS0CAT reporter

construct [8]. The integrity of the reporter constructs was

confirmed by direct sequencing.

2.3. UVB Radiation Source

As the UVB source, a bank of 7 fluorescent sunlamps

(FL20SE.30; Toshiba Medical Supply, Tokyo, Japan)

emitting rays of 275 - 305 nm and peaking at 305 nm

was used [9]. The radiation dose was measured using a

radiometer (UVR-3036/S; Clinical Supply, Kakamigahara,

Japan).

2.4. Transient Transfection Experiments

Transient transfection was performed using Trans IT trans-

fection reagent (PanVera, Madison, WI, USA). Briefly,

60% confluent HaCaT cells were placed in a 60-mm dish

and then incubated with 0.5 g reporter gene and the

transfection reagent for 6 h. For monitoring the transfec-

tion efficiency, the cells were cotransfected with the

RSV-β-galactosidase expression vector. The cells were

treated with various concentrations of cytokines for 24 h.

The cells were also irradiated with UVB, followed by

further incubation with the medium for 24 h. The treated

cells were rinsed twice with phosphate buffered saline

and then lysed in 200 ml of reporter lysisbuffer (Promega,

Madison, WI, USA). As positive control, we used the

transfected cells that were incubated with 10 M of

cadmium.

2.5. Chloramphenicol Acetyltransferase (CAT)

Assays

The CAT activity, an indicator of the promoter activity,

was determined by incubation with [14C]-chloramphenicol

[8]. The β-galactosidase activity of all the samples were

measured, and each CAT activity value was corrected for

the β-galactosidase activity in the corresponding cell

culture transfected in parallel. CAT activity was quanti-

fied by measuring the amount of [14C]-chloramphenicol

converted to the monoacetylated form. The promoter

activity was expressed as the rate of CAT activity of the

sample to that of the positive control (10 M of cad-

mium).

3. RESULTS

3.1. Effect of IL-1α, IL-6, and IL-10 (Figure 1)

First, we introduced the MT promoter construct into the

HaCaT cells and treated them with various concentra-

tions of IL-6, IL-1, and IL-10. After the addition of

IL-1α, the promoter activity of the MT gene increased in

a dose-dependent manner and reached the maximum at a

concentration of 10 ng/ml. IL-6 induced the promoter

activity at an even lower concentration than IL-1α. On

the other hand, IL-10 did not activate the promoter.

3.2. Effect of UVB (Figure 2)

Next, we examined the effect of UVB radiation on the

promoter activity of the MT gene. We harvested the Ha-

CaT cells 24 h after UVB irradiation. The results showed

(a) (b)

(c)

Figure 1. Effects of IL-1α, IL-6, and IL-10 on the promoter

activity of the MT gene. We introduced the MT promoter con-

struct into the HaCaT cells and treated them with various con-

centrations of IL-1α (a), IL-6 (b), and IL-10 (c). The activity

levels are expressed as mean (SD).

Figure 2. Effects of UVB on the promoter activity of the

MT gene. We introduced the MT promoter construct into

the HaCaT cells and harvested the cells 24 h after UVB

irradiation. The activity levels are expressed as mean (SD).

H. Narumi et al. / Advances in Bioscience and Biotechnology 4 (2013) 896-899

898

that UVB had no or little enhancing effect on the pro-

moter activity at doses ranging from 5 to 30 mJ/cm2.

Furthermore, we collected the treated cells several times

after irradiation, but we could not observe any strong

induction of the promoter activity.

4. DISCUSSION

IL-1α has many physiological functions in the immune,

metabolic, and hematopoietic systems. Keratinocytes are

a major source of IL-1α. As a proinflammatory cytokine,

IL-1α is involved in inflammatory and allergic skin dis-

eases such as psoriasis and contact dermatitis. It also

plays an important role in many recently defined autoin-

flammatory diseases [10]. IL-6 acts both as a pro- and

anti-inflammatory cytokine. It is also secreted by kerati-

nocytes, and it stimulates the immune response in both

normal and abnormal skin conditions [11]. Furthermore,

IL-1α and IL-6 are known to increase the MT gene ex-

pression and thereby, its protein expression. In this study,

we first examined the effects of IL-1α and IL-6 on the

MT promoter activity in epidermal keratinocytes. Our

results show that both cytokines induced promoter activ-

ity in a dose-dependent manner. We also investigated the

effect of IL-10 on MT expression, and as expected, we

did not find any significant effect of IL-10 on the pro-

moter activity.

The epidermis is the outermost layer of the skin. Kerati-

nocytes form a majority, i.e., about 95%, of the epider-

mis’ cells and are major targets of solar radiation. UVB

benefits humans by catalyzing the production of vitamin

D, but it also causes sunburn, photoaging, and skin can-

cers. UVB is responsible for the production of many cy-

tokines in the keratinocytes [12]. Furthermore, UVB is

known to induce MT expression in the epidermal kerati-

nocytes, and IL-6 is a possible mediator of MT induction

by UV radiation [13]. Therefore, we investigated whether

UVB radiation could induce MT promoter activity. Our

results showed, interestingly, that UVB radiation has no

or little effects on the promoter activity, although IL-6

clearly induced the promoter activity of the MT gene in

our study. This discrepancy implies non-transcriptional

mechanisms such as an increase in mRNA stability, sug-

gesting complex molecular regulation of the MT gene.

5. ACKNOWLEDGEMENTS

The authors would like to thank Ms Yuka Toyomaki, Mrs Yukiko

Tamura, Mrs Yuriko Takagi, and Ms Nanako Seitoh for their excellent

technical assistance. This work was supported in part by Grants-in-Aid

from the Ministry of Education, Science, Sports, and Culture of Japan.

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