Biochemical and Biophysical Research Communications 418 (2012) 457–463

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Smad, but not MAPK, pathway mediates the expression of type I collagen inradiation induced ﬁbrosis
Hiroyuki Yano a,c, Ryoji Hamanaka b, Miki Nakamura b, Hideaki Sumiyoshi a, Noritaka Matsuo a,
Hidekatsu Yoshioka a,⇑abc

Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japan
Cell Biology, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japan
Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593, Japana r t i c l e

i n f oArticle history:
Received 21 December 2011
Available online 18 January 2012
Keywords:
Radiation induced ﬁbrosis
Collagen
TGF-b
Smad pathway
Extracellular matrix

a b s t r a c t
Radiation induced ﬁbrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue ﬁbrosis is the excessive accumulation of collagen and other extracellular matrix components.
This study investigated how ionizing radiation affects the expression level and signal pathway of type I
collagen. Real time RT-RCR showed that both a1and a2 chain of type I collagen mRNA were elevated from48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I
collagen marker were elevated at 72 h. TGF-b1 mRNA was elevated earlier than those of type I collagengenes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-b receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen.
The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelledusing a mutant construct of Smad binding site of a2(I) collagen gene. However, the MAPK pathways, p38,
ERK1/2 and JNK, were not affected with speciﬁc inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced ﬁbrosis.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
Fibrosis is the excessive accumulation of collagen and otherextracellular matrix (ECM) components following breakdown in
the normal balance of ECM synthesis and degradation. Ionizing
radiation is a highly effective modality for local control of tumor
growth. However ionizing radiation can induce normal tissue toundergo ﬁbrogenesis [1]. Radiation induced ﬁbrosis (RIF) is assumed to occur as a result of a coordinated response to ionizing
radiation involving several cytokines and growth factors, ﬁbroblast
proliferation and differentiation, and also remodeling of the ECM[2–4]. However, the intracellular signaling and mechanism of gene
expression that promote the RIF process are not yet fully
understood.
Collagens, which are the major constituents of extracellularmatrices, are critical for the formation and function of the organs
in the body [5]. There are 46 human collagen genes, each encoding
procollagen molecules that coalesce to form long triple helices [6].

Abbreviations: RIF, radiation induced ﬁbrosis; ECM, extracellular matrix; ELISA,
enzyme-linked immunosorbent assay.
⇑ Corresponding author. Fax: +81 97 586 5674.
E-mail address: hidey@oita-u.ac.jp (H. Yoshioka).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2012.01.039

Type I collagen is the major component of the ECM, which is a heterotrimer composed of coordinately expressed two a1 chains andone a2 chain. Their expression is modulated by various cytokines[7]. Transforming growth factor (TGF)-b is a member of a superfamily of multifunctional cytokines which function during development, wound repair, and other pathologic processes [8]. TGF-b
is a potent inducer of ECM protein synthesis and accumulationand has been implicated as the key mediator of various ﬁbrosis[9,10]. Therefore, TGF-b strongly contributes to ﬁbrotic disorders
as various ﬁbrogenous diseases, hepatic cirrhosis, diabetic
nephropathy, and rheumatoid arthritis.
TGF-b mediates signals through pairs of speciﬁc plasma membrane receptors (types I and II serine/threonine kinases). TGF-b
binds and activates the type II receptor, which then phosphorylates
the type I receptor. The active receptor complex then phosphorylates the so-called R (receptor)-Smad2 or Smad3. The phosphorylation of Smad2/3 propagates the signal by binding with Smad4(a so-called co-Smad). This complex translocates into the nucleus
and binds transcriptional co-activators such as p300 andCreb-binding-protein (CBP), which regulate the transcriptional
activity of various TGF-b target genes [11]. Furthermore, recent
evidence has indicated that TGF-b transduces through anotherpathway – mitogen activated protein kinase (MAPK) [12,13] and458

H. Yano et al. / Biochemical and Biophysical Research Communications 418 (2012) 457–463

cross-talk between the MAPK and Smad pathway in response to
TGF-b signaling have been suggested [14].
This study investigated how ionizing radiation affects theexpression level and signal pathway of type I collagen, which is
associated with the RIF process.
2. Materials and methods
2.1. Cell culture
The cell lines used in this study were mouse ﬁbroblast NIH-3T3cell and mouse embryonic ﬁbroblast cells.
The mouse embryonic ﬁbroblast cells were isolated fromembryonic mice. The ICR mice were purchased from commercial
sources (Yoshitomi, Fukuoka, Japan). The animals were treated inaccordance with the Oita University Guidelines for the Care andUse of Laboratory Animals based on the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
The cells were cultured in Dulbecco’s Modiﬁed Eagle’s Medium(DMEM) containing 10% heat-inactivated fetal bovine serum (FBS;
Sanko Junyaku, Tokyo, Japan) at 37 °C in a 5% CO2/air environment.
2.2. Ionizing irradiation of cells in culture
The cells were irradiated with 137Cs c-ray using a Gammacell 40(MDS Nordion, Ottawa, Canada) at a dose rate of approximately
1.0 Gy/min. The cells were irradiated to the total does called forin the experimental design for each experiment. The radiation
doses used in this experiment was 10 Gy. Ionizing irradiation
was performed at the initial point of 0 h, when ionizing irradiation
was completed. Before irradiation, the cells were incubated in
DMEM supplemented with 10% FBS for 24 h. The effect of ionizing
irradiation on type I collagen expression levels and the signal pathway was conﬁrmed at various times.
2.3. RNA isolation and real-time PCR
Total RNA was isolated from cultured cells using Isogen (Nippon
Gene, Tokyo, Japan) according to the manufacturer’s protocol. RNA
was quantiﬁed by optical density (A260) and stored at 80 °C. One
microgram of total RNA was reverse transcribed by random primer
using ReverTra Ace (Toyobo, Tokyo, Japan). Real-time PCR was performed using a LightCycler Taqman Master (Roche, Indianapolis,
IN, USA). The thermal cycling conditions included 1cycle at 95 °C
for 10 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for
1 s. The relative mRNA expression levels were normalized againstthat of GAPDH gene from the same RNA preparations, using acomparative threshold cycle method. The primer sets are listed
in Table S1.
2.4. Measurement of the Cross-linked N-telopeptides of type I collagen(NTx)
The cells were placed in 35 mm dishes. Twenty mL of the medium was used in subsequent assays. The concentration of NTx in
the medium was measured with an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s protocol(Cusabio Biotech, Wuhan, China).

calcium phosphate precipitation methods. Plasmid pRL-TK vector(Promega, Madison, WI, USA) was always cotransfected as an internal control for transfection efﬁciency. After additional cultivationfor 24 h, the transfected cells were irradiated with dose of 10 Gy.
At 72 h after ionizing irradiation, the cells were harvested, lysed,
centrifuged to pellet the debris, and subjected to luciferase assay.
The luciferase activities were measured as chemiluminescence ina luminometer (Lumat LB 9507, Perkin–Elmer Life Sciences, Waltham, MA, USA) using the Dual Luciferase Reporter Assay System(Promega) according to the manufacturer’s protocol.

2.6. Preparation of cell lysates and Western blot analysis
The cells were treated with ionizing irradiation, washed twicewith ice-cold phosphate-buffered saline (PBS), and lysed on ice
in RIPA buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM
EDTA, 1% NP-40, 0.5% Na-deoxycholate) containing protease andphosphatase inhibitors (1 mM phenylmethylsulfonyl ﬂuoride,
1 mM Na3VO4, 1 mM NaF, 1 g/mL aprotinin, 1 g/mL leupeptin,
1 g/mL pepstatin). Cell extracts were then centrifuged at 15,000g
for 10 min at 4 °C, supernatants were collected, and protein content was determined using a bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL, USA). Fresh cell extracts
were prepared in sample buffer (0.125 M Tris–HCl [pH 6.8]) 4%
SDS, 20% glycerol, 0.002% bromophenol blue, and 10% 2-mercaptoethanol). Samples were separated by SDS–PAGE and electroblotted onto a polyvinylidene diﬂuoride (PVDF) membrane. Equal
loading and appropriate transfer of each lane was conﬁrmed bystaining the PVDF membrane with the ponceau S solution (Sigma–Aldrich, St. Louis, MO, USA). The membrane were blocked with
5% skim milk or 5% BSA in Tris-buffered saline containing 0.1%
Tween-20 for 1 h at room temperature, incubated with antiSmad2/3 antibody, or anti-phospho-Smad2/3 antibody overnightat 4 °C, and then incubated in the horseradish peroxidase (HRP)conjugated antirabbit IgG for 1 h at room temperature. The signals
were enhanced using a chemiluminescence system (ECL plus;
Amersham Pharmacia Biotech, Buckinghamshire, UK) and exposed
to X-ray ﬁlm.

2.7. Reagents
SB431542, a speciﬁc inhibitor of TGF-b type I receptor, was purchased (Sigma). ERK1/2 inhibitor (PD98059), p38 MAPK inhibitor(SB203580) and JNK inhibitor (SP600125) were purchased from
Calbiochem (San Diego, CA, USA). The cells were preincubated withthose reagents for 1 h before ionizing radiation.

2.8. siRNA transfection
The siRNA cocktails targeting mouse MAPK (ERK 1/2, p38
MAPK) or mouse Smad3 were purchased (Sigma). The cells weretransfected using Lipofectamine 2000 (Invitrogen Co., Carlsbad,
CA, USA) to achieve a ﬁnal siRNA concentration of 50 nM. The speciﬁcity of MAPK (ERK 1/2, p38 MAPK) and mouse Smad3 knockdown was conﬁrmed by a real-time PCR reaction.

2.9. Statistical analysis
2.5. Transient transfection and luciferase assays
Plasmid constructs used in these experiments were previously
described [15]. The cells were plated at a density of 2  105 per
35-mm dish 18 h before transfection. For transient transfection,
10 lg of plasmid DNA was transfected into these cells by using

The values are presented as the mean ± SD of multiple independent tests and Student’s t-test was used to evaluate the statisticaldifferences between groups, and a p < 0.05 was considered to besigniﬁcant. Each experiment was performed at least three times
and individual samples were run in triplicate.

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459

Fig. 1. Effect of irradiation with 10 Gy on the expression of type I collagen. Real time RT-PCR for collagen a1(I) (A) and a2(I) (B) chain genes before (0 h) and after radiation at
6, 24, 48 and 72 h in NIH3T3. Each value is normalized to that before radiation (white column). Asterisks indicate statically signiﬁcant in comparison to the value before
radiation (A and B). Real time RT-PCR for collagen a1(I) (C) and a2(I) (D) using mouse embryonic ﬁbroblast. The gray and white columns in each time point show treatment
with and without radiation. Each value is normalized to that before radiation. Luciferase assay for collagen a1(I) (E) and a2(I) (F) chain genes. Each value is normalized to thevalue without radiation at 6 h after radiation. ELISA for measurement of concentration of NTx in culture medium of NIH3T3 (G). Asterisks indicate statically signiﬁcant incomparison to the values with and without radiation at each time point (C–G). All date represent the mean ± SD of results in three independent experiments. ⁄p < 0.05.

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H. Yano et al. / Biochemical and Biophysical Research Communications 418 (2012) 457–463

Fig. 2. Effect of irradiation with 10 Gy on the expression of TGF-b and Smad. (A) Real time RT-PCR for TGF-b1before (0 h) and after radiation at 24, 48 and 72 h in NIH3T3.
Each value is normalized to that before radiation (white column). Asterisks indicate statically signiﬁcant in comparison to the value before radiation. (B) Real time RT-PCR for
TGF-b1 and TGF-b2 at 72 h in NIH3T3. Each value is normalized to the value without radiation of TGF-b1. (C) Western blotting for Smad3 and phospho-Smad3. Relative
expression a1(I) chain (D) and a2(I) chain (E) treated with and without TGF-b receptor inhibitor SB431542 (10 lM) at 72 h after radiation in NIH3T3. Effect of SB431542 on
phosphorylation of Smad3 using Western blotting (F) and luciferase activity of collagen a2(I) gene (G). Luciferase assay using Smad mutant construct of a2(I) chain collagenpromoter (H) .Each value is normalized to that without radiation and with SB431542 (D, E and G) or without radiation and with wild construct (WT) (H). Asterisks indicate
statically signiﬁcant compared to the value without radiation (B, D, E, G and H). Date represent the mean ± SD of results in three independent experiments. ⁄p < 0.05. (F).

H. Yano et al. / Biochemical and Biophysical Research Communications 418 (2012) 457–463

3. Results
3.1. Up-regulation of type I collagen expression in NIH-3T3 cells andmouse embryonic ﬁbroblast on ionizing radiation
The expression of a1 and a2 chain of type I collagen genes were
initially examined by real-time PCR in NIH-3T3 cells (Fig. 1A and B).
They were almost no change at 24 h and increased at 48 and 72 h
after irradiation. Similarly, they were up-regulated at 72 h in mouseembryonic ﬁbroblast (Fig. 1C and D).
A luciferase assay was performed using the constructs of the basal promoter of the a1and a2 chain of type I collagen gene [15].
The both activity did not changed at 6 h and increased at 72 h in
NIH3T3 cells (Fig. 1E and F).
The concentration of N-telopeptide (NTx) from type I collagenmolecule in the medium of NIH3T3 cells was measured using ELISA
to examine whether the increase of collagen mRNA expression was
followed by an increase in collagen protein synthesis, (Fig. 1G). The
data were consistent with the mRNA and promoter activity(Fig. 1G).

461

TGF-b2, which was also increased after ionizing radiation, in
NIH3T3 (Fig. 2B).
TGF-b signals from the cell surface are transduced to the nucleus by Smad2/3 and 4 complexes. The phosphorylation of
Smad2/3 is crucial for this downstream signaling cascade. The levels of Smad3 and phospho-Smad3 were measured using Western
blotting. The level of phospho-Smad3 was signiﬁcantly increased
at 72 h after irradiation (Fig. 2C).
The expression of type I collagen was measured after treating
cells with SB431542, a speciﬁc inhibitor of the TGF-b receptor. This
suppressed the increased expression of type I collagen chains after
ionizing radiation (Fig. 2D and E). The level of phospho-Smad3 wasalso suppressed with treatment of SB431542 at 72 h after irradiation using Western blotting (Fig. 2F). Furthermore, the promoter
activity of a2(I) collagen gene after ionizing radiation was suppressed with SB431542 (Fig. 2G). Similarly, the luciferase activity
was examined using a mutant Smad binding site construct(Smad-mut/Luc) [15]. The effect of ionizing radiation was notinhibited using the mutant construct (Fig. 2H).
3.3. MAPK pathway on the type I collagen expression after ionizing
radiation

3.2. Effects of ionizing radiation on the expression of TGF-b that
regulates collagen expression
Many studies have suggested that several growth factor andcytokines released from ﬁbroblast play an important role in theexpression of type I collagen [11]. TGF-b is a potent regulator oftype I collagen synthesis. Real time RT-PCR was performed to assess the affect of TGF-b on type I collagen expression after ionizing
radiation. TGF-b1 mRNA was already elevated at 24 h (Fig. 2A),
while those of a1 and a2 chain of type I collagen were not(Fig. 1A and B). TGF-b1 expression was still elevated at 72 h
after ionizing radiation and approximately 20 times higher thanFinally, study investigated whether the MAPK pathway is involved in the expression of type I collagen after ionizing radiation.
The MAPK pathway was inhibited with PD98059, SB203580 and
SP600125, which are speciﬁc inhibitors for ERK1/2, p38 MAPK,
and JNK, respectively. These inhibitor treatments had very little effect on the type I collagen mRNA expression induced by ionizing
radiation (Fig. 3A). In addition, these inhibitors had very little effecton the transcriptional activity of type I collagen following ionizing
radiation (Fig. 3B).
Similarly, ERK1/2, p38 MAPK and Smad3 were selectively
inhibited by speciﬁc siRNAs. These siRNAs speciﬁcally inhibited

Fig. 3. Effect of MAPK pathway on type I collagen mRNA expression induced by ionizing radiation. Relative mRNA expression of a1(I) collagen (A) and luciferase activity ofa2(I) collagen gene (B) treated with or without ERK inhibitor PD98059 (10 lM), p38 MAPK inhibitor SB203580 (10 lM) and JNK inhibitor SP600125 (10 lM). Relative mRNA
expression of a1(I) collagen treated with and without siRNA targeted against ERK1/2, p38 and Smad3(C). The data are after at 72 h in NIH3T3 cells with and without radiation.
Each value is normalized to the value without radiation at 72 h. Date represents the mean ± SD of results in three independent experiments. ⁄p < 0.05.

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H. Yano et al. / Biochemical and Biophysical Research Communications 418 (2012) 457–463

Fig. 4. Schematic illustration of signaling after radiation in NIH3T3 cells.

expression by 50–70% both before and after radiation (Fig. S1). The
inhibition by siRNA-mediated knockdown of ERK1/2 and p38
MAPK had no affect on the expression of type I collagen, while thatby siRNA Smad3 suppressed the expression of type I collagen induced by ionizing radiation (Fig. 3C).

4. Discussion
The present study examined the effect of ionizing radiation on
the expression of type I collagen associated with ﬁbrosis in theﬁbroblast. Ionizing radiation increased the levels of type I collagenmRNA expression and protein synthesis as well as the promoter
activity. The increased expression was seen at 48–72 h, but not
24 h after irradiation. These changes indicate that the effect of ionizing radiation on the expression of type I collagen is a rather delayed response.
TGF-b is a member of a superfamily of multifunctional cytokines which is potent inducer of collagen gene expression duringﬁbrosis. Therefore, TGF-b is considered as a master switch for theformation of RIF. The effect of ionizing radiation on TGF-b expression was seen at 24 h, and it seemed to be earlier than that of collagen expression. The TGF-b signal pathway was inhibited using
TGF-b receptor inhibitor and speciﬁc siRNA associated with inducing expression of collagen gene after irradiation. Smads operate
downstream of the TGF-b superfamily. The luciferase activity was
inhibited when the Smad binding site was mutated. This result
indicates that ionizing radiation activates Smad mediated TGF-bdependent transcriptional activity and that this signal pathway
contributes to type I collagen gene expression in irradiated cells(Fig. 4). On the other hand, TGF-b also transduces signals throughmitogen activated protein kinase (MAPK). The ERK1/2, p38 MAPK
and JNK inhibitors did not affect the expression of type I collagengene induced by ionizing radiation. In addition, the inhibition by
speciﬁc siRNA-mediated knockdown of ERK1/2 and p38 MAPK
had no effect on type I collagen mRNA expression. These results
indicates that ionizing radiation up-regulated type I collagen
expression through the TGF-Smad signal pathway, and that

ERK1/2 and p38 MAPK pathway are not involved in ionizing radiation induced of type I collagen expression in NIH3T3.
However, another mechanism might also work on RIF. Connective tissue growth factor (CTGF) is a matricellular protein that
promotes ﬁbroblast proliferation and ECM production, and is adownstream target of TGF-b signaling [16]. Previously studies reported the effect of CTGF on the expression of type I collagen. The
selective expression of CTGF promotes systemic tissue ﬁbrosis
in vivo [17]. A preliminary experiment showed the up-regulationof CTGF mRNA after radiation and it was suppressed when the
TGF-b signal pathway was inhibited. Therefore, CTGF might bealso involved in RIF via the direct or indirect pathway. Furthermore, the involvement of micro RNAs, which are small, noncoding
and approximately 20-nucleotide-length RNAs, were demonstrated in the regulation of the gene expression [18]. Roderburg
et al. showed that miR-29 mediates the regulation of liver ﬁbrosis
and is part of a signaling molecule involving TGF-b in activatedhepatic stellate cells [19]. This pathway might be also involved
in RIF as well. Further studies will clarify these mechanisms,
which could be useful for the development of novel therapeuticstrategies against RIF.
Acknowledgments
We thank Ms. S. Adachi for her valuable technical support, andthe staff members of Divisions of Life science Research, Laboratory
Animal Science and Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, where
we performed some experiments. This work was supported by
Grants-In-Aid for Scientiﬁc Research (No. 20390402 to H.Y.) from
the Ministry of Education, Culture, Sports, Science, and Technologyof Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2012.01.039.

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