Copyright # Blackwell Munksgaard 2003

Experimental Dermatology 2003: 12 (Suppl. 2): 35–42
Blackwell Munksgaard . Printed in Denmark

EXPERIMENTAL DERMATOLOGYISSN 1479-1250

Tretinoin reverses upregulation of matrix
metalloproteinase-13 in humankeloid-derived fibroblasts
Uchida G, Yoshimura K, Kitano Y, Okazaki M, Harii K. Tretinoin
reverses upregulation of matrix metalloproteinase-13 in humankeloid-derived fibroblasts.
Exp Dermatol 2003: 12 (Suppl. 2) : 35–42. # Blackwell Munksgaard, 2003

Gentaro Uchida, Kotaro
Yoshimura, Yukie Kitano,Mutsumi Okazaki and Kiyonori
Harii

Abstract: Keloids are skin abnormalities that are characterized byexcessive deposition of collagen bundles in the dermis. Patients withkeloids complain not only about their cosmetic appearance, but also
about continuous itching and/or tenderness associated with chronic
inflammation. Degradation of extracellular matrix (ECM) may be
upregulated, associated with the expansion of keloids into circumferential
skin, and high metabolic activity of keloid tissues may be due to increased
matrix metalloproteinase (MMP) activity. Based on these hypotheses, we
examined differences in expression of MMP-1, MMP-8, and MMP-13
between keloid-derived and normal dermal fibroblasts. Since retinoids arepotent inhibitors of MMPs in the treatment of photoaged skin andcancers, we also examined whether or not tretinoin affects MMP
expression of keloid-derived fibroblasts. The results of real-timepolymerase chain reaction and ELISA demonstrated significant
upregulation of MMP-13 and significant downregulation of MMP-1 and
MMP-8 in keloid-derived fibroblasts, at both mRNA and protein levels.
MMP-1 mRNA expression in the control group was significantly
upregulated after the addition of tretinoin, whereas no significant change
was observed in the keloid group. MMP-8 mRNA expression in thecontrol group was significantly upregulated by tretinoin, with the peak at
12 h, while no significant change was observed in the keloid-derived
fibroblasts. In contrast, the remarkably elevated MMP-13 mRNA
expression in the keloid group was significantly suppressed, with the peaksuppression at 12 h after addition of tretinoin, while MMP-13 mRNA
expression in the control group was not significantly changed. The
decrease in MMP-1 and MMP-8 may contribute to accumulation of type I
and type III collagen in keloid tissues, and this mechanism may be
modulated by molecular interaction with MMP-13. Tretinoin appeared toreverse the abnormal expression profile of MMPs in keloid-derived
fibroblasts, such as markedly elevated expression of MMP-13, partly
through inactivation of AP-1 pathway. The present results suggest that
tretinoin may be clinically useful to improve the chronic inflammation
seen in keloids and prevent expansion of keloid tissues into circumferential
normal skin.

Department of Plastic and Reconstructive
Surgery, Graduate School of Medicine, Universityof Tokyo, Tokyo, Japan

Introduction
Keloids are skin abnormalities characterized byexcessive deposition of collagen bundles in thedermis. In keloids, the normal wound healing process is derailed from the normal, resulting inimpairment of the balance between production
and degradation of extracellular matrix (ECM)

Keywords: chemoprevention – fibroblast –keloid – matrix metalloproteinases (MMPs) –tretinoin (all-trans retinoic acid)
Kotaro Yoshimura MD
Department of Plastic and Reconstructive
Surgery
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo
Bunkyo-ku
Tokyo 113-8655
Japan.
Tel: þ81 3 5800 8670
Fax: þ81 3 5800 6929
E-mail: yoshimura-pla@h.u-tokyo.ac.jp

components such as collagens (1). Since fibroblasts
play a leading part in production of ECMs, it isthought that there is a difference in the cellularfunction between keloid-derived fibroblasts andnormal ones. However, accumulating data haveshown that there is no significant difference in
culture growth, cell size, population density, andkaryotype between these fibroblasts (2).

35

Uchida et al.

During normal wound repair, type III collagenappears at day 2–3, followed by type I collagen atday 6–7 (3). The total amount of type I and III
collagen increases over time, whereas the proportionof type III collagen decreases from 60% at 1 week
after wounding, to 28% in the mature scar (4). In
keloids, however, the relative amount of type III
collagen remains high compared with normal scars
or normal skin (5). The ratio of a-1(I)-procollagen(a precursor of type I collagen) mRNA to a-1(III)procollagen (a precursor of type III collagen)mRNA is markedly elevated in keloid-derived fibroblasts compared with that in normal-tissue-derived
fibroblasts in vitro (6). The same tendency is also
observed in keloid tissues in vivo (7). There seems
to be a discrepancy between excessive accumulationof type III collagen in keloid tissues and an elevatedmRNA level of type I procollagen in biosynthesis,
which remains unknown. This discrepancy is explicable if cytologic aberrations occur at the level ofcollagen fiber degradation, especially type III collagen. However, previous studies have only shown anormal (8), decreased (9), or increased (10) collagenase activity (more accurately, degradation activity of type I collagen); no studies have demonstrated
altered expression of each type of collagenase in
keloid tissues or keloid-derived fibroblasts.
Currently, collagenases are categorized as matrix
metalloproteinase (MMPs), which are a group of
endoproteinases with a divalent Zn2þ at the active
site involved in ECM remodeling. MMP-1 (alsoknown as interstitial collagenase or collagenase1), MMP-8 (neutrophil collagenase, collagenase2), and MMP-13 (collagenase 3) are the only
mammalian enzymes recognized for their uniqueability to cleave the triple helical domain of fibrillar
collagen types I, II, and III (11). However, eachcollagenase differs in the extent to which it cleaves
these fibrillar collagen subtypes in vitro. MMP-1
preferentially degrades type III collagen, whereas
MMP-8 has its greatest activity on type I collagen(12). Neither MMP-1 nor MMP-8 appears to haveany significant activity against type II and IV
collagen. MMP-13 is the most recently discovered
human collagenase, which can degrade all fibrillar
collagen subtypes with almost equal efficacy, and is
the only collagenase with significant activity
against type II and IV collagen (13).
Before establishing the novel classification ofcollagenases described above, fibroplastic lesions
due to deposition of ECM components such as
collagen fibers had been classified into two groups;
increased-level and decreased-level, according totheir collagenase activities. Rheumatoid arthritis,
osteoarthritis, periodontal diseases, otitis media
cholesteatoma, and malignant tumors belong to36

the former, while pulmonary fibrosis, hepatic
fibrosis, hepatic cirrhosis, and systemic sclerosis
belong to the latter (11). We thought that, todecide the direction of treatment for keloids, it
was essential to determine whether keloids belong
to the former group or the latter one. The activityof MMPs is regulated at three levels; transcription,
zymogen activation, and inhibition of proteolytic
activity (11). Regarding regulation at the transcriptional level, most MMPs are induced throughactivation of nuclear AP-1 transcription factor(14–16). The AP-1-dependent activation of inducible MMPs is potently inhibited by glucocorticoids(17) and retinoids (18) at the transcriptional level.
For regulation at the extracellular zymogen activation level, latent precursors or zymogens of most
MMPs are proteolytically activated via exposureof the catalytic site (19). As for the regulation at
the level of inhibition of proteolytic activity, nonspecific inhibitors such as a2-macroglobulin anda1-antiprotease, and specific inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), areresponsible for the inhibition (20).
It has been reported that MMP-1 and MMP-8
activities are upregulated in skin photoaged byrepeated exposure to ultraviolet irradiation(21,22). However, tretinoin suppressed upregulated MMP-1 in photoaged skin at the transcriptional level, probably via anti-AP-1 effects (23).
The activity of MMPs is also intimately correlated with the invasive or metastatic ability ofmalignant tumor cells (24,25). For skin malignancies especially, degradation of ECM is the first step
to local invasion and metastasis. Thus, basic andclinical studies have been performed with the aimof chemoprevention of ECM degradation inmalignant melanoma, basal cell carcinoma, andsquamous cell carcinoma (26–28), and chemoprevention of cell growth. Retinoids are the subject ofincreasing interest as an effective means of controlling upregulated MMP activity of malignant
tumor cells and inhibiting the advancement oftumors (27). It has been reported that retinoids
suppress MMP-1 and MMP-8 activity in thesemalignant tumor cells in vitro (28).
Thus, we hypothesized that degradation of
ECM may be upregulated during the expansionof keloids into circumferential skin, and that thehigh metabolic activity of keloid tissues (29) may
be due to increased MMP activity, which may
contribute to the continuous itching and/or tenderness associated with chronic inflammation seen
in keloids (30). Based on these hypotheses, we
examined differences in expression of MMP-1,
MMP-8, and MMP-13 between keloid-derived
and normal dermal fibroblasts. Since retinoids

Tretinoin reverses upregulation of MMP-13 in human keloid-derived fibroblasts

are potent inhibitors of MMPs in the treatment ofphotoaged skin and cancers, as described above,
we also examined whether or not tretinoin affects
MMP expression of keloid-derived fibroblasts.
Materials and methods
Clinical specimens
Twelve specimens of keloid (keloid group), diagnosed on thebasis of their clinical appearance, anatomic location, etc., were
excised at the Department of Plastic and Reconstructive Surgery,
University of Tokyo Hospital. As a control group, 12 normalskin samples, matched to the site of predilection for keloids(scapular area, shoulder, and upper arm), were also excised during the plastic surgery.
Part of each tissue sample was used to establish a primary cell
culture, and the rest was used for histopathologic diagnosis. All
keloid samples displayed the diagnostic histopathologic characteristics of keloids. No hypertrophic scars were included in thematerials. The clinical data of the keloid group and the control
group are shown in Table 1. No significant difference in age
between the two groups was observed (unpaired Student’s t-test;
P ¼ 0.4907). All the biopsies were taken in accordance with the
Declaration of Helsinki.

Primary dermal fibroblast cultures
The primary dermal fibroblast cultures from the keloids (n ¼ 12)
and control skin samples (n ¼ 12) were established using explants(31). For primary culture of keloid fibroblasts, marginal portionsof keloid lesions were used. Briefly, after removal of the reticularlayer of the dermis and epidermis from total skin samples, thesurface side of the papillary layer was attached to the culture dish,then the culture medium was added and a cell culture was started(37 C, 5% CO2). Subculture was performed 2 weeks after primary culture, when cell culture reached 60–70% confluence.
Human fibroblasts were isolated from the same skin specimens
used for explants after they were separated from the epithelium,
and grown in fibroblast growth medium (FGM), which consistsof Dulbecco’s modified Eagle’s medium, 0.6 mg/ml glutamine,and 10% fetal calf serum. Since the primary culture of dermal
fibroblasts contained a small amount of keratinocytes, passages
3–5 were used for the experiment.

Measurement of MMP mRNA expression by
real-time PCR
Real-time reverse transcriptase polymerase chain reaction (realtime PCR) assays (32) using SYBR1 Green chemistry (33,34)

were performed with a sequence detection system (ABI PRISM1
7700; PE Biosystems, Foster City, CA, USA) to quantify the
MMP-1, MMP-8, and MMP-13 mRNA expression.
The fibroblasts of the keloid group and normal group wereseeded at the density of 5  106 cells on a 100-mm Petri dish in
10 ml of culture medium. Forty-eight hours after seeding, theculture medium of each dish was changed to medium containing
1 mmol/l tretinoin. Total RNA was obtained with RNeasy1 Mini
Kit (Qiagen, Hilden, Germany) as described previously (35), at
0, 6, 12, 24 and 48 h after the medium change. In order to eliminate
any residual genomic DNA, RNase-Free DNase Set (Qiagen)was also used. The concentration of each RNA sample wasmeasured with a spectrophotometer (V-530 UV/VIS; Jasco,Tokyo, Japan).
A reverse transcriptase reaction was performed using RNA
PCR Kit (AMV Ver.2.1; TaKaRa, Tokyo, Japan). We added
5 mg RNA to 100 ml of reaction mixture (final concentrations
5 mmol/l MgCl2, 1 mmol/l dNTP mixture, 1 U/ml RNase inhibitor, 0.125 mmol/l oligo dT-Adaptor primer, 10 mmol/l Tris-HCl,
50 mmol/l KCl, pH 8.3) containing 25 U AMV Reverse Transcriptase XL, which was incubated at 42 C for 30 min, followed
by inactivation of the enzyme at 99 C for 5 min with Program
Temp Control System PC-700 (Astec, Fukuoka, Japan). The
control reaction was performed simultaneously with an otherwise
identical reaction, but without reverse transcriptase.
Real-time PCR was performed on 96-well optical reaction
plates (ABI PRISM; PE Biosystems). Sequences of each oligonucleotide primers are shown in Table 2. All PCR reaction mixturescontained 25 ml of TaqMan SYBR1 Green PCR Master Mix(2) (PE Biosystems,), 0.25 ml of forward primer (10 pmol/ml),
0.25 ml of reverse primer (10 pmol/ml), 4 ml of each diluted sample,
and 20.5 ml of double distilled water per well. PCR amplification of
the identical sample was performed with both specific primer pairsof the target MMP gene and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene on the same reaction plate. The
PCR reaction comprised 40 cycles, consisting of denaturing at
95 C (15 s), then annealing/extension at 60 C (1 min). In order toeliminate the possibility of contamination of genomic DNA during extraction of total RNA, the RNA extract before reverse
transcription was amplified in the same way as the control, andno amplification was detected.

Measurement of secreted MMP protein by
enzyme-linked immunosorbent assay
The fibroblasts, derived from keloid tissue or normal skin, wereseeded at the density of 5  106 cells on a 100-mm Petri dish in
10 ml of culture medium as described above. The culture medium
Table 2. Oligonucleotide primers used in the real-time PCR amplification of
MMPs

Gene

GenBank
accession
numberHuman MMP-1

X05231

Table 1. Profiles of skin samples used in the experiments

NumberAge (years)(mean  SD)
Sex (M/F)
Body sites

Control group(normal skin)

Keloid group12
17–51 (32.8  9.5)

12
8–58 (29.0  15.9)

6/6
Scapular region (9),upper arm (2),shoulder (1)5/7
Scapular region (4),upper arm (2)shoulder (2) chest (2),forearm (1), ear (1)No significant difference was observed between the control and the keloidgroup (P ¼ 0.4907).

Primer sequence1

Nucleotide
coordinates

ACGGATACCCCAAGGACATCT
1139–1159
CTCAGAAAGAGCAGCATCGATATG 1192–1215
Human MMP-8 J05556
ACCAAAGAGATCACGGTGACAA
559–580
TGAGCATCTCCTCCAATACCTTG
633–655
Human MMP-13 X75308
CCTGGAGCACTCATGTTTCCTAT
710–732GACTGGATCCCTTGTACATCGTC
773–795
81–99
Human GAPDH2 NM_17701 GAAGGTGAAGGTCGGAGTCGAAGATGGTGATGGGATTTC
287–306
1
All primer sequences are written from 5’ to 3’. For each primer pair, the top
sequence is sense and the bottom sequence is antisense. 2GAPDH is humanglyceraldehyde-3-phosphate dehydrogenase and was used as a housekeeping
gene (i.e. a control).

37

Uchida et al.
of each dish of the experimental group was changed to a medium
containing 1 mmol/l tretinoin (with 10 ml of ethanol as a vehicle),or to that containing only 10 ml vehicle, at 48 h after seeding.
Before assay, 2 ml of each culture supernatant was concentrated
by freeze-drying (Freeze Dryer FRD-mini; Asahi Technoglass,Tokyo, Japan). Freeze-dried supernatants were dissolved in theassay buffer for the enzyme-linked immunosorbent assay(ELISA) system (0.03 mol/l H3PO4, 0.1 mol/l NaCl, 1% bovine
serum albumin, 0.01 mol/l EDTA). For the MMP-1 assay, 10
concentrated samples were prepared, and for MMP-8 and MMP13, 20 concentrated samples were prepared.
The culture supernatant of each dish was collected 96 h
after the medium change. The BIOTRAK ELISA MMP-1,
MMP-8, and MMP-13 System (Amersham Pharmacia Biotech,
Buckinghamshire, U.K.) was used for measurement of MMP-1,
MMP-8, and MMP-13 protein levels in each culture supernatant,respectively. Standard and concentrated (10) samples wereincubated in microtiter wells precoated with a primary mouseantihuman MMP-1 monoclonal antibody followed by a secondary rabbit antihuman MMP-1 polyclonal antibody. The resulting
antigen–antibody complex was detected using horseradish
peroxidase-labeled donkey antirabbit IgG, and the conjugate
quantified by a colorimetric reaction with 3,30 ,5,50 -tetramethylbenzidine substrate. After stopping the reaction with 100 ml of
1 mol/l sulphilic acid, the resultant color was read at 450 nm witha microplate reader (Model 550; Bio-Rad Laboratories, Hercules,
CA, USA). All samples were assayed in duplicate, and the concentration of the target protein in each sample was determined byinterpolation from the standard curve.

Statistical analysis
All data are presented as mean + standard error. The data were
statistically analyzed using Student’s t-test. Differences in the
keloid and normal groups were tested using a paired t-test. Differences between the keloid and control groups were tested using
an unpaired t-test. P < 0.05 was considered significant.

Results
MMP mRNA expression in keloid-derived
fibroblasts and normal-skin-derived fibroblasts
MMP-1, MMP-8, and MMP-13 mRNA expression in the keloid group and the control groupwere measured by real-time PCR system, and theresults are demonstrated in Fig. 1.

A

B

The normalized MMP-1 mRNA expression(MMP-1/GAPDH) was significantly downregulated in keloid-derived compared with normal
fibroblasts (P ¼ 0.0001), and the fold change vs.
the average of the control group was 0.32 + 0.02
(mean + standard error). Similarly, the normalized MMP-8 mRNA expression was significantly
downregulated in keloid-derived fibloblasts(P ¼ 0.0120); the fold change vs. the average ofthe control group was 0.29 + 0.02. However, thenormalized MMP-13 mRNA expression was significantly elevated in keloid-derived fibroblasts(P < 0.0001); the fold change vs. the average ofthe control group was 21.21 + 1.24. No agerelated difference was observed in MMP expression in either normal or keloid-derived fibroblasts
in this study using non-ultraviolet (UV)-exposedskin.
Effects of tretinoin on MMP mRNA expression in
keloid-derived fibroblasts and normal-skin-derived
fibroblasts.
Effects of tretinoin on MMP-1, MMP-8, and
MMP-13 mRNA expression over time were also
examined by real-time PCR, and the results areshown in Fig. 2. MMP-1 and MMP-8 mRNA
expression in the control group were significantly
upregulated, with the peak at 12 h after addition oftretinoin (2.03 + 0.03 and 250.80 + 4.98, respectively) (P < 0.0001), whereas no significant change
was observed in the keloid group within 24 h after
the addition of tretinoin. In contrast, markedly
elevated MMP-13 mRNA expression in the keloidgroup was significantly suppressed by tretinoinwith the peak suppression at 12 h (1.29 + 0.04)(P ¼ 0.0003). MMP-13 mRNA expression in thecontrol group was not significantly changed bytreatment with tretinoin.

C

Figure 1. MMP-1, -8, and -13 (A, B, and C) mRNA expression in keloid-derived fibroblasts and normal dermal fibroblasts, quantified
by real-time PCR. Normalized MMP mRNA expression were calculated as MMP mRNA/GAPDH mRNA. The values of the keloidgroup were presented as the fold change in normalized MMP mRNA expression relative to the average value of the control group(normal dermal fibroblasts). Values are means + SE. *P < 0.05 vs. normal dermal fibroblasts. NF: normal dermal fibroblasts (n ¼ 12),
KF: keloid-derived fibroblasts (n ¼ 12).

38

Tretinoin reverses upregulation of MMP-13 in human keloid-derived fibroblasts
A

B

C

Figure 2. Sequential changes in MMP-1, -8, and -13 (A, B, and C) mRNA expression in keloid-derived fibroblasts and normal dermal
fibroblasts quantified by real-time PCR. Normalized MMPs mRNA expression were calculated as MMP mRNA/GAPDH mRNA. The
values of the keloid group were presented as the fold change in normalized MMP mRNA expression relative to the average value of thecontrol group (normal dermal fibroblasts) at 0 h. Values are means + SE. * P < 0.05 vs. normal dermal fibroblasts at each time point.
# P < 0.05 vs. before treatment with tretinoin. NF: normal dermal fibroblasts (n ¼ 12), KF: keloid-derived fibroblasts (n ¼ 12).

MMP protein levels in the culture supernatants andeffects of tretinoin on them
MMP-1, MMP-8, and MMP-13 protein levels in theculture supernatants in the keloid and control
groups, and effects of tretinoin on these, were examined by ELISA. The results are shown in Fig. 3.
MMP-1 protein expression was significantly
lower in the keloid (1.04 + 0.03 ng/ml) than in
the control group (6.16 + 0.10 ng/ml) (P < 0.0001).
Similarly, the MMP-8 protein level was
significantly lower in the keloid (11.54 + 0.24 pg/ml)
than in the control group (15.36 + 0.29 pg/ml)(P ¼ 0.0043). However, the MMP-13 protein level
was significantly elevated in the keloid group(17.53 + 0.33 pg/ml) in contrast to the control
group (6.71 + 0.10 pg/ml) (P < 0.0001).
In both groups, tretinoin treatment for 96 h significantly elevated the MMP-1 (3.35 + 0.07 ng/ml,
8.22 + 0.09 ng/ml; P < 0.0001, P ¼ 0.0019) and
MMP-8 (21.21 + 0.22 pg/ml, 30.13 + 0.37 pg/ml;
P < 0.0001 for both) protein levels. However, themarkedly elevated MMP-13 protein level in the
keloid group was significantly decreased after
treatment with tretinoin for 96 h (8.56 + 0.20 pg/ml)(P < 0.0001, while the MMP-13 protein level inABthe control group was modestly suppressed bytretinoin (6.23 + 0.08 pg/ml) (P ¼ 0.0415).
Discussion
MMP-1, MMP-8, and MMP-13 all degrade type I
and type III collagen. Among the three MMPs,
MMP-1 and MMP-8 most effectively degrade
type III and type I collagen, respectively. The decreasein MMP-1 and MMP-8 may partly contribute to
the accumulation of type I and type III collagenin keloid tissues, and this mechanism may be
modulated by molecular interaction with MMP-13.
MMP-13 is an abnormal collagenase subtype
that has been found in the bottom of chronic
ulcers, where angiogenesis and fibrosis occur (36).
On the other hand, MMP-1 and MMP-8 are considered to be ‘normal’ collagenase subtypes, whichappear in normal wound-healing process (12,37).
Before the discovery of MMP-13, reports had
shown rather conflicting results concerning thecollagenase activity in degradation of type I or
total collagen in keloid tissues; some reports had
shown normal (8), or decreased (9), and others
showed increased (10) activity of collagenase.
C

Figure 3. MMP-1, -8, and -13 (A, B, and C) protein levels in the culture supernatants of the keloid group and the control groupmeasured by ELISA. The effects of tretinoin was examined after treatment of 1 mmol/l tretinoin for 96 h. Values are means + SE.
* P < 0.05 vs. the control group (without addition of tretinoin). # P < 0.05 vs. before treatment with tretinoin. NF: normal dermal
fibroblasts (n ¼ 12), KF: keloid-derived fibroblasts (n ¼ 12).

39

Uchida et al.

These variable results may be partly due to different areas of keloid tissue, for example, a marginalor a central area, being used. In our preliminary
study, MMP-13 mRNA expression was found to
be markedly higher in marginal than central areasof keloid tissues (data not shown). In the present
study, comparison of MMP expression was
performed using a marginal area of each keloid
sample.
Our study has demonstrated a significant
increase in MMP-13 expression and a decrease in
expression of MMP-1 and MMP-8 in keloidderived fibroblasts, in both mRNA and protein
levels. The remodeling of the surrounding matrix
by MMP-13 may interfere in the normal degradation process of wound healing in keloid tissues,
and may initiate the negative feedback mechanism
to transcription of MMP-1 and MMP-8, which act
in the normal wound-healing process. Thesemechanisms could be related to the chronic inflammation and infiltration into circumferential normal skin seen in keloid tissues.
To correct the abnormal wound-healingmechanism mentioned above, we assumed that
retinoids are potent additives, and then investigated the influences of tretinoin on abnormal
MMP expression of keloid tissues. The present
study revealed that addition of tretinoin to theculture media caused significant downregulationof MMP-13 in keloid-derived fibroblasts at bothmRNA and protein levels, and significant upregulation of MMP-8 in normal dermal fibroblasts.
Although mRNA expression of MMP-1 was notclearly affected in the keloid-derived fibroblasts
after treatment with tretinoin, upregulation of
MMP-1 and MMP-8, and downregulation of
MMP-13 at the protein level, may suggest that
tretinoin reverses the specific changes in the
MMP expression profile of keloids. We also examined mRNA expression of four subtypes of TIMP
(TIMP-1, -2, -3, and -4); all of these subtypes wereupregulated in keloid-derived fibroblasts, but we
did not detect any significant changes after treatment with tretinoin (data not shown).
A small number of previous studies reported
effects of retinoids on primary cultured humandermal fibroblasts. Daly et al. (38) demonstrated
that tretinoin significantly reduces collagen production of human primary cultured fibroblasts.
Abergel et al. (39) reported that tretinoin and isotretinoin significantly inhibit degradation activity
of type I collagen fibers in keloid-derived fibroblasts. On the other hand, in the field of cancer cellstudy, degradation of type I and type IV collagen,and invasion into collagen matrix was reported tobe significantly inhibited by retinoids (28). The

40

results of our study and those in the literaturesuggest that a remarkable inhibition of degradation of type I collagen by tretinoin is presumably
due to a strong inhibition of MMP-13 expression
by tretinoin, which negates the upregulation of
MMP-8.
A previous study using human skin in vivo suggested that MMP-1, but not MMP-13 were upregulated in human keratinocytes and fibroblasts by
UV irradiation, MMP-8 was upregulated inhuman neutrophils by UV, and only MMP-1 upregulated by UV was blocked by tretinoin (22). Ourin vitro study using fibroblasts taken from nonUV-exposed skin (keloid-prone location) showedthat only MMP-13 was significantly suppressed bytretinoin.
Expression of MMP-1 and MMP-13 are known
to be induced at the transcriptional level by avariety of growth factors (14), and these extracellular stimuli result in activation of the nuclear AP1 trascription factor complex, which binds to theAP-1 cis-regulatory element in the promoter
region of the relevant MMP gene and potently
activates its transcription (15). This AP-1dependent activation of inducible MMPs ispotently inhibited by glucocorticoids (17) and tretinoin (18) at the transcriptional level. The present
results reveal that MMP-13 is upregulated inkeloid-derived fibroblasts, and that this upregulation of MMP-13 is inhibited at the transcriptional
level by tretinoin, suggesting that this upregulationin keloids is induced via the AP-1 pathway. However, exactly how tretinoin upregulates MMP-1
and MMP-8 in keloid-derived fibroblasts and innormal dermal fibroblasts remains unknown.
Further investigations of the regulatory mechanisms are necessary.
Conclusion
In this study, it is suggested that MMPs are abnormally regulated in keloid tissues and chronic
ulcers, and that these abnormal changes may bereversed by treatment with retinoids. Tretinoin
may improve the chronic inflammation seen in
keloids and prevent expansion of keloid tissues
into circumferential normal skin.
Since 1999, we have been performing clinical
trials with tretinoin aqueous gel (0.1–0.4%) for
treatment of keloids. Our preliminary results
demonstrate that topical application of tretinoin
on keloids has unique advantages. In most cases,
itching and/or tenderness of the lesions disappeared after topical tretinoin (in preparation),
although the volume-suppressing effects on the
fibrosis were quite modest. We assume that theTretinoin reverses upregulation of MMP-13 in human keloid-derived fibroblasts

effects of tretinoin on MMP expression resulted insuppression of chronic inflammation, and prevention of growth and invasion of keloid tissues. Considering the limited clinical improvements andside-effects generated by existing techniques, theclinical use of topical tretinoin looks promising.
Thus, the molecular mechanisms of the regulationof MMPs deserve further investigation. The resultsof this study may be helpful in developing morechemically stable synthesized retinoids, which specifically reverse the abnormal expression of MMPs
and prevent cell growth in keloids, with minimumside-effects.
Acknowledgements
This study was supported in part by Grants-in-Aid for Fundamental Research A(2) 12307039 and B(2) 12470374 from theJapanese Ministry of Education, Culture, Sports, Science, andTechnology.

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