Download Growth regulation of skin fibroblasts

Journal of Dermatological Science 24 Suppl. 1 (2000) S70 – S77
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Growth regulation of skin fibroblasts
Kazuhiko Takehara *
Department of Dermatology, Kanazawa Uni6ersity School of Medicine, 13 -1 Takara-machl, Kanazawa 920 -8641, Japan
Abstract
The growth of skin fibroblasts is regulated in a complex manner by various growth factors. Representative growth
factors are platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth
factor-b (TGF-b), and connective tissue growth factor (CTGF). These growth factors have various biological
activities besides growth regulation of skin fibroblasts, and are involved in wound healing and in the pathogenesis of
various disorders. For example, PDGF and CTGF stimulate chemotaxis of skin fibroblasts, b-FGF stimulates
angiogenesis, and TGF-b stimulates production of matrix proteins. First, the properties of these growth factors are
reviewed briefly. Our skin fibrosis model in newborn mice are also described here. In 1986, Roberts et al. reported
that subcutaneous injection of TGF-b in newborn mice caused granulation tissue formation followed by fibrosis
(Roberts et al. Proc Natl Acad Sci USA 1986;83:4167 – 71). We conducted similar experiments, and found that
TGF-b1, b2 or b3 caused skin fibrosis after 3 consecutive days of injection; this change was transient and disappeared
after 7 consecutive days of injection. In contrast, irreversible fibrosis was observed upon stimultaneous injection of
TGF-b and b-FGF or TGF-b and CTGF, or TGF-b injection for the first 3 days and b-FGF or CTGF injection for
the next 4 days (Shinozaki et al. Biochem Biophys Res Commun 1997;237:292 – 7; Mori et al. J Cell Physiol
1999;181:153–9). These observations suggest that TGF-b induces skin fibrosis and b-FGF or CTGF maintains it in
various skin fibrotic disorders. In the 21st century, we speculate that cocktails of various growth factors may permit
subtle growth regulation of skin fibroblasts; such technology would have applications in the treatment of many skin
diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Skin fibroblasts; Growth regulation; Growth factors
1. Introduction
The abnormal growth or function of skin
fibroblasts causes fibrotic disorders such as scleroderma, keloid and hypertrophic scars. We have
been studying the role of growth factors in the
* Tel.: +81-76-2652343; fax: + 81-76-2344270.
E-mail address: [email protected] (K. Takehara).
pathogenesis of scleroderma and our results suggest that certain growth factors and cytokines
play key roles in the occurrence of scleroderma
[1–10]. In other words, fibrotic disorders of various organs, including skin, can be considered to
be a result of excessive healing (overhealing). As
shown in Figs. 1 and 2, the early sclerotic skin of
scleroderma clinically shows a shiny appearance,
and histologically there is an abundant accumulation of collagen bundles. If we could clarify in
detail the mechanisms of skin fibrosis, it might be
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K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
possible to modulate the growth regulation and
function of skin fibroblasts in such a way as to
prevent skin aging and to maintain young-appearing skin over a long period.
In the 21st century, we speculate that the use of
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cocktails of various growth factors may enable
subtle regulation of the growth of skin fibroblasts,
which will be applicable to the treatment of various skin diseases, as well as to the prevention, or
at least delay, of skin aging.
Fig. 1. Clinical features of typical early scleroderma. Chest skin has a shiny appearance.
Fig. 2. The left panel shows histological change of early edematous scleroderma. The right panel shows histological change of
advanced sclerotic stage of scleroderma.
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K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
Table 1
Bioactive peptides and the fields in which they were discovered
Peptide hormones (classical endocrinology)
Cytokines
Interleukins (immunology)
Interferons (virology)
Colony stimulating factors (hematology)
Tumor necrosis factors (oncology)
Growth factor (cell biology)
Others
Endothelin, etc.
2. Cytokines and growth factors
Cytokines are a group of regulatory molecules
that function as mediators of cell communication
in both normal and pathologic conditions. Cytokines are low-molecular-weight (glyco)proteins
that are produced by a variety of cells. Via interaction with specific cell surface receptors, they
exert multiple biological functions. Mediators in
the cytokine family are interleukins, hematopoietic growth factors, interferons, tumor necrosis
factors, and growth factors, as shown in Table 1.
These regulatory proteins appear to participate in
the mediation of inflammatory as well as immune
reactions, and some may influence tumor growth.
Growth factors were originally regarded only as
regulators of cell proliferation. The family of
growth factors includes epidermal growth factor
(EGF), platelet-derived growth factor (PDGF),
basic fibroblast growth factor (b-FGF), transforming growth factor-b (TGF-b) and connective
tissue growth factor (CTGF). In addition to controlling the growth of normal and malignant cells,
as well as matrix protein formation, some growth
factors are important mediators of cell surface
alteration and immunoregulation.
Several growth factors are essential for normal
wound healing and clinical application of such
growth factors for impaired wound healing has
already been tried [11 – 13].
origins of these growth factors and their target
cells are shown in Table 2.
3.1. PDGF
In 1974, it was discovered that platelets were
the main source of mitogenic activity found in
whole blood serum and missing in plasma. The
growth factor with this mitogenic activity was
called PDGF [14]. PDGF has a molecular weight
of 28 77–35 000. It is made up of two chains,
which share 60% homology. The A chain has a
molecular weight of 14 000 and the B chain has a
molecular weight of 17 000. The B chain has a
high degree of homology with v-sis oncogene.
The PDGF receptor is found on cells that show
a mitogenic response to PDGF. Thus, the receptor is present on fibroblasts, glial cells and vascular smooth-muscle cells. The PDGF receptor has
an extracellular ligand-binding domain, and an
intracellular effector domain with tyrosine kinase
activity, and the peptide-receptor complex is internalized by endocytosis. The receptor is downgraded by PDGF binding. PDGF is chemotactic
for fibroblasts and smooth-muscle cells, at low
concentrations [15].
Recently, PDGF B chain products were approved by the FDA as a wound repair drug for
Table 2
Characteristics of growth factors relevant to wound healing
Factor
Producing cells
Target cells
PDGF
Platelets
Fibroblasts (mitogen,
chemotaxis)
b-FGF
TGF-b
Fibroblasts
3. Major growth factors in wound healing
The major growth factors in wound healing are
PDGF, b-FGF, TGF-b and CTGF. The cellular
Endothelial cells
Macrophages
Smooth muscle
cells
Keratinocytes
Macrophages
Endothelial cells
Platelets
Macrophages
CTGF
Endothelial cells
Fibroblasts
Fibroblasts (mitogen)
Endothelial (angiogenesis)
Fibroblasts (collagen
synthesis)
Endothelial (growth
inhibition, matrix synthesis)
Fibroblasts (mitogen)
Endothelial (angiogenesis)
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
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Fig. 3. The effects of various growth factors in the serum-free culture system of skin fibroblasts. b-FGF had the most potent growth
stimulatory effect among these growth factors.
diabetic ulcers in the United States. Clinical trials
for the treatment of impaired wound healing,
including diabetic ulcers, are being planned in
Japan.
3.2. b-FGF
b-FGF, by stimulating division and migration
of fibroblasts, endothelial cells and epithelial cells,
promotes formation of new capillaries and granulation tissue. In our serum-free experimental systems, b-FGF is the most potent growth stimulator
among the various growth factors (Fig. 3). b-FGF
has a strong homology with hst-1 oncogene.
Clinical trials of b-FGF for treatment of impaired wound healing in Japan have shown that
this agent is very effective for skin ulcers, and this
product is expected to be approved for clinical
usage by the Japanese FDA.
produced as a latent form. The detailed mechanisms of activation of TGF-b in vivo have not
been clarified.
In general, the biological activities of TGF-b
are very strong, and therefore the molecule is
probably produced as an inactive form in order to
block excessive TGF-b action [18].
Serine I threonine kinase is involved in TGF-b
signal transduction, which is quite different from
3.3. TGF-b
TGF-b is a 25 kd homodimer. Five distinct
isoforms were found, of which three are present in
humans. TGF-b1, b2 and b3 exhibit similar, but
not identical biological activities in vitro. TGF-b
acts as a growth inhibitor for most cell types (Fig.
4) [16] and stimulates extracellular matrix production, including type I collagen, type III collagen
and fibronectin, in fibroblasts [17]. This stimulatory effect on ECM production is much stronger
than those of other cytokines. One of the most
important characteristics of TGF-b is that it is
Fig. 4. Effects of various growth factors on endothelial cell
growth. Confluent RHECs were trypsinized, and 1 ×104 cells
were suspended in DMEM containing 10% FCS and plated in
24-well plates. Following cell attachment overnight, the
medium was changed to DMEM containing 2.5% FCS without growth factor (), with 1 ng/ml TGF-b () with 0.25
ng/ml TGF-b (), with 10 ng/ml EGF (), with 10 ng/ml
FGF (), or with both 1 ng/ml TGF-b and 10 ng/ml EGF
( × ) (day 0). Cell numbers were determined on days 3, 6 and
9. (Takehara et al. [16]).
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K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
Fig. 5. CTGF is an autocrine growth factor and is produced
by skin fibroblasts only when they are stimulated with TGF-b.
the case of other growth factors such as PDGF
and EGF, which use tyrosine kinase.
TGF-b has attracted much attention as a key
factor in various fibrotic disorders [19], because it
potently increases ECM production by many
cells.
3.4. CTGF
CTGF was identified originally from cultured
human umbilical vein endothelial cells by Grotendorst and his group at Miami University [20].
This factor is also produced by skin fibroblasts,
but only when they are stimulated with TGF-b
[21]. CTGF is recognized by anti-PDGF polyclonal antibody and CTGF c-DNA was cloned
using anti-PDGF antibody. This factor exhibits
PDGF-like chemotactic and mitogenic activities
and binds to PDGF receptors, though CTGF has
little peptide sequence homology to PDQF A or B
chains.
Fig. 5 summarizes the relationship between
TGF-b and CTGF. CTGF acts as an autocrine
growth factor when skin fibroblasts are stimulated
with TGF-b.
4. An animal model of skin fibrosis by exogenous
injection of TGF-b and other growth factors
We recently established an animal model of
skin fibrosis by exogeneous injection of TGF-b
and other growth factors [22,23]. In 1986,
Roberts et al. reported that TGF-b injection
into newborn mice caused granulation tissue
formation and skin fibrosis [24]. We initially
tried to establish an animal model of skin
fibrosis by TGF-b injection using a similar
method to that described by Roberts et al. [24].
We injected 800 ng of TGF-b1, b2 or b3 into
subcutaneous tissue of newborn mice for 7 days.
The details were described elsewhere [22,23]. The
results of these experiments are summarized in
Table 3.
4.1. Response to application of single growth
factors
Three days of TGF-b injection resulted in a
firm infiltrated lesion at the injection site. Histologic examination revealed granulated tissue formation (Fig. 6A), consisting of lymphocytes,
histiocytes and fibroblasts. After 7 days of consecutive injections, however, this response was not
detectable either macroscopically or microscopically (Fig. 6B).
CTGF injection caused slight edema and
some cell infiltration. Similarly, b-FGF injection
caused slight edematous granulated tissue formation.
4.2. Response to simultaneous and serial
applications of two growth factors
Simultaneous injection of TGF-b plus CTGF
or TGF-b plus b-FGF resulted in fibrotic tissue
formation (Fig. 6C), consisting of fibroblast aggregation and ECM deposition, which persisted
for up to 14 days, even though the injections were
discontinued on day 7. To examine further the
tissue response, two different growth factors were
injected serially, viz. TGF-b on days 1–3 followed
by CTGF on days 4–7. Serial injections of CTGF
after TGF-b caused fibrotic tissue formation. This
response was relatively weak but persisted for at
least 14 days, even though we stopped the injection of growth factors on day 7. Injecting b-FGF
after TGF-b caused increased edematous granulated tissue formation that remained for 10 days.
Injection of CTGF or b-FGF before TGF-b did
not cause any significant change compared with
TGF-b alone.
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
4.3. Meaning of our results
The results of our study clearly demonstrate
that single application of any growth factor is not
sufficient to induce persistent fibrosis, despite continuous injections. Instead, interaction of plural
growth factors seems to be necessary for the
induction of persistent fibrosis in this animal
model. Our observations on serial application of
different growth factors suggest that TGF-b plays
an important role in inducing granulation and
fibrotic tissue formation. The other growth factors, CTGF and b-FGF, are important in maintaining fibrosis. In contrast, injections in the
reverse order, viz. CTGF followed by TGF-b or
b-FGF followed by TGF-b did not produce any
significant response. Hence, we conclude that induction of persistent fibrosis most likely requires
two factors: an induction factor such as TGF-b
and a maintenance factor such as CTGF. B-FGF
may act synergistically by inducing CTGF, because our results suggest that either CTGF
mRNA expression or the CTGF protein itself is
required to develop persistent fibrotic changes.
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We then focused on the action of CTGF as a
candidate mediator to maintain skin fibrosis, because TGF-b selectively stimulates CTGF expression of fibroblasts. CTGF, which is known to
increase fibroblast proliferation and ECM synthesis, has recently been proposed to be a TGF-bspecific downstream mediator. We previously
reported that CTGF mRNA expression is
strongly correlated with tissue fibrosis in
systemic sclerosis and other skin fibrotic disorders
[7,8,25].
5. Two-step fibrosis hypothesis in systemic
sclerosis
Based on the results with TGF-b and CTGF
described above, we hypothesized that a two-step
process of fibrosis occurs in scleroderma.We think
that TGF-b induces fibrosis in the early stage of
systemic sclerosis and then CTGF acts to maintain tissue fibrosis. TGF-b induces CTGF
mRNA, but some additional factor is required for
continuous CTGF mRNA expression.
Table 3
Histological responses to single, simultaneous and serial injections of different growth factors into newborn micea
Injection schedule
Histological change
Days 1–3
Days 4–7
Days 8–10
Day 4
Day 8
Day 11
Single injection
TGF-b
CTGF
b-FGF
PBS
TGF-b
CTGF
b-FGF
PBS
−
−
−
−
++
+−
+
−
−
−
+−
−
−
−
−
−
TGF-b+CTGF
TGF-b+b-FGF
−
−
+++
+++
+++*
+++*
+++*
+++*
CTGF
b-FGF
TGF-b
TGF-b
TGF-b
TGF-b
b-FGF
CTGF
−
−
−
TGF-b
−
TGF-b
−
−
++
++
+−
+−
+
+
+−
+
+++*
+++*
++
++
++
++
+
−
++
++
−
+
−
+
−
−
Simultaneous injection
TGF-b+CTGF
TGF-b+b-FGF
Serial injection
TGF-b
TGF-b
CTGF
CTGF
b-FGF
b-FGF
CTGF
b-FGF
a
−, no change; +−, slight edema and some cell infiltration; +, edematous granulation tissue; ++, granulation tissue consisting
of lymphocytes, histiocytes, and fibroblasts; +++, fibrotic tissue consisting of fibroblast aggregation and extracellular matrix
deposition, and asterisks denotes marked fibrosis. See the text for the concentrations of different growth factors injected.
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K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
Acknowledgements
This article is based on a presentation given at
the Shiseido Science Symposium 277, held on
April 15, 2000, Tokyo. The major part of these
investigations were conducted by collaborators at
Tokyo University (Drs Atsuyuki Igarashi,
Yoshinao Soma, Nobukazu Hayashi and Takashi
Kakinuma) and at Kanazawa University (Drs
Mikio Shinozaki, Toshifumi Mori, Shigeru
Kawara and Shinichi Sato).
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Fig. 6. An animal model of skin fibrosis induced by exogenous
growth factor injections. (A) After 3 consecutive days of
injections of TGF-b2. (B) After 7 consecutive days of injections of TGF-b2. (C) Combination of TGF-b 3 and b-FGF
for 7 consecutive injections.
In the future, substances that block CTGF
activities, such as anti-CTGF neutralizing antibody or CTGF antisense molecules are likely to
be candidates for therapeutic use to treat systemic
sclerosis. Some preliminary experiments using our
animal model are in progress in our laboratory.
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