REVIEW
Transcriptional proﬁling of the scleroderma ﬁbroblast reveals
a potential role for connective tissue growth factor (CTGF) in
pathological ﬁbrosis
Andrew Leask
Centre for Rheumatology, Royal Free and University College Medical School, University College London(Royal Free Campus), London, UK(Received for publication on December 15, 2003)

Abstract. The cause of ﬁbrotic disease is unknown. We have undertaken transcriptional proﬁling ofdermal ﬁbroblasts cultured from patients with the ﬁbrotic disease scleroderma (systemic sclerosis, SSc)
to identify genes overexpressed in ﬁbrosis and have explored their contribution to the ﬁbrotic phenotype. Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, isoverexpressed in SSc ﬁbroblasts. In adult skin, CTGF is not normally expressed in dermal ﬁbroblasts.
However, CTGF is induced during the wound healing response and is constitutively overexpressed byﬁbroblasts present in ﬁbrotic lesions. The overexpression of CTGF present in ﬁbrotic lesions contributes to the phenotype of scleroderma in that CTGF promotes matrix deposition, and ﬁbroblast adhesion and proliferation. In animal models, whereas either TGFb or CTGF alone produce only a transientﬁbrotic response, CTGF and TGFb act together to promote sustained ﬁbrosis. Thus the constitutive
overexpression of CTGF by ﬁbroblasts present in ﬁbrotic lesions would be expected to directly contribute to chronic, persistent ﬁbrosis. (Keio J Med 53 (2): 74–77, June 2004)
Key words: CTGF, scleroderma, ﬁbrosis, TGFb

Introduction

CTGF is Overexpressed in Scleroderma Fibroblasts

De novo synthesis of connective tissue occurs during
the wound healing process. Normally this response isappropriately terminated; however, if the wound healing process occurs unabated, excessive deposition of
extracellular matrix (ECM) occurs resulting in the formation of scar tissue.1 Excessive scarring results inthe progressive, pathological scarring characteristic ofﬁbrotic disease. One example of such a connective
tissue disease is systemic sclerosis (scleroderma; SSc)
which affects the skin as well as internal organs.2,3There is no effective treatment for this disorder, in part
because the etiology of this disease is unknown. Thus
to identify appropriate targets for therapeutic intervention, it is necessary ﬁrst to identify proteins overexpressed in ﬁbrotic disease and then to assess therole that these proteins might have in the ﬁbrotic phenotype.

By differential display and Western blot analyses,
we found that connective tissue growth factor (CTGF,CCN2) is constitutively overexpressed in dermal ﬁbroblasts isolated from SSc lesions.4 In vitro, CTGF promotes ﬁbroblast proliferation, matrix production, andgranulation tissue formation.5–7 CTGF also promotes
cell adhesion and migration in a wide variety of cell
types.8 Human foreskin ﬁbroblasts adhere to CTGF
through integrina6b1.8 In addition, a heparin-binding
domain of CTGF, present in the carboxy-terminal region of CTGF, is important for CTGF-mediated ﬁbroblast proliferation and adhesion.7,9 Therefore, CTGF
promotes adhesion in a seemingly unique integrin- andHSPG-dependent fashion.
One of the most compelling pieces of evidence
showing CTGF can independently induce matrix is thatan expression vector encoding CTGF transfected into

Presented at the 1333rd Meeting of the Keio Medical Society in Tokyo, November 27, 2003.
Reprint requests to: Dr. Andrew Leask, Centre for Rheumatology, Royal Free and University College Medical School, University CollegeLondon (Royal Free Campus), Rowland Hill Street, London UK NW3 2PF, email: a.leask@rfc.ucl.ac.uk
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Keio J Med 2004; 53 (2): 74–77

75

The TGFb
TGF induction of CTGF requires Smad and TEF
binding elementsb
SEAP reporter
Smad

SEAP reporter

TEF

TGF
TG response

-805
-244
-166
Smad mut
TK-244 to -76-166 to -76-166 to -116
TEF 5’ mut
TEF 3’ mut
TEF 5’ mut
TEF 3’ mut
Fig. 1 Mapping of response elements in the CTGF promoter necessary for its induction by TGFb. Smad ¼ Smad binding element, TEF ¼ TEFbinding element, Star ¼ construct which is TGFb responsive, X ¼ mutated binding site, TK ¼ minimal herpes simplex virus thymidine kinasepromoter, SEAP ¼ secreted enhanced alkaline phosphatase reporter gene. NIH 3T3 ﬁbroblasts were transfected with CTGF promoter/reporter constructs, and reporter gene expression was determined. The Smad and TEF elements are required for the ability of the CTGF promoter to respond to TGFb. For details, see refs (13, 14).

ﬁbroblasts can activate a cotransfected reporter construct driven by the type I collagen promoter.4 In vivo,
CTGF acts with TGFb to induce a sustained ﬁbrotic
response, as although subcutaneous injection of TGFb
into neonatal mice caused a transient ﬁbrotic response and injection of CTGF alone had little effect,
co-injection of CTGF and TGFb resulted in persistentﬁbrosis.10
CTGF Gene Regulation: CTGF Overexpression inScleroderma Fibroblasts is Independent of Its TGFb
Response Element but Dependent on Sp1
CTGF is not normally expressed in skin unless induced, for example during the normal wound repair
process.11 The proﬁbrotic protein TGFb induces CTGF
expression in dermal ﬁbroblasts, but not in epidermal
keratinocytes.6,11–13 Although the TGFb induction ofCTGF requires Smad3 and a functional Smad binding
element in the CTGF promoter acting with a consensus
TEF binding element (Fig. 1),13,14 the elevated expression of the CTGF promoter in SSc ﬁbroblasts
seems to be independent of its Smad response element14 and TGFb response element.13 Instead, theelevated expression of the CTGF promoter in SScﬁbroblasts requires a Sp1 binding element in the CTGF
promoter, which is not required for the ability of CTGF
to respond to TGFb (Fig. 2).13,15 Sp1 regulates a wide
variety of matrix genes16 suggesting that elevation ofmatrix genes in lesional SSc ﬁbroblasts may be due toelevated Sp1 binding activity.
Hypothesis: CTGF and TGFb Act to Promote a
Sustained Fibrotic Phenotype
Histological studies examining the distribution ofCTGF and TGFb mRNAs in skin sections showedthat in diffuse SSc lesions TGFb is overexpressed in theleading inﬂammatory edge of the lesion, but not in thelesional area itself.17 CTGF expression shows an expression pattern correlating with the severity of ﬁbrosis;that is, CTGF expression is abundant in ﬁbrotic lesions,even in the absence of markedly elevated amountsof TGFb ligand.18 Taken together, in contrast to thesituation in normal dermal ﬁbroblasts whereby CTGF
is not normally expressed unless induced by TGFb
through the TGFb response element, the persistentlevel of CTGF observed in lesional SSc ﬁbroblasts

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Leask A: (CTGF) in pathological ﬁbrosis

NormalFibroblasts,
No TGFβCTGF

No CTGF

CTGF

CTGF

Sp1
SclerodermaFibroblasts,
No TGFβFig. 2 Sp1 is required for the overexpression of the CTGF promoter in SSc ﬁbroblasts. The minimal CTGF promoter containing only a functional Sp1 element is required for the elevated level of CTGF promoter expression in SSc ﬁbroblasts. Removal of the Sp1 element in the promoter or blockade of Sp1 activity by mithramycin suppresses elevated CTGF promoter and protein expression in SSc ﬁbroblasts. For details,
see ref (15).

seems to be independent of TGFb ligand.
Based on these results, we hypothesize that in thenormal wound healing response, the up-regulation of
CTGF is dependent on the TGFb response element of
the CTGF promoter, and is therefore subject to thecontrols that normally negatively regulate the TGFbinduced wound healing response.19 In pathological ﬁbrosis, however, ﬁbroblasts display an elevated levelof CTGF expression that is independent of the TGFbresponse element of the CTGF promoter.14,15 Thus
we believe that, whereas TGFb is essential for the initiation of ﬁbrosis, the persistent, TGFb-independent
CTGF expression characteristic of ﬁbrotic lesions perpetuates the ﬁbrotic response.
Conclusion
The observations concerning CTGF expression andfunction have led to the intriguing notion that CTGF
represents a novel, molecular target for therapeutic intervention in ﬁbrotic disease.20–23 As further researchis conducted, the precise functional role of CTGF in
cellular function as well as the mechanism of CTGF
action should emerge. This process should result in thedevelopment of novel methods for anti-ﬁbrotic intervention to prevent dermal scarring and alleviate thesymptoms of ﬁbrosis.
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