Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Apr 6.
Published in final edited form as: J Invest Dermatol. 2010 Feb 25;130(6):1514–1523. doi: 10.1038/jid.2010.15

Antagonistic effect of the matricellular signaling protein CCN3 on TGF-β- and Wnt-mediated fibrillinogenesis in systemic sclerosis and Marfan syndrome

Raphael Lemaire 1,2, Giuseppina Farina 1, Julie Bayle 1, Michael Dimarzio 1, Sarah A Pendergrass 3, Ausra Milano 3, Michael L Whitfield 3, Robert Lafyatis 1
PMCID: PMC3071475  NIHMSID: NIHMS284592  PMID: 20182440

Abstract

Abnormal fibrillinogenesis is associated with connective tissue disorders (CTDs), including Marfan syndrome (MFS), systemic sclerosis (SSc) and Tight-skin (Tsk) mice. We have previously shown that TGF-β and Wnt stimulate fibrillin-1 assembly and that fibrillin-1 and the developmental regulator CCN3 are both highly increased in Tsk skin. We investigated the role of CCN3 in abnormal fibrillinogenesis in Tsk mice, MFS, and SSc. Smad3 deletion in Tsk mice decreased CCN3 overexpression, suggesting that TGF-β mediates at least part of the effect of Tsk fibrillin on CCN3 and consistent with a synergistic effect of TGF-β and Wnt in vitro on CCN3 expression. Disruption of fibrillin-1 assembly by MFS fibrillin decreased CCN3 expression and skin from patients with early diffuse SSc showed a strong correlation between increased CCN3 and fibrillin-1 expression, suggesting that CCN3 regulation by fibrillin-1 extends to these CTDs. Diffuse SSc skin and sera also showed evidence of increased Wnt activity, implicating a Wnt stimulus behind this correlation. CCN3 overexpression markedly repressed fibrillin-1 assembly and also blocked other TGFβ- and Wnt-regulated profibrotic gene expression. Together these data indicate that CCN3 counter-regulates positive signals from TGF-β and Wnt for fibrillin fibrillogenesis and profibrotic gene expression.

INTRODUCTION

Microfibrils, 10–12 nm in diameter are important structural and functional components of the extracellular matrix of a wide range of connective tissues, including skin, bones and aorta. The largest components of microfibrils are the fibrillins, including fibrillin-1 and -2 (Sakai et al., 1986; Zhang et al., 1994). The association of mutations in the fibrillin-1 gene with connective tissue disorders (CTDs) illustrates the important function of fibrillin-1 in tissue development and extracellular matrix remodeling. Marfan syndrome (MFS), an autosomal hereditary CTDs with prominent manifestations in the skeletal, cardiovascular and ocular systems, is caused by fibrillin-1 mutations and is associated with reduced amount of fibrillin-1 deposited in the matrix (Lemaire et al., 2006). Fibrillin-1 mutations are also present in populations with the highest reported prevalence of SSc, a CTD characterized by skin fibrosis and increased amounts of fibrillin deposited in the deep reticular dermis (Davis et al., 1999; Fleischmajer et al., 1991). How mutations in fibrillin-1 induce matrix remodeling is unclear. The study of fibrillin-deficient mice suggests that mutant fibrillin matrix may lead to faulty growth factor signaling. Aneurysm caused by the C1039G fibrillin-1 mutation in a mouse model of MFS is associated with decreased levels of microfibrils and increased levels of TGF-β signaling (Ng et al., 2004).

We have recently shown that skin fibrosis, bone overgrowth and heart enlargement caused by a large duplication within the fibrillin-1 gene in the Tsk mouse model of SSc is developmentally regulated and associated with dramatically increased fibrillin-1 and Wnt levels. Further we showed that Wnts stimulate fibrillin-1 matrix assembly (Bayle et al., 2008). However, the most highly upregulated gene expression by microarray analysis of Tsk skin was CCN3, a growth factor belonging to the CCN (Cyr-61/Ctgf/Nov) family of developmental regulators (Brigstock, 1999; Lau and Lam, 1999; Perbal, 2001). We show here that overexpression of fibrillin-1 in Tsk skin is temporally and spatially associated with a similar overexpression of CCN3, suggesting that CCN3 regulates Tsk matrix remodeling. Further, we show that TGF-β and Wnt may work synergistically to stimulate CCN3 in Tsk skin. However, in contrast to TGF-β and Wnt, we show that CCN3 represses fibrillin-1 matrix assembly. We also show that mRNA levels of Wnt2 and the Wnt-regulated gene, WISP1, are increased in the skin of patients with diffuse cutaneous SSc, and that the expression of CCN3 and fibrillin-1 correlates strongly in early diffuse SSc skin and in an in vitro model of MFS, suggesting that CCN3 may represent a counter-regulatory mechanism to the stimulating effects of TGF-β and Wnt on fibrillin fibrillinogenesis in CTDs.

RESULTS

Spatial and temporal correlation of CCN3 and fibrillin-1 overexpression in Tsk mice

Our recent microarray data have shown that, along with fibrillin-1, CCN3 features the highest increase among over-expressed genes in skin of Tsk mice (Bayle et al., 2008). Here, we confirmed these microarray data using northern and western blot analysis of CCN3 in Tsk compared to wild-type skin of 6-week of age mouse littermates. CCN3 was increased about 15-fold at the mRNA level (Fig. 1A, left panel) and 16-fold at the protein level (Fig. 1A, right panel). Immunochemistry showed that this dramatic increase in CCN3 was mainly located in the hypodermis of Tsk skin (Fig. 1B), similar to what is seen for fibrillin-1 (Lemaire et al., 2004b). A strong parallel between increased expression of CCN3 and increased fibrillin-1 expression was seen in the skin, as well as in skeletal muscle, heart, and bones of Tsk mice (Fig. 1C and 1D). Although CCN3 expression was highest in the brain, fibrillin-1 expression was very low in this tissue and neither CCN3 or fibrillin-1 showed any alteration in levels of expression. Both CCN3 and fibrillin-1 also showed developmental regulation and were overexpressed in Tsk skin as soon as 18.5 day embryonic age and thereafter (Fig. 1E). The spatial and temporal correlation of CCN3 and fibrillin-1 overexpression in Tsk mice suggested an important functional relationship of these two genes for the onset of the Tsk pathology.

Figure 1. Spatial and temporal correlation of CCN3 overexpression in Tsk mice.

Figure 1

Open in a new tab

(A) Tsk and wild-type (WT) skins were analyzed for CCN3 mRNA and protein expression, respectively by northern blot (left panel) and western blot (right panel). (B) Tsk and WT skin samples were analyzed for CCN3 by immunohistochemistry - sf: hypodermis. (C and D) Brain, spleen, skeletal muscle, heart, skin, kidneys and bones from Tsk and WT mice were analyzed for both fibrillin and/or CCN3 mRNAs by northern blot. (E) Tsk and WT skin samples were analyzed for both fibrillin and/or CCN3 at 18.5 embryo age stage and 4 weeks after birth by northern blot.

Deletion of Smad3 in Tsk mice reduces CCN3 and fibrillin overexpression in Tsk mice

We assessed whether TGF-β contributes to the effect of Tsk fibrillin on CCN3 expression by looking at CCN3 expression in Tsk mice deleted of Smad3, a key TGF-β intracellular signaling molecule. Deletion of a single Smad3 allele did not alter overexpression of CCN3 in Tsk skin compared to wild-type skin (Fig. 2A, Smad3 +/−, WT vs. TSK). However, deletion of both Smad3 alleles in Tsk skin largely reduced CCN3 overexpression (Fig. 2A, Smad3 −/−, WT vs. TSK). Interestingly, both mutant and wild type fibrillin followed the same expression pattern, as deletion of both Smad3 alleles in Tsk skin also largely reduced fibrillin-1 overexpression (Fig. 2A, Smad3 −/−, WT vs. TSK), suggesting that TGF-β contributes to increased CCN3 and fibrillin-1 in Tsk skin. However, the importance of TGF-β appears to be restricted to early developmental time points as blocking TGF-β after birth using neutralizing anti-TGF-β antibodies or induced deletion of the TGF-β receptor after birth did not produce reproducible changes in CCN3 expression (R. Lafyatis, unpublished observations). This is consistent with the observation that the Tsk phenotype is detectable at birth. To further investigate the potential for TGF-β to drive increased CCN3 in Tsk skin, we assessed the effect of TGF-β on CCN3 expression in vitro. Treatment of cultured fibroblasts with TGF-β alone did not stimulate CCN3 expression. Since canonical Wnt signaling can interact with the Smad pathway (Furuhashi et al., 2001; Gadue et al., 2006) and we have recently shown that Wnt signaling is altered in Tsk skin (Bayle et al., 2008), we then tested the effect Wnt3a on CCN3 expression. Treatment of fibroblasts with Wnt3a stimulated CCN3 mRNA expression by 3-fold, and TGF-β acted synergistically with Wnt3 to induce CCN3 expression by more than 6-fold (Fig. 2B). These data suggested that the TGF-β and Wnt may co-mediate the stimulating effect of Tsk fibrillin on CCN3 expression.

Figure 2. Deletion of Smad3 in Tsk mice reduces CCN3 and fibrillins overexpression in Tsk mice.

Figure 2

Open in a new tab

(A) Skin samples from Tsk and WT mice deleted of one Smad3 allele (Smad3+/−) or two Smad3 alleles (Smad3+/+) were analyzed for both CCN3 and fibrillin-1 mRNAs by northern blot (upper panel). CCN3 (lower left panel) and fibrillin (lower right panel) mRNAs were quantified by phosphorimaging analysis. (B) Cultured mouse embryonic fibroblasts (MEF) were left untreated or treated with TGF-β (2 ng/ml), Wnt3a (50 ng/ml), or both for 20 hours and then analyzed for CCN3 mRNA expression by northern blot.

Disruption of fibrillin-1 matrix assembly by MFS-like fibrillin correlates with decreased expression of CCN3

Based on the correlation between increased CCN3 and fibrillin-1 expression in Tsk skin, heart and muscle, suggesting a function of CCN3 in fibrillinopathy, we then assessed CCN3 expression in MFS, a human fibrillinopathy caused by mutations in the fibrillin-1 gene. For these studies, we used an in vitro model of MFS recently developed by our group that features a doxycycline (dox)-regulated system allowing cultured fibroblasts to conditionally overexpress a Marfan-like fibrillin deletion mutant where the C-terminal region is replaced at amino acid 2780 by EGFP. Overexpression of this MFS fibrillin mutant prevents matrix assembly of wild-type fibrillin-1 (Lemaire et al., 2007a) similar to what is seen in Marfan tissues. Briefly, in the presence of dox, expression of the MFS fibrillin mutant is repressed (Fig. 3A, a) and associated with deposition of a complex and thick fibrillin network, as wild-type fibrillin-1 assembles within the matrix (Fig. 3A, c, red staining). However in the absence of dox, MFS fibrillin is overexpressed (Fig. 3A, b, intracellular green staining), preventing matrix assembly of wild-type fibrillin-1 (Fib. 3A, d). Expression of MFS fibrillin and therefore disruption of fibrillin matrix assembly was associated with markedly decreased CCN3 mRNA expression (Fig. 3 B, dox + vs. dox −). This decrease in CCN3 was MFS-fibrillin dose dependent (Fig. 3B, low MFS fibrillin producing MEF-MSF1 cell line vs. high MFS fibrillin producing MEF-MSF2 cell line).

Figure 3. Disruption of fibrillin-1 matrix assembly by MFS-like fibrillin correlates with decreased expression of CCN3.

Figure 3

Open in a new tab

(A) MEF-MFS cells conditionally overexpressing EGFP-tagged MFS fibrillin were cultured for 1 week with or without dox. Total fibrillin-1 (red) and MFS fibrillin (green) were co-analyzed, respectively by immunofluorescence using polyclonal antibody to fibrillin-1 and EGFP fluorescence. Nuclei were stained with Hoechst (blue) (B) MFS fibrillin and CCN3 mRNA were analyzed by northern blot in parallel in two MEF-MFS cell lines (MEF-MFS1 and MEF-MFS2).

Expression of CCN3 and fibrillin-1 expression correlates in early diffuse SSc skin

Diffuse SSc skin shows increased levels of fibrillin-1 microfibrils in the deep dermis (Davis et al., 1999; Fleischmajer et al., 1991). Therefore, we looked at whether the correlation between CCN3 and fibrillin-1 seen in murine Tsk and human MFS fibrillinopathies could be extended to SSc. Microarray analysis of CCN3 and fibrillin-1 mRNA levels in skin was performed using forearm lesional and back non-lesional biopsies from 8 patients with early diffuse SSc, 9 patients with late diffuse SSc, 7 patients with limited SSc and 6 healthy control individuals (Fig. 4A, left panel) (Milano et al., 2008). Microarray expression showed no significant difference between CCN3 or fibrillin-1 expression in skin from patients with limited or diffuse SSc (Fig. 1A supplemental, right panel). However, the average levels of CCN3 and fibrillin-1 in early diffuse SSc skin were slightly increased compared to healthy skin, and CCN3 expression correlated highly with fibrillin-1 expression (R2 = 0.80, p=0.002, Fig. 1B supplemental, upper left panel), unlike late diffuse SSc, limited SSc and healthy skin (Fig. 1B supplemental, respectively upper right, lower left and lower right panels). To more quantitatively assess this relationship, RNA from skin samples from patients with early diffuse SSc were analyzed for CCN3 and fibrillin-1 expression by RT-PCR. CCN3 showed 2.2- and fibrillin-1 2.4-fold greater expression in dSSc skin compared to healthy control skin (Fig. 4). More strikingly, however, and confirming the microarray data, CCN3 expression correlated highly with fibrillin-1 expression (Fig. 4, R2=0.51)

Figure 4. Correlation between increased CCN3 and Fibrillin-1 expression in SSc skin.

Figure 4

Open in a new tab

Top panels: mRNA expression of CCN3 (left panel) and fibrillin-1 (right panel) in lesional (20 biopsies) and control skin (6 biopsies). Fold-change shown on the graph is normalized to mRNA expression to one of the healthy controls. The average fold-change of CCN3 in SSc skin (1.88) compared to the average fold-change in control, healthy skin (0.86), was increased by 2.2-fold (p<0.05). The average fold-change of fibrillin-1 in SSc skin (1.86) compared to the average fold-change in control, healthy skin (0.66), was increased by 2.4-fold (p<0.01). The bottom panel shows the correlation between fibrillin-1 and CCN3 expression (R2=0.51)

Wnt activity is increased in diffuse SSc

We have previously shown that Tsk skin expresses increased levels of Wnt and Wnt-regulated genes. In order to assess whether Wnt stimulation of fibrillin-1 and CCN3 might underlie the correlation between fibrillin-1 and CCN3 in diffuse SSc skin, we looked at Wnt signaling and activity in diffuse SSc. For these studies we analyzed mRNA expression of 84 Wnt-related genes in forearm skin biopsies from 12 diffuse SSc patients and 5 healthy individuals using Wnt Signaling Pathway Real-Time PCR array. Statistical analysis using a t-test corrected for multiple hypothesis testing (significance analysis of microarrays, SAM, q < 0.01%, http://www-stat.stanford.edu/~tibs/SAM/) showed statistically significant changes in expression for 9 genes in SSc compared to healthy skin, (Fig. 5A, upper left panel). Among these genes, 3 genes showed increased expression, including Sfrp4, Wnt2, and Wisp1 (Wnt-inducible protein 1, also called CCN4) (Fig. 5A, upper right panel). This expression pattern is similar to that seen in Tsk skin, which shows increased levels of Sfrp4, Wnt2, and Wisp2 (also called CCN5) (Bayle et al., 2008). Interestingly, Wnt 2 and Sfrp4 (a Wnt inhibitor) significantly correlated (Fig. 5A, lower left panel), suggesting that these two molecules might control net levels of Wnt activity in SSc. SSc skin also showed significant decreases in 6 genes, including Wif1 and Frzb. These two inhibitors of Wnt activity showed the two largest decreases, possibly also contributing to increased net Wnt activity in diffuse SSc skin (Fig. 5A, lower right panel). This is consistent with increased levels of Wisp1, the only Wnt-regulated gene attested on these arrays and possibly a biomarker of Wnt activity.

Figure 5. Wnt activity is increased in diffuse SSc.

Figure 5

Open in a new tab

(A) Skin biopsies from patients with diffuse SSc (n=12) and from control healthy individual (n=5) were analyzed for expression of 84 genes of the Wnt signaling pathway by real-time PCR array. Expression of 9 genes differed significantly in SSc skin compared to control skin (SAM, q < 0.01%) (upper left panel), including 3 genes with increased expression in SSc skin (upper right panel) and 6 genes in healthy skin (lower right panel). Scatter plot graph showing high correlation between Sfrp4 and Wnt2 expression in SSc and healthy skin is presented in lower left panel. (B) Sera from 25 SSc patients (n=25) and healthy individuals (n=8) were analyzed for Wnt activity using LSL luciferase reporter cells.

To further investigate the possibility that Wnt activity is increased in SSc, we measured Wnt activity in sera of 24 patients with diffuse SSc and 8 healthy individuals using Wnt reporter LSL cells (Blitzer and Nusse, 2006). Diffuse SSc sera showed a modest but statistically significant 1.56-fold increase in Wnt activity compared healthy individual sera (p=0.00001) (Fig. 5B). Since Wnt activity was higher in diffuse SSc skin and Wnt regulates both fibrillin-1 (Bayle et al., 2008) and CCN3 (Fig. 2B), Wnt represents a good candidate to mediate the correlation between fibrillin-1 and CCN3 in early SSc skin.

CCN3 overexpression represses fibrillin-1 matrix assembly

Based on the above data showing a strong correlation between CCN3 and fibrillin-1 in Tsk mice, and early diffuse SSc, we hypothesized that an important function of CCN3 is to regulate fibrillin-1 levels in connective tissues. We analyzed the effect of overexpression of CCN3 on fibrillin-1 using dox-regulated cell lines conditionally overexpressing CCN3. In the absence of dox treatment, cells overexpressed CCN3. In the presence of dox, CCN3 was entirely repressed (Fig. 6A and 6B). Cells not expressing exogenous CCN3 showed a strong and extended fibrillin-1 network in the matrix (Fig. 6B, a, red staining). In contrast, cells expressing high levels of endogenous CCN3 showed a poor and limited fibrillin-1 matrix network (Fig. 6B, b). Northern blot analysis showed that this strong repressive effect of CCN3 on fibrillin-associated matrix was independent of fibrillin-1 expression, as CCN3 overexpression did not affect fibrillin-1 mRNA levels (Fig. 6C) or protein levels (data not shown), suggesting that CCN3 affects fibrillin-1 assembly. Since fibrillin-1 is the main component of elastic fibers, we also looked at the effect of CCN3 elastic fibers assembly synthesized from exogenous tropoelastin-EGFP. Consistent with the effect of CCN3 on fibrillin-1, overexpression of CCN3 strongly reduced elastic fibers assembly (Fig. 6B, c v. d, green staining).

Figure 6. CCN3 overexpression represses fibrillin-1 matrix assembly.

Figure 6

Open in a new tab

MEF cells conditionally overexpressing CCN3 were cultured with or without dox for 1 week, and then analyzed for CCN3 mRNA and protein expression, respectively by northern blot (left panel) and western blot (right panel). (B) In parallel cultures, fibrillin-1 (red) and elastic fiber (green) were co-analyzed using immunofluorescence and EGFP fluorescence, respectively. Nuclei were stained with Hoechst (blue) (C) Fibrillin mRNA expression was also analyzed in parallel by northern blot.

CCN3 inhibits TGFβ- and Wnt-induced gene expression

To gain further insight into the mechanism of CCN3 inhibition of TGFβ- and Wnt-induced fibrillogenesis, we examined the effect of CCN3 on expression of genes regulated by these cytokines in mouse fibroblasts. Wnt3a upregulated MEF cell expression of CCN3 as shown above (Fig. 2B), and connective tissue growth factor (CTGF/CCN2), endothelin-1 (ET-1) and smooth muscle actin (SMA) as reported previously (Chen et al., 2007). CCN3 completely blocked Wnt-induced CCN3, partially blocked Wnt-induced ET-1, and had little effect on Wnt-induced SMA and CTGF (Fig. 7A). CCN3 also partially inhibited TGFβ-induced CCN2 expression (Fig. 7B). In addition, CCN3 also strongly inhibited the Wnt-inducible TOPFLASH promoter (Fig. 7C), however had no effect on TGFβ-induced promoter activity of the PAI-1 promoter-reporter in TMLC cells (data not shown).

Figure 7. Regulation of Wnt- and TGFβ-induced gene expression by CCN3.

Figure 7

Open in a new tab

Panel A: Mouse embryonic fibroblasts (MEF) treated 24 hours with Wnt3a (50 ng/ml), CCN3 (10 μg/ml), neither or both analyzed for CCN3, CTGF (CCN2), Endothelin-1 (ET-1) and smooth muscle actin (SMA) gene expression by RT-PCR. Panel B: MEF cells treated 24 hours with TGFβ (2 ng/ml), CCN3 (10 μg/ml), both or neither analyzed for CTGF/CCN2 expression by RT-PCR. Panel C: Luciferase activity of Wnt promoter-reporter (LSL) cells, treated 24 hours with Wnt3a (0.08 or 8 ng/ml), CCN3 (10 μg/ml), or Wnt3a (8ng/ml) and CCN3 (10 μg/ml).

DISCUSSION

Defining the factors that mediate fibrillin expression and matrix assembly is a necessary step for better undertanding CTDs. We have previously shown that TGFβ regulates fibrillin-1 assembly (Kissin et al., 2002), consistent with data showing that increased TGFβ activity contributes to the pathology of MFS (Neptune et al., 2003). In Tsk mice the role of TGFβ in altered fibrillogenesis is likely required at a point during early mouse skin development that ends or is largely attenuated before or shortly after birth, based on our inability to block increased CCN3 expression using strategies targeting 5 day and older mice. This is consistent with our observation that the Tsk phenotype and increased CCN3 expression are detectable at birth. In SSc many studies have suggested that TGFβ mediates tissue fibrosis, and it may also account for increased fibrillin-microfibrils in the deep dermis of SSc skin. However, other cytokines may also contribute to altered fibrillin deposition in these diseases. We have recently shown that Wnt signaling is altered in Tsk mice, specifically in fibrillin-containing tissues, including skin, heart and skeletal muscles, suggesting that Wnts may mediate tissue fibrosis in Tsk mice. Accordingly, we have also shown that Wnt3a stimulates matrix assembly of fibrillin-1, independently of fibrillin-1 transcription (Bayle et al., 2008). A role for Wnts in skin fibrosis is further suggested by studies showing that Wnt3a induces CTGF, TGFβ and ET-1 (Chen et al., 2007). Interestingly, we show here that Wnt2 and a marker of Wnt activity in the skin, Wisp1, are increased and genes associated with Wnt inhibition, Wif1 and Frzb, decreased in SSc skin. In addition we show that serum Wnt activity is modestly but significantly increased in SSc patient sera. Although changes in Wnt activity in serum are modest, they are similar in magnitude to changes seen in aged mice associated with fibrosis of muscles (Brack et al., 2007). Together these data suggest that Wnt may contribute to the increase in fibrillin-microfibril assembly in the deep dermis of SSc skin, and are consistent with past observations suggesting a Wnt signature in scleroderma skin (Gardner et al., 2006). Wnts have other profibrotic activities including their direct effects on fibrotic gene expression (Chen et al., 2007), but also effects on cell differentiation (Brack et al., 2007), indicating potential important roles beyond altered fibrillogenesis in SSc.

Wnt and TGF-β signaling pathways can interact through several mechanisms (Furuhashi et al., 2001; Gadue et al., 2006; Nishita et al., 2000), possibly accounting for synergistic effects of these cytokines on CCN3 expression. The best-defined involves smad4 (a “co-Smad” important in signaling by TGF-β stimulated Smad2/3) forming a complex with Lef1/Tcf and β-catenin (Nishita et al., 2000). Another study has shown the interaction of Smad4 with Lef1/Tcf regulates downstream gene expression in the absence of TGF-β (Lim and Hoffmann, 2006).

Along with TGF-β and Wnt, CCN3 may represent another signaling pathway important in fibrillinopathies. We show here a high correlation between increased fibrillin-1 and increased CCN3 expression in Tsk skin, early diffuse SSc skin and in an in vitro model of MFS. Consistent with a role of CCN3 in fibrillinogenesis, we also show that CCN3 overexpression represses fibrillin-1 matrix assembly. Interestingly, this repressive function of CCN3 is in contrast to the stimulating activity of TGF-β and Wnt on fibrillin assembly, suggesting that CCN3 provides a counter regulatory mechanism to the stimulating effect of TGF-β and Wnt on fibrillin-1 assembly in fibrillinopathies (see Fig. 8). Tight control exerted in concert by TGF-β and Wnt (positive regulators) and CCN3 (negative regulator) on fibrillin-1 matrix assembly may account for the correlation between fibrillin-1 and CCN3 expression in early diffuse SSc skin. This counter-regulatory role of CCN3 appears to extend beyond fibrillogenesis and includes other Wnt and TGFβ targets, including CCN3 itself, and ET1 and CCN2, respectively. The effect of CCN3 on CCN2 expression may be particularly important, as recent observations suggest that CCN3 inhibition of CCN2 may lead to downregulation of TGFβ-mediated fibrosis (Riser et al., 2009).

Figure 8. Counter-regulatory role of CCN3 in matrix formation.

Figure 8

Open in a new tab

TGFβ and Wnt stimulate fibrillin and collagen matrix, while CCN3 blocks effects of both the cytokines. Fibrillin matrix upregulates CCN3, providing a homeostatic feedback mechanism for controlling extracellular matrix.

Our data provide some insights into the mechanism of how CCN3 downregulates TGFβ and Wnt signals. Despite the effect of smad3 deletion on fibrillin expression in Tsk mice, it seems unlikely that CCN3 downregulates TGFβ activity through smad2/3, as the PAI-1 promoter-reporter construct in TMLC cells is activated by smad2/3 (Chen et al., 1996), but was not affected by CCN3. Recently published observations support this interpretation, showing that CCN3 inhibits TGFβ-mediated signals, but does not alter smad3 phosphorylation (Riser et al., 2009). Smad-independent signaling provides many other targets for such regulation (Derynck and Zhang, 2003). Strong inhibition of the TOP-Flash promoter-reporter in LSL cells by CCN3, suggests that CCN3 downregulates Lef1/Tcf, the primary signaling transcription factor in canonical Wnt signaling regulating this promoter (Oosterwegel et al., 1991)

The function of CCN3 in fibrillin microfibril assembly described in this study is in line with a broader role of CCN3 in matrix biology and CTDs, as targeted disruption of CCN3 in mice causes abnormal development of bones, heart and skeletal muscles (Lin et al., 2005), the same tissues that are altered in Tsk mice.

In conclusion, this report defines an important and novel function of CCN3 in extracellular homeostasis that is relevant to CTDs, including SSc and MFS. Further understanding of the interaction of CCN3 with TGF-β and Wnt should provide new insight into these sometimes lethal diseases.

MATERIALS AND METHODS - Supplemental

Patients and controls

All patients with diffuse and limited SSc met the American College of Rheumatology criteria for SSc. Early diffuse SSc was defined by disease duration < 3 years based of onset of non-Raynaud's symptoms. The control healthy individuals had no history of skin disease. Skin punch biopsies were obtained from lesional regions of the dorsal mid-forearm and non-lesional regions of the back. Biopsies were performed after patient written consent and with approval of the Institutional Review Board for Human Studies. The study was conducted according to the Declaration of Helsinki Principles.

Mice

Heterozygous Tight skin (B6Fbn1Tsk+/+Pldnpa) mice originally purchased from Jackson Laboratory (Bar Harbor, ME) were bred to the B6 mice harboring the closely linked pallid mutation pa (B6+Pldnpa/+Pldnpa) and identified by genotyping and manual assessment of skin tightness over the back.

Cells and plasmids

Dox-regulated cells overexpressing CCN3 were developed from a mouse embryonic fibroblast line (MEF 3T3, Clontech, Palo Alto, CA) that harbors the pTET-Off regulator, plasmid expressing a tet-controlled transactivator (tTA) protein that binds to promoter tet-responsive element (TRE). These MEF cells were transfected with pTRE2-CCN3, a vector supporting dox-regulated TRE-driven expression of CCN3. pTRE2-CCN3 was constructed by inserting a 2.3-kb mouse CCN3 cDNA (MGC-5979, ATCC, Manassas, VA) as a blunted Sal I / Not I fragment into pTRE2 (Clontech) at blunted BamH I / Not I sites.MEF-MFS cells conditionally overexpressing MFS fibrillin were developed by transfecting MEF cells with pTRE-MFS-Fbn-EGFP, a vector supporting dox-regulated TRE-driven expression of the a c-terminus truncated fibrillin-1 mutant (Lemaire et al., 2007b).

Cell transfection

Cell lines were transfected using lipofectamine plus reagent (Invitrogen, Carlsbad, CA). After transfection, cells were selected for 12–14 days using G418 (100 μg/ml) and Hygromycin (60 μg/ml). 40 to 50 selected cell colonies were then ring-cloned, expanded, and analyzed for the transfected gene by northern blot and western blot.

Immunofluorescence

Cells were grown in Lab-Tek 8-chamber culture slides (Nalge Nunc International, Naperville, IL), fixed for 10 min in paraformaldehyde at room temperature, blocked for 30 min with 3% BSA in TBS, and then incubated for 2 h with polyclonal rabbit Ab9543 antisera directed against mouse fibrillin-1 (gift of Lynn Sakai) (Lemaire et al., 2004a). Cells were washed 3 times with TBS for 5 min, incubated for 1 h with a rhodamine-conjugated donkey anti-rabbit IgG at a 1/200 dilution. Specific fluorescence signals were then visualized by an Olympus BH-2 microscope under fluorescent light and captured with high resolution Olympus DP70 camera. Exposure times were set manually and were identical between dox + and dox − conditions.

Immunohistochemistry

Tissues were fixed overnight to several days in buffered 4% paraformaldehyde and then embedded in paraffin. Sections were cut, mounted on uncoated slides, and hydrated by passage through xylene and graded ethanols. Sections were incubated with polyclonal rabbit anti-sera directed against CCN3 (1/1000 dilution, gift of B. Perbal) for 2 hours in a humidified chamber, and washed 3 times with TBS for 10 minutes. Sections were then incubated for 1 hour with anti-rabbit alkaline phosphatase labeled polymer and stained using Fast Red (Envision, Dako Laboratory, Carpinteria, CA). Tissues were counterstained in Mayer's Hematoxylin (DAKO) and viewed under an Olympus Olympus DP70 camera.

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting

Cells were directly homogenized and lysed in SDS-PAGE buffer/2% beta–mercaptoethanol. Proteins were resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose, and then incubated with primary polyclonal rabbit antisera directed against mouse CCN3 (1/500 dilution). The secondary antibody consisted of a secondary horseradish peroxidase-conjugated donkey anti-mouse IgG antibody (1/10000). Signal was detected using Supersignal West Pico or Femto Chemiluminescent Reagent (Pierce, Rockford, IL) followed by autoradiography.

RNA analysis

Total RNA was extracted and purified from mouse or human tissues using RNeasy Total RNA kit (Qiagen, Valencia, CA), as previously described (Bayle et al., 2008). RNAs were then separated by electrophoresis through a 1M formaldehyde/1% agarose gel and blotted onto a nylon membrane (Hybond-H, Amersham Biosciences, Piscataway, NJ). Blotted RNAs were then hybridized to random primed 32P-labeled cDNA probes for either mouse fibrillin-1 and CCN3, as previously described (Kissin et al., 2002). Signals were analyzed and quantified using a Phosphoimager (Cyclone, Packard Biosciences, Waltham, MA).

RT-PCR and Wnt RT-PCR array

cDNAs were synthesized from 0.05 μg of total skin RNA using Superscript II RNase H− reverse transcriptase and random primers (Invitrogen Life Technologies, Rockville, MD). Primers for quantitative real-time PCR were designed using Primer Express software (Applied BioSystems Inc., ABI) and synthesized by Integrated DNA Technologies (IDT). The following primers were used to detect human Fibrillin: forward, 5' CCA ATG GAG CAG ATA TCG ATG A; reverse, 5' TTC GGC AAA CAT CGT GAA TAA C; human CTGF forward, 5' TGT GTG ACG AGC CCA AGG A 3'; reverse, 5' TCT GGG CCA AAC GTG TCT T C 3'; human TGFβ forward, 5' CCC TGC CCC TAC ATT TGG A, reverse, 5' CCG GGT TAT GCT GGT TGT ACA; human Endothelin-1 (ET-1) forward, 5' AGG AAG AAA AAT CAG AAG AAG TTC AGA; reverse, 5' CTC CGA CCT GGT TTG TCT TAG G, human Smooth muscle actin (SMA) forward, 5' GCC AAC CGG GAG AAA ATG A; reverse, 5'CGC CTG GAT AGC CAC ATA CA. Relative quantification of human Fibrillin, CTGF, ET-1, TGFβ, SMA was measured using SYBR Green chemistry. Taqman primers for human CCN3/NOV, human control 18S rRNA, mouse SMA, ET-1, CTGF, TGFβ and mouse control GAPDH were purchased from ABI. TaqMan Master Mix chemistry was used to detect gene expression in an ABI Prism 7300 Sequence Detector as recommended by the supplier (ABI). Expression was normalized to18S rRNA (human) or GAPDH (mouse) and relative expression of each gene calculated using ΔΔCt formula, choosing a healthy donor sample as the control (Livak and Schmittgen, 2001).

Wnt Signaling Pathway PCR Array (SA Biosciences) was performed according to the supplied manufacturer protocol using SSc and healthy skin cDNAs prepared as above carried out on an ABI Prism 7700 RT-PCR machine with results normalized to five housekeeping controls included on the plate.

TGFβ and Wnt promoter-reporter assays

70% confluent TMLC (TGFβ, (Abe et al., 1994)) or LSL cells (Wnt, (Blitzer and Nusse, 2006)) were treated in DMEM containing 1% FBS and growth factor(s) to be tested. For assays of human sera, 10% sera was added. After 24 hours, cells were rinsed with PBS, lysed with Reporter Lysis Buffer (Luciferase Assay System, Promega) and 10 μL of each sample vortexed with 50 μL of Luciferase Assay Reagent in a disposable cuvette for light measurement using a luminometer (Turner TD-20e Luminometer).

Supplementary Material

Supplementary Files

ACKNOWLEDGMENTS

The authors thank Dr. Roel Nusse for LSL cells and Dr. Daniel Rifkin for TMLC cells. Support for the work in this manuscript was provided by National Institute of Arthritis and Musculoskeletal and Skin Diseases to R. Lafyatis (NIAMS - R01AR051089). A. Milano and M.L.Whitfield were supported by a grant from the Scleroderma Research Foundation. S. Pendergrass received support from a Hulda Irene Duggan Arthritis Investigator Award from the Arthritis Foundation to M.L.Whitfield and from NIH Autoimmunity and Connective Tissue Biology Training Grant (AR007576) from the National Institute of Arthritis, Musculoskeletal and Skin diseases.

REFERENCES

  1. Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB. An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem. 1994;216:276–284. doi: 10.1006/abio.1994.1042. [DOI] [PubMed] [Google Scholar]
  2. Bayle J, Fitch J, Jacobsen K, Kumar R, Lafyatis R, Lemaire R. Increased expression of Wnt2 and SFRP4 in Tsk mouse skin: role of Wnt signaling in altered dermal fibrillin deposition and systemic sclerosis. J Invest Dermatol. 2008;128:871–881. doi: 10.1038/sj.jid.5701101. [DOI] [PubMed] [Google Scholar]
  3. Blitzer JT, Nusse R. A critical role for endocytosis in Wnt signaling. BMC cell biology. 2006;7:28. doi: 10.1186/1471-2121-7-28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, Keller C, et al. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science. 2007;317:807–810. doi: 10.1126/science.1144090. [DOI] [PubMed] [Google Scholar]
  5. Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev. 1999;20:189–206. doi: 10.1210/edrv.20.2.0360. [DOI] [PubMed] [Google Scholar]
  6. Chen S, McLean S, Carter DE, Leask A. The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts. Journal of cell communication and signaling. 2007;1:175–183. doi: 10.1007/s12079-007-0015-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Y, Lebrun JJ, Vale W. Regulation of transforming growth factor beta- and activin-induced transcription by mammalian Mad proteins. Proc Natl Acad Sci U S A. 1996;93:12992–12997. doi: 10.1073/pnas.93.23.12992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis EC, Blattel SA, Mecham RP. Remodeling of elastic fiber components in scleroderma skin. Connect Tissue Res. 1999;40:113–121. doi: 10.3109/03008209909029107. [DOI] [PubMed] [Google Scholar]
  9. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–584. doi: 10.1038/nature02006. [DOI] [PubMed] [Google Scholar]
  10. Fleischmajer R, Jacobs L, Schwartz E, Sakai LY. Extracellular microfibrils are increased in localized and systemic scleroderma skin. Lab Invest. 1991;64:791–798. [PubMed] [Google Scholar]
  11. Furuhashi M, Yagi K, Yamamoto H, Furukawa Y, Shimada S, Nakamura Y, et al. Axin facilitates Smad3 activation in the transforming growth factor beta signaling pathway. Mol Cell Biol. 2001;21:5132–5141. doi: 10.1128/MCB.21.15.5132-5141.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gadue P, Huber TL, Paddison PJ, Keller GM. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103:16806–16811. doi: 10.1073/pnas.0603916103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gardner H, Shearstone JR, Bandaru R, Crowell T, Lynes M, Trojanowska M, et al. Gene profiling of scleroderma skin reveals robust signatures of disease that are imperfectly reflected in the transcript profiles of explanted fibroblasts. Arthritis Rheum. 2006;54:1961–1973. doi: 10.1002/art.21894. [DOI] [PubMed] [Google Scholar]
  14. Kissin EY, Lemaire R, Korn JH, Lafyatis R. Transforming growth factor beta induces fibroblast fibrillin-1 matrix formation. Arthritis Rheum. 2002;46:3000–3009. doi: 10.1002/art.10621. [DOI] [PubMed] [Google Scholar]
  15. Lau LF, Lam SC. The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res. 1999;248:44–57. doi: 10.1006/excr.1999.4456. [DOI] [PubMed] [Google Scholar]
  16. Lemaire R, Bayle J, Lafyatis R. Fibrillin in Marfan syndrome and tight skin mice provides new insights into transforming growth factor-beta regulation in systemic sclerosis. Curr Opin Rheumatol 2006. 2006;18:582–587. doi: 10.1097/01.bor.0000245719.64393.57. [DOI] [PubMed] [Google Scholar]
  17. Lemaire R, Bayle J, Mecham RP, Lafyatis R. Microfibril-associated MAGP-2 stimulates elastic fiber assembly. J Biol Chem. 2007a;282:800–808. doi: 10.1074/jbc.M609692200. available online at http://www.jbc.org/cgi/reprint/282/801/800. [DOI] [PubMed] [Google Scholar]
  18. Lemaire R, Bayle J, Mecham RP, Lafyatis R. Microfibril-associated MAGP-2 stimulates elastic fiber assembly. J Biol Chem. 2007b;282:800–808. doi: 10.1074/jbc.M609692200. [DOI] [PubMed] [Google Scholar]
  19. Lemaire R, Farina G, Kissin E, Shipley JM, Bona C, Korn JH, et al. Mutant fibrillin-1 from Tsk mice increase extracellular matrix incorporation of MAGP-2 and type I collagen. Arthritis Rheum. 2004a;50:915–926. doi: 10.1002/art.20053. [DOI] [PubMed] [Google Scholar]
  20. Lemaire R, Korn JH, Shipley JM, Lafyatis R. Fibulin-2 and fibulin-5 alterations in tsk mice associated with disorganized hypodermal elastic fibers and skin tethering. J Invest Dermatol. 2004b;123:1063–1069. doi: 10.1111/j.0022-202X.2004.23471.x. [DOI] [PubMed] [Google Scholar]
  21. Lim SK, Hoffmann FM. Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c-myc expression in the absence of TGF-beta signaling. Proc Natl Acad Sci U S A. 2006;103:18580–18585. doi: 10.1073/pnas.0604773103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin CG, Chen CC, Leu SJ, Grzeszkiewicz TM, Lau LF. Integrin-dependent functions of the angiogenic inducer NOV (CCN3): implication in wound healing. J Biol Chem. 2005;280:8229–8237. doi: 10.1074/jbc.M404903200. [DOI] [PubMed] [Google Scholar]
  23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  24. Milano A, Pendergrass SA, Sargent JL, George LK, McCalmont TH, Connolly MK, et al. Molecular subsets in the gene expression signatures of scleroderma skin. PLoS ONE. 2008 doi: 10.1371/journal.pone.0002696. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud B, et al. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nat Genet. 2003;33:407–411. doi: 10.1038/ng1116. [DOI] [PubMed] [Google Scholar]
  26. Ng CM, Cheng A, Myers LA, Martinez-Murillo F, Jie C, Bedja D, et al. TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. J Clin Invest. 2004;114:1586–1592. doi: 10.1172/JCI22715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nishita M, Hashimoto MK, Ogata S, Laurent MN, Ueno N, Shibuya H, et al. Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer. Nature. 2000;403:781–785. doi: 10.1038/35001602. [DOI] [PubMed] [Google Scholar]
  28. Oosterwegel M, van de Wetering M, Dooijes D, Klomp L, Winoto A, Georgopoulos K, et al. Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers. J Exp Med. 1991;173:1133–1142. doi: 10.1084/jem.173.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perbal B. NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues. Mol Pathol. 2001;54:57–79. doi: 10.1136/mp.54.2.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Riser BL, Najmabadi F, Perbal B, Peterson DR, Rambow JA, Riser ML, et al. CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease. Am J Pathol. 2009;174:1725–1734. doi: 10.2353/ajpath.2009.080241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol. 1986;103:2499–2509. doi: 10.1083/jcb.103.6.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhang H, Apfelroth SD, Hu W, Davis EC, Sanguineti C, Bonadio J, et al. Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices. J Cell Biol. 1994;124:855–863. doi: 10.1083/jcb.124.5.855. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Files
NIHMS284592-supplement-Supplementary_Files.pdf (522.6KB, pdf)

RESOURCES