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Antagonistic Effect of the Matricellular Signaling Protein CCN3 on TGF-β- and Wnt- Mediated Fibrillinogenesis in Systemic Sclerosis and Marfan Syndrome 

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Presentation on theme: "Antagonistic Effect of the Matricellular Signaling Protein CCN3 on TGF-β- and Wnt- Mediated Fibrillinogenesis in Systemic Sclerosis and Marfan Syndrome "— Presentation transcript:

1 Antagonistic Effect of the Matricellular Signaling Protein CCN3 on TGF-β- and Wnt- Mediated Fibrillinogenesis in Systemic Sclerosis and Marfan Syndrome  Raphael Lemaire, Giuseppina Farina, Julie Bayle, Michael Dimarzio, Sarah A. Pendergrass, Ausra Milano, Michael L. Whitfield, Robert Lafyatis  Journal of Investigative Dermatology  Volume 130, Issue 6, Pages (June 2010) DOI: /jid Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Spatial and temporal correlation of CCN3 overexpression in Tsk mice. (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 (scale bar=100μM)). (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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Deletion of Smad3 in Tsk mice reduces CCN3 and fibrillins overexpression in Tsk mice. (a) Skin samples from Tsk and WT mice deleted 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 (2ngml−1), Wnt3a (50ngml−1), or both for 20hours and then analyzed for CCN3 mRNA expression by northern blot. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Disruption of fibrillin-1 matrix assembly by MFS-like fibrillin correlates with decreased expression of CCN3. (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 by immunofluorescence using polyclonal antibody to fibrillin-1 and EGFP fluorescence, respectively. Nuclei were stained with Hoechst (blue, bar=10μM). (b) MFS fibrillin and CCN3 mRNA were analyzed by northern blot in parallel in two MEF-MFS cell lines (MEF-MFS1 and MEF-MFS2). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Correlation between increased CCN3 and Fibrillin-1 expression in SSc skin. Expression of CCN3 (a) and fibrillin-1 (b) 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 with 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 with the average fold change in control, healthy skin (0.66), was increased by 2.4-fold (P<0.01). Correlation between fibrillin-1 and CCN3 expression (c, R2=0.51). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Wnt activity is increased in diffuse SSc. (a) Skin biopsies from patients with diffuse SSc (n=12) and from control healthy individuals (n=5) were analyzed for expression of 84 genes of the Wnt-signaling pathway by real-time PCR array. Expression of nine genes differed significantly in SSc skin compared with control skin (SAM, q<0.01%) (upper left panel), including three genes with increased expression in SSc skin (upper right panel) and six 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. *P<0.05. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 CCN3 overexpression represses fibrillin-1 matrix assembly. (a) 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, bar=10μM). (c) Fibrillin mRNA expression was also analyzed in parallel by northern blot. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Regulation of Wnt- and TGFβ-induced gene expression by CCN3. (a) Mouse embryonic fibroblasts (MEF) treated 24hours with Wnt3a (50ngml−1), CCN3 (10μgml−1), neither, or both were analyzed for CCN3, CTGF (CCN2), endothelin-1 (ET-1), and smooth muscle actin (SMA) gene expression by RT-PCR. (b) MEF cells treated 24hours with TGFβ (2ngml−1), CCN3 (10μgml−1), neither, or both were analyzed for CTGF/CCN2 expression by RT-PCR. (c) Luciferase activity of Wnt promoter-reporter (LSL) cells, treated 24hours with Wnt3a (0.08 or 8ngml−1), CCN3 (10μgml−1), or Wnt3a (8ngml−1) and CCN3 (10μgml−1). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Counter-regulatory role of CCN3 in matrix formation. TGFβ and Wnt stimulate fibrillin and collagen matrix, whereas CCN3 blocks effects of both the cytokines. Fibrillin matrix upregulates CCN3, providing a homeostatic feedback mechanism for controlling extracellular matrix. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

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