Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-β Signaling Overactivation

Carmela Fusco,Paola Parrella,Giuseppina Annicchiarico,Orazio Palumbo,Paolo Graziano,Grazia Nardella,Raffaela Barbano,Bartolomeo Augello,Francesca Boccafoschi,Lucia Micale,Marco Castori,Luca Fusaro,Angelantonio Notarangelo,Carmela De Meco

doi:10.3390/ijms21145141

Abstract

Transforming growth factor β (TGF-β) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-β results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-β signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient’s fibroblasts were employed. We hypothesized an impairment of TGF-β signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient’s fibroblasts to losartan led to the partial restoration of normal transforming growth factor β (TGF-β) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-β signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-β signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

Highlights

The hyperactivation of transforming growth factor beta (TGF-β) mediated signaling pathways stimulates fibrosis and, as such, it has been demonstrated or postulated in multiple acquired and congenital human disorders
We explored the expression of pro-fibrotic genes, the matrix metalloproteinase-2 (MMP2) enzymatic activity, cell motility, and proliferation, as well as the in vitro effects of losartan exposure on the Transforming growth factor β (TGF-β) levels in patient’s fibroblasts
As extracellular signal-regulated kinase 1/2 (ERK1/2) activation controls the levels of the transcription factor nuclear factor-kB (NFkB) [27], we explore the effect of ERK1/2 activation on NFkB levels by performing an immunoblotting analysis

Summary

Introduction

The hyperactivation of transforming growth factor beta (TGF-β) mediated signaling pathways stimulates fibrosis and, as such, it has been demonstrated or postulated in multiple acquired and congenital human disorders. Stiff skin syndrome (SSS, MIM 184900) is an ultrarare and untreatable disorder characterized by generalized or localized progressive non-inflammatory fibrosis of the skin, which turns harder and thicker. While the generalized form of SSS is a widespread and bilateral disorder, localized or segmental SSS has unilateral or monomelic presentation with consequences limited to the affected body segment. Loeys et al demonstrated germline heterozygous variants in the Fibrillin 1 gene (FBN1) as responsible for generalized SSS [12]. Reported in 4 families only, genotype–phenotype correlations seem strict, as SSS-specific FBN1 variants alter both microfibrillar assembly and microfibrill–integrin interactions, which contribute to TGF-β signaling dysregulation. The authors demonstrated a dramatic increased deposition of collagen, elastin, and fibrillin 1 in patients’ skin [12]

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