Abstract
The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis.In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.
Highlights
The Enamel Renal Syndrome (ERS) is a rare autosomal recessive disorder caused by loss-of-function mutations in the FAM20A Golgi associated secretory pathway pseudokinase gene (OMIM#611062)
Whereas in control gingival fibroblasts (GFs) FAM20A was essentially localized in discoidal vesicles, most likely secretory ones, in the mutant GFs FAM20A was exclusively detected in HPA positive, cis-Golgi structures (Supplementary Figures 1A–D)
We identified an ERS-specific protein signature composed of 109 dysregulated proteins including the overexpressed COL8A1, HAPLN1, Netrin 1, Transforming growth factor beta 2 (TGFb2) and Gremlin 1 as well as the under-represented Pentraxin 3 (PTX3), C9, BGN, Serpine1 and Serpine2, ANXA2 and ANXA5
Summary
The Enamel Renal Syndrome (ERS) is a rare autosomal recessive disorder caused by loss-of-function mutations in the FAM20A Golgi associated secretory pathway pseudokinase gene (OMIM#611062). Compound heterozygous mutations in FAM20B are believed to cause lethal short limb dysplasia [10]. FAM20C is the Golgi associated secretory pathway kinase responsible for phosphorylating most of the secreted phosphoproteins on the SxE/pS motif. Loss of function mutations in the FAM20C gene cause the Raine syndrome (RS, OMIM#259775), a rare autosomal recessive disorder, generally leading to a lethal osteosclerotic bone dysplasia. In non-lethal RS forms, hypophosphatemic rickets, neurological disorders, amelogenesis imperfecta (AI) and gingival overgrowth were reported [11]. In vitro FAM20A forms a complex with FAM20C and promotes its kinase activity [13]. The in vivo function of FAM20A remains elusive it is interesting to note that Fam20A inactivation in the mouse has been associated with calcifications of muscular arteries in various organs such as heart and kidney [14]
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