SMAD3 mutation in LDS3 causes bone fragility by impairing the TGF-β pathway and enhancing osteoclastogenesis
Ahmed El-Gazzar,
Heeseog Kang,
Nadja Fratzl-Zelman,
Emma Webb,
Aileen M. Barnes,
Milena Jovanovic,
Sarju G. Mehta,
Vipan Datta,
Vrinda Saraff,
Ryan K. Dale,
Frank Rauch,
Joan C. Marini,
Wolfgang Högler
Affiliations
Ahmed El-Gazzar
Department of Paediatrics and Adolescent Medicine, Johannes Kepler University Linz, Linz, Austria
Heeseog Kang
Section on Heritable Disorders of Bone and Extracellular Matrix, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Nadja Fratzl-Zelman
Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Medical Department Hanusch Hospital, Vienna, Austria; Vienna Bone and Growth Center, Vienna, Austria
Emma Webb
Department of Paediatrics, Jenny Lind Children's Hospital, Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK; Norwich Medical School, University of East Anglia, Norwich, UK
Aileen M. Barnes
Section on Heritable Disorders of Bone and Extracellular Matrix, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Milena Jovanovic
Section on Heritable Disorders of Bone and Extracellular Matrix, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Sarju G. Mehta
East Anglian Regional Medical Genetics Service, Addenbrookes Hospital, Cambridge, UK
Vipan Datta
Department of Paediatrics, Jenny Lind Children's Hospital, Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK
Vrinda Saraff
Department of Endocrinology and Diabetes, Birmingham Women's and Children's Hospital NHS Foundation Trust, Birmingham, UK
Ryan K. Dale
Bioinformatics and Scientific Programming Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA
Frank Rauch
Shriners Hospital for Children-Canada, Montreal, QC H4A 0A9, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 0C7, Canada
Joan C. Marini
Section on Heritable Disorders of Bone and Extracellular Matrix, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Wolfgang Högler
Department of Paediatrics and Adolescent Medicine, Johannes Kepler University Linz, Linz, Austria; Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; Corresponding author at: Department of Paediatrics and Adolescent Medicine, Johannes Kepler University Linz, Kepler University Hospital, Krankenhausstrasse 26-30, 4020 Linz, Austria.
Loss-of-function mutations in SMAD3 cause Loeys-Dietz syndrome type 3 (LDS3), a rare autosomal-dominant connective tissue disorder characterized by vascular pathology and skeletal abnormalities. Dysregulation of TGF-β/SMAD signaling is associated with abnormal skeletal features and bone fragility. To date, histomorphometric and ultrastructural characteristics of bone with SMAD3 mutations have not been reported in humans and the exact mechanism by which SMAD3 mutations cause the LDS3 phenotype is poorly understood. Here, we investigated bone histomorphometry and matrix mineralization in human bone with a SMAD3 mutation and explored the associated cellular defect in the TGF-β/SMAD pathway in vitro. The index patient had recurrent fractures, mild facial dysmorphism, arachnodactyly, pectus excavatum, chest asymmetry and kyphoscoliosis. Bone histomorphometry revealed markedly reduced cortical thickness (−68 %), trabecular thickness (−32 %), bone formation rate (−50 %) and delayed mineralization. Quantitative backscattered electron imaging demonstrated undermineralized bone matrix with increased heterogeneity in mineralization. The patient's SMAD3 mutation (c.200 T > G; p.I67S), when expressed from plasmid vectors in HEK293 cells, showed reduced phosphorylation and transcription factor activity compared to normal control and SMAD3 (p.S264Y), a gain-of-function mutation, somatic mosaicism of which causes melorheostosis. Transfection study of the patients' SMAD3 (p.I67S) mutation displayed lower luciferase reporter activity than normal SMAD3 and reduced expression of TGF-β signaling target genes. Patient fibroblasts also demonstrated impaired SMAD3 protein stability. Osteoclastogenic differentiation significantly increased and osteoclast-associated genes, including ACP5 (encoding TRAP), ATP6V0D2, and DCSTAMP, were up-regulated in CD14 (+) peripheral blood mononuclear cells (PBMCs) with the SMAD3 (p.I67S) mutation. Upregulation of osteoclastogenic genes was associated with decreased expression of TGF-β signaling target genes. We conclude that bone with the SMAD3 (p.I67S) mutation features reduced bone formation, and our functional studies revealed decreased SMAD3 activation and protein stability as well as increased osteoclastogenesis. These findings enhance our understanding of the pathophysiology of LDS3 caused by SMAD3 mutations. Emerging therapies targeting in the TGF-β/SMAD pathway also raise hope for treatment of LDS3.
