Abstract
Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn’t require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues.
Highlights
FAM20A, FAM20B and FAM20C are the three structurally-related members of the “family with sequence similarity 20” (FAM20) proteins[1]
Extracellular FAM20C is absent in mouse embryonic fibroblast (MEF) cultures derived from Fam20a KO mice, but present in cultures derived from WT mouse embryonic fibroblasts (MEFs), demonstrating that FAM20A is required for FAM20C extracellular localization
Our results showed that the extent of mineralization of MC3T3-E1 cells treated with the conditioned media from Fam20a KO MEF cells was markedly lesser than that of MC3T3-E1 cells with the conditioned media from Fam20a WT MEF cells
Summary
FAM20A, FAM20B and FAM20C are the three structurally-related members of the “family with sequence similarity 20” (FAM20) proteins[1]. Non-sense or missense mutations in the FAM20A gene were identified that are associated with an autosomal recessive type of Amelogenesis Imperfecta (AI), resulting in premature termination or amino acid changes of the peptides (OMIM#614253). It has been demonstrated that secretion of FAM20C (by overexpression) occurs regardless of its kinase activity, because a catalytically inactive FAM20C (D478A) mutant, but not most of other Raine syndrome-associated missense FAM20C mutants, is still detected in the conditioned media[23]. This suggests the presence of another molecule(s) which likely determines the FAM20C localization. To the best of our knowledge, our data demonstrate for the first time that FAM20A regulates FAM20C localization and assists in the extracellular function of FAM20C
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