Elsevier

Bone

Volume 67, October 2014, Pages 145-151
Bone

Original Full Length Article
Functional analysis of mutant FAM20C in Raine syndrome with FGF23-related hypophosphatemia

https://doi.org/10.1016/j.bone.2014.07.009Get rights and content

Highlights

  • In vitro analysis showed that mutant FAM20C found in Rain syndrome have decreased kinase activities compared to wild type (WT) FAM20C.

  • WT FAM20C protein increased DMP1 promoter activity, while mutant FAM20C proteins did not.

  • Inactivating mutations in FAM20C cause FGF23-related hypophosphatemia by decreasing transcription of DMP1.

Abstract

Raine syndrome is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype. Either homozygous or compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) have been reported to cause this syndrome. Recently, it was reported that fibroblast growth factor 23 (FGF23)-related hypophosphatemia was found in patients with non-lethal Raine syndrome, and Fam20c conditional knockout mice presented Fgf23-related hypophosphatemic rickets. To clarify the mechanism of how FAM20C regulates FGF23, we performed functional analysis of mutant FAM20C proteins reported in Raine syndrome. We analyzed 6 mutant FAM20C proteins (T268M, P328S, R408W, D451N, D478A, and R549W) for their distributions, kinase activities, and effects on dentin matrix protein (DMP1) promoter activity. We also analyzed the effect of Fam20c knockdown on Dmp1 and Fgf23 mRNA levels in UMR-106 cells. As a result, all the mutant FAM20C proteins showed decreased kinase activities compared to wild-type (WT) FAM20C, and most of them also showed impaired secretion. Overexpression of WT FAM20C increased DMP1 promoter activity in Saos-2 cells while mutant FAM20C did not. Fam20c knockdown decreased Dmp1 mRNA and increased Fgf23 mRNA in UMR-106 cells. In conclusion, our results suggest that FAM20C suppresses FGF23 production by enhancing DMP1 expression, and inactivating mutations in FAM20C cause FGF23-related hypophosphatemia by decreasing transcription of DMP1.

Introduction

Raine syndrome is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype. It was first reported in 1989 by Raine et al. as a lethal osteosclerotic bone dysplasia [1]. Later, Simpson et al. found that mutations in family with sequence similarity 20, member C (FAM20C) is responsible for this syndrome [2], and non-lethal cases were reported afterwards [3], [4], [5].

FAM20C is a member of a four-jointed family of protein kinases. Four-jointed is a kinase that localizes to the Golgi in Drosophila and phosphorylates the secretory proteins [6]. Among four-jointed family members, only FAM20C and FAM20A have been shown to be associated with human diseases. FAM20A is one of the close relatives of FAM20C in a phylogenic tree, and is also a protein kinase. FAM20A is expressed mainly in the parathyroid glands and the cells comprising the tooth and gingivae, such as ameloblasts, odontoblasts, dental pulp cells, and suprabasal cells of the gingivae, and mutations in FAM20A cause amelogenesis imperfecta (AI) and gingival hyperplasia syndrome in humans [7].

FAM20C was shown to be expressed in a wide variety of tissues [8]. In the skeletal and dental tissues, FAM20C was detected in the osteoblasts, odontoblasts, and ameloblasts, and was proposed to act as an inhibitor for mineralization during osteogenesis [9]. It is also shown that secretory calcium-binding phosphoprotein (SCPP) members whose genes cluster on chromosome 4 in humans, have multiple S-x-E/pS motifs and are phosphorylated by FAM20C [10]. The small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) are a subfamily of the SCPPs and comprise dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), and bone sialoprotein (BSP). The contribution of these proteins to mineralization is complicated. Some proteins such as OPN are considered to inhibit the mineralization process [11], while others such as DMP1 are reported to promote tissue mineralization [12]. Therefore, these phosphorylated secretory proteins may regulate proper growth/formation of hydroxyapatite crystals. Actually, abnormal phosphorylation of these proteins has been shown to be responsible for the deranged mineralization in Raine syndrome [13], [14].

Besides the abnormal mineralization, the association with fibroblast growth factor 23 (FGF23)-related hypophosphatemia has been shown in patients with Raine syndrome. In 2013, whole-exome sequencing revealed compound heterozygous mutations in FAM20C in Norwegian brothers with FGF23-related hypophosphatemia, dental anomalies, and ectopic calcifications [5].

FGF23 is a hormone that regulates phosphate homeostasis [15]. Several types of FGF23-related congenital hypophosphatemic rickets have been reported. Among them, X-linked hypophosphatemic rickets (XLHR: OMIM#307800) is believed to be the most prevalent form of genetic hypophosphatemic rickets and is caused by inactivating mutations in the phosphate-regulating endopeptidase homolog, X-linked (PHEX) gene [16]. Autosomal dominant hypophosphatemic rickets (ADHR: OMIM#193100) is caused by missense mutations in the FGF23 gene [17], which prevent the proteolytic cleavage of FGF23 protein into inactive fragments [18], [19], and autosomal recessive hypophosphatemic rickets 1 and 2 (ARHR1: OMIM#241520 and ARHR2: OMIM#613312) are caused by inactivating mutations in DMP1 [20], [21] and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) [22], [23], respectively.

Along with the FGF23-related hypophosphatemia in the Norwegian brothers with compound heterozygous mutations in FAM20C [5], coexisting hypophosphatemia have been reported in other non-lethal patients with Raine syndrome [3], [4]. These results suggest the possibility that FAM20C is another candidate gene that regulates FGF23 production. In addition, in vivo analysis of Fam20c knockout mice confirmed the association between Fam20c and Fgf23 [24]. Fam20c conditional knockout mice, in which exons 6–9 were ablated, developed Fgf23-related hypophosphatemic rickets but not osteosclerosis [24]. It is noteworthy that down regulation of Dmp1 was observed in the calvaria of Fam20c conditional knockout mice [24]. Since DMP1 is a target for phosphorylation by FAM20C [13] and Dmp1 knockout mice present Fgf23-related hypophosphatemic rickets [20], Dmp1 might be a key regulator of Fgf23 in Fam20c knockout mice. However, it is not clear how mutations in FAM20C result in elevation of FGF23 in patients with Raine syndrome.

Therefore, we performed functional analysis of mutant FAM20C proteins to clarify the mechanism of FGF23-related hypophosphatemia in patients with Raine syndrome.

Section snippets

Construction of vectors

Human FAM20C cDNA was amplified by RT-PCR from peripheral blood leukocytes and cloned into pcDNA 3.1/myc-His A (Invitrogen) with In-Fusion HD Cloning Kit (Clontech). Human OPN cDNA was amplified by PCR with pDest490-OPN-a (Addgene plasmid 17590) as a template and similarly cloned into pSecTag/FRT/V5-His-TOPO vector (Invitrogen). Human DMP1 cDNA was amplified by RT-PCR from peripheral blood leukocytes and cloned into either pcDNA 3.1/myc-His A or pcDNA3 mCherry LIC cloning vector (6B) (Addgene

Functional analysis of FAM20C

The kinase domain of FAM20C protein is from 354 to 565 amino residues, and D478A [13] is an artificially created kinase-dead mutant (Fig. 2A). In addition to this mutant, we analyzed 4 types of mutant proteins (T268M, P328S, R408W, and D451N) which were found in patients with non-lethal Raine syndrome who also showed hypophosphatemia [3], [4], [5]. We also included one mutant protein (R549W) which was found in a patient with lethal Raine syndrome [2]. When expressed in HEK293 cells, WT FAM20C

Discussion

Previous studies have reported that mutant FAM20C proteins found in patients with Raine syndrome showed either decreased kinase activity, impaired secretion, or both [13], [14]. To clarify the characteristics of mutant FAM20C proteins which are important in deciding the phenotypes of patients, such as lethal or non-lethal, and normophosphatemic or hypophosphatemic, we performed in vitro analysis of six types of mutant FAM20C proteins on their distributions and kinase activities. Among them,

Acknowledgments

This study was supported in part by grants from the Ministry of Health, Labour and Welfare, Japan (H23-Nanchi-007).

References (35)

  • K. Narayanan et al.

    Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation

    J Biol Chem

    (2006)
  • J. Raine et al.

    Unknown syndrome: microcephaly, hypoplastic nose, exophthalmos, gum hyperplasia, cleft palate, low set ears, and osteosclerosis

    J Med Genet

    (1989)
  • M.A. Simpson et al.

    Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia

    Clin Genet

    (2009)
  • M. Fradin et al.

    Osteosclerotic bone dysplasia in siblings with a Fam20C mutation

    Clin Genet

    (2011)
  • S.H. Rafaelsen et al.

    Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification

    J Bone Miner Res

    (2013)
  • H.O. Ishikawa et al.

    Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains

    Science

    (2008)
  • D. Nalbant et al.

    FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    BMC Genomics

    (2005)
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