Altered sulfation status of FAM20C-dependent chondroitin sulfate is associated with osteosclerotic bone dysplasia

Abstract

Raine syndrome, a lethal osteosclerotic bone dysplasia in humans, is caused by loss-of-function mutations in FAM20C; however, Fam20c deficiency in mice does not recapitulate the human disorder, so the underlying pathoetiological mechanisms remain poorly understood. Here we show that FAM20C, in addition to the reported casein kinase activity, also fine-tunes the biosynthesis of chondroitin sulfate (CS) chains to impact bone homeostasis. Specifically, FAM20C with Raine-originated mutations loses the ability to interact with chondroitin 4-O-sulfotransferase-1, and is associated with reduced 4-sulfation/6-sulfation (4S/6S) ratio of CS chains and upregulated biomineralization in human osteosarcoma cells. By contrast, overexpressing chondroitin 6-O-sulfotransferase-1 reduces CS 4S/6S ratio, and induces osteoblast differentiation in vitro and higher bone mineral density in transgenic mice. Meanwhile, a potential xylose kinase activity of FAM20C does not impact CS 4S/6S ratio, and is not associated with Raine syndrome mutations. Our results thus implicate CS 4S/6S ratio imbalances caused by FAM20C mutations as a contributor of Raine syndrome etiology.

Fig. 1. FAM20C can regulate GAG abundance.

a Biosynthetic pathways for CS and HS GAG chains. Multiple glycosyltransferases and distinct kinase/phosphatase(s) contribute to the synthesis of the common tetrasaccharide linkage region and repeating disaccharide region characteristic of CS and HS chains. Transient phosphorylation of the Xyl residue by XYLKs, including FAM20B, enhances linkage tetrasaccharide synthesis. Before subsequent GAG assembly by distinct biosynthetic machinery for CS and HS chains, the Xyl residue is dephosphorylated by a 2-phosphoxylose phosphatase (PXYLP). This XYLK-enhanced GAG biosynthesis determines the number of CS and HS chains attached to core proteins. XylT xylosyltransferase, GalT-I β1,4-galactosyltransferase-I, GalT-II β1,3-galactosyltransferase-II, GlcAT-I β1,3-glucuronyltransferase-I. b, c Constitutive overexpression (OE) of FAM20C in HeLa cells elevated the levels of (b) CS and (c) HS chains, whereas its knockdown (KD) abrogated both GAG levels (b, c) compared with the mock controls (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). d, e Gel filtration elution profiles of (d) CS and (e) HS chains from FAM20C-related stable HeLa cell lines. Numbered arrowheads (200 k, 65 k, 37 k, and 18 k) indicate the elution position of 200-, 65-, 37-, and 18-kDa saccharides derived from size-defined commercial dextran, respectively. Results represent the average of two series of independent experiments. f The 4S/6S ratio of CS chains from FAM20C-related stable HeLa cell lines (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in b, c, and f are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig. 2. FAM20C-mediated control of CS biosynthesis is exerted by its physical interaction with C4ST-1.

a Schematic diagram of sulfation pathways for the chondroitin backbone. Characteristic CS disaccharide units, including A, C, D, and E, are sequentially formed under the control of CS-specific sulfotransferases such as C4ST-1, C4ST-2, and C6ST-1. b Gel filtration elution profiles of CS chains from mouse fibroblastic L or sog9 cells overexpressing wild-type FAM20B or FAM20C (Data are derived from the single experiment). Numbered arrowheads (200 k, 65 k, 37 k, and 18 k) indicate the elution position of 200-, 65-, 37-, and 18-kDa saccharides derived from size-defined commercial dextran, respectively. c Pulldown (PD) and immunoblotting (IB) analysis of culture medium from COS-1 cells co-expressing soluble forms of His-tagged C4ST-1 and FLAG-tagged FAM20 proteins. Data are obtained from three independent experiments and representative images are shown. d Sulfotransferase activities of C4ST-1 alone (Mock) and that from cells co-transfected with the individual FAM20 proteins (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). e The 4S/6S ratio of CS chains from HeLa cell lines overexpressing wild-type or mutant FAM20C proteins (n = 4 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in d and e are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig. 3. FAM20C mutants enhance biomineralization in human osteosarcoma cells.

a, b Expression of FAM20C, FAM20B, and C4ST-1 in stable Saos-2 cell lines overexpressing individual FAM20 proteins at the a transcriptional and b translational levels. GAPDH was used as an internal standard in (a), and as a loading control in (b). Data are obtained from three independent experiments and representative images are shown. c The 4S/6S ratio of CS chains from the stable Saos-2 cell lines in (a) and (b) (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). d, e Biomineralization level of the 21-day cultures of the stable Saos-2 cell lines in (a) and (b) was assessed by Alizarin red staining (d), followed by colorimetric quantification of the dye extracts derived from the stained cultures (e, n = 4 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in c and e are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig. 4. C6ST-1 overexpression potentiates mouse osteoblast differentiation.

a The 4S/6S ratio of CS chains from parental, mock-transfected (Mock), or C6ST-1 overexpressing (C6ST-1 OE #1 and #2) MC3T3-E1 cells (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). b ALP staining and mRNA expression for ALP (Akp2) in 14-day cultures of parental, mock, or C6ST-1 OE cells (#1 and #2) (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). c Mineralized nodule formation in 21-day cultures of parental, mock, or C6ST-1 OE cells (#1 and #2) was assessed by Alizarin red staining. Data are obtained from three independent experiments and representative images are shown. (d) Adhesion of intact, ChABC-pretreated, or EDTA-treated MC3T3-E1 cells (parental or C6ST-1 OE #1 and #2) to plates precoated with recombinant N-cadherin or cadherin-11 (n = 10 fields from 3 independent experiments, for each condition, Tukey–Kramer multiple comparison method). e CS-C modulated the cadherin-mediated intracellular signaling pathways, including ERK1/2, Smad3, and Smad1/5/8, and upregulated Akp2 expression in semi-confluent, parental MC3T3-E1 cultures in the presence of an isotype control antibody (control Ab). Pretreatment with neutralizing antibodies for N-cadherin and cadherin-11 (cadherin Ab) abolished the CS-C effects (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in a, b, d, and e are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig. 5. High bone mass phenotypes in C6ST1 transgenic mice.

a The length of long bones (femurs and tibias) from 16-week-old wild-type (WT) or C6ST1 transgenic (TG) male mice (n = 5 bones from total, each from different litters, unpaired Student’s t-test, two-sided). b–i Micro-CT analysis of tibias from 16-week-old WT or C6ST1 TG male mice. b Medial, longitudinal section through a µCT-generated three-dimensional reconstruction of a tibia. c Micro-CT reconstruction of the trabecular region of tibial metaphysis. Micro-CT-based measurements of d trabecular bone volume to total volume (BV/TV), e trabecular thickness (Tb. Th.), f trabecular number (Tb. N.), and g trabecular separation (Tb. Sp.). d–g n = 3 bones from the total, each from different litters, unpaired Student’s t-test, two-sided). h Micro-CT reconstruction of the cortical region of the tibial diaphysis. i Micro-CT-based measurement of cortical thickness (Co. Th.). (n = 3 bones from total, each from different litters, unpaired Student’s t-test, two-sided). j–m Decreased 4S/6S ratio of CS chains promotes osteoblastogenesis. j The 4S/6S ratio of CS chains from WT or C6ST1 TG BMSCs (n = 3 cultures from 3 independent mice litters, for each genotype, unpaired Student’s t-test, two-sided). k–m Osteoblastic potential of BMSCs isolated from WT or C6ST1 TG male mice. BMSCs were maintained in a differentiation medium (DM) for 21 days in the absence (−) or presence (+) of ChABC. ALP staining (k) and Alizarin red staining (l) were performed on 7-day and 21-day cultures of BMSCs, respectively. Expression of Akp2 mRNA in 7-day cultures of BMSCs (m, n = 3 cultures from 3 independent mice litters, for each genotype, Tukey–Kramer multiple comparison method). Data in a, d–g, i, j, and m are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig. 6. Non-enzymatic functions of FAM20C in the control of CS biosynthesis and its involvement in Raine syndrome etiology.

In physiological states (a), FAM20C can physically interact with C4ST-1 and augment the enzymatic activity of C4ST-1, leading to increased length of the individual CS chains via cooperative actions with glycosyltransferases (N-acetylgalactosaminyltransferases; ChGns)^, and to an elevated 4S/6S ratio. In contrast b, lethal Raine syndrome mutations in FAM20C cause functional uncoupling between FAM20C and C4ST-1, perturbing the C4ST-1-mediated chain elongation process and sulfation balance of CS chains. The consequent decrease in the 4S/6S ratio may be involved in the manifestation of chondroitin 6-sulfation-dependent skeletal phenotypes, which might be recapitulated by a gain-of-function of FAM20B.