1. Identification and Functional Characterization of RSPO2 as a Susceptibility Gene for Ossification of the Posterior Longitudinal Ligament of the Spine 
Masahiro Nakajima, Ikuyo Kou, Hirofumi Ohashi, Shiro Ikegawa 
The American Journal of Human Genetics 
Volume 99, Issue 1, Pages (July 2016)
DOI: /j.ajhg
Copyright © 2016 American Society of Human Genetics Terms and Conditions

2. Figure 1 RSPO2 Inhibits Chondrocyte Differentiation of ATDC5 Cells
(A) ATDC5 cells were differentiated after transfection with RSPO2. Overexpression of RSPO2 inhibited Col2a1, Acan, and Sox9 mRNA expression.
(B) ATDC5 cells were differentiated for 7 days and then cultured with recombinant RSPO2 for 2 days. Recombinant RSPO2 inhibited chondrocyte differentiation.
(C) ATDC5 cells were differentiated for 14 days with (black bars) or without (white bars) recombinant RSPO2 (10 ng/ml). Recombinant RSPO2 inhibited expression of Col2a1, Acan, and Sox9 mRNA.
(D) ATDC5 cells were differentiated for 7 days and then cultured with RSPO2-neutralizing antibodies for 2 days. RSPO2-neutralizing antibodies stimulated expression of Col2a1, Acan, and Sox9 mRNA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus control by Student’s t test. Data represent the mean ± SD of values from triplicate assays.
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2016 American Society of Human Genetics Terms and Conditions

3. Figure 2 RSPO2 Enhances Wnt-β-Catenin Signaling Pathway in ATDC5 Cells
(A) RSPO2 enhanced Wnt3a-induced inhibition of expression of Col2a1, Acan, and Sox9 mRNA in ATDC5 cells. ATDC5 cells were differentiated for 7 days and then cultured with Wnt3a (10 ng/ml) and RSPO2 (10 ng/ml). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by Student’s t test. Data represent the mean ± SD of values from triplicate assays.
(B) RSPO2 enhanced Wnt3a-induced Wnt-β-catenin signaling. ATDC5 cells were transfected with Topflash and Fopflash reporter vectors and cultured with Wnt3a and RSPO2 (10 ng/ml) as indicated. Results are expressed as the ratio of Topflash over Fopflash activity. ∗∗∗p < versus corresponding RSPO2 (−) by Student’s t test. Data represent the mean ± SD of values from four transfections.
(C) Inhibitory effects of RSPO2-neutralizing antibodies on Wnt-β-catenin signaling in ATDC5 cells. ATDC5 cells were transfected with Topflash and Fopflash reporter vectors and cultured with Wnt3a (50 ng/ml) and RSPO2-neutralizing antibodies (RSPO2 Ab) as indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus corresponding RSPO2 Ab (−) by Student’s t test.
(D) IWR-1, an inhibitor of the canonical Wnt pathway, blocked the RSPO2-induced decrease in expression of genes encoding chondrocyte differentiation markers. ATDC5 cells were differentiated for 7 days and then cultured with (black bars) or without (white bars) recombinant RSPO2 for 2 days. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < versus RSPO2 alone by Student’s t test.
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2016 American Society of Human Genetics Terms and Conditions

4. Figure 3
Role of RSPO2 and the Susceptibility SNP in Pathogenesis of OPLL
(A) Localization of RSPO2 promoter activity. SNP rs is contained within the core promoter.
(B) Reporter assays in mesenchymal stem cells. Top: schematic of the luciferase reporter construct with rs Bottom: RSPO2 promoter activity for the risk allele (C) of rs was significantly lower than that of the non-risk allele (T) when C/EBPβ was co-expressed. Data represent the mean ± SD of values from four transfections. ∗p < 0.05 versus the T allele.
(C) EMSAs with nuclear protein extracts from the C/EBPβ-transfected chondrosarcoma cell line HCS2/8. There was higher affinity for the probe with the T allele (lane 3, white triangle) than for the probe with the C allele (lane 4) of rs (sense: 5′-TCTATCCCCCGCACTYTGGAAAGCCCGAAGA-3′; antisense: 5′-TCTTCGGGCTTTCCARAGTGCGGGGGATAGA-3′). Binding of the T allele probe was competed away by an unlabeled probe (250-fold excess; lane 5). The black triangle indicates the supershifted band generated with antibodies to C/EBPβ.
(D) Effect of rs on RSPO2 expression in fibroblasts. The y axis shows the log2 expression of RSPO2 values per rs genotype (x axis). Subjects carrying TC and CC genotypes had significantly lower RSPO2 expression than those with a TT genotype.
(E) RSPO2 might be a gatekeeper of ectopic ossification of ligaments. In the early stage of ligament regeneration, RSPO2 blocks ectopic ossification of ligaments by enhancing Wnt-β-catenin signal that blocks chondrocyte differentiation of ligament MSCs.
The American Journal of Human Genetics  , DOI: ( /j.ajhg )
Copyright © 2016 American Society of Human Genetics Terms and Conditions