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Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling.

ABSTRACT: Heterozygous missense mutations in IHH result in Brachydactyly type A1 (BDA1; OMIM 112500), a condition characterized by the shortening of digits due to hypoplasia/aplasia of the middle phalanx. Indian Hedgehog signaling regulates the proliferation and differentiation of chondrocytes and is essential for endochondral bone formation. Analyses of activated IHH signaling in C3H10T1/2 cells showed that three BDA1-associated mutations (p.E95K, p.D100E and p.E131K) severely impaired the induction of targets such as Ptch1 and Gli1. However, this was not a complete loss of function, suggesting that these mutations may affect the interaction with the receptor PTCH1 or its partners, with an impact on the induction potency. From comparative microarray expression analyses and quantitative real-time PCR, we identified three additional targets, Sostdc1, Penk1 and Igfbp5, which were also severely affected. Penk1 and Igfbp5 were confirmed to be regulated by GLI1, while the induction of Sostdc1 by IHH is independent of GLI1. SOSTDC1 is a BMP antagonist, and altered BMP signaling is known to affect digit formation. The role of Penk1 and Igfbp5 in skeletogenesis is not known. However, we have shown that both Penk1 and Igfbp5 are expressed in the interzone region of the developing joint of mouse digits, providing another link for a role for IHH signaling in the formation of the distal digits.

SUBMITTER: Guo S

PROVIDER: S-EPMC6275863 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling.

Guo Shengzhen S Zhou Jian J Gao Bo B Hu Jianxin J Wang Hongsheng H Meng Junwei J Zhao Xinzhi X Ma Gang G Lin Chuwen C Xiao Yue Y Tang Wei W Zhu Xuming X Cheah Kathryn S E KS Feng Guoying G Chan Danny D He Lin L

Cellular & molecular biology letters 20091220 1

Heterozygous missense mutations in IHH result in Brachydactyly type A1 (BDA1; OMIM 112500), a condition characterized by the shortening of digits due to hypoplasia/aplasia of the middle phalanx. Indian Hedgehog signaling regulates the proliferation and differentiation of chondrocytes and is essential for endochondral bone formation. Analyses of activated IHH signaling in C3H10T1/2 cells showed that three BDA1-associated mutations (p.E95K, p.D100E and p.E131K) severely impaired the induction of t ...[more]

PMID: 20024692

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Project description:BACKGROUND:Brachydactyly type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes. It is the first recorded disorder of the autosomal dominant Mendelian trait. Indian hedgehog (IHH) gene is closely associated with BDA1, which was firstly mapped and identified in Chinese families in 2000. Previous studies have demonstrated that BDA1-related mutant IHH proteins affected interactions with its receptors and impaired IHH signaling. However, how the altered signaling pathway affects downstream transcriptional regulation remains unclear. RESULTS:Based on the mouse C3H10T1/2 cell model for IHH signaling activation, two recombinant human IHH-N proteins, including a wild type protein (WT, amino acid residues 28-202) and a mutant protein (MT, p.E95k), were analyzed. We identified 347, 47 and 4 Gli1 binding sites in the corresponding WT, MT and control group by chromatin immunoprecipitation and the overlapping of these three sets was poor. The putative cis regulated genes in WT group were enriched in sensory perception and G-protein coupled receptor-signaling pathway. On the other hand, putative cis regulated genes were enriched in Runx2-related pathways in MT group. Differentially expressed genes in WT and MT groups indicated that the alteration of mutant IHH signaling involved cell-cell signaling and cellular migration. Cellular assay of migration and proliferation validated that the mutant IHH signaling impaired these two cellular functions. CONCLUSIONS:In this study, we performed integrated genome-wide analyses to characterize differences of IHH/Gli1 downstream regulation between wild type IHH signaling and the E95K mutant signaling. Based on the cell model, our results demonstrated that the E95K mutant signaling altered Gli1-DNA binding pattern, impaired downstream gene expressions, and leaded to weakened cellular proliferation and migration. This study may help to deepen the understanding of pathogenesis of BDA1 and the role of IHH signaling in chondrogenesis.

Project description:BackgroundBrachydactyly type A1 (BDA1) is an autosomal dominant disorder characterized by uniform shortening of the middle phalanges in all digits. It is associated with variants in the Indian Hedgehog (IHH) gene, which plays a key role in endochondral ossification. To date, heterozygous pathogenic IHH variants involving several codons, which are restricted to a specific region of the N-terminal active fragment of IHH, have been reported. The purpose of this study was to identify the pathogenic variant in a Japanese family with BDA1 and to evaluate its pathogenesis with regard to previous reports.MethodsThe proband, a 9-year-old boy, his siblings, and his father had shortened digits and a short stature of variable severity. Based on physical examinations, radiographic findings and family history, they were diagnosed with BDA1. This family is the first case of an isolated malformation in Japan. Sanger sequencing of IHH was performed on these individuals and on the proband's unaffected mother. The significance of the variants was assessed using three-dimensional analysis methods.ResultsSanger sequencing showed a novel IHH heterozygous variant, NM_002181.4:c.544_549delTCAAAG(p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del].. These two residues are located outside the cluster region considered a hotspot of pathogenic variants. Three-dimensional modelling showed that S182 and K183 are located on the same surface as other residues associated with BDA1. Analysis of residue interactions across the interface between IHH and its interacting receptor protein revealed the presence of hydrogen bonds between them.ConclusionsWe report a novel variant, NM_002181.4:c.544_549delTCAAAG (p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del] in a Japanese family with BDA1. Indeed, neither variations in codons 182 or 183 nor with such two-amino-acid deletions in IHH have been reported previously. Although these two residues are located outside the cluster region considered a hotspot of pathogenic variants, we speculate that this variant causes BDA1 through impaired interactions between IHH and target receptor proteins in the same manner as other pathogenic variants located in the cluster region. This report expands the genetic spectrum of BDA1.

Dataset Information

Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling.

Publications

Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling.

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