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. 2009 Dec 20;15(1):153–176. doi: 10.2478/s11658-009-0040-2

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

Shengzhen Guo 1,2,3, Jian Zhou 1,2, Bo Gao 1,2,3, Jianxin Hu 1,2,3, Hongsheng Wang 1,2, Junwei Meng 1,2, Xinzhi Zhao 1,2, Gang Ma 1,2, Chuwen Lin 1,2, Yue Xiao 1,2, Wei Tang 1,2, Xuming Zhu 1,2, Kathryn SE Cheah 3, Guoying Feng 4, Danny Chan 3,7,✉, Lin He 1,2,5,6,✉
PMCID: PMC6275863  PMID: 20024692

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.

Key words: Indian Hedgehog, Brachydactyly type A1, Microarray, EMSA

Full Text

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Abbreviations used

AKP2

alkaline phosphatase 2

BDA1

Brachydactyly type A1

BMP

bone morphogenic protein

BMPR1A

bone morphogenetic protein receptor type 1A

BMPR1B

bone morphogenetic protein receptor type 1B

BOC

brother of CDO

CDO

cysteine dioxygenase

COL2A1

procollagen type II, alpha 1

DHH

Desert hedgehog

EMSA

electrophoretic mobility shift assays

GAPDH

glyceraldehydes-3-phosphate dehydrogenase

GDF5

growth differentiation factor 5

GLI1

GLI-Kruppel family member GLI1

IGFBP5

insulin-like growth factor-binding 5

IHH

Indian hedgehog

IHOG

interference hedgehog

PENK1

preproenkephalin 1

PTCH

patched homologue 1

PTHrP

parathyroid hormone-related peptide

ROR2

receptor tyrosine kinase-like orphan receptor 2

SHH

Sonic hedgehog

SMO

smoothened homologue (Drosophila)

SOSTDC1

sclerostin domain containing 1

Footnotes

These authors contributed equally to this work

Contributor Information

Danny Chan, Phone: 852-28199482, FAX: 852-28551254, Email: chand@hkusua.hku.hk.

Lin He, Phone: 86-21-62822491, FAX: 86-21-62822491, Email: helin@bio-x.cn.

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