Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

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

Cellular & Molecular Biology Letters volume 15, pages 153–176 (2010)Cite this article

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.

Abbreviations

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

References

  1. Nusslein-Volhard, C. and Wieschaus, E. Mutations affecting segment number and polarity in Drosophila. Nature 287 (1980) 795–801.

    Article  PubMed  CAS  Google Scholar 

  2. Levin, M., Johnson, R.L., Stern, C.D., Kuehn, M. and Tabin, C. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82 (1995) 803–814.

    Article  PubMed  CAS  Google Scholar 

  3. Riddle, R.D., Johnson, R.L., Laufer, E. and Tabin, C. Sonic Hedgehog mediates the polarizing activity of the ZPA. Cell 75 (1993) 1401–1416.

    Article  PubMed  CAS  Google Scholar 

  4. Echelard, Y., Epstein, D.J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J.A. and McMahon, A.P. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75 (1993) 1417–1430.

    Article  PubMed  CAS  Google Scholar 

  5. Bitgood, M.J., Shen, L. and McMahon, A.P. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr. Biol. 6 (1996) 298–304.

    Article  PubMed  CAS  Google Scholar 

  6. Vortkamp, A., Lee, K., Lanske, B., Segre, G.V., Kronenberg, H.M. and Tabin, C.J. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273 (1996) 613–622.

    Article  PubMed  CAS  Google Scholar 

  7. Chen, Y. and Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell (1996) 87 553–563.

    Article  PubMed  CAS  Google Scholar 

  8. Marigo, V., Johnson, R.L., Vortkamp, A. and Tabin, C.J. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180 (1996) 273–283.

    Article  PubMed  CAS  Google Scholar 

  9. Lee, J., Platt, K.A., Censullo, P. and Ruiz i Altaba, A. Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 124 (1997) 2537–2552.

    PubMed  CAS  Google Scholar 

  10. Pathi, S., Rutenberg, J.B., Johnson, R.L. and Vortkamp, A. Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation. Dev. Biol. 209 (1999) 239–253.

    Article  PubMed  CAS  Google Scholar 

  11. Laufer, E., Nelson, C.E., Johnson, R.L., Morgan, B.A. and Tabin, C. Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79 (1994) 993–1003.

    Article  PubMed  CAS  Google Scholar 

  12. Lanske, B., Karaplis, A.C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., Karperien, M., Defize, L.H., Ho, C., Mulligan, R.C., Abou-Samra, A.B., Juppner, H., Segre, G.V. and Kronenberg, H.M. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273 (1996) 663–666.

    Article  PubMed  CAS  Google Scholar 

  13. Bitgood, M.J. and McMahon, A.P. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172 (1995) 126–138.

    Article  PubMed  CAS  Google Scholar 

  14. St-Jacques, B., Hammerschmidt, M. and McMahon, A.P. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13 (1999) 2072–2086.

    Article  PubMed  CAS  Google Scholar 

  15. Gao, B. and He, L. Answering a century old riddle: brachydactyly type A1. Cell. Res. 14 (2004) 179–187.

    Article  PubMed  CAS  Google Scholar 

  16. Byrnes, A.M., Racacho, L., Grimsey, A., Hudgins, L., Kwan, A.C., Sangalli, M., Kidd, A., Yaron, Y. and Lau, Y.L. Brachydactyly A-1 mutations restricted to the central region of the N-terminal active fragment of Indian Hedgehog. Eur. J. Hum. Genet. 17 (2009) 1112–1120.

    Article  PubMed  CAS  Google Scholar 

  17. Gao, B., Guo, J., She, C., Shu, A., Yang, M., Tan, Z., Yang, X., Guo, S., Feng, G. and He, L. Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1. Nat. Genet. 28 (2001) 386–388.

    Article  PubMed  CAS  Google Scholar 

  18. McLellan, J.S., Zheng, X., Hauk, G., Ghirlando, R., Beachy, P.A. and Leachy, D.J. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455 (2008) 979–983.

    Article  PubMed  CAS  Google Scholar 

  19. Gao, B., Hu, J., Stricker, S., Cheung, M., Ma, G., Law, K.F., Witte, F., Briscoe, J., Mundlos, S., He, L., Cheah, K.S. and Chan, D. A mutation in Ihh that causes digit abnormalities alters its signaling capacity and range. Nature 458 (2009) 1196–2000.

    Article  PubMed  CAS  Google Scholar 

  20. Hall, T.M., Porter, J.A., Beachy, P.A. and Leahy, D.J. A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog. Nature 378 (1995) 212–216.

    Article  PubMed  CAS  Google Scholar 

  21. Guo, S., Shi, Y., Zhao, X., Duan, S., Zhou, J., Meng, J., Yang, Y., Gu, N., Feng, G., Liu, H., Zhu, S. and He, L. No genetic association between polymorphisms in the AMPA receptor subunit GluR4 gene (GRIA4) and schizophrenia in the Chinese population. Neurosci. Lett. 369 (2004) 168–172.

    Article  PubMed  CAS  Google Scholar 

  22. Aoyama, S., Kase, H. and Borrelli, E. Rescue of locomotor impairment in dopamine D2 receptor-deficient mice by an adenosine A2A receptor antagonist. J. Neurosci. 20 (2000) 5848–5852.

    PubMed  CAS  Google Scholar 

  23. Spinella-Jaegle, S., Rawadi, G., Kawai, S., Gallea, S., Faucheu, C., Mollat, P., Courtois, B., Bergaud, B., Ramez, V., Blanchet, A.M., Adelmant, G., Baron, R. and Roman-Roman, S. Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. J. Cell. Sci. 114 (2001) 2085–2094.

    PubMed  CAS  Google Scholar 

  24. Nakamura, T., Aikawa, T., Iwamoto-Enomoto, M., Iwamoto, M., Higuchi, Y., Pacifici, M., Kinto, N., Yamaguchi, A., Noji, S., Kurisu, K. and Matsuya, T. Induction of osteogenic differentiation by hedgehog proteins. Biochem. Biophys. Res. Commun. 237 (1997) 465–469.

    Article  PubMed  CAS  Google Scholar 

  25. Pathi, S., Pagan-Westphal, S., Baker, D.P., Garber, E.A., Rayhorn, P., Bumcrot, D., Tabin, C.J., Blake Pepinsky, R. and Williams, K.P. Comparative biological responses to human Sonic, Indian, and Desert hedgehog. Mech. Dev. 106 (2001) 107–117.

    Article  PubMed  CAS  Google Scholar 

  26. Chen, M.H., Li, Y.J., Kawakami, T., Xu, S.M. and Chuang, P.T. Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev. 18 (2004) 641–659.

    Article  PubMed  CAS  Google Scholar 

  27. Laurikkala, J., Kassai, Y., Pakkasjarvi, L., Thesleff, I. and Itoh, N. Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot. Dev. Biol. 264 (2003) 91–105.

    Article  PubMed  CAS  Google Scholar 

  28. Walterhouse, D.O., Yoon, J.W. and Iannaccone, P.M. Developmental pathways: sonic hedgehog-patched-GLI. Environ. Health Perspect. 107 (1999) 167–171.

    Article  PubMed  CAS  Google Scholar 

  29. Yoon, J.W., Kita, Y., Frank, D.J., Majewski, R.R., Konicek, B.A., Nobrega, M.A., Jacob, H., Walterhouse, D. and Iannaccone, P. Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation. J. Biol. Chem. 277 (2002) 5548–5555.

    Article  PubMed  CAS  Google Scholar 

  30. Agren, M., Kogerman, P., Kleman, M.I., Wessling, M. and Toftgard, R. Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene 330 (2004) 101–114.

    Article  PubMed  CAS  Google Scholar 

  31. Farabee, W.C. hereditary and sexual influence in meristic variation: a study of digital malformations in man. Thesis, Harvard University, 1903.

  32. Hellemans, J., Coucke, P.J., Giedion, A., De Paepe, A., Kramer, P., Beemer, F. and Mortier, G.R. Homozygous mutations in IHH cause acrocapito-femoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips. Am. J. Hum. Genet. 72 (2003) 1040–1046.

    Article  PubMed  Google Scholar 

  33. Lee, J.J., Ekker, S.C., von Kessler, D., Porter, J.A., Sun, B.I. and Beachy, P.A. Autoproteolysis in hedgehog protein biogenesis. Science 266 (1994) 1528–1537.

    Article  PubMed  CAS  Google Scholar 

  34. Porter, J.A., Ekker, S.C., Park, W.J., von Kessler, D.P., Young, K.E., Chen, C.H., Ma, Y., Woods, A.S., Cotter, R.J., Koonin, E.V. and Beachy, P.A. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86 (1996) 21–34.

    Article  PubMed  CAS  Google Scholar 

  35. Casali, A. and Struhl, G. Reading the Hedgehog morphogen gradient by measuring the ratio of bound to unbound Patched protein. Nature 431 (2004) 76–80.

    Article  PubMed  CAS  Google Scholar 

  36. Fuse, N., Maiti, T., Wang, B., Porter, J.A., Hall, T.M., Leahy, D.J. and Beachy, P.A. Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for patched. Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 10992–10999.

    Article  PubMed  CAS  Google Scholar 

  37. Ekker, S.C., McGrew, L.L., Lai, C.J., Lee, J.J., von Kessler, D.P., Moon, R.T. and Beachy, P.A. Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis. Development 121 (1995) 2337–2347.

    PubMed  CAS  Google Scholar 

  38. Yao, S., Lum, L. and Beachy, P. The ihog cell-surface proteins bind Hedgehog and mediate pathway activation. Cell 125 (2006) 343–357.

    Article  PubMed  CAS  Google Scholar 

  39. Tenzen, T., Allen, B.L., Cole, F., Kang, J.S., Krauss, R.S. and McMahon, A.P. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Dev. Cell. 10 (2006) 647–656.

    Article  PubMed  CAS  Google Scholar 

  40. Zhang, W., Kang, J.S., Cole, F., Yi, M J. and Krauss, R.S. Cdo functions at multiple points in the Sonic Hedgehog pathway, and Cdo-deficient mice accurately model human holoprosencephaly. Dev. Cell. 10 (2006) 657–665.

    Article  PubMed  CAS  Google Scholar 

  41. Lin, X. Functions of heparan sulfate proteoglycans in cell signaling during development. Development 131 (2004) 6009–6021.

    Article  PubMed  CAS  Google Scholar 

  42. Kang, J.S., Mulieri, P.J., Miller, C., Sassoon, D.A. and Krauss, R.S. CDO, a robo-related cell surface protein that mediates myogenic differentiation. J. Cell. Biol. 143 (1998) 403–413.

    Article  PubMed  CAS  Google Scholar 

  43. Dahn, R.D. and Fallon, J.F. Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling. Science 289 (2000) 438–441.

    Article  PubMed  CAS  Google Scholar 

  44. Minina, E., Wenzel, H.M., Kreschel, C., Karp, S., Gaffield, W., McMahon, A.P. and Vortkamp, A. BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation. Development 128 (2001) 4523–4534.

    PubMed  CAS  Google Scholar 

  45. Wu, Q., Zhang, Y. and Chen, Q. Indian Hedgeog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation. J. Biol. Cem. 276 (2001) 35290–35296.

    Article  CAS  Google Scholar 

  46. Lehmann, K., Seemann, P., Stricker, S., Sammar, M., Meyer, B., Suring, K., Majewski, F., Tinscert, S., Grzeshcik, K., Muller, D., Knaus, P., Nurnberg, P. and Mundlos, S. Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2. Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 12277–12282.

    Article  PubMed  CAS  Google Scholar 

  47. Polinkovsky, A., Robin, N., Thomas, J.T., Irons, M., Lynn, A., Goodman, F.R., Reardon, W., Kant, S.G., Brunner, G., van der Burgt, I., Chitayat, D., McGaugran, J., Donnai, D., Luyten, F.P. and Warman, M.L. Mutations in CDMP1cause autosomal dominant brachydactyly type C. Nat. Genet. 17 (1997) 18–19.

    Article  PubMed  CAS  Google Scholar 

  48. Seemann, P., Schwappacher, R., Kjaer, KW., Krakow, D., Lehmann, K., Dawson, K., Stricker, S., Pohl, J., Ploger, F., Staub, E., Nickel, J., Sebald, W., Knaus, P. and Mundlos, S. Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2. J. Clin. Invest. 115 (2005) 2373–2381.

    Article  PubMed  CAS  Google Scholar 

  49. Olney, R.C. and Mougey, E.B. Expression of the components of the insulin-like growth factor axis across the growth-plate. Mol. Cell. Endocrinol. 156 (1999) 63–71.

    Article  PubMed  CAS  Google Scholar 

  50. Salih, D.A., Tripathi, G., Holding, C., Szestak, T.A., Gonzalez, M.I., Carter, E.J., Cobb, L.J., Eisemann, J.E. and Pell, J.M. Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice. Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 4314–4319.

    Article  PubMed  CAS  Google Scholar 

  51. Sekiya, I., Vuoristo, J.T., Larson, B.L. and Prockop, D.J. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chon-drogenesis. Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 4397–4402.

    Article  PubMed  CAS  Google Scholar 

  52. Allan, G.J., Zannoni, A., McKinnell, I., Otto, W.R., Holzenberger, M., Flint, D.J. and Patel, K. Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis, with IGF binding protein (IGFBP)-5 expression subject to regulation by Sonic hedgehog. Anat. Embryol. (Berl) 207 (2003) 73–84.

    Article  CAS  Google Scholar 

  53. Allan, G.J., Flint, D.J., Darling, S.M., Geh, J. and Patel, K. Altered expression of insulin-like growth factor-1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity. Anat. Embryol. (Berl) 202 (2000) 1–11.

    Article  CAS  Google Scholar 

  54. Konig, M., Zimmer, A.M., Steiner, H., Holmes, P.V., Crawley, J.N., Brownstein, M.J. and Zimmer, A. Pain responses, anxiety and aggression in mice deficient in pre-proenkephalin. Nature 383 (1996) 535–538.

    Article  PubMed  CAS  Google Scholar 

  55. Brunet, L.J., McMaon, J.A., McMaon, A.P. and Harland, R.M. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280 (1998) 1455–1457.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Shengzhen Guo, Jian Zhou, Bo Gao, Jianxin Hu, Hongsheng Wang, Junwei Meng, Xinzhi Zhao, Gang Ma, Chuwen Lin, Yue Xiao, Wei Tang, Xuming Zhu & Lin He

  2. Bio-X Center, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, 200030, China

    Shengzhen Guo, Jian Zhou, Bo Gao, Jianxin Hu, Hongsheng Wang, Junwei Meng, Xinzhi Zhao, Gang Ma, Chuwen Lin, Yue Xiao, Wei Tang, Xuming Zhu & Lin He

  3. Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong, China

    Shengzhen Guo, Bo Gao, Jianxin Hu, Kathryn S.E. Cheah & Danny Chan

  4. Shanghai Institutes of Mental Health, Shanghai, 200030, China

    Guoying Feng

  5. Institutes of Biomedical Sciences, Fudan University, Ming De Building, 138 Yi Xue Yuan Road, Shanghai, 200032, PR China

    Lin He

  6. Bio-X Center, Shanghai Jiao Tong University, Cental Little White House, 1954 Huashan Road, Shanghai, 200030, China

    Lin He

  7. Department of Biochemistry, The University of Hong Kong, 3/F, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China

    Danny Chan

Authors
  1. Shengzhen Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bo Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jianxin Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongsheng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Junwei Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinzhi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gang Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuwen Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yue Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Wei Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Xuming Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kathryn S.E. Cheah
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Guoying Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Danny Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Lin He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Danny Chan or Lin He.

Additional information

These authors contributed equally to this work

Rights and permissions

Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Reprints and permissions

About this article

Cite this article

Guo, S., Zhou, J., Gao, B. et al. Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling. Cell Mol Biol Lett 15, 153–176 (2010). https://doi.org/10.2478/s11658-009-0040-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-009-0040-2

Key words

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us