DESARROLLO DE LA SESION:

calverial cells further suggested that Wnt5a signaling is important during osteogenesis, and

Wnt5a might act upstream of those key osteoblast transcription factors. This could have an

indirect effect on the observed decreased proliferation of Wnt5a

calvarial cells. Wnt5a has been

implicated in control of cell growth (Prockop Gregory and Spees 2003). Osteoprogenitors

derived from Wnt5a

mice were senescent and showed reduced cell growth. Genes controlling

cell cycle, cell division, cell cycle progression regulation, mitotic cell cycle, and cell

proliferation were altered as suggested by the microarray results in this study.

Wnt5a is considered as a non-canonical Wnt that mediates downstream signaling through JNK or Ca2+_{/PKC. The MAPK signaling pathway was altered in Wnt5a deficient mouse calvarial cells, which} suggests that Wnt5a signaling may influence the MAPK/JNK pathway. Further detailed investigation is required to ascertain this hypothesis. On the other hand, several components involved in Ca2+_/PKC pathway were also regulated in Wnt5a-/- _{cells, such as phospholipase C, protein phosphatase 3,} calcium/calmodulin-dependent protein kinase I gamma (Camk1g), Camk4, and PKC.

In addition, Wnt5a may affect the canonical Wnt signaling pathway. In the microarray study, at least 26 of the Wnt/β-catenin pathway involved genes were altered in Wnt5a deficient cells. It has been shown that Sfrp2 binds to canonical Wnts and inhibit β-catenin-mediated signaling (Galli et al 2006). In this study, Sfrp2 was shown to be downregulated when Wnt5a activity was diminished. It suggested that Wnt5a might antagonize canonical Wnt signaling, which has been demonstrated previously by other investigators (Ishitani et al 2003; Olson and Gibo 1998; Topol et al 2003; Torres et al 1996).

The hedgehog (Hh) family of genes, including sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh), encode signaling molecules that regulate multiple functions during organ development and in adult tissues. Hh signal is mediated by binding to the thansmembrane receptor Patched. Upon binding to Hh, Patched releases its inhibition of Smoothened (Smo), which is then free to

transmit its signal downstream and consequently control of Gli family transcription factors. Fox family genes are known to be regulated by Hh signaling. They are the major mediators of the function of Hh signaling in craniofacial development (Jeong et al 2004). In the Wnt5a-/- _{cells, the levels of several genes} involved in Hh signaling were altered. Included were Dhh, Gas1, FoxC1, FoxC2, FoxD1, FoxG1, Gli3 and Gli5, suggesting some interaction between Hh signaling and Wnt5a signaling. Hh could be downstream target of Wnt5a signaling and requires further investigation.

In conclusion, Wnt5a expression occurs in lineage specific manner. Knock-out cells reveal Wnt5a expression affects cell cycle progression and is required for cell growth. The knock-out model also suggests a role for non-canonical Wnt (Wnt5a) signaling during osteogenesis.

Acknowledgements

We thank Dr. Yingzi Yang (National human genome research institute, National institute of health, Besthesda, MD, USA) for providing us the Wnt5a+/- _mice.

References (Chapter III):

Aubin JE, Bonnelye E. (2000) Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption. Osteoporos Int 11:905-913.

Austin TW, Solar GP, Ziegler FC, Liem L, Matthews W. (1997) A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells. Blood 89:3624-3635.

Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. (2001) Controlling the false discovery rate in behavior genetics research. Behav Brain Res 125:279-284.

Boutros M, Paricio N, Strutt DI, Mlodzik M. (1998) Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94:109-118.

Brandon C, Eisenberg LM, Eisenberg CA. (2000) WNT signaling modulates the diversification of hematopoietic cells. Blood 96:4132-4141.

Carlson MR, Bryant SV, Gardiner DM. (1998) Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs. J Exp Zool 282:715-723.

Chimal-Monroy J, Montero JA, Ganan Y, Macias D, Garcia-Porrero JA, Hurle JM. (2002) Comparative analysis of the expression and regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in micromass cultures. Dev Dyn 224:314-320.

Church VL, Francis-West P. (2002) Wnt signalling during limb development. Int J Dev Biol 46:927-936. Cooper LF, Harris CT, Bruder SP, Kowalski R, Kadiyala S. (2001) Incipient analysis of mesenchymal stem-cell-derived osteogenesis. J Dent Res 80:314-320.

Crowe R, Zikherman J, Niswander L. (1999) Delta-1 negatively regulates the transition from

prehypertrophic to hypertrophic chondrocytes during cartilage formation. Development 126:987-998. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747-754.

Galli LM, Barnes T, Cheng T, Acosta L, Anglade A, Willert K, et al. (2006) Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3. Dev Dyn 235:681-690.

Gordon MD, Nusse R. (2006) Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 281:22429-22433.

Gregory CA, Singh H, Perry AS, Prockop DJ. (2003) The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 278:28067- 28078.

Gregory CA, Gunn WG, Reyes E, Smolarz AJ, Munoz J, Spees JL, et al. (2005) How Wnt signaling affects bone repair by mesenchymal stem cells from the bone marrow. Ann N Y Acad Sci 1049:97-106. Hartmann C, Tabin CJ. (2000) Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development 127:3141-3159.

Ishitani T, Kishida S, Hyodo-Miura J, Ueno N, Yasuda J, Waterman M, et al. (2003) The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol 23:131-139.

Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. (1997) Osteogenic differentiation of purified, culture- expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295-312.

Janderova L, McNeil M, Murrell AN, Mynatt RL, Smith SR. (2003) Human mesenchymal stem cells as an in vitro model for human adipogenesis. Obes Res 11:65-74.

Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP. (2004) Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev 18:937-951.

Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP. (2000) Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein- dependent and -independent pathways. Development 127:543-548.

Katoh M. (2002) Regulation of WNT signaling molecules by retinoic acid during neuronal differentiation in NT2 cells: threshold model of WNT action (review). Int J Mol Med 10:683-687.

Komaki M, Katagiri T, Suda T. (1996) Bone morphogenetic protein-2 does not alter the differentiation pathway of committed progenitors of osteoblasts and chondroblasts. Cell Tissue Res 284:9-17.

Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, et al. (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-764.

Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT. (2000) The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 16:279-283.

Lako M, Lindsay S, Lincoln J, Cairns PM, Armstrong L, Hole N. (2001) Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation. Mech Dev 103:49-59.

Li C, Xiao J, Hormi K, Borok Z, Minoo P. (2002) Wnt5a participates in distal lung morphogenesis. Dev Biol 248:68-81.

Lickert H, Kispert A, Kutsch S, Kemler R. (2001) Expression patterns of Wnt genes in mouse gut development. Mech Dev 105:181-184.

Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.

Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4:415-428.

Monkley SJ, Delaney SJ, Pennisi DJ, Christiansen JH, Wainwright BJ. (1996) Targeted disruption of the Wnt2 gene results in placentation defects. Development 122:3343-3353.

Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. (2002) The novel zinc finger- containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17-29.

Nusse R. (2005) Wnt signaling in disease and in development. Cell Res 15:28-32.

Olson DJ, Gibo DM. (1998) Antisense wnt-5a mimics wnt-1-mediated C57MG mammary epithelial cell transformation. Exp Cell Res 241:134-141.

Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, et al. (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone

development. Cell 89:765-771.

Pandur P, Lasche M, Eisenberg LM, Kuhl M. (2002) Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418:636-641.

Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147.

Prockop DJ, Gregory CA, Spees JL. (2003) One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A 100 Suppl 1:11917-11923.

Reya T, Clevers H. (2005) Wnt signalling in stem cells and cancer. Nature 434:843-850.

Robledo RF, Rajan L, Li X, Lufkin T. (2002) The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev 16:1089-1101.

Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, et al. (2000) Inhibition of adipogenesis by Wnt signaling. Science 289:950-953.

Ryu JH, Chun JS. (2006) Opposing roles of WNT-5A and WNT-11 in interleukin-1beta regulation of type II collagen expression in articular chondrocytes. J Biol Chem 281:22039-22047.

Sarray S, Berthet V, Calvete JJ, Secchi J, Marvaldi J, El-Ayeb M, et al. (2004) Lebectin, a novel C-type lectin from Macrovipera lebetina venom, inhibits integrin-mediated adhesion, migration and invasion of human tumour cells. Lab Invest 84:573-581.

Sheldahl LC, Park M, Malbon CC, Moon RT. (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr Biol 9:695-698.

Skillington J, Choy L, Derynck R. (2002) Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 159:135-146.

Smyth GK, Michaud J, Scott HS. (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21:2067-2075.

Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y. (2003) Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol 162:899-908.

Torres MA, Yang-Snyder JA, Purcell SM, DeMarais AA, McGrew LL, Moon RT. (1996) Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development. J Cell Biol 133:1123-1137.

Tusher VG, Tibshirani R, Chu G. (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121.

Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. (1996) Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273:613-622.

Yamaguchi TP, Bradley A, McMahon AP, Jones S. (1999) A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126:1211-1223.

Yamanaka H, Moriguchi T, Masuyama N, Kusakabe M, Hanafusa H, Takada R, et al. (2002) JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. EMBO Rep 3:69-75.

Yang Y, Topol L, Lee H, Wu J. (2003) Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130:1003-1015.

CHAPTER IV