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日本学術振興会 先端拠点形成事業 ・21世紀COE共催
ABJS(Advanced Bone and Joint Science) 国際セミナー(第2回)

2004年10月 25日 (月) 11:00- 15:00

Masaki Noda&s_comma; MD&s_comma; PhD
Tokyo Medical and Dental University
Introduction
Roel Nusse&s_comma; PhD
Stanford University
Signaling by Wnt Protein During Development and
Regeneration
Yukiko Gotoh&s_comma; PhD
University of Tokyo
Fate Regulation of Neural Precursor Cells in Embryonic Forebrain
Keiichi Ozono&s_comma; MD&s_comma; PhD
Osaka University
Lrp6 Plays an Important Role in Somitogenesis and Osteogenesis
Signaling by Wnt Proteins During Development and Regeneration
Roel Nusse&s_comma; PhD
Signals that control how embryonic cells become specified during development
fall into a relatively small number of families&s_comma; including the Wnt&s_comma; BMP and
Hedgehog families. These proteins act between cells during growth and
differentiation. During the regeneration and renewal of adult tissue&s_comma; for
example in the skin and in the gut&s_comma; the same signals control how stem cells
divide

Our laboratory has a long-standing interest in how Wnt proteins work during
embryogenesis and other processes. There is substantial evidence that
signaling by Wnt proteins is important in the control over stem cells&s_comma; and
Wnt proteins might therefore become important reagents to expand stem cells
or to guide them to differentiate properly. In order to exploit the use of
Wnt proteins in such experiments&s_comma; it is important to isolate them in an
active form. Although Wnt proteins are secreted from cells&s_comma; secretion is
usually inefficient and previous attempts to characterize active Wnt
proteins have been hampered by their high degree of insolubility.

To address this problem&s_comma; we expressed several Wnt proteins in cell lines and
generated antibodies to monitor Wnt protein secretion into the medium.
Following a protein purification protocol designed in our lab&s_comma; we were then&s_comma;
for the first time&s_comma; isolated an active Wnt molecule. The purified protein
induces appropriate expression of target genes in cell lines and in explants
of animal tissue. We have now been able to purify 4 members of the Wnt
protein family and have seen that they have diverse activities.

With the purified proteins&s_comma; we could also establish that Wnt proteins are
modified by fatty acids&s_comma; which makes them very hydrophobic. By eliminating
the lipid from the Wnt protein&s_comma; we could establish that this modification is
actually important for activity&s_comma; as the non-modified Wnt has reduced
signaling function.

We tested the possible activity of Wnt protein on a variety of stem cells.
Mesenchymal cells isolated from mouse limb buds are undifferentiated but
have the ability&s_comma; in cell culture&s_comma; to differentiate into chondrocytes&s_comma; cells
that form cartilage. Wnt protein&s_comma; when added to the primitive mesenchymal
cells&s_comma; inhibits differentiation and leads instead to proliferation of the
cells. To understand how Wnt proteins can control cell proliferation and
differentiation&s_comma; it is important to identify target genes. To that end&s_comma; we
have employed the microarray technology. Using mesenchymal cells and
purified Wnt protein&s_comma; we found a set of interesting genes activated within a
fairly short time&s_comma; suggesting that all these genes are direct targets of Wnt
signaling. Several of the target genes we identified&s_comma; such as the Msx genes&s_comma;
have been implicated in stem cell control and we are extending these studies
to experiments in which we use different Wnt proteins and various stem cells
systems.

In a collaboration with the lab of Dr. Irving Weissman at Stanford&s_comma; we found
that the purified Wnt protein maintains and expands hematopoietic stem
cells. These finding&s_comma; plus the wide range of biological activities
attributed to members of the Wnt gene family&s_comma; indicates that the
availability of Wnt proteins as active growth factors may become a
significant resource in tissue engineering.



References



Reya&s_comma; T.&s_comma; Duncan&s_comma; A. W.&s_comma; Ailles&s_comma; L.&s_comma; Domen&s_comma; J.&s_comma; Scherer&s_comma; D. C.&s_comma; Willert&s_comma; K.&s_comma;
Hintz&s_comma; L.&s_comma; Nusse&s_comma; R.&s_comma; and Weissman&s_comma; I. L. (2003). A role for Wnt signalling
in self-renewal of haematopoietic stem cells. Nature 423&s_comma; 409-414.



Willert&s_comma; K.&s_comma; Brown&s_comma; J. D.&s_comma; Danenberg&s_comma; E.&s_comma; Duncan&s_comma; A. W.&s_comma; Weissman&s_comma; I. L.&s_comma;
Reya&s_comma; T.&s_comma; Yates&s_comma; J. R.&s_comma; and Nusse&s_comma; R. (2003). Wnt proteins are
lipid-modified and can act as stem cell growth factors. Nature 423&s_comma; 448-452.



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