Department of
Cell Biology and Anatomy
Outline and Research Objectives
When our institution was reformed from School of Medicine to Graduate School of
Medicine, Department of Cell Biology and Anatomy has been built with the transition fromthe previous department of Anatomy. Therefore, directions of researches in this department
are molecular cell biology based on structural cell biology.
At the same time this department has high responsibility for education of medical students, students of other faculties, and master course and Ph.D. course students (totally 518
hours per year). The teaching covers lectures and laboratory courses of gross anatomy, neuroanatomy, histology, cell biology and developmental biolgy.
Main subjects of researches are focused on 1) the mechanisms of intracellular transport,
2) the mechanism of cell morphogenesis, and 3) roles of cytoskeleton on development.
To solve these problems we combine structural biological approaches such as various
kinds of light microscopy, immunocytochemistry, quick freeze deep etch electron
microscopy, cryoelectron microscopy, X-ray crystallography with molecular biology, biochemistry, molecular biophysics, electrophysiology and molecular genetics.

Faculties and Students
Professor and Chair

Nobutaka Hirokawa, M. D., Ph.D.
(from 1983~)
Associate Professor Yoshimitsu Kanai, M. D., Ph. D.
Takao Nakata, M. D., Ph. D.
Lecturer
Yasuko Noda, M. D., Ph. D.
Sumio Terada, M. D., Ph. D.
Associate .................................8
Postdoctoral Fellow ................2 (Japan),1 (France),.................................................1 (China)
Graduate Students ..................7 (Japan), 2 (China)
Research Student ....................3 (China), 1 (Korea)
Secretary & Technician ...........4

Past Research, Current Research and Major
Accomplishments
I. Rapid Freeze Deep Etch Electron Microscopy
and Identification of New Groups of Crossbridge
Structures Among Microtubules, Intermediate
Filaments, Actin and Membranous Organelles.
Hirokawa has developed a new microscopic
method which rapidly freeze cells and tissues by making contact with a pure copper block cooled with liquid nitrogen (-190 C) or helium (-269 C) in Japan. He
further developed this method in USA with Dr. John
Heuser and visualised as yet unknown aspects of specialized membrane structures such as Gap Junction,
Tight Junction, and Neuromuscular Junctions. Further
he discovered a group of new filamentous crossbridges among microtubules, intermediate filaments,

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actin filalments and membranous organelles.
Combining immunocytochemistry, biochemistry, andin vitro reconstitution he proved the chemical natureof these structures as 1) fodrin and myosin in brushborders of intestinal cells, a model of cellular cortex,
2) MAP1A, MAP1B, MAP2 and Tau in microtubule
domains in neuronal axon and dendrites, 3) elongated
C-terminus of Neurofilament M and H proteins inneurofilament domains in neuronal axons, 4)Synapsin I associated with actin and synaptic vesicles
in presynaptic terminals.
II. Discovery of Kinesin Superfamily Proteins,
KIFs and Elucidation of Mechanism ofIntracellular Transport.
Hirokawa discovered various kinds of new filamentous structures between distinct kinds of membranous organelles and microtubules. He predicted
these short crossbridges to be microtubule associatedmotor proteins carrying cargo vesicles along the
microtubules.
The nerve axon is frequently very long (frequently~1m long). Because of lack of protein synthesis
machinery in the axon most of the proteins necessary
in the axon and synaptic terminals ought to be transported down the axon as various kinds of membranous organelles and protein complexes after the synthesis in the cell body so that intracellular transport isfundamental for neuronal morphogenesis and functioning. At the same time because similar mechanism
exists in every kinds of cells nerve cells serve as agood model system to elucidate this mechanism.
Using molecular biology we discovered most ofkinesin superfamily motor proteins, KIFs which movealong microtubule rails by hydrolysing ATP. Recently
we identified all 45 kif genes in mammalian such as
human and mouse. We have uncovered the structure,dynamics and functions of many members of KIFs
usig molecular cell biological, biophysical, structural
biological and molecular genetical approaches.
KIFs in Axonal Transport
KIF1A and KIF1Bβ transport synaptic vesicle precursors anterogradely from cell biology to synaptic
terminals. They are fundamental for neuronal functioning and survivals. Further, we showed using
molecular genetic approaches that KIF1Bβ is a responsible gene of human hereditary neuropathy CharcotMarie-Tooth type 2A and proved that CMT2A is due
to haploinsufficiency of functional KIF1Bβ anddecrease of synaptic vesicle protein transport.
KIF1Bβ+/- mice serve as a model for CMT2A and
we found a way of diagnosis and potential therapy ofthe symptom of this desease. KIF1B a and KIF5A, B, C
redundantly convey mitochondria anterogradely.
KIF3, composed of KIF3A and KIF3B heterdimer andassociated protein KAP3, transports vesicles associated with a fodrin and important for neurites elongation through KAP3-α fodrin interaction. KIF2 andKIF4 are expressed specifically in juvenile neurons
and functioning for neurite extension.
Receptor Transport in Dendrites
Our studies revealed the mechanism of transportof receptors in dendrites.
KIF17 transports NMDA type glutamate receptor
containing vesicles in dendrites toward microtubule
plus ends through interaction between KIF17 C-terminal tail-Mint 1 (mLin10)- CASK (mLin2)- Velis (mLin7)
and NR2B subunit of NMDA receptor. Furthermore,
we showed by transgenic mouse strategy that KIF17
plays a significant role on memory and learning.
KIF5A, B, C convey AMPA type glutamate receptor
containing vesicles towards microtubule plus end
through the interaction between KIF5 heavy chain GRIP1 (glutamate receptor interacting protein 1)-GluR
2 subunit of AMPA type glutamate receptors.
These studies elucidated also for the first timelong standing questions how KIFs recognize and bindto cargoes by showing KIF tail - scaffolding protein
complex or adaptor protein complex- membrane protein interaction is a typical way. This idea was supported by our other study revealing that KIF13A
transports Mannose 6- phosphate Receptor containing
vesicles from Golgi to plasma mambrane through theinteraction between KIF13A tail - AP-1 adaptor protein complex and Mannose 6 phosphate receptors.
Our study about the transport of AMPA type receptor
by KIF5 also elucidated a mechanism how motor pro-tein determines its direction, axon vs dendrites.
GRIP1 steers KIF5 to dendrites via KIF5 heavy chain GRIP1 interaction, while JSAP1 steers KIF5 to axon
via KIF5 light chain- JSAP1 interaction.
KIF3 Determines Left-Right Asymmetery of OurBody
Our gene targeting study of KIF3 (KIF3A-/-, andKIF3B-/-) siginificantly contributued for the elucidation of mechanism of determination of left-rightasymmetry of our body, a very important hot problem
in developmental biology. Our study uncovered thatKIF3 motor is essential for left-right determination of
our body through intraciliary transportation of protein complexes for the ciliogenesis of motile primarycilia that generate leftward unidirectional flow of
extraembryonic fluid, "nodal flow", which could produce a concentration gradient of putative secreted
morphogen in the extraembryonic fluid along the leftright axis in the node of early embryo, very importantregion for the determination of our body plan.
Mechanism of Motility of Monomeric Motor,
KIF1A
We have discovered monomeric motors KIF1 subfamily such as KIF1A, KIF1B α and β. Because motors
previously identified are all dimers such as conventional kinesin (KIF5), dyneins and myosin the prevailing hypothesis for how the motor moves processively
for certain distance along rails was the hand overhand model, which means a motor needs two legs tomove as we walk on the rail. We, however, discovered
monomeric motor such as KIF1A. Because themonomeric motor is the simplest motor it is a goodmodel system to study the basic mechanism of motility of KIFs. We showed using molecular biophysics
approach that a single KIF1A motor can move processively for more than 1µm on microtubules.
Furthermore, our study revealed that the processive
movement of KIF1A is based on the biased Brownian
movement. This is the first clear experimental
demonstration showing that motor protein can moveby the biased Brownian movement. We further elucidated why single leg motor can move processively
along microtubules without dissociation. We foundpolylysin loop (K-loop) in loop12 and showed that
this K-loop is critical for processive movement
through the interaction with C-terminal flexible endof tubulin (E-hook) especially at the weak binding
state (ADP state) by biophysics, and cryoelectron
microscopy.
We further analysed how the plus end bias can be
emerged by combination of cryoelectron microscopy
and X-ray crystallography. We found that counterclock wise 20 degree rotation of motor domain from
ADP to ATP like state is critical for plus end biased

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KIF review(Hirokawa 1998)

KIF17 transports NMDA
receptors in dendrites(Setou et al. 2000)

Kinesin structure(Hirokawa et al. 1989)

Photobleaching recovery
study of fluorescenttubulin (Okabe et al. 1990)

KIF1Atransports synaptic
vesicle precursor(Okada et al. 1995)

Tau induced microtubule
bundles and processelongation (Kanai et al. 1989)

KIF1B is responsible geneof hereditary neuropathy(Zhao et al., 2001)

Tau/MAP1B double
knockout mice
(Takei et al. 2000)

Cryo EM study of KIF1A motor
KIF1A monomer moves domain-microtubule complex(Kikkawa et al. Cell 2000)
processively by biased
Brownian movement(Okada &Hirokawa Science 1999)
KIFs transport various kinds ofcargoes in axon and dendrites

KIFs recognize and bind functional membraneproteins, through KIF
tail-scaffolding protein/adaptor protein complex interaction (Setou et al.
Science 2000, Nakagawa et al. Cell 2001, Setou et al. Nature 2002)

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X-ray crystallography and cryoEM studyof monomeric KIF1A motor domain(Kikkawa et al. Nature 2001)

movement of KIF1A monomeric motor.
Mechanism of Slow Axonal Transport
Cytoskeletal proteins such as tubulin, actin andneurofilament triplet proteins (NFH, NFM, NFL) andsome cytosolic proteins were known to be transported down the axon slowly at 1~2mm/day. Using 1)
microinjection of fluorescein labelled cytoskeletal proteins and subsequent analysis by fluorescent photobleach recovery, 2) microinjection of caged-fluoresceinlabelled cytoskeletal proteins and subsequent analysis
by UV-photoactivation in cultured neurons weshowed most of cytoskeletal proteins such as tubulin
and actin are transported as small oligomers. Further,recently using microinjection of fluorescein labelledtubulin and cytosolic proteins into squid giant axons
and subsequent analysis with fluorescence confocal
laser scanning microscopy and fluorescence correlation spectroscopy combined with microinjection ofanti KIF antibodies we demonstrated that tubulin istransported as small oligomers by conventional
kinesin (KIF5) as a motor.
III. Microtubule Associated Proteins (MAPs) andMechanism of Neuronal Morphogenesis.
Hirokawa identified various kinds of filamentous
structures associated with microtubules by the quick
freeze deep etch electron microscopy. The next questions were what are the chemical nature and what are
the functions of these new structures. The microtubule associated proteins (MAPs) identified in brains
were good candidates. Therefore, we have studied
MAPs. We biochemically isolated major MAPs ofmammalian brains such as MAP1A, MAP1B, MAP2
and tau, and studied molecular structure and localization. All these MAPs were filamentous flexible strutures from 185 to 50nm in length dependent on the
difference of molecular weight. Immunocytochemistry
and in vitro reconstitution comfirmed that these
MAPs are components of filamentous structures associated with microtubules in neurons, To know thefunction of MAPs, we expressed tau and MAP2 in
non neuronal cells such as fibroblasts and Sf9 cells by
cDNA transfection. These studies showed that tau
and MAP2 induce microtubule polymerization andbundling and process extention. They also revealed
that C-terminal domain are critical for microtubule
polymerization and N-terminal projection domain
determines the different spacings between adjacentmicrotubules in microtubule domains in axon vs dendrites. These data suggest the important role of tau
and MAP2 in formation of axon and dendrites. In
order to elucidate function of MAPs in vivo we generated single knockout mice of tau, MAP1B, and MAP2
and analysed them. Generally, the phenotypes of single knockout mice were subtle. However, tau/MAP1B

and MAP2/MAP1B double knockout mice showed
predominant disturbance of axonal elongation anddendritic elongation respectively. Cellular bases ofthese phenotypes are suppression of microtubule stability and bundle formation in growth cones of axonand dendrites leading to the inhibition of proper neurite extention and neuronal cell migration. Thus, ourstudies revealed Tau/MAP1B and MAP2/MAP1B synergistically play fundamental roles in axonal and dendritic formation respectively.

Future Prospects
The further studies in near future will include following subjects as the continuation of on going
researches.
I. Mechanism of Intracellular Transport
1) The functions of new KIFs especially in neurons,
epithelial cells and fibroblasts to elucidate yet
unknown mechanism of intracellular transport ofvarious kinds of membranous organelles, protein
complexes and mRNA.
2) Understanding how each KIFs recognize and bindtheir specific cargoes.
3) The studies how the binding and unbinding of KIFs
with cargoes are regulated.
4) The mechanism how neurons determine the direction of transport by KIFs, toward axon vs dendrites.
5) The study of relationship between KIFs and neuronal functions such as memory and learning by
molecular genetic approach.
6) The relationship between KIFs and diseases using
molecular genetical approaches.
7) The detailed mechanism of motility of KIF1A motorusing structural biology and molecular biophysics.

II. Mechanism of Neuronal Morphophogenesis
and MAPs
Using molecular genetical approaches we will
unravel the function of MAP1A, one member of major
MAPs whose function has been unknown.

Research Grants
1. Center of Excellence (COE) grant from the Ministryof Education, Culture, Science and Sports, "Molecular
Cell Biological and Molecular Genetical Study of theCytoskeleton: Mechanism of Intracellular Transport,
Signal Transmission and Cellular Morphogenesis."
From April 1996 to March 2001. 1,885,000,000 yen
2. Grant in Aid for Basic Research, "Molecular Cell
Biology of New Molecular Motors, KIFs," from the
Ministry of Education, Culture, Science and Sports.
From April 1996 to Mach 1999. 34,500,000 yen
3. Center of Excellence (COE) grant from the Ministryof Education, Culture, Science, Sports andTechnology, "Mechanism of Intracellular Transport

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:Molecular Cel Biology, Structure Biology andMolecular Genetics."From April 2001 to March 2006.
2,020,000,000 yen

Publication list
1. Hirokawa N and Kirino T. An ultrastructural study ofnerve and glia cells by freeze-substitution. Journal of
Neurocytology 9, 243-254, 1980.
2. Hirokawa N. Crosslinker system between neurofilaments, microtubules and membranous organelles infrog axons revealed by quick freeze, freeze fracture,
deep etching method. Journal of Cell Biology 94, 129142, 1982.
3. Hirokawa N, and Heuser J. E. The inside and outsideof gap junction membranes visualized by deep etching. Cell 30, 395-406, 1982.
4. Hirokawa N, Cheney R, and Willard M B. Location ofa protein of the fodrin-spectrin-TW 260/240 family
in the mouse intestinal brush border. Cell 32, 953965, 1983.
5. Hirokawa N, M. A. Glicksman, and M. B. Willard.
Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. Journal of
Cell Biology 98, 1523-1536, 1984.
6. Shiomura Y and HirokawaN. The molecular structureof microtubule-associated protein 1A (MAP1A) invivo and in vitro. An immunoelectron microscopy
and quick-freeze, deep-etch study. Journal of
Neuroscience 7, 1461-1469, 1987.
7. Hirokawa N, Hisanaga S and Shiomura Y. MAP2 is acomponent of crossbridges between microtubules
and neurofilaments in vivo and in vitro. Quickfreeze, deep etch immunoelectron microscopy andreconstitutio studies. Journal of Neuroscience 8,2769-2779, 1988.
8. Hirokawa N, Pfister K K, Yorifuji H, Wagner MC,
Brady S T and Bloom GS. Submolecular domains ofbovine brain kinesin identified by electron
microscopy and monoclonal antibody decoration.
Cell 56, 867-878, 1989.
9. Kanai Y, Takemura R, Ohshima T, Mori H, Ihara Y,
Yanagisawa M, Masaki T and Hirokawa N.
Expression of multiple tau isoforms and microtubule
bundle formation in fibroblasts transfected with asingle tau cDNA. Journal of Cell Biology 109, 11731184, 1989.
10. Sato-Yoshitake R, Shiomura Y, Miyasaka H and
Hirokawa N. Microtubule-associated protein
1B:Molecular structure,localization, and its phosphorylation-dependent expression in developing neurons. Neuron 3, 229-238, 1989.
11. Okabe S and Hirokawa N. Turnover of fluorescently
labeled tubulin and actin in the axon. Nature 343,
479-482, 1990.
12. Hirokawa N, Yoshida T, Sato-Yoshitake R, andKawashima T. Brain dynein (MAP1C) localizes onboth anterogradely and retrogradely transported
membranous organelles. Journal of Cell Biology 111,
1027-1037, 1990.

12

13. Hirokawa N, Sato-Yoshitake R, Kobayashi N, Pfister
KK, Bloom GH, and Brady ST. Kinesin associateswith anterogradely transported membranousorganelles in vivo. Journal of Cell Biology 114, 295302, 1991.
14. Aizawa H, Sekine Y, Takemura R, Zhang Z, Nangaku
M and Hirokawa N. Kinesin family in murine central
nervous system. Journal of Cell Biology 119, 12871296, 1992.
15. Chen J, Kanai Y, Cowan NJ and Hirokawa N.
Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons.
Nature 360, 674-677, 1992.
16. Hirokawa N. Microtubule organization and dynamics
dependent on microtubule-associated proteins.
Current Opinion in Cell Biology 6, 74-82, 1994.
17. Harada A, Oguchi K, Okabe S, Kuno J, Terada S,
Ohshima T, Sato-Yoshitake R, Takei Y, Noda T and
Hirokawa N. Altered microtubule organization insmall-calibre axons of mice lacking tau protein.
Nature 369, 488-491, 1994.
18. Kondo S, Sato-Yoshitake R, Noda Y, Aizawa H,
Nakata T, Matsuura Y and Hirokawa N. KIF3A is a
new microtubules-based anterograde motor in thenerve axon. Journal of Cell Biology 125, 1095-1107,
1994.
19. Sekine Y, Okada Y, Kondo S, Aizawa H, Takemura R
and Hirokawa N. A novel microtubule-based motor
protein (KIF4) for organelle transports, whose
expression is regulated developmentally. Journal of
Cell Biology 127, 187-202, 1994.
20. Nangaku M, Sato-Yoshitake R, Okada Y, Noda Y,
Takemura R, Yamazaki H, and Hirokawa N. KIF1B; A
novel microtubule plus end-directed monomericmotor protein for transport of mitochondria. Cell 79,
1209-1220, 1994.
21. Noda Y, Sato-Yoshitake R, Kondo S, Nangaku M and
Hirokawa N. KIF2 is a new microtubule-based
anterograde motor that transports membranousorganelles distinct from those carried by kinesin
heavy chain or KIF3A/B. Jounal of Cell Biology 129,
157-167, 1995.
22. Okada Y, Yamazaki H, Sekine Y, and Hirokawa N.
The neuron-specific kinesin superfamily protein
KIF1A is a unique monomeric motor for anterograde
axonal transport of synaptic vesicle precursors. Cell81, 769-780, 1995.
23. Kikkawa M, Ishikawa T, Wakabayashi T and
Hirokawa N. Three-dimensional structure of thekinesin head-microtubule complex. Nature 376, 274276. 1995.
24. Yamazaki H, Nakata T, Okada Y, and Hirokawa N.
KIF3A/B: A heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed
motor for membrane organelle transport. Journal of
Cell Biology 130, 1387-1399, 1995.
25. Hirokawa N. Organelle transport along microtubulesthe role of KIFs (Kinesin superfamily proteins).
Trends in Cell Biology 6, 135-141, 1996.

26. Terada S, Nakata T, Peterson A and Hirokawa N.
Visualization of slow axonal transport in vivo.
Science 273, 784-788, 1996.
27. Saito N, Okada Y, Noda Y, Kinoshita Y, Kondo S andHirokawa N. KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for dendritictransport of multivesicular body-like organelles.
Neuron 18, 425-438, 1997.
28. Takei Y, Kondo S, Harada A, Inomata S, Noda T and
Hirokawa N. Delayed development of nervous system in mice homozygous for disrupted microtubuleassociated protein 1B (MAP1B) gene. Journal of Cell
Biology 137, 1615-1626, 1997.
29. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport.
Science 279, 519-526, 1998.
30. Nakata T, Terada S and Hirokawa N. Visualization of
the dynamics of synaptic vesicle and plasma membrane proteins in living axons. Journal of Cell Biology
140, 659-674, 1998.
31. Yonekawa Y, Harada A, Okada Y, Kanai Y, Takei Y,
Funakoshi T, Terada S, Noda T and Hirokawa N.
Defect in synaptic vesicle precursor transport andneuronal cell death in KIF1A motor protein-deficientmice. Journal of Cell Biology 141, 431-441, 1998.
32. Tanaka Y, Kanai Y, Okada Y, Nonaka S, Takeda S,
Harada A and Hirokawa N. Targeted disruption ofmouse conventional kinesin heavy chain, KIF5B,
results in abnormal perinuclear clustering of mitochondria. Cell 93, 1147-1158, 1998.
33. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A,
Kanai Y, Kido M and Hirokawa N. Randamization ofleft-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in micelacking KIF3B motor protein. Cell 95, 829-837, 1998.
34. Okada Y and Hirokawa N. A processive single-headed motor: kinesin superfamily protein, KIF1A.
Science 283, 1152-1157, 1999.
35. Takeda S, Yonekawa Y, Tanaka Y, Okada Y, Nonaka
S and Hirokawa N. Left-right asymmetry and kinesin
superfamily protein KIF3A: New insights in determination of laterality and mesoderm induction by
Kif3A-/- mice analyses. Journal of Cell Biology 145,
825-836, 1999.
36. Okada Y, Nonaka S, Tanaka Y, Saijoh Y, Hamada H
and Hirokawa N. Abnormal nodal flow precedes
situs inversus in iv and inv mice. Molecular Cell 4,459-468, 1999.
37. Okada Y and Hirokawa N. Mechanism of the singleheaded processivity : Diffusional anchoring between"k-loop" of kinesin and the C-terminus of tubulin.
Proceedings of the National Academy of Science of
the USA 97, 640-645, 2000.
38. Kikkawa M, Okada Y and Hirokawa N. 15Å resolution model of the monomeric kinesin motor: KIF1A.
Cell 100, 241-252, 2000.
39. Takeda S, Yamazaki H, Seog D-H, Kanai Y, Terada S
and Hirokawa N. KIF3 motor transports fodrin-associating vesicles important for neurite building.
Journal of Cell Biology 148, 1255-1266, 2000.

40. Setou M, Nakagawa T, Seog D-H and Hirokawa N.
Kinesin superfamily motor protein KIF17 and mLin10 in NMDA receptor-containing vesicle transport.
Science 288, 1796-1802, 2000.
41. Takei Y, Teng J, Harada A and Hirokawa N. Defects
in axonal elongation and neuronal migration in micewith disrupted tau and map1b genes. Journal of Cell
Biology 150, 989-1000, 2000.
42. Terada S, Kinjo M and Hirokawa N. Oligomeric tubulin in large transporting complex is transported via
kinesin in squid giant axons. Cell 103, 141-155,
2000.
43. Nakagawa T, Setou M, Seog D-H, Ogasawara K,
Dohmae N, Takio K and Hirokawa N. A novel
kinesin superfamily motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane
through direct interaction with AP-1 complex. Cell
10, 569-581, 2000.
44. Kikkawa M, Sablin EP, Okada Y, Yajima H, Fletterick
RJ and Hirokawa N. Switch-based mechanism ofkinesin motors. Nature 411, 439 - 445, 2001.
45. Zhao C, Takita J, Tanaka Y, Setou M, Nakagawa T,
Takeda S, Hong W-Y, Terada S, Nakata T, Takei Y,
Saito M, Tsuji S, Hayashi Y and Hirokawa N.
Charcot-Marie-Tooth Disease type 2A caused bymutation in a microtubule motor KIF1Bβ. Cell 105,587-597, 2001.
46. Miki H, Setou M, Kanashiro K and Hirokawa N. All
kinesin superfamily protein, KIF, genes in mouseand human. Proceedings of the National Academy of
Science of the USA 98, 7004-7011, 2001.
47. Teng J, Takei Y, Harada A, Nakata T, Chen J and
Hirokawa N. Synergistic effects of MAP2 and
MAP1B knockout in neuronal migration, dendritic
outgrowth, and microtubule organization. Journal of
Cell Biology 15, 65-76, 2001.
48. Noda Y, Okada Y, Saito N, Setou M, Xu Y, Zhang Z
and Hirokawa N. KIFC3, a microtubule minus enddirected motor for the apical transport of annexin
XIIIb-associated Triton-insoluble membranes.
Journal of Cell Biology 155, 77-88, 2001.
49. Setou M, Seog D-H, Tanaka Y, Kanai Y, Takei Y,
Kawagishi M and Hirokawa N. Glutamate-receptorinteracting protein GRIP1 directly steers kinesin to
dendrites. Nature 417, 83-87, 2002.
50. Harada A, Teng J, Takei Y, Oguchi K and Hirokawa
N MAP2 is required for dendrite elongation, PKA
anchoring in dendrites, and proper PKA signal transduction. Journal of Cell Biology 15, 541-549, 2002.

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Department of
Molecular Biology
Outline and Research Objectives
This department was established in 1893 as the first Biochemistry Department in Japan.
Since then, this department has been devoted to biochemical studies on vitamin, oxidative
phosphorylation, lipid, carbohydrate and nucleic acid metabolisms, making remarkable contributions to advancements in medical biochemistry. After H. Okayama succeeded Chair in
1992, research has been focused on cancer, the cell cycle and differentiation in order tounderstand the molecular mechanism of malignant transformation and growth control.

Faculties and Students
Professor and Chair Hiroto Okayama, MD & PhD (1992~)
Associate .................................3
Postdoctoral Fellow ................1
Graduate Student....................5
Research Student ....................5
Secretary .................................2

Past Research and Major Accomplishments
Development of high-efficiency full-length cDNA
cloning method and eukaryotic expression cDNA
cloning system
Most of our past and current research stemmed
from the development of the full-length cDNA
cloning method and the cDNA expression cloning
vector system that was done in the early 1980s by
Hiroto Okayama and Paul Berg (Fig. 1)(1,2).
The full-length cDNA cloning method and thecDNA expression libraries constructed thereby havebeen used to clone numerous functional genes in laboratories over the world, the earliest one of which is
the human Lesch-Nyhan syndrome causative gene (3).
The cDNA expression cloning vector system was originally designed to clone genes on the basis of the

14

functions they express in a wide range of host cells,
from fission yeast up to mammalian cells. This wasenabled by development of highly efficient methods
for introducing cDNA libraries into mammalian cells
and fission yeast and construction of vectors specialized for library transduction into fission yeast (Fig. 1)(8, 10, 11). One of the most successful applications of
this expression cloning system was isolation ofhuman orthologues of several key cell cycle regulators
initially found in fission yeast. Paul Nurse cloned thehuman cdc2 cDNA from one of our expression
libraries on the basis of trans-complementation of afission yeast temperature-sensitive mutant of cdc2+
essential for mitosis, and we cloned human orthologues of wee1+ and cdc25+ genes (13, 14), which critically regulate Cdc2 kinase during this transition, as
described below.

Cell cycle control
Cdc2 kinase is a protein kinase required for the
onset of mitosis, initially found in fission yeast, but
present in all eukaryotes. This kinase, associated withcyclins, is regulated by inhibitory tyrosine phosphorylation and activating dephosphorylation by Wee1
kinase and Cdc25 phosphatase, constituting a coreelement of DNA damage-responsive cell cycle checkpoint control for the G2-M transition in fission yeast.

The identification of human orthologues (cdc2, wee1
and cdc25B) of these fission yeast cell cycle regulators
demonstrated the evolutionary conservation of a cell
cycle control system up to mammals (13, 14, 30).
Using the same expression cloning system, we also
identified an additional mammalian homologue(cdc25A) of cdc25+ gene, which was found to play an
unexpected role (23).
This gene is expressed in early G1 and required forthe cell cycle start. Search for targets for this novel
phosphatase led us to discover that Cdk4, a cyclindependent kinase essential for the cell cycle start, isregulated by tyrosine phosphorylation and that its
regulation constitutes a key part of DNA damageinduced G1 checkpoint arrest during cell cycle entry
from quiescence (23, 28, 37), revealing the presence of
mirror image mechanisms for controlling DNA damage-responsive G1 and G2 checkpoint arrest in mammals (Fig. 2).
In addition to the studies on mammalian cell cycle
control, we identified genes for 11 fission yeast novel
cell cycle factors and cyclin G a novel mammalian
cyclin (22), which control the G1-S transition, DNAreplication checkpoint or the formation and maintenance of sister chromatid cohesion (20, 24-27, 29, 33,
35, 40, 42-44, 48). Recent studies on two newly identified factors Eso1 and Pds5 involved in the formationof sister chromatid cohesion led us to discover thatsister chromatid cohesion is formed and maintainedby a highly unexpected mechanism, which functions
as a molecular zipper and thereby promotes the formation of cohesions only between sister chromatids(48). This finding provided a new clue to the understanding of the mechanism for the genetic instability
associated with malignant transformation.

Differentiation control
One of the aspects of the cell we have been interested in understanding concerning carcinogenesis isdifferentiation control. To this end, we also used fission yeast as a model organism and discovered fivenovel genes controlling the commitment to differenti-

ation. They encode Esc1 a MyoD-like helix-loop-helix
protein, Nrd1 an RNA-binding protein, Phh1 a stress
MAP kinase similar to p38, Rcd1 an evolutionally conserved protein and Pas1 and Cyc17 novel cyclins (21.
26, 31, 33, 36, 43, 44). These proteins were found to
be a modifier of cyclin AMP signaling, a determinantof the threshold to nutrient starvation for the commitment to differentiation, a stress signal transducer
as an absolute requirement for the differentiationcommitment, a key mediator of nitrogen starvation
signal and factors that ensure mutual exclusivenessbetween the cell cycle start and the commitment todifferentiation. In addition, using trans-complementation cloning, we isolated a human functional counterpart of the RNA-binding protein with predicted functions, which were shown in hematopoietic cells (38).
Furthermore, we recently found that the mammalian counterpart of Rcd1 is a novel transcriptional
cofactor mediating retinoic acid-induced cell differentiation and is deeply involved in mouse lung development (50). These findings not only reveal remarkableconservation of the differentiation commitment system as well as the key elements that control the commitment step from yeast up to mammals (Fig. 3), butalso greatly help understand the highly complex
mechanism controlling the differentiation commitment in mammals.

Anchorage-independent cell cycle start: a key
mechanism for malignant transformation
One of the fundamental phenotypes that distinguish malignant from benign cells is the metastatic
capability, which is underlain by the acquirement of
the ability to perform S phase entry in the absence ofanchorage. In the late 1980s, we began studies tounderstand the nature and mechanism of the acquirement of this unique property upon malignant transformation, by using growth factor-transformable NRK
cells as a model system. The generally held concept
was that anchorage-independent S phase entry is amere consequence of excessive activation of growthsignaling or defects in a growth arrest system, which15

are caused by activation of oncogenes and inactivationof recessive oncogenes. For the past 10 years, westudied genetically and cytologically the properties of
oncogenically stimulated NRK cells and came to theseveral remarkable conclusions: 1) In NRK cells, epidermal growth factor and platelet-derived growth factor signals merge into a common pathway composedof CrkII, Ras and Raf-1, through which many oncogenes induce transformation; 2) Activation of thispathway induces anchorage-independent S phaseentry in a synchronized fashion, but is dispensable forcell proliferation in the presence of anchorage; 3)
Oncogenic stimulation recruits Cdk6 to participate inan essential step of anchorage-independent S phase
onset; 4) Activation of G1 cyclin-dependent kinases(Cdk4/6, Cdk2) is insufficient to induce anchorageindependent S phase onset (15, 16, 32, 39). Recently,
we found that Cdc6, a key protein for the activationof replication origins, requires anchorage or oncogenic
stimulation for its expression, which is regulated at
the levels of transcription and proteolysis via a calpain-like protease, and moreover, that G 1 cyclindependent kinases and Cdc6 constitute key targets forcontrolling the G1-S transition by anchorage and oncogenic signals (Fig. 4)(49).
We believe that this latest finding would provide a16major breakthrough toward the understanding of themechanisms for anchorage loss-invoked restriction of
S phase onset and oncogenically induced anchorageindependent S phase onset, the latter being responsible for the metastatic capability of malignant cells. In
addition, all these findings taken together raise thepossibility that oncogenic transformation is not merely resulted from a failure to arrest due to excessive
growth stimulation or defective cell cycle arrest systems, but from constitutive activation of a dormant
mechanism that is present in normal cells and enables
the cell cycle start in the absence of anchorage (46).

Current Research
We are currently devoting most of our efforts tothe understanding of how Cdc6 expression is regulated by anchorage and oncogenic signals.

Requirement for PI3 kinase in Cdc6 expression
It has been known that phosphoinositol-3 (PI3)kinase is required for the cell cycle start and one of its
targets is p27 cyclin-dependent kinase inhibitor. We
recently found that entirely independent from p27
downregulation, Cdc6 requires PI3 kinase for its
expression during anchorage-dependent, but notanchorage-independent, S phase entry. Treatment ofcells with a specific PI3 kinase inhibitor shuts off
Cdc6 expression despite E2F activation, but interestingly this shutoff is overridden by oncogenic stimulation. Further analysis is underway to identify how PI3
kinase and anchorage signal cooperate to express
Cdc6.

Identification of the protease responsible for theCdc6 degradation and its regulation
The most crucial point in understanding the regulatory mechanism of Cdc6 expression by anchorageand oncogenic stimulation is identification of the
responsible protease and its regulatory mechanism(Fig.4). To this end, a human cdc6 cDNA was isolated,tagged with a histidine tag and transcribed in vitro
and expressed in reticulocyte lysates, and theexpressed His-tagged Cdc6 protein was purified byaffinity chromatography in order to establish an invitro assay system. Analysis of the nature of the putative protease suggests that the cells arrested in G1 byanchorage loss contain the proteolytic activity that
seems to be responsible for Cdc6 degradation.
Analysis of the nature of this protease and its regulation is currently in progress.
Cdk6-cyclin D3 as a highly potent sensitizer ofcells to chemical and physical transformation
We recently found that among D-type cyclin (D1,
D2, D3) and partner kinase (Cdk4, Cdk6) combinations, the Cdk6-D3 complex is unique and can evades
inhibition by p27 and p21, consequently enabling thiscomplex to control proliferation competence under
growth suppressive conditions (47). This finding led
us to investigate the possible effects of the elevatedexpression of this complex on cell’s susceptibility tomalignant transformation, and we have found that 25 fold overexpression of Cdk6-D3 elevates 103-106
folds the susceptibility of rodent fibroblasts to UV
irradiation- or 3-methylcholanthrene-induced malignant transformation. Analysis is in progress to understand this sensitization mechanism.

Future Prospects
There are three key questions regarding malignant
transformation: 1) What is the mechanism by whichtransformed cells acquire the ability to performanchorage-independent S phase onset as a basis formetastatic capability?; 2) What causes the genetic
instability of cancer, which is believed to be a driving
force for malignant progression and what are the key
cell cycle factors activated or inactivated during thisprocess?; 3) What is the mechanism for dedifferentiation of malignant cells? We believe that the series ofour research will certainly offer critical clues to solvethese questions. We particularly believe that the finding that Cdc6, a key factor for the activation of replication origins, absolutely requires anchorage or oncogenic stimulation for its expression is a major breakthrough to understanding the central mechanism of
oncogenically induced anchorage-independent S
phase onset.

Research Grants
1. H. Okayama, Grant-in-Aid for Scientific Research (A),
JSPS, 1992-present. ¥10,000,000 a year.
2. H. Okayama, Grant-in-Aid for Scientific Research on
Priority Areas “Advanced Cancer Research”, MEXT,
1992-1999. ¥32,000,000 a year.
3. H. Okayama, Grant-in-Aid for Scientific Research on
Priority Areas “Cell Cycle Control”, MEXT, 2000-present. ¥60,000,000 a year.

Select Publications
1. Okayama, H., and Berg, P. High efficiency cloning offull length cDNA. Mol. Cell. Biol. 2, 161-170, 1982.
2. Okayama, H., and Berg, P. A cDNA cloning vector
that permits expression of cDNA inserts in mammalian cells. Mol. Cell. Biol. 3, 280-289, 1983.
3. Jolly, D. J., Okayama, H., Berg, P., Esty, A. C., Filpula,D., Bohlen, P., Johnson, G., and Friedman, T.
Isolation and characterization of a full length,
expressible cDNA for human hypoxanthine guanine
phosphoribosyl transferase. Proc. Natl. Acad. Sci.
USA 80, 477-481, 1983.
4. Chin, D. J., Gil, G., Russell, D. W., Liscum, L.,
Luskey, K., Basu, S., Okayama, H., Berg, P.,
Goldstein, J. L., and Brown, M. Nucleotide sequenceof 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, a glycoprotein of endoplasmic reticulum. Nature
308, 613-617, 1984.
5. Yang, Y-C., Okayama, H., and Howley, P. Bovine
papillomavirus contains multiple transforming genes.
Proc. Natl. Acad. Sci. USA 82, 1030-1034, 1985.
6. Okayama, H., Kawaichi, M., Brownstein, M., Lee, F.,
Yokota, T., and Arai, K. High-efficiency cloning offull length cDNA; Construction and screening of
cDNA expression libraries for mammalian cells.
Methods Enz. 154, 3-28, 1987.
7. Chen, C., and Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol.
Cell. Biol. 7, 2745-2752, 1987.
8. Okayama, H., and Chen, C. Calcium phosphate mediated gene transfer into established cell lines.
Methods Mol. Biol. pp. 15-21, 1990.
9. Tanaka, K., Miura, N., Satokata, I., Miyamoto, I.,
Yoshida, M. Sato, Y., Kondo, S., Yasui, A., Okayama,
H., and Okada, Y. A cDNA sequence of xeroderma
pigmentosum group A gene encoding a protein withzinc finger structure. Nature 348, 73-76, 1990.

17

10. Inoue, H., Nojima, H., and Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23-28, 1990.
11. Okazaki, K., Okazaki, N., Kume, K., Jinno, S.,
Tanaka, K., and Okayama. H. High-frequency transformation method and library transducing vectors
for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nuc. Acids
Res. 18, 6485-6489, 1990.
12. Takamizawa, A., Mori, C., Fuke, I., Manabe, S.,
Murakami, S., Fujita, J., Onishi, E., Andoh, T.,
Yoshida, I., and Okayama, H. Structure and organization of the hepatitis C virus genome isolated fromhuman carriers. J. Virol. 65, 1105-1113, 1991.
13. Nagata, A., Igarashi, M., Jinno, S., Suto, K., andOkayama, H. An additional homolog of the fission
yeast cdc25+ gene occurs in human and is highly
expressed in some cancer cells. New Biologist 3, 959968, 1991.
14. Igarashi, M., Nagata, A., Jinno, S., Suto, K., andOkayama, H. Wee1+-like gene in human cells Nature
353, 80-83, 1991.
15. Masuda, A., Kizaka-Kondoh, S., Miwatani, H.,
Terada, Y., Nojima, H., and Okayama, H. Signaltransduction cascade shared by epidermal growth
factor and platelet-derived growth factor is a major
pathway for oncogenic transformation in NRK cells.
New Biologist 4, 489-503, 1992.
16. Kume, K., Jinno, S., Miwatani, H., Kizaka-Kondoh, S.,
Terada, Y., Nojima, H., and Okayama, H. Oncogenic
signal-induced ability to enter S phase in the absenceof anchorage is the mechanism for the growth oftransformed NRK cells in soft agar. New Biologist 4,
504-511, 1992.
17. Kimura, T., Tanizawa, O., Mori, K., Brownstein, M.,
and Okayama, H. Structure and expression of ahuman oxytocin receptor. Nature 356, 526-529,
1992.
18. Tatsuka, M., Mitsui, H., Wada, M., Nagata, A.,
Nojima, H., and Okayama, H. Elongation factor-1agene determines susceptibility to transformation.
Nature 359, 333-336, 1992.
19. Kizaka-Kondoh, S., Sato, K., Tamura, K., Nojima, H.,
and Okayama, H. Raf-1 protein kinase is an integral
component of the oncogenic signal cascade sharedby epidermal growth factor and platelet-derived
growth factor. Mol. Cell. Biol. 12, 5078-5086, 1992.
20. Tanaka, K., Okazaki, K., Okazaki, N. Ueda, T.
Sugiyama, A., Nojima, H. and Okayama, H. A new
cdc gene required for S phase entry of
Schizosaccharomyces pombe encodes a protein similar to the cdc10+ and SWI4 gene products. EMBO J.
11, 4923-4932, 1992.
21. Benton, B. K., Reid, M. S. and Okayama, H. A
Schizosaccharomyces pombe gene that promotes
sexual differentiation encodes a helix - loop - helix
protein with homology to MyoD. EMBO J. 12, 135143, 1993.
22. Tamura, K., Kanaoka, Y., Jinno, S., Nagata, A., Ogiso,
Y., Shimizu, K., Hayakawa, T., Nojima, H., and18

Okayama, H. Cyclin G: A new mammalian cyclinwith a potential tyrosine phosphorylation site.
Oncogene 8, 2113-2118, 1993.
23. Jinno, S., Suto, K., Nagata, A., Igarashi, M., Kanaoka,
Y., Nojima, H., and Okayama, H. Cdc25A is a novel
phosphatase functioning early in the cell cycle.
EMBO J. 13, 1549-1556, 1994.
24. Sugiyama, A., Tanaka, K., Okazaki, K., Nojima, H.,
and Okayama, H. A zinc finger protein controls theonset of premeiotic DNA synthesis of fission yeast ina Mei2-independent cascade. EMBO J. 13, 1881-1887,
1994.
25. Miyamoto, M., Tanaka, K., and Okayama, H. res2+, a
new member of cdc10+/SWI4 family, controls the∞gstart∞h of mitotic and meiotic cycles in fission
yeast. EMBO J. 13, 1873-1880, 1994.
26. Obara-Ishihara, T., and Okayama, H. A B-type cyclin
regulates conjugation via interaction with cell cycle“start” genes in fission yeast. EMBO J. 13, 1863-1872,
1994.
27. Murakami, H., and Okayama, H. A kinase from fission yeast responsible for blocking mitosis in S
phase. Nature 374, 817-819, 1995.
28. Terada, Y., Tatsuka, M., Jinno, S., and Okayama, H.
Requirement for tyrosine phosphorylation of Cdk4 in
G1 arrest induced by ultraviolet irradiation. Nature
367, 358-362, 1995.
29. Nakashima, N., Tanaka, K., Sturm, S., and Okayama,
H. Fission Yeast Rep2 is a putative transcriptional
activator subunit for the cell cycle “start” function of
Res2-Cdc10. EMBO J. 14, 4794-4802, 1995.
30. Okayama, H., Nagata, A., Jinno, S., Murakami, H.,
Tanaka, K., and Nakashima, N. Cell cycle control infission yeast and mammals identification of new regulatory mechanisms. 1996, Adv. Cancer Re. 69, 1762.
31. Kato, Jr., T., Okazaki, K., Murakami, H., Stettler, S.,
Fantes, A., P., and Okayama, H. Stress signal, mediated by a Hog1-like MAP kinase, controls sexual
development in fission yeast. FEBS Let. 378, 207212, 1996.
32. Kizaka-Kondoh, S., Matsuda, M., and Okayama, H.
CrkII signals from epidermal growth factor receptor
to Ras. Proc. Natl. Acad. Sci. USA 93, 12177-12182,
1996.
33. Yamaguchi, S., Murakami, H., and Okayama, H. A
WD repeat protein controls the cell cycle and differentiation by negatively regulating Cdc2/B-type cyclin
complexes. Mol. Biol. Cell 8, 2475-2486, 1997.
34. Okazaki, N., Okazaki, K., Watanabe, Y., KatoHayashi, M., Yamamoto, M., and Okayama, H. Novel
factor highly conserved among eukaryotes controls
sexual development in fission yeast. Mol. Cell. Biol.
18, 887-895, 1998.
35. Tahara, S., Tanaka, K., Yuasa, Y., and Okayama, H.
Functional domains of Rep2, a transcriptional activator subunit for Res2-Cdc10, controlling the cell cycle
start. Mol. Biol. Cell 9, 1577-1588, 1998.
36. Tsukahara, K., Yamamoto, H., and Okayama, H. An
RNA binding protein negatively controlling differen-tiation in fission yeast. Mol Cell. Biol. 18, 4488-4498,
1998.
37. Jinno, S., Hung, S.-C., and Okayama, H. Cell cycle
start from quiescence controlled by tyrosine phosphorylation of Cdk4. Oncogene 18, 565-577, 1999.
38. Yamamoto, H., Tsukahara, K., Kanaoka, Y., Jinno, S.,
and Okayama, H. Isolation of a mammalian homologue of a fission yeast differentiation regulator. Mol.
Cell. Biol. 19, 3829-3841, 1999.
39. Jinno, S., Hung, S.-C., Yamamoto, Y., Lin, J., Nagata,A., and Okayama, H. Oncogenic stimulation recruits
Cdk6 in the cell cycle start in rat fibroblast. Proc.
Natl. Acad. Sci. USA 96, 13197-13202, 1999.
40. Suto, K., Murakami, H., Nagata, A., and Okayama, H.
A double-strand Break repair component is essential
for S phase completion in fission yeast cell cycling.
Mol. Biol. Cell 10, 3331-3343, 1999.
41. Masuda, M., Nagai, Y., Oshima, N., Tanaka, K.,
Murakami, H., Igarashi, H., and Okayama H. Genetic
Studies with the Fission Yeast Schizosaccharomyces
pombe Suggest Involvement of Wee1, Ppa2, andRad24 in Induction of Cell Cycle Arrest by Human
Immunodeficiency Virus Type 1 Vpr. J. Virol. 74,
2636-2646, 2000.
42. Tanaka, K., Yonekawa, T., Kawasaki, Y., Kai, M.,
Furuya, K., Iwasaki, M., Murakami, H., Yanagida, M.,
and Okayama, H. Fission yeast Eso1p is required forestablishing chromatid cohesion during S phase.
Mol. Cell. Biol. 20, 3459-3469, 2000.
43. Yamaguchi, S., Okayama, H. and Nurse, P. Fission
yeast Fizzy-related protein Srw1p is a G(1)-specificpromoter of mitotic cyclin B degradation. EMBO J.
19, 3968-3977, 2000.
44. Tanaka, K., and Okayama, H. A Pcl-like cyclin activates the Res2p-Cdc10p cell cycle `start’ transcriptional factor in fission yeast. Mol. Biol. Cell 11, 28452862, 2000.
45. Nakamura, E., Isobe, H., Tomita, N., Sawamura, M.,
Jinno, S., and Okayama, H. Funtionalized fullerene
as an artificial vector for transfection. Angew. Chem.
112, 4424-4427, 2000.
46. Jinno, S., Lin, J., Yageta, M., and Okayama, H.
Oncogenic cell cycle start control. Mut. Res. 477, 2329, 2001.
47. Lin, J., Jinno, S., and Okayama H. Cyclin D3-Cdk6
complex evades inhibition by inhibitor proteins anduniquely controls cell’s growth competence.
Oncogene 20, 2000-2009, 2001.
48. Tanaka, K., Hao, Z., Kai, M. and Okayama, H.
Establishment and maintenance of sister chromatid
cohesion in fission yeast by a unique mechanism.
EMBO J. 20, 5779-5790, 2001.
49. Jinno, S., Yageta, M., Nagata, A. and Okayama, H.
Cdc6 requires anchorage for its expression.
Oncogene 21, 1777-1784, 2002.
50. Hiroi, N., Jinno, S., and Okayama, H. Mammalian
Rcd1 is a novel transcriptional cofactor that mediates
retinoic acid-induced cell differentiation. EMBO J. 21,
5235-5244, 2002.

19

Department of
Cellular Signaling
Outline and Research Objectives
When cells are stimulated, by the serial actions of various enzymes, biologically active
lipid molecules are produced and released. They include prostaglandins, leukotrienes,
platelet-activating factor (PAF), lysophosphatidic acid (LPA) etc., collectively termed lipid
mediators (Fig 1). In concert with various neurotransmitters and hormones, these lipid
mediators are playing important roles in self-defenses and maintenance of homeostasis.
Unlike the biogenic amines or peptide mediators, lipid mediators are not stored in granules,
but they are biosynthesized from precursor lipids when necessary. To elucidate the functions of lipid mediators, we isolated enzymes involved in the biosyntheses and metabolismof lipid mediators, isolated G-protein-coupled receptors, and identification of intracellularsignaling. We are especially interested in identifying the specific roles of lipid mediators in
inflammatory responses as well as neuronal functions. We generated several transgenic orknockout mice and analysed their roles in vivo. By mass spectrometric analyses, we alsomeasured dynamic changes in membrane lipid compositions, the significance of thesechanges being in general signaling pathway. In the postsequece of whole human genome,
the intensive studies of protein regulation as well as behavior of small lipid metabolites areof the highest importance.

Faculties and Students
Professor
Takao Shimizu, M.D., Ph.D. (1991-)
Associate Professor Takehiko Yokomizo, M.D., Ph.D.
Associate .................................3
Postdoctoral Fellows...............4
Graduate Students ................12
Research Students...................7
Secretary .................................2

Past Research and Major Accomplishments
We have purified and cDNA cloned various

enzymes involved in the biosyntheses and metabolism of lipid mediators. Leukotriene A4 hydrolase,
one of the key enzyme in the biosynthesis of chemotactic leukotrienes, was cloned and characterized in
our laboratory. This achievement was the first successful example of molecular cloning of the enzymes
in the field of lipid mediators. We also obtained PAF
receptor, as the first successful example of receptor
cloning of lipid mediators, followed by cloning receptors of various lipid mediators. We determined theregulation of these enzymes by Ca-dependent intracellular translocation, phosphorylation by various kinases, and other posttranslational modifications. We also

Membrane phospholipids

20

Phospholipids:

Lyso-phospholipids:

Eicosanoids:

Platelet-activating factor
Psychosine
Sphingophosphocholine

Lyso-phosphatidylcholine
Lyso-phosphatidic acid
Sphigosine 1-phosphate
2-Arachidonoyl-glycerol

Prostaglandins
Thromboxanes
Leukotrienes
Lipoxinsgenerated PAF receptor-overexpressing mice, PAF
receptor-deficient mice, leukotriene receptor-deficient
mice, and the mice deficient in cytosolic phospholipase A2 (cPLA2), which are involved in the synthesesof eicosanoids and PAF. By analyzing the phenotypesof these mice, we reported that the mediators are
involved in the pathogenesis of various inflammatory
and immune disorders, as well as normal physiology
including reproduction and synaptic plasticity.
Independently, one of the Associate is working onaxon guidance molecules, by establishing knockout
mice of guidance molecules (Sema 3A), and analysesof their multiple phenotypes.

Current Research
Followings are ongoing projects in our laboratory.
1. Elucidation of enzymes in the biosyntheses oflipid mediators, their regulation and mechanismof intracellular translocation by stimuli.
2. Deorphaning projects of putative lipid mediator
GPCRs, which include indentificaion of novel lipid
mediators (Fig. 2).
3. Elucidation of GPCR-sorting mechanism in polarized cells (kidney epithelial cells, neuron etc.)
4. Phenotype analyses of various genetic engineered
mice. Understanding of the molecular mechanismof individual phenotypes in depth.

21

5. Establishment of novel gene-targeted mice(enzymes and receptors), and their congenic line
(B6, Balb/c, DBA etc.)
6. Identification of Sema3A knockout mice, in nervous systems, olfactory systems, bone formation.

3. 1999-2002 Grant from the Ministry of Education,
Science, Culture, Sports, and Technology of Japan (A,B, and C)
4. 1997 Priority Area A, from the Ministry of
Education, Science, Culture, Sports, and Technologyof Japan.
5. 1996-2000 Ministry of Welfare, Health and Labor.

Future Prospects
We will pursue following different projects, leadby each faculty member.
1. Identification of the roles of lipid mediators in thecentral nervous system. For this purpose, we will
analyze the localization and subcellular localization of enzymes involved in the biosyntheses oflipid mediators. It is the most important to determine, how and which direction (either axon or
dendrite), these enzyme cause translocation in a
stimulus-dependent manner. The mice deficient of
enzymes or receptors of lipid mediators are useful
to identify the roles of mediators in the central
nervous systems. Some link has been reported
between Sema3A axon guidance molecules andlipid mediators. By the use of double knockout
mice (Sema3A and lipid mediators), more confirmative and direct evidence will be obtained.
2. We have so far obtained various phenotypes ofgenetic engineered mice, but molecular mechanisms underlying these phenotypes remain mostly
unknown. The link between lipid mediators andhormones, neurotransmitters, and cytokines will
be analyzed to obtain a whole view.
3. By mass spectrometric analyses, we like to findout the temporal and positional dynamic changesof membrane compositions including Raft andcaveolae. Also, mass spectrometry attached with
HPLC will aid in the identification of novel natural
lipid mediators (deorphaning project). For these
purposes, we recently built a new research groupcalled Department of Lipid Metabolome, by donation. We have collected several excellent lipid biochemists, and mass spectrometer specialists.
Metabolome (metabolomics) is a new researchstrategy after genome and proteome research. The
study is to analyze in detail small-sized molecules(metabolites), and in combination with proteomics, we will gain a more broad view how cells
adapt to a new environment, change membrane
fluidity, and cause metabolic changes, which finally yield to gene expression.

Research Grants
1. 1998-2003 CREST of Japan Science and Technology
Corporation
2. 2001-2004 PREST of Japan Science and Technology
Corporation22

Select Publications (50 among 128 publications between 1991 and 2002)
1. Ito, N., Yokomizo, T., Sasaki, T., Kurosu, H.,
Penninger, J., Kanaho, Y., Katada, T., Hanaoka, K.,
and Shimizu, T. Requirement of phosphatidylinositol
3-kinase-activation and Ca influx for leukotriene B4induced enzyme release. J. Biol. Chem. in press,
2002.
2. Stromgaard, K., Saito, D.R., Shindou, H., Ishii, S.,
Shimizu, T., and Nakanishi, K. Ginkgolide derivatives for photolabeling studies: preparation and pharmacological evaluation. J. Med. Chem. 45,4038-46,
2002.
3. Wong, D.A., Uozumi, N., Kita, Y., and Shimizu, T.
Discrete role for cytosolic phospholipase A2-alpha inplatelets: Studies using single and double mutant
mice of cytosolic and group IIA-secretory phospholipase A2. J. Exp. Med. 196,349-57, 2002.
4. Ogasawara, H., Ishii, S., Yokomizo, T., Kakinuma, T.,
Komine, M., Tamaki, K., Shimizu, T., and Izumi, T.
Characterization of mouse cysteinyl leukotriene
receptors, mCysLT1 and mCysLT2: Differential
pharamacological properties and tissue distribution.
J. Biol. Chem. 277, 18763-18768, 2002.
5. Nagase, T., Uozumi, N., Ishii, S., Kita, Y., Yamamoto,H., Ohga, E., Ouchi, Y. and Shimizu, T. A pivotal roleof cytosolic phospholipase A2 in bleomycin-induced
pulmonary fibrosis. Nature Med. 8, 480-484, 2002.
6. Ohshima, N., Ishii, S., Izumi, T., and Shimizu, T.
Receptor-dependent metabolism of platelet-activating factor in murine macrophages. J. Biol. Chem. 277,9722-9727, 2002.
7. Nagase, T., Ishii, S., Shindou, H., Ouchi, Y., andShimizu, T. Airway hyperresponsiveness in transgenic mice overexpressing platelet activating factor
receptor is mediated by an atropine-sensitive pathway. Am. J. Respir. Crit. Care Med. 165, 200-205,
2002.
8. Yokomizo, T., Izumi, T., and Shimizu, T. Leukotriene
B4: metabolism and signal transduction. Arch.
Biochem. Biophys. 385, 231-241, 2001.
9. Yokomizo, T., Kato, K., Hagiya, H., Izumi, T., andShimizu, T. Hydroxyeicosanoids bind to and activate
the low affinity leukotriene B4 receptor, BLT2. J.
Biol. Chem. 276, 12454-12459, 2001.
10. Wu, C., Stojanov, T., Chami, O., Ishii, S., Shimizu, T.,
Li, A., and O’Neill, C. Evidence for the autocrine
induction of capacitation of mammalian spermatozoa. J. Biol. Chem. 276, 26962-26968, 2001.
11. Yamashita, A., Kawagishi, N., Miyashita, T.,
Nagatsuka, T., Sugiura, T., Kume, K., Shimizu, T.,and Waku, K. ATP-independent fatty acyl-coenzyme
A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic
acid catalyzed by acyl-coenzyme A:lysophosphatidic
acid acyltransferase. J. Biol. Chem. 276, 26745-26752,
2001.
12. Kim, Y. J., Noguchi, S., Hayashi, Y. K., Tsukahara, T.,
Shimizu, T., and Arahata, K. The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates
muscle-specific gene expression. Hum. Mol. Genet.
10, 1129-1139, 2001.
13. Fukunaga, K., Ishii, S., Asano, K., Yokomizo, T.,
Shiomi, T., Shimizu, T., and Yamaguchi, K. Single
nucleotide polymorphism of human platelet-activating factor receptor impairs G-protein activation. J.
Biol. Chem. 276, 43025-43030, 2001.
14. Aihara, M., Ishii, S., Kume, K., and Shimizu, T.
Interaction between neurone and microglia mediatedby platelet-activating factor. Genes Cells 5, 397-406,
2000.
15. Noiri, E., Yokomizo, T., Nakao, A., Izumi, T., Fujita,T., Kimura, S., and Shimizu, T. An in vivo approach
showing the chemotactic activity of leukotriene B(4)in acute renal ischemic-reperfusion injury. Proc. Natl.
Acad. Sci. USA 97, 823-828, 2000.
16. Nagase, T., Uozumi, N., Ishii, S., Kume, K., Izumi, T.,
Ouchi, Y., and Shimizu, T. Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2. Nat. Immunol. 1, 42-46, 2000.
17. Kato, K., Yokomizo, T., Izumi, T., and Shimizu, T.
Cell-specific transcriptional regulation of humanleukotriene B(4) receptor gene. J. Exp. Med. 192, 413420, 2000.
18. Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., andShimizu, T. A second leukotriene B(4) receptor,
BLT2. A new therapeutic target in inflammation andimmunological disorders. J. Exp. Med. 192, 421-432,
2000.
19. Takaku, K., Sonoshita, M., Sasaki, N., Uozumi, N.,
Doi, Y., Shimizu, T., and Taketo, M. M. Suppressionof intestinal polyposis in Apc(delta 716) knockout
mice by an additional mutation in the cytosolic phospholipase A(2) gene. J. Biol. Chem. 275, 3401334016, 2000.
20. Nakatani, N., Uozumi, N., Kume, K., Murakami, M.,
Kudo, I., and Shimizu, T. Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast
cells. Biochem. J. 352 Pt 2, 311-317, 2000.
21. Okamoto, H., Takuwa, N., Yokomizo, T., Sugimoto,N., Sakurada, S., Shigematsu, H., and Takuwa, Y.
Inhibitory regulation of Rac activation, membrane
ruffling, and cell migration by the G protein-coupledsphingosine-1-phosphate receptor EDG5 but not
EDG1 or EDG3. Mol. Cell Biol. 20, 9247-9261, 2000.
22. Kobayashi, K., Ishii, S., Kume, K., Takahashi, T.,
Shimizu, T., and Manabe, T. Platelet-activating factor
receptor is not required for long-term potentiation in

the hippocampal CA1 region. Eur. J. Neurosci. 11,
1313-1316, 1999.
23. Nagase, T., Ishii, S., Kume, K., Uozumi, N., Izumi, T.,
Ouchi, Y., and Shimizu, T. Platelet-activating factor
mediates acid-induced lung injury in genetically engineered mice. J. Clin. Invest. 104, 1071-1076, 1999.
24. Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T.,
Iino, M., Itoh, H., and Shimizu, T. Critical duration of
intracellular Ca2+ response required for continuoustranslocation and activation of cytosolic phospholipase A2. J. Biol. Chem. 274, 5163-5169, 1999.
25. Ishii, I., Saito, E., Izumi, T., Ui, M., and Shimizu, T.
Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Roleof cytoplasmic tail phosphorylation in each process.
J. Biol. Chem. 273, 9878-9885, 1998.
26. Hoshino, M., Izumi, T., and Shimizu, T. Leukotriene
D4 activates mitogen-activated protein kinasethrough a protein kinase Calpha-Raf-1-dependent
pathway in human monocytic leukemia THP-1 cells.
J. Biol. Chem. 273, 4878-4882, 1998.
27. Ishii, S., Kuwaki, T., Nagase, T., Maki, K., Tashiro,F., Sunaga, S., Cao, W. H., Kume, K., Fukuchi, Y.,
Ikuta, K., Miyazaki, J., Kumada, M., and Shimizu, T.
Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating
factor receptor. J. Exp. Med. 187, 1779-1788, 1998.
28. Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume,
K., Shimizu, T., and Sugiyama, Y. Hepatic expressionof multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats.
Mol. Pharmacol. 53, 1068-1075, 1998.
29. Ishii, S., Nagase, T., Tashiro, F., Ikuta, K., Sato, S.,
Waga, I., Kume, K., Miyazaki, J., and Shimizu, T.
Bronchial hyperreactivity, increased endotoxin
lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor
receptor. EMBO. J. 16, 133-142, 1997.
30. Kume, K., and Shimizu, T. Platelet-activating factor(PAF) induces growth stimulation, inhibition, andsuppression of oncogenic transformation in NRK
cells overexpressing the PAF receptor. J. Biol. Chem.
272, 22898-22904, 1997.
31. Ishii, I., Izumi, T., Tsukamoto, H., Umeyama, H., Ui,
M., and Shimizu, T. Alanine exchanges of polaramino acids in the transmembrane domains of aplatelet-activating factor receptor generate both constitutively active and inactive mutants. J. Biol. Chem.
272, 7846-7854, 1997.
32. Honda, Z., Suzuki, T., Hirose, N., Aihara, M.,
Shimizu, T., Nada, S., Okada, M., Ra, C., Morita, Y.,
and Ito, K. Roles of C-terminal Src kinase in the initiation and the termination of the high affinity IgE
receptor-mediated signaling. J. Biol. Chem. 272,
25753-25760, 1997.
33. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., andShimizu, T. A G-protein-coupled receptor forleukotriene B4 that mediates chemotaxis. Nature
387, 620-624, 1997.

23

34. Uozumi, N., Kume, K., Nagase, T., Nakatani, N.,
Ishii, S., Tashiro, F., Komagata, Y., Maki, K., Ikuta,
K., Ouchi, Y., Miyazaki, J., and Shimizu, T. Role ofcytosolic phospholipase A2 in allergic response andparturition. Nature 390, 618-622, 1997.
35. Yokomizo, T., Ogawa, Y., Uozumi, N., Kume, K.,
Izumi, T., and Shimizu, T. cDNA cloning, expression,and mutagenesis study of leukotriene B4 12-hydroxydehydrogenase. J. Biol. Chem. 271, 2844-2850,
1996.
36. Ferby, I. M., Waga, I., Hoshino, M., Kume, K., andShimizu, T. Wortmannin inhibits mitogen-activatedprotein kinase activation by platelet-activating factor
through a mechanism independent of p85/p110-type
phosphatidylinositol 3-kinase. J. Biol. Chem. 271,
11684-11688, 1996.
37. Kishimoto, S., Shimadzu, W., Izumi, T., Shimizu, T.,
Fukuda, T., Makino, S., Sugiura, T., and Waku, K.
Regulation by IL-5 of expression of functional
platelet-activating factor receptors on humaneosinophils. J. Immunol. 157, 4126-4132, 1996.
38. Mori, M., Aihara, M., Kume, K., Hamanoue, M.,
Kohsaka, S., and Shimizu, T. Predominant expression of platelet-activating factor receptor in the rat
brain microglia. J. Neurosci. 16, 3590-3600, 1996.
39. Mutoh, H., Fukuda, T., Kitamaoto, T., Masushige, S.,
Sasaki, H., Shimizu, T., and Kato, S. Tissue-specificresponse of the human platelet-activating factor
receptor gene to retinoic acid and thyroid hormone
by alternative promoter usage. Proc. Natl. Acad. Sci.
USA 93, 774-779, 1996.
40. Watanabe, T., Waga, I., Honda, Z., Kurokawa, K.,
and Shimizu, T. Prostaglandin F2 alpha stimulates
formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-proteincoupled pathway. J. Biol. Chem. 270, 8984-8990,
1995.
41. Honda, Z., Takano, T., Hirose, N., Suzuki, T., Muto,
A., Kume, S., Mikoshiba, K., Itoh, K., and Shimizu, T.
Gq pathway desensitizes chemotactic receptorinduced calcium signaling via inositol trisphosphate
receptor down-regulation. J. Biol. Chem. 270, 48404844, 1995.
42. Shirasaki, H., Nishikawa, M., Adcock, I. M., Mak, J.
C., Sakamoto, T., Shimizu, T., and Barnes, P. J.
Expression of platelet-activating factor receptor
mRNA in human and guinea pig lung. Am. J. Respir.
Cell Mol. Biol. 10, 533-537, 1994.
43. Takano, T., Honda, Z., Sakanaka, C., Izumi, T.,
Kameyama, K., Haga, K., Haga, T., Kurokawa, K., andShimizu, T. Role of cytoplasmic tail phosphorylationsites of platelet-activating factor receptor in agonistinduced desensitization. J. Biol. Chem. 269, 2245322458, 1994.
44. Honda, Z., Takano, T., Gotoh, Y., Nishida, E., Ito, K.,
and Shimizu, T. Transfected platelet-activating factor
receptor activates mitogen-activated protein (MAP)kinase and MAP kinase kinase in Chinese hamster
ovary cells. J. Biol. Chem. 269, 2307-2315, 1994.

24

45. Ferby, I. M., Waga, I., Sakanaka, C., Kume, K., andShimizu, T. Wortmannin inhibits mitogen-activatedprotein kinase activation induced by platelet-activating factor in guinea pig neutrophils. J. Biol. Chem.
269, 30485-30488, 1994.
46. Bito, H., Mori, M., Sakanaka, C., Takano, T., Honda,
Z., Gotoh, Y., Nishida, E., and Shimizu, T. Functional
coupling of SSTR4, a major hippocampal somatostatin receptor, to adenylate cyclase inhibition,arachidonate release and activation of the mitogenactivated protein kinase cascade. J. Biol. Chem. 269,
12722-12730, 1994.
47. Bito, H., Nakamura, M., Honda, Z., Izumi, T.,
Iwatsubo, T., Seyama, Y., Ogura, A., Kudo, Y., andShimizu, T. Platelet-activating factor (PAF) receptor
in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons. Neuron 9, 285-294, 1992.
48. Nakamura, M., Honda, Z., Izumi, T., Sakanaka, C.,
Mutoh, H., Minami, M., Bito, H., Seyama, Y.,
Matsumoto, T., Noma, M., and Shimizu, T.
Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J. Biol.
Chem. 266, 20400-20405, 1991.
49. Honda, Z., Nakamura, M., Miki, I., Minami, M.,
Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito,K., Miyamoto, T., and Shimizu, T. Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. Nature 349, 342-346, 1991.
50. Watanabe, T., Yatomi, Y., Sunaga, S., Miki, I., Ishii,A., Nakao, A., Higashihara, M., Seyama, Y., Ogura,
M., Saito, H., Kurokawa, K., and Shimizu, T.
Characterization of prostaglandin and thromboxane
receptors expressed on a megakaryoblastic leukemia
cell line, MEG-01s. Blood 78, 2328-2336, 1991.

Department of Physiological
Chemistry and Metabolism
Outline and Research Objectives
The Department of Physiological Chemistry and Nutrition, the predecessor of the present department, was founded in 1952. The successive professors were as follows;
The first professor:
Haruhisa Yoshikawa (1952~1969)
The second professor: Osamu Hayaishi (1970~1974)
The third professor: Yoshitake Mano (1974~1982)
The fourth professor: Yousuke Seyama (1983~2001)
During the past 50 years, these professors lead high-quality researches in the field of biochemistry, metabolism and nutrition and greatly contributed to scientific progresses inJapan. Upon the restructuring of the university system in 1997, the department was
renamed ‘Department of Physiological Chemistry and Metabolism’ as one unit of theSpecialty of Molecular Cell Biology. In 2002, Dr. Hiroki Kurihara was designated as a successor of Professor Yousuke Seyama, and started new researches on developmental biology andtechnology as well as regenerative medicine.

Faculties and Students
Professor and Chair Hiroki Kurihara, M.D., Ph.D. (2002~)
Associate .................................3
Postdoctoral Fellow ................1
Graduate Student....................2
Secretary .................................1

Past Research and Major Accomplishments
1. Vascular Biology(1) Biology of Vasoactive Peptidesi) Endothelin
a. Discovery and Basic Characterization ofEndothelin
Our researches started with contribution to thediscovery of endothelin (ET-1), an endotheliumderived vasoactive peptide, with Drs. Masashi
Yanagisawa, Tomoh Masaki and colleagues in
1987. Then, we studied the role of ET-1 in vascularphysiology and pathophysiology and revealed its
vasoconstrictive effect on in vivo canine coronary
arteries and induction of the ET-1 gene by somecytokines and flow-induced mechanical stress invascular endothelial cells.
b. Endothelin-1 Knockout Mice
In 1991, we introduced the technique of genetargeting in mice to our lab to further analyze thebiological implication of ET-1. As a result, wefound two novel and important roles of ET-1. First,
ET-1 proved to lower blood pressure when it actsin the central nervous system although it mainly
acts as a pressor in the periphery. ET-1 also modulates stress responses in the central nervous system by regulating catecholamine metabolism.
Second, ET-1 proved to be essential for embryogenesis. The phenotype of mice lacking ET-1
involved craniofacial and cardiac neural crestderived structures including the branchial archesand great vessels. Indeed, ET-1 was the firstvasoactive substance bound to G protein-coupled
receptors that proved to be involved in embryonicdevelopment (See 2-1)).
c. Endothelin-1 Overexpressing MiceWe found that the expression of ET-1 and its
processing enzyme ECE-1 is increased in the lesionof human atherosclerosis and experimental vascular injury. The effect of overexpressed ET-1 wasfurther analyzed in transgenic mice and proved tocause mesangial hyperplasia and glomerulosclerosis in the kidney. This result was indicative of thestimulating effect of ET-1 on cellular proliferation
and remodeling in vivo.
ii) Adrenomedullin
a. Adrenomedullin Overexpressing Mice
Adrenomedullin (AM), a vasodilating peptide,can be regarded as a counterpart of ET-1 in vascular tone regulation. We investigated the in vivo
effect of AM by producing transgenic mice overexpressing AM and found NO-dependent hypotension and resistance to endotoxin shock (decreasein lethality) in transgenic mice. These results suggested that AM may lower blood pressure by stim-

25

ulating NO production and that AM may protect
tissues and organs from shock-induced damage.
b. Adrenomedullin Knockout Mice
The biological importance of AM was further
investigated by gene targeting. We demonstrated
that AM is indispensable for the vascular morphogenesis during embryonic development and for
postnatal regulation of blood pressure by stimulating NO production, confirming the hypothesis
derived from AM overexpression mice.
iii) CGRPCGRP is structural related to AM and shares thecommon receptor CRLR. To investigate systematically the differential role of the AM/CGRP family
members, we established alphaCGRP knockout
mice. The resultant phenotype revealed thatalphaCGRP contributes to the regulation of cardiovascular function through inhibitory modulation of
sympathetic nervous activity and that AM and
CGRP have distinct physiological roles.
(2) Transgenic techniques
During gene manipulation studies in mice, we
found that the ET-1 gene promoter is useful for vesselselective gene expression. This promoter was used
not only to overexpress ET-1 and AM but also tomake endothelial NO synthase and 15-lipoxygenase
overexpressing mice by our collaborators with fruitfulresults. Recently, we realized targeting gene expression in vascular smooth muscle cells by using the ETA receptor (ETAR) gene promoter.
2. Developmental Biology(1) Neural Crest Development
Craniofacial and cardiovascular defects in ET-1
knockout embryos gave a clue to the elucidation of
mechanisms for branchial arch formation contributedby neural crest cells. By subsequent analysis, werevealed that ET-1 regulates several downstream
genes such as HAND1/2 and Goosecoid transcription
factors to contribute to cranial/cardiac neural crest
development. Recently, we established ETAR promoter::GFP and ETAR promoter::TVA (avian retrovirus
receptor) to visualize and isolate ETAR+ neural crest
cells and to transfer genes specifically to them via
retroviral vectors. These systems serve as very usefultools for the analysis of neural crest-derived branchialarch development.
(2) Vascular Development
ET-1 knockout mice also demonstrated defects in
the great vessels and cardiac outflow tract, to which
cardiac neural crest cells largely contribute. From thisresult, we found that ET-1 is important in vascularsmooth muscle cell development originated from neu-

26

ral crest cells and that the ET-1to HAND2 signaling
pathway seems to be critical. The outcome from AM
knockout mice showed that AM is important in theformation of basement membrane during angiogenesis to stabilize vascular network.
3. Collaborative Works(1) Biology of Metalloproteinase
In collaboration with Dr. Kouji Matsushima (Dept.
of Molecular Preventive Medicine), we exploited thebiological implication of ADAMTS-1, a member of the
ADAM-type metalloproteinase family, by gene targeting. We found that ADAMTS-1 is important for normal growth, fertility and organ developemnt (the kidneys, adrenal glands, adipose tissues etc.).
(2) Biology of Antimicrobial Peptides
In collaboration with Drs. Yasuyoshi Ouchi and
Takahide Nagase (Dept. of Geriatrics), we are studying
about defensins, endogenous antimicrobial peptides.
We have identified several novel types of human andmouse beta-defensins which are expressed skeletal
muscle and epididymis.

Current Research
1. Vascular Biology
We have converged our previous achievement ofvascular biology on the aspect of developmental biology including vascular development as described
below.
2. Developmental Biology(1) Neural Crest Development
Extending our achievement of ET-1 knockout
mice, we are studying the mechanism how the intercellular and intracellular signaling system related ET-1
is involved in neural crest development and branchialarch formation because it may give a clue to theinsight how signaling interactions can change cellularbehavior to lead to morphogenesis. For this purpose,
we have established GFP and TVA transgenic mice as
described and are now establishing some other mice
which will be useful for the analysis of branchial archformation. We are also performing a systematic
screening for differential gene expression by DNA
microarray followed by in situ hybridization. By thismethod, several important genes are to be obtained.
We are also studying about neural crest differentiation using murine neural crest cell culture and Sendai
virus vectors developed by Drs. Yoshiyuki Nagai andAtsushi Kato. In our preliminary result, we realized
nearly 100 % gene transfer into neural crest cells andare analyzing the effect of some genes on neural crestdifferentiation. Using some markers, we are also trying to isolate neural crest stem cells.
We are further looking for the mechanism how
some transcription factors important for neural crest
development can exert their effects. For this purpose,
we are performing yeast two-hybrid screening using
Pax3 and HAND2 as baits. We have obtained several
interesting clones and are analyzing their molecularcharacteristics.
(2) Vascular Development
Above-mentioned studies concerning neural crest
differentiation include the issue of vascular smoothmuscle cell differentiation and we are trying to identify the target genes of the ET-1-HAND2 pathway thatis important for smooth muscle differentiation. Twohybrid studies also involve this theme. Abnormalities
in vascular formation in AM knockout mice are also
being investigated further concerning the mechanism.
In addition, we have started studies on vascularendothelial cell differentiation using the embryonicstem cell system and are looking for possible interaction between neural crest-derived smooth muscle precursors and endothelial precursors. It may clarify anovel cellular interaction mechanism which can stimulate vascular formation.
3. Collaborative Works
In collaboration with Drs. Yasuyoshi Ouchi and
Takahide Nagase (Dept. of Geriatrics), we are starting
further study on defensins using gene manipulationin mice.

Future Prospects(1) Basic research
Our future study will focus mainly on neural crestand vascular development. Especially, major questionsare (1) how do intracellular mechanisms modulate
stem cell behavior including fate determination anddifferentiation, and (2) how are cell behaviors integrated into morphogenesis and organogenesis. By
studying neural crest development as a model, we
expect that we can approach these general issues. For
our coming research, we are introducing some newtechniques such as nuclear transfer.
(2) Applied research
Our current researches on vascular developmentusing embryonic stem cells are close to appliedresearches in the field of regenerative medicine. On
this background together with nuclear transfer technique, we are planning translational researches togenerate vessels and other tissues and organs related

neural crest cells.

Research Grants
1. JSPS (Japan Society for the Promotion of Science)
Research for the Future Program (2000.4~2004.3)
total 240,000,000 yen.
2. Grants-in-Aid for Scientific Research from JSPS,
Scientific Research (B)(2002.4~2004.3) total 14,900,000 yen
3. Grants-in-Aid for Scientific Research from JSPS,
Exploratory Research (C)(2002.4~2004.3) total 9,900,000 yen
4. Grants-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture, Japan,
Priority Areas Research (A), Allotted.
(2000.4~2001.3) total 1,800,000 yen.
5. the Research Grant for Cardiovascular Diseases (14C1) from the Ministry of Health and Welfare(2002.4~2005.3) total 6,300,000 yen.

Select Publications
1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y,
Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T.
A novel vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988.
2. Komuro I, Kurihara H, Sugiyama T, Yoshizumi M,
Takaku F, Yazaki Y. Endothelin stimulates c-fos andc-myc expression and proliferation of vascularsmooth muscle cells. FEBS Lett. 238: 249-252, 1988.
3. Kurihara H, Yoshizumi M, Sugiyama T, Takaku F,
Yanagisawa M, Masaki T, Hamaoki M, Kato H,
Yazaki Y. Transforming growth factor-ß stimulates
the expression of endothelin mRNA by vascularendothelial cells. Biochem. Biophys. Res. Commun.
359: 1435-1440, 1989.
4. Kurihara H, Yoshizumi M, Sugiyama T, Yamaoki K,
Nagai R, Takaku F, Satoh H, Inui J, Yanagisawa M,
Masaki T, Yazaki Y. The possible role of endothelin1 in the pathogenesis of coronary vasospasm. J.
Cardiovasc. Pharmacol. 13(Suppl. 5): S132-S137,
1989.
5. Kurihara H, Yamaoki K, Nagai R, Yoshizumi M,
Takaku F, Satoh H, Inui J, Yazaki Y. Endothelin: apotent vasoconstrictor associated with coronary
vasospasm. Life Sci. 44: 1937-1943, 1989.
6. Goto K, Kasuya Y, Matsuki N, Takuwa Y, Kurihara H,
Ishikawa T, Kimura S, Yanagisawa M, Masaki T.
Endothelin activates the dihydropyridine-sensitive,
voltage-dependent Ca2+-channel in vascular smoothmuscle. Proc. Natl. Acad. Sci. U.S.A. 86: 3915-3918,
1989.
7. Ouchi Y, Kim S, Souza AC, Iijima S, Hattori A, Orimo
H, Yoshizumi M, Kurihara H, Yazaki Y. Central effectof endothelin on blood pressure in conscious rats.
Am J. Physiol. 256: H1747-H1751, 1989.
8. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F,
Yanagisawa M, Masaki T, Yazaki Y. Hemodynamic

27

shear stress stimulates endothelin production by cultured endothelial cells. Biochem. Biophys. Res
Commun. 161: 859-864, 1989.
9. Yoshizumi M, Kurihara H, Morita T, Yamashita T,
Oh-hashi Y, Sugiyama T, Takaku F, Yanagisawa M,
Masaki T, Yazaki Y. Interleukin-1 induces the production of endothelin-1 by cultured endothelial cells.
Biohem. Biophys. Res. Commun. 166: 324-329, 1990.
10. Morita T, Kurihara H, Yoshizumi M, Maemura K,
Sugiyama T, Nagai R, Yazaki Y. Polymorphonuclear
leukocytes have dual effects on endothelin-1: theinduction of endothelin-1 mRNA expression in vascular endothelial cells and modification of theendothelin-1 molecule. Heart Vessels 8: 1-6, 1993.
11. Morita T, Kurihara H, Maemura K, Yoshizumi M,
Yazaki Y. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells. J.
Clin. Invest. 92: 1706-1712, 1993.
12. Morita T, Yoshizumi M, Kurihara H, Maemura K,
Nagai R, Yazaki Y. Shear stress increases heparinbinding epidermal growth factor-like growth factor
mRNA levels in human vascular endothelial cells.
Biocem. Biophys. Res. Commun. 197: 256-262, 1993.
13. Kurihara Y*, Kurihara H*, Suzuki H, Kodama T,
Maemura K, Nagai R, Oda H, Kuwaki T, Cao W-H,
Kamada N, Jishage K, Ouchi Y, Azuma S, Toyoda Y,
Ishikawa T, Kumada M, Yazaki Y. Elevated bloodpressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368: 703-710, 1994.
(*equal contribution)
14. Maemura K, Kurihara H, Kurihara Y, Nagai R, Yazaki
Y. Isolation and characterization of vascular endothelial cells derived from mice lacking endothelin-1.
Biochem. Biophys. Res. Commun. 201: 538-545,
1994.
15. Maemura K, Kurihara Y, Morita H, Kurihara H,
Yazaki Y. Renal endothelin and hypertension - reply.
Nature 372: 50, 1994.
16. Morita T, Kurihara H, Maemura K, Yoshizumi M,
Nagai R, Yazaki Y. Role of Ca2+ and protein kinase C
in shear stress-induced actin depolymerization andendothelin-1 gene expression. Circ. Res. 75: 630-636,
1994.
17. Harats D*, Kurihara H*, Belloni P, Oakley H, Ziober
A, Ackley D, Cain G, Kurihara Y, Lawn R, Sigal E.
Targeting gene expression to the vascular wall intransgenic mice using the murine preproendothelin-1
promoter. J. Clin. Invest. 95: 1335-1344, 1995.
(*equal contribution)
18. Kurihara Y, Kurihara H, Oda H, Maemura K, Nagai R,
Ishikawa T, Yazaki Y. Aortic arch malformations andventricular septal defect in mice deficient in endothelin-1. J. Clin. Invest. 96: 293-300, 1995.
19. Maemura K, Kurihara H, Kurihara Y, Oda H,
Ishikawa T, Copeland NG, Gilbert DJ, Jenkins NA,
Yazaki Y. Sequence analysis, chromosomal locationand developmental expression of the mouse preproendothelin-1 gene. Genomics 31: 177-184, 1996.

28

20. Kuwaki T, Cao W-H, Kurihara Y, Kurihara H, Ling GY, Onodera M, Ju K-H, Yazaki Y, Kumada M.
Impaired ventilatory responses to hypoxia andhypercapnia in mutant mice deficient in endothelin1. Am. J. Physiol. 270: R1279-R1286, 1996.
21. Reid K, Turnley AM, Maxwell GD, Kurihara Y,
Kurihara H, Bartlett PF, Murphy M. Multiple roles forendothelin 3 in melanocyte development: Regulationof progenitor number and stimulation of differentiation. Development 122: 3911-3919, 1996.
22. Minamino T, Kurihara H, Takahashi M, Shimada K.
Maemura K, Oda H, Ishikawa T, Uchiyama T,
Tanzawa K, Yazaki Y. Endothelin converting enzyme(ECE-1) expression in the rat vascular injury modeland human coronary atherosclerosis. Circulation 95:
221-230, 1997.
23. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka
M, Jishage K, Ueda O, Sakaguchi H, Higashi T,
Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada
Y, Honda M, Kurihara H, Aburatani H, Doi T,
Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura
H, Yazaki Y, Horiuchi S, Takahashi K, Kruijt JK, vanBerkel TJC, Steinbrecher UP, Ishibashi S, Maeda N,
Gordon S, Kodama T. A role for macrophage scavenger receptors in atherosclerosis and susceptibility
to infection. Nature 386: 292-296, 1997.
24. Kurihara H, Kurihara Y, Maemura K, Yazaki Y. The
role of endothelin-1 in cardiovascular development.
Ann. NY. Acad. Sci. 811: 168-177, 1997.
25. Morita H, Taguchi J, Kurihara H, Kitaoka M, Kaneda
H, Kurihara Y, Maemura K, Shindo T, Minamino T,
Ohno M, Yamaoki K, Ogasawara K, Aizawa T, Suzuki
S, Yazaki Y. Genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) as a risk factor for coronary artery disease. Circulation 95: 20322036, 1997.
26. Kuwaki T, Kurihara H, Cao WH, Kurihara Y,
Unekawa M, Yazaki Y, Kumada M: Physiological roleof brain endothelin in the central autonomic control:from neuron to knockout mice. Prog. Neurobiol. 51:
545-579, 1997.
27. Nagase T, Kurihara H, Kurihara Y, Aoki-Nagase T,
Fukuchi Y, Yazaki Y, Ouchi Y. Airway hyperresponsiveness to methacholine in mutant mice deficient inendothelin-1. Am. J. Respir. Crit. Care Med. 157: 560564, 1998.
28. Kitano Y, Kurihara H, Kurihara Y, Maemura K, Ryo
Y, Yazaki Y, Harii K. Gene expression of bone matrix
proteins and endothelin receptors in endothelin-1deficient mice revealed by in situ hybridization. J.
Bone Miner. Res. 13: 237-244, 1998.
29. Thomas T, Kurihara H, Yamagishi H, Kurihara Y,
Yazaki Y, Olson EN, Srivastava D. A signaling cascade involving endothelin-1, dHAND and Msx1 regulates development of neural crest-derived branchialarch mesenchyme. Development 125: 3005-3014,
1998.
30. Morita H, Kurihara H, Tsubaki S, Sugiyama T,
Hamada C, Kurihara Y, Shindo T, Oh-hashi Y,
Kitamura K, Yazaki Y. Methylenetetrahydrofolatereductase (MTHFR) gene polymorphism andischemic stroke in Japanese. Arterioscler. Thromb.
Vasc. Biol. 18: 1465-1469, 1998.
31. Ling GY, Cao WH, Onodera M, Ju KH, Kurihara H,
Kurihara Y, Yazaki Y, Kumada M, Fukuda Y, Kuwaki
T. Renal sympathetic nerve activity in mice: comparison between mice and rats and between normal andendothelin-1 deficient mice. Brain Res. 808: 238-249,
1998.
32. Ohashi Y, Kawashima S, Hirata K, Yamashita T,
Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y,
Yokoyama M. Hypotension and reduced nitric oxideelicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J. Clin.
Invest. 102: 2061-2071, 1998.
33. Morita H, Kurihara H, Sugiyama T, Kurihara Y,
Shindo T, Oh-hashi Y, Yazaki Y. D919G polymorphism of the methionine synthase gene. Association
with homocysteine metabolism and late-onset vascular diseases in the Japanese population. Arterioscler.
Thromb. Vasc. Biol. 19: 298-302, 1999.
34. Kuwaki T, Ling GY, Onodera M, Ishii T, Nakamura
A, Ju KH, Cao WH, Kumada M, Kurihara H, Kurihara
Y, Yazaki Y, Ohuchi T, Yanagisawa M, Fukuda Y.
Endothelin in the central control of cardiovascularand respiratory functions. Clin. Exp. Pharmacol.
Physiol. 26: 989-994, 1999.
35. Kurihara H, Kurihara Y, Nagai R, Yazaki Y.
Endothelin and neural crest development. Cell. Mol.
Biol. 45: 639-651, 1999.
36. Nagase T, Kurihara H, Kurihara Y, Aoki-Nagase T,
Nagai R, Ouchi Y. Disruption of ET-1 gene enhances
pulmonary responses to methacholine via functional
mechanism in knockout mice. J. Appl. Physiol. 87:
2020-2024, 1999.
37. Shindo T, Kurihara H, Maemura K, Kurihara Y,
Kuwaki T, Izumida T, Minamino N, Ju K-H, Morita
H, Oh-hashi Y, Kumada M, Kangawa K, Nagai R,
Yazaki Y. Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature.
Circulation 101: 2309-2316, 2000.
38. Kurihara Y*, Kurihara H*, Morita H, Cao W-H, Ling
G-Y, Kumada M, Kimura S, Nagai R, Yazaki Y,
Kuwaki T. Role of endothelin-1 in stress response in
the central nervous system. Am. J. Physiol. 279:
R515-R521, 2000. (*equal contribution)
39 Shindo T, Kurihara H, Kuno K, Yokoyama H, Wada
T, Kurihara Y, Imai T, Wang Y, Ogata M, Oh-hashi Y,
Morita H, Ishikawa T, Nagai R, Yazaki Y,
Matsushima K. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility andorgan morphology and function. J. Clin. Invest. 105:1345-1352, 2000.
40 Harats D, Shaish A, George J, Mulkins M, Kurihara
H, Levkovitz H, Sigal E. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice.
Arterioscler. Thromb. Vasc. Biol. 20: 2100-2105,
2000.

41 Morita H, Kurihara H, Yoshida S, Saito Y, Shindo T,
Oh-hashi Y, Kurihara Y, Yazaki Y, Nagai R. Dietinduced hyperhomocysteinemia exacerbates neointima formation in rat carotid arteries after baloon
injury. Circulation 103: 133-139, 2001.
42 Ho MCY, Lo ACY, Kurihara H, Yu ACH, Chung SK.
Endothelin-1
protects
astrocytesfromhypoxic/ischemic injury. FASEB J. 15: 618-626, 2001.
43 Imai T, Morita T, Shindo S, Nagai R, Yazaki Y,
Kurihara H, Suematsu M, Katayama S. Vascularsmooth muscle cell-directed overexpression ofhuman heme oxygenase-1 elavates blood pressure
through attenuation of NO-induced vasodilation in
mice. Circ. Res. 89:55-62, 2001.
44 Yamaguchi Y, Fukuhara S, Nagase T, Tomita T,
Hitomi S, Kimura S, Kurihara H, Ouchi Y. A novel
mouse ß-defensin, mBD-6, predominantly expressedin skeletal muscle. J. Biol.Chem. 276:31510-31514,
2001.
45 Imai Y, Shindo T, Maemura K, Kurihara Y, Nagai R,
Kurihara H. Evidence for the physiological andpathological roles of adrenomedullin from genetic
engineering in mice. Ann. NY. Acad. Sci. 947:26-34,
2001.
46 Shindo T, Kurihara Y, Nishimatsu H, Moriyama N,
Kakoki M, Wang Y, Imai Y, Ebihara A, Kuwaki T, Ju
K-H, Minamino N, Kangawa K, Ishikawa T, Fukuda
M, Akimoto Y, Kawakami H, Imai Y, Imai T, Morita
H, Yazaki Y, Nagai R, Hirata Y, Kurihara H. Vascularabnormalities and elevated blood pressure in micelacking adrenomedullin gene. Circulation 104:19641971, 2001.
47 Oh-hashi Y, Shindo T, Kurihara Y, Imai T, Wang Y,
Morita H, Imai Y, Kayaba Y, Nishimatsu H, Suematsu
Y, Hirata Y, Yazaki Y, Nagai R, Kuwaki T, Kurihara
H. Elevated sympathetic nervous activity in mice
deficient in aCGRP. Circ. Res. 89:983-990, 2001.
48 Shindo T, Kurihara H, Maemura K, Kurihara Y, Ueda
O, Suzuki H, Kuwaki T, Ju K-H, Wang Y, Morita H,
Oh-hashi Y, Kumada M, Nagai R, Yazaki, Kimura K.
Renal damage and salt-dependent hypertension inaged transgenic mice overexpressing endothelin-1. J.
Mol. Med. 80:105-116, 2002.
49 Imai Y, Shindo T, Sata M, Memura K, Saito Y,
Akishita M, Osuga J, Ishibashi S, Tobe K, Maekawa
H, Oh-hashi Y, Morita H, Kurihara Y, Yazaki Y, Nagai
R, Kurihara H. Resistance to neointimal hyperplasia
and fatty streak formation in adrenomedullin overexpression mice. Arterioscler. Thromb. Vasc. Biol.
22: 1310-1315, 2002.
50. Yamaguchi Y, Nagase T, Makita R, Fukuhara S,
Tomita T, Tominaga T, Kurihara H, Ouchi Y.
Identification of multiple novel epididymis-specificb-defensin isoforms in humans and mice. J.
Immunol. 169: 2516-2523, 2002.

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