April 28, 2003
Purification of Signaling Protein May Boost Tissue Engineering
The purification of a powerful signaling molecule that coaxes cells
to mature may also signal the beginning of a new era in tissue
engineering.
In newly published studies in the journal Nature, researchers
show that purified Wnt protein, long known as a potent trigger of
development and cell proliferation, can also cause blood-forming stem
cells to proliferate.
“With these studies, we can now imagine isolating and expanding a patient’s stem cells using activated Wnt proteins before they are treated with chemotherapy which destroys their immune system.”
Roel Nusse
The discovery suggests novel ways to enhance stem cells to restore
the blood-forming systems of cancer patients whose cells have been
destroyed by chemotherapy.
The research on Wnt activation and its role promoting the
development of blood-forming (hematopoietic) stem cells was reported in
two related articles published online on April 28, 2003, in
Nature.
Howard Hughes Medical Institute investigator Roel
Nusse and Karl Willert at Stanford University School of Medicine
led one team that reported that the addition of a lipid molecule is
necessary for the activation of Wnt proteins. Co-authors on that
article include Jeff Brown and Esther Danenberg, as well as researchers
in the laboratory of Irving Weissman, also of Stanford Medical School.
Weissman led a second group of scientists who reported in Nature that
activated Wnt is crucial for self-renewal of hematopoietic stem cells.
Lead authors on that paper were Tannishtha Reya and Andrew Duncan, now
at Duke University Medical Center.
According to Nusse, researchers had not isolated active Wnt proteins
before — a long sought-after goal since the proteins play important
roles in embryonic development and in controlling the proliferation of
stem cells. Nusse and Harold Varmus, President of Memorial
Sloan-Kettering Cancer Center and a member of the HHMI medical advisory
board, were members of one of the research teams that discoverered Wnt
genes. They identified Wnt genes as oncogenes activated in mouse breast
cancer.
Pluripotent embryonic stem cells are immature cells that have the
capability to mature into a wide range of blood and tissue cells. They
may to hold the key to restoring compromised immune systems and even
regenerating tissues and organs damaged by disease or trauma. Even
though such stem cells have been isolated, inducing them to proliferate
for use in treatment has been only marginally successful.
“It has always been predicted that the Wnt proteins could act
as growth factors that could be added to cells and direct them into a
particular growth pathway without genetically changing the
cells,” said Nusse. Central to this process, he said, has been
purifying and characterizing the active form of Wnt proteins, which has
proven especially frustrating.
“We now understand that this protein is modified by the
attachment of a lipid after it is produced, which gives it a tendency
to stick to cell membranes where it is active,” said Nusse.
“However, that means the protein also was not soluble and would
stick to containers, so standard purification techniques didn't
work.”
Nusse and his colleagues developed techniques that used detergents
to render the protein soluble, and also robust cell assays that would
measure the biological activity of the protein. “It was a real
challenge to get the methods to work,” said Nusse,
“requiring all kinds of biochemical tricks.”
In their latest work, Nusse and his colleagues showed that one
member of the Wnt protein family, mouse Wnt3a, is activated by the
attachment of a lipid called palmitoyl to a particular amino acid on
the protein. The lipid is necessary for activation, as is the presence
of the attached amino acid, cysteine, which is conserved in all Wnt
proteins, said Nusse. The researchers may well have found the key
mechanism of activation for the multitude of Wnt proteins.
Next, Nusse, Weissman and their colleagues tested the effects of the
activated Wnt protein on hematopoietic stem cells. They found that the
protein greatly enhanced stem cell proliferation in the test tube,
while maintaining the stem cells in their immature state. The
researchers also observed that Wnt-treated stem cells retained their
activity and were able to reconstitute the blood-forming systems of
mice that had been irradiated to destroy their hematopoietic cells.
The accompanying Nature paper by Reya, Duncan, Weissman and
their colleagues demonstrated that the Wnt signaling pathway plays a
crucial role in hematopoietic stem cell self-renewal. The researchers
showed that the protein specifically affected by Wnt — beta catenin —
is necessary for stem cell proliferation, as is the Wnt protein
itself.
“With these studies, we can now imagine isolating and
expanding a patient's stem cells using activated Wnt proteins before
they are treated with chemotherapy which destroys their immune
system,” said Nusse. “Those proliferated cells could then
provide a powerful way to restore the hematopoietic system. And since
Wnt is a specific growth factor and doesn't fundamentally alter the
nature of the cells, there is no danger that the cells will take on
unwanted properties.”
According to Nusse, over-activation of the Wnt signaling pathway due
to genetic mutation has been implicated in some cancers. Thus, he said,
discovery of the nature of Wnt activation in cells will enable
researchers to mimic the cancer process experimentally, to study its
mechanism.
Nusse and his colleagues are now studying other Wnt proteins to
determine whether they require the same mechanism for activation and
whether they, too, can trigger proliferation of other types of stem
cells. In some ways, Nusse and his colleagues have a head start - in
collaboration with HHMI investigator Jeremy
Nathans at The Johns Hopkins University School of Medicine they
have already identified the specific cell surface receptors, known as
Frizzleds, to which the Wnt proteins bind to activate the cells.
“We're trying also to understand why cells respond to
particular Wnt proteins by looking at the expression of the specific
receptor for the Wnts,” said Nusse.
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