From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
endothelial junctions. It is possible that other
related GTPases, like Rap1A, may compensate in this aspect.
Conflict-of-interest disclosure: The authors
declare no competing financial interests. ■
REFERENCES
1. Bos JL. Linking Rap to cell adhesion. Curr Opin Cell
Biol. 2005;17:123-128.
2. Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM, et al. Rap1b is required for normal platelet
function and hemostasis in mice. J Clin Invest. 2005;115:
680-687.
3. Fukuhara S, Sakurai A, Yamagishi A, et al. Vascular
endothelial cadherin-mediated cell-cell adhesion regulated
by a small GTPase, Rap1. J Biochem Mol Biol. 2006;39:
132-139.
4. Glading A, Han J, Stockton RA, Ginsberg MH. KRIT1/CCM1 is a Rap1 effector that regulates endothelial cell
cell junctions. J Cell Biol. 2007;1798:247-254.
● ● ● HEMOSTASIS
Comment on Carmona et al, page 2640
Epac1 activation and progenitor-cell
homing
---------------------------------------------------------------------------------------------------------------Domenico Ribatti
UNIVERSITY OF BARI MEDICAL SCHOOL
The article by Carmona and colleagues in this issue of Blood demonstrates that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential.
ndothelial progenitor cells (EPCs) were
initially described by Asahara and coworkers in 1997,1 and the past few years have
seen a rapid expansion of our knowledge of
EPC biology. Prior to the discovery of this cell
type, new-vessel formation was believed to
result from the proliferation of existing endothelial cells. These findings have overturned
the previous dogma that vasculogenesis can
occur only during embryogenesis.
Little is known about the mechanisms of
homing and differentiation of EPCs. Homing
is a complex process, involving the arrest of
circulating cells within tumor microvessels,
extravasation into the interstitium, and incorporation into new vessels, and is dependent on
an interplay between chemokines, chemokine
receptors, intracellular signaling, adhesion
molecules (selectins and integrins), and proteases. Existing data support the involvement
of integrins in the homing of EPCs and progenitor cells to sites of active neovascularization. Chavakis et al have demonstrated that
human adult peripheral-blood– derived EPCs,
murine adult bone marrow– derived EPCs,
and bone marrow– derived hematopoietic progenitor/stem cells express ␤2 integrin.2
In an experimental study published in this
issue of Blood, Carmona and colleagues investigated whether pharmacological activation of
Epac1, a nucleotide exchange protein for Rap1
previously implicated in leukocyte adhesion
E
blood 1 M A R C H 2 0 0 8 I V O L U M E 1 1 1 , N U M B E R 5
and chemotaxis, can improve the adhesive and
migratory capacity of distinct progenitor-cell
populations. The authors demonstrated that
stimulation of Epac1 by a cAMP analog increased Rap1 activity and stimulated the adhesion of ex vivo– expanded human EPCs,
CD34⫹ hematopoietic progenitor cells, and
mesenchymal stem cells. Furthermore, Epac1
activation induced the ␤1- and ␤2-integrin–
dependent migration of EPCs on fibronectin
and fibrinogen, respectively. Finally, prestimulation of EPCs with the Epac1 activator
increased homing to ischemic muscles and the
neovascularization-promoting capacity of intravenously injected EPCs in the murine
model of hind-limb ischemia.
Overall, these data provide insights into
the regulation of integrin activity in progenitor
cells, and unravel a new possibility: to activate
integrin-dependent homing in progenitor cells
by stimulation of Epac1. The molecular and
cellular mechanisms underlying EPC recruitment and differentiation are not completely
understood, and remain as one of the central
issues in stem-cell biology. Gaining knowledge of the molecular mechanisms that allow
homing of different progenitor-cell subpopulations to sites of neovascularization is important for the development of new, specific
therapeutic strategies concerning the efficacy
of cell-based therapies in patients with ischemic diseases, the ability to vascularize various engineered tissues, and the inhibition of
EPC-mediated tumor neovascularization.
Conflict-of-interest disclosure: The author
declares no competing financial interests. ■
REFERENCES
1. Asahara T, Murohara T, Sullivan A, et al. Isolation of
putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964-967.
2. Chavakis E, Aicher A, Heeschen C, et al. Role of beta
2-integrins for homing and neovascularization capacity
of endothelial progenitor cells. J Exp Med.
2005;201:63-72.
● ● ● IMMUNOBIOLOGY
Comment on Chen et al, page 2685
Unveiling
ZAP-70’s plan B
---------------------------------------------------------------------------------------------------------------Amélie Montel-Hagen, Rita Vicente, and Naomi Taylor
INSTITUT DE GÉNÉTIQUE MOLÉCULAIRE DE
MONTPELLIER
In this issue of Blood, Chen and colleagues demonstrate that ZAP-70 enhances
BCR signaling in B-CLL cells by promoting phosphorylation of the ITAMs in the
Ig signaling subunit independently of its kinase activity.
he finding that ZAP-70 is often expressed
in the B-cell chronic lymphocytic leukemia (B-CLL) cells of patients who, in general,
have a more aggressive disease course was one
of the first clinically relevant data to emerge
from gene-expression microarray technology.1
Before then, the ZAP-70 protein tyrosine kinase was thought to be expressed only in Tlineage cells, associated with phosphorylated
T
immunoreceptor tyrosine– based activation
motifs (ITAMs) of the T-cell receptor ␨ chain
following receptor engagement.
Chen, Kipps, and their colleagues have
been instrumental in furthering our understanding of the unexpected association between ZAP-70 expression and poor B-CLL
prognosis. They determined that ZAP-70
enhances Ig receptor signaling in B-CLL
2501
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
cells.2,3 This finding was particularly surprising because B-CLL cells express the related
Syk protein whose kinase activity is 100-fold
higher than that of ZAP-70,4 and which,
unlike ZAP-70, can be activated in an Srckinase–independent fashion.5
In this issue of Blood, Chen and colleagues
shed light on this issue, demonstrating that the
2 functional SH2 domains of ZAP-70 are required for enhanced IgM signaling in B-CLL
cells, whereas the ZAP-70 SH1 kinase domain
is dispensable. In addressing the mechanism(s)
underlying this effect, Chen and coworkers
eliminate the possibility that ZAP-70 facilitates Ig-receptor signaling by competing for
binding to c-Cbl, an E3 ubiquitin ligase that
targets proteins for proteosomal degradation.
Indeed, the enhancing role of ZAP-70 is independent of its ability to interact with c-Cbl.
The second hypothesis that the authors go
on to establish is that ZAP-70, but not Syk,
enhances phosphorylation of the ITAMs in
the Ig signaling subunit, the CD79a/CD79b
(Ig-␣/Ig-␤) heterodimer. They find significantly higher levels of CD79b phosphorylation following anti-␮ treatment in ZAP-70 –
expressing B-CLL cells than in their ZAP70 –negative counterparts. This hypothesis is
partially at odds with the conventional “ordered” model of TCR/BCR signaling,
wherein phosphorylation of ITAM tyrosines
is mediated solely by Src-family kinases. However, several lines of evidence had already suggested that, at least in T cells, this simplified
model might be flawed. In murine thymocytes
as well as human T cells, ZAP-70, but not Syk,
was shown to promote phosphorylation of
ITAMs in the TCR-␨ chain, an effect that was
also independent of ZAP-70 kinase activity.6,7
The authors’ “plan B” proposes that ZAP-70
enhances BCR signaling in B-CLL cells by promoting phosphorylation of the ITAMs in the Ig
signaling subunit. In this context, it is notable
that Hou et al8 recently showed that BCR phosphorylation and internalization are mutually
exclusive events. Specifically, the same tyrosine
residues in the BCR complex regulate 2 distinct
fates; phosphorylation results in signaling,
whereas the absence of phosphorylation results
in receptor internalization.8 While it is crucial to
determine whether this phenomenon occurs in
B-CLL cells, support for this proposition comes
from the recent demonstration that introduction
of ZAP-70 into the ZAP-70 –negative BJAB
lymphoma cell line leads to decreased ligandmediated BCR internalization, even though ectopic ZAP-70 has very low catalytic activity.9
Furthermore, using an analog-sensitive Syk
mutant, a very recent study showed that the
length of time for which anti–IgM-BCR complexes are retained at the cell surface significantly
shapes signaling fates; NF␬B is immediately
activated, whereas activation of NFAT requires
that BCR complexes and the associated Syk molecule be phosphorylated for more than 1 hour.10
These effects might be expected to be more pronounced in B-CLL cells, in which levels of
CD79b are significantly lower than on normal
B lymphocytes.11,12 In this model, downstream
BCR signaling in B-CLL cells is mediated by the
Syk kinase, but optimal cell-surface expression
and phosphorylation of the BCR requires the
presence of the ZAP-70 kinase (see figure).
Many questions remain, including the phosphorylation status of the Ig-␣/Ig-␤ heterodimer,
the relative association of ZAP-70 versus Syk with
the Ig-␣/Ig-␤ heterodimer in B-CLL cells as well
as in normal B cells, the ZAP-70–dependent enhancement of signaling in B-CLL cells but not
normal B cells, and finally, the mechanisms responsible for the distinct role of ZAP-70 in promoting
CD79b phosphorylation in B-CLL cells. Irrespective of the answers, the present work by Chen and
colleagues unveils a new ZAP-70–dependent
pathway wherein the catalytic activity of ZAP-70
does not appear to contribute to the aggressive
nature of ZAP-70⫹ B-CLL. This has significant
ramifications for the development of new therapeutic strategies for B-CLL patients, targeting
ZAP-70 and/or the BCR itself.
A.M.-H. and R.V. contributed equally to this
work and are listed alphabetically.
Conflict-of-interest disclosure: The authors
declare no competing financial interests. ■
REFERENCES
1. Rosenwald A, Alizadeh AA, Widhopf G, et al. Relation
of gene expression phenotype to immunoglobulin mutation
genotype in B cell chronic lymphocytic leukemia. J Exp
Med. 2001;194:1639-1647.
2. Chen L, Widhopf G, Huynh L, et al. Expression of
ZAP-70 is associated with increased B-cell receptor signaling
in chronic lymphocytic leukemia. Blood. 2002;100:4609-4614.
3. Chen L, Apgar J, Huynh L, et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia.
Blood. 2005;105:2036-2041.
4. Latour S, Chow L, Veillette A. Differential intrinsic
enzymatic activity of Syk and Zap-70 protein- tyrosine kinases. J Biol Chem. 1996;271:22782-22790.
5. Zoller KE, MacNeil IA, Brugge JS. Protein tyrosine
kinases Syk and ZAP-70 display distinct requirements
for Src family kinases in immune response receptor signal transduction. J Immunol. 1997;158:1650-1659.
6. Ashe JM, Wiest DL, Abe R, Singer A. ZAP-70 protein
promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(⫹)8(⫹) thymocytes with limiting p56(lck). J Exp Med. 1999;189:1163-1168.
7. Steinberg M, Adjali O, Swainson L, et al. T-cell receptorinduced phosphorylation of the zeta chain is efficiently promoted by ZAP-70 but not Syk. Blood. 2004;104:760-767.
8. Hou P, Araujo E, Zhao T, et al. B cell antigen receptor
signaling and internalization are mutually exclusive events.
PLoS Biol. 2006;4:e200.
9. Gobessi S, Laurenti L, Longo PG, Sica S, Leone G,
Efremov DG. ZAP-70 enhances B-cell-receptor signaling
despite absent or inefficient tyrosine kinase activation in
chronic lymphocytic leukemia and lymphoma B cells.
Blood. 2007;109:2032-2039.
10. Oh H, Ozkirimli E, Shah K, Harrison ML, Geahlen
RL. Generation of an analog-sensitive Syk tyrosine kinase
for the study of signaling dynamics from the B cell antigen
receptor. J Biol Chem. 2007;282:33760-33768.
ZAP-70 enhances BCR signaling in B-CLL cells by promoting CD79 phosphorylation. In the proposed model, the
increased levels of CD79a and CD79b phosphorylation in B-CLL cells expressing ZAP-70 are dependent on the
presence of the 2 ZAP-70 SH2 domains. The mechanisms via which ZAP-70 enhances phosphorylation of
CD79a/CD79b are not yet known, but may be the result of a direct interaction between the ZAP-70 SH2 domains
and the phosphorylated ITAMs. The phosphorylation of CD79a/CD79b would result in an augmented phosphorylation of Syk and activation of downstream signaling cascades while limiting BCR internalization. In the absence
of ZAP-70, the decreased phosphorylation of CD79a/CD79b would limit Syk phosphorylation and downstream
signaling but would result in significantly higher levels of BCR internalization.
11. Vuillier F, Dumas G, Magnac C, et al. Lower levels of
surface B-cell-receptor expression in chronic lymphocytic
leukemia are associated with glycosylation and folding defects
of the mu and CD79a chains. Blood. 2005;105:2933-2940.
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1 MARCH 2008 I VOLUME 111, NUMBER 5
12. Zomas AP, Matutes E, Morilla R, Owusu-Ankomah K,
Seon BK, Catovsky D. Expression of the immunoglobulinassociated protein B29 in B cell disorders with the monoclonal
antibody SN8 (CD79b). Leukemia. 1996;10:1966-1970.
blood
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2008 111: 2501-2502
doi:10.1182/blood-2007-12-128348
Unveiling ZAP-70's plan B
Amélie Montel-Hagen, Rita Vicente and Naomi Taylor
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