NEWS AND VIEWS
remain to be defined. Identifying targets will be ubiquitin conjugation is still far from being
essential for fully understanding how E2 acti-
vation and karyopherin-mediated localization
might be used to regulate ubiquitin conjugation
to specific substrates.
In summary, the findings of Plafker and col-
leagues reveal a fascinating new mechanism
through which ubiquitin conjugation may be
regulated by E2 activation and nuclear import,
and they demonstrate that the regulation of
Hearing the messenger: Ins(1,4,5)P3 and deafness
Roberto Bruzzone and Martine Cohen-Salmon
Genetic studies have conclusively linked connexin channels to human diseases, but the nature of the signals that are disrupted
by channel mutations has remained elusive. A recent study has taken advantage of a deafness-causing mutation to suggest that
permeability to inositol trisphosphate, the Ca2+-mobilizing messenger, is crucial for normal hearing.
The first genetic disease associated with con-
nexin mutations was discovered about ten years
ago, and since then eight more connexin genes
have been implicated in human hereditary dis-
orders affecting different organs1. Despite all the
information provided by animal models and by
electrophysiological studies, a notable progress
into understanding the sequence of events that
occurs in connexin-based disorders has been
lacking. One of the reasons is perhaps inherent
to the uniqueness of connexin channels, which
allow the exchange not only of ions, but also of
small molecules between the cytoplasmic envi-
ronment of neighbouring cells. Although the
finding that most connexin mutations result in
a loss of function suggests that it is a deficit of
intercellular communication that leads to the
pathological phenotype1, a lingering question
has remained unanswered: what is it exactly
that needs to be exchanged? Ions? Metabolites?
Second messengers? On page 63 of this issue2,
a study by Beltramello et al. has delivered the
first answer by pinning down one of the usual
suspects, the Ca2+-releasing messenger inosi-
tol trisphosphate (Ins(1,4,5)P3), thus provid-
ing a plausible hypothesis to account for the
pathogenesis of DFNB1, the most frequent
form of deafness.
Roberto Bruzzone and Martine Cohen-Salmon are in
the Department of Neuroscience, Institut Pasteur,
75015 Paris, France.
e-mail: bruzzone@pasteur.fr
14
Jan 05 N&V Final.indd 14
272, 10895–10903 (1997).
fully understood.
1. Pickart, C. M. Annu. Rev. Biochem. 70, 503–533
(2001).
2. Fang, S. & Weissman, A. M. Cell. Mol. Life Sci. 61,
1546–1561 (2004).
3. Plafker, S. M., Plafker, K. S., Weissman, A. M. & Macara,
I. G. J. Cell Biol. 167, 649–659 (2004).
4. Plafker, S. M. & Macara, I. G. EMBO J. 19, 5502–5513
(2000).
5. Stephen, A. G., Trausch-Azar, J. S., Handley-Gearhart,
P. M., Ciechanover, A. & Schwartz, A. L. J. Biol. Chem.
Although inherited deafness is genetically
heterogeneous, mutations in the gene encod-
ing connexin 26 (Cx26) have been shown to
account for a large proportion of cases in every
population tested, and two other connexins,
Cx30 and Cx31, have also been linked to sen-
sorineural hearing loss3. In the cochlea, which
is the auditory organ, Cx26 and Cx30 proteins
exhibit overlapping expression patterns4 that
define two main communication compart-
ments (Fig. 1): an epithelial network, compris-
ing supporting cells and adjacent epithelial
cells, and a connective network, both of which
participate in the regulation of the unique ionic
composition of extracellular fluids.
A key aspect of the physiology of the mam-
malian cochlea is the ionic composition of
the extracellular environment that surrounds
the hair cells, the cells that sense sound and
initiate signals to afferent nerve fibres. Their
basolateral surface is bathed in perilymph, a
fluid with normal extracellular salt composi-
tion (high Na+, low K+), but their apical surface
is contacted by endolymph, which has a salt
composition that resembles intracellular fluids
(high K+, low Na+). Another unique feature is
the very high trans-epithelial potential between
the endolymphatic and perilymphatic compart-
ments, which is necessary to trigger the influx
of K+ into hair cells that depolarizes them after
sound stimulation. K+ is, in turn, transported
into the interstitial space of the organ of Corti,
NATURE CELL BIOLOGY VOLUME 7 | NUMBER 1 | JANUARY 2005
©2005 Nature Publishing Group
6. Weis, K. Cell 112, 441–451 (2003).
7. Hamilton, M. H., Tcherepanova, I., Huibregtse, J. M. &
McDonnell, D. P. J. Biol. Chem. 276, 26324–26331
(2001).
8. Tao, W. & Levine, A. J. Proc. Natl Acad. Sci. USA 96,
6937–6941 (1999).
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& Schwartz, A. L. J. Biol. Chem. 271, 15608–15614
(1996).
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29037 (1999).
11. LaCasse, E. C. & Lefebvre, Y. A. Nucleic Acids Res. 23,
1647–1656 (1995).
where it is partly taken up by cochlear support-
ing cells5. Hence, Cx26 and Cx30 intercellular
channels between epithelial cells are believed
to delineate an intercellular pathway to remove
the excess K+ that results from auditory signals,
providing a spatial buffering function that is
similar to astrocytic gap junctions in the brain.
Such a role for gap junctions in the cochlea has
been further supported by the finding that mice
with a targeted deletion of Cx26 in the epithe-
lial network were deaf due to degeneration of
the organ of Corti6. Because cell death occurred
at the time of hearing onset, it was thought to
result from impaired K+ disposal.
There are some problems with this K+
hypothesis. The foremost being the presence
in the same epithelial network of another con-
nexin, Cx30, which is perfectly capable of car-
rying K+ currents7, but cannot functionally
compensate for the absence of Cx26 (ref. 6).
The inability of other connexins to function-
ally compensate for specific connexin mutants
is a common feature of connexin-based disor-
ders (the crystalline lens is another example1),
and has been interpreted as an indication that
a specific complement of connexin channels
is required to meet the physiological needs of
a specific tissue. We now know that all con-
nexins are not made equal; there are differ-
ences in size and ionic selectivity, differences
in the rules of compatibility between available
partners, and distinct gating mechanisms.
10/12/04 5:28:11 pm
 NEWS AND VIEWS
a
mutation alters the channel permeability to a
second messenger molecule. The authors turned
their attention to Ca2+ signalling because Ca2+
waves, transfer of Ins(1,4,5)P3 and gap junc-
Perilymph tions have long been linked together (although
K+ 3−5 mM
other mechanisms may be implicated)12. They
used state-of-the-art techniques to demonstrate
Endolymph that V84L channels exhibit an impaired perme-
K+
150 mM
ability to Ins(1,4,5)P3 that results in a reduced
+80 mV ability to propagate Ca2+ waves. The evidence
can be summarized as follows. First, compari-
son between Cx26 wild-type and V84L in pairs
of transfected HeLa cells (a well-characterized
expression system) shows that they are indis-
tinguishable in terms of unitary conductance
Perilymph
K+ and open time probability, meaning that pairs
3−5 mM with similar macroscopic conductance would
Endolymph have a similar number of open channels at any
b K+ Cl− K+ Cl− K+ Cl− given time. Under these controlled conditions,
permeability to Lucifer Yellow — a fluorescent
K+ K+ tracer that is discriminated by certain connexin
Ca2+ Ca2+ Ca2+
K+ K+ channels13 — was also comparable. In contrast,
K+ when Ins(1,4,5)P3 was delivered to an individual
cell through a patch pipette, notable differences
Ins(1,4,5)P3 Ins(1,4,5)P3 Ins(1,4,5)P3 Ins(1,4,5)P3 were observed in the magnitude and kinetics
of Ca2+ changes in connected cells. Beltramello
et al. further calculated that the apparent per-
meability coefficient of V84L for Ins(1,4,5)P3 is
only 8% of that of wild-type Cx26, thus estab-
lishing that the mutated channels must have a
ATP ATP ATP
structural modification that reduces the passage
Cx26 wild-type
of Ins(1,4,5)P3, without compromising that of
Figure 1 Proposed role of Ins(1,4,5)P3 propagation through connexin channels in auditory transduction. other ions. Furthermore, they found that per-
(a) Schematic representation of a transverse section of the cochlear duct. The endolymphatic (yellow) meability of Cx30 to Ins(1,4,5)P3 was signifi-
compartment is delimited by tight junctions (red line). Perilymph (blue) bathes the rest of the cochlea. cantly lower than that of Cx26. Although we
Cx26 and Cx30 are co-expressed in the supporting cells of the epithelial network (green) and in the
connective-tissue network (brown). Yellow, the bone forming the otic capsule; red, capillaries. Sensory
do not know the precise molecular composition
transduction depends on a K+ current (black arrows) that flows from the endolymph through sensory of gap-junction channels in supporting cells,
hair cells (blue rods). K+ is subsequently recycled and different routes have been proposed (dashed whether they are mostly homotypic or mixed,
lines). (b) During sound stimulation, K+ is released by hair cells and enters supporting cells by means Beltramello et al. argued that Ins(1,4,5)P3 per-
of a K+/Cl− co-transporter, Kcc4 (black circle)5. K+ is then removed (black arrows) through gap-junction
channels. Cx26 wild-type (orange) also permits the intercellular passage (black arrows) of Ins(1,4,5)P3
meability would be significantly reduced in the
and the propagation of Ca2+ waves. Although the precise mechanism of the initial increase of this second presence of two V84L alleles.
messenger has not been elucidated, the result is the long-range activation of a K+/Cl− efflux system How can a change in the diffusion of
that recycles K+ from supporting cells into the endolymph (green arrows), preventing ionic overload. Ins(1,4,5)P3 be related to deafness? Here the
Intercellular waves also require activation of purinergic receptors (black) by a paracrine route (red
arrows). Whether ATP release is mediated by connexin hemi-channels (cyan) or by another mechanism answer is not so straightforward. To their credit,
remains to be investigated. The mutated V84L channels exhibit a permeability defect that greatly Beltramello et al. pushed their observations using
compromises both the transfer of Ins(1,4,5)P3 (but not of K+ and other ions) and the magnitude of Ca2+ organotypic cultures of the rat cochlea, and found
changes. These alterations could lead to a reduction of co-transport activity, accumulation of K+ ions in
that: first, Ins(1,4,5)P3 induced the propagation of
the extracellular space that surrounds hair and supporting cells and, further, to their excitotoxic death.
Ca2+ waves in supporting cells; and second, such
These characteristics result in selective per- ment of connexins expressed. The identifica- waves required both diffusion of Ins(1,4,5)P3
meability of distinct connexins to endog- tion in DFNB1 patients of a recessive Cx26 through gap junctions as well as release of ATP
enous metabolites and second messengers8–10. mutation, V84L, that did not appreciably affect into the extracellular medium. As Ca2+ activates
Although the degree of these differences is not basic channel properties, seemed to be the ideal a combined K+/Cl efflux from supporting cells,
on the order of the exquisite selectivity of most tool to look for molecules that would be specifi- they propose that the permeability defect of
ion channels, the transient nature of second cally excluded from the mutated channels. V84L channels may interfere with K+ recycling.
messenger elevation suggests that spreading The study by Beltramello et al. documents for The ensuing perturbation of the ionic balance of
of signals would be dependent on the comple- the first time that a disease-causing connexin cochlear fluids would lead to the accumulation of

NATURE CELL BIOLOGY VOLUME 7 | NUMBER 1 | JANUARY 2005 15

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Jan 05 N&V Final.indd 15 10/12/04 5:28:16 pm

 NEWS AND VIEWS

K+ ions and, further, to excitotoxic death of hair
and supporting cells.
Of course, there are still a number of unre-
solved issues that should be addressed to vali-
date this model. First of all it should be borne
in mind that, although the recorded Ca2+
waves resemble those triggered by mechani-
cal damage or pharmacological stimulation
of purinergic receptors14, there is no evidence
that such intercellular signalling between
supporting cells occurs physiologically dur-
ing audition. In vivo imaging studies will be
needed to address this crucial issue. Then,
if the model is correct, one would predict
that a reduction in the propagation of Ca2+
waves should occur in mice with targeted
inactivation of Cx26 in the epithelial network.
Conversely, inactivation of Cx30, which has
a low Ins(1,4,5)P3 permeability and cannot
functionally compensate for the loss of Cx26,
should not interfere with Ca2+ waves. Finally,
replacement of Cx26 with V84L should gener-
ate mice with hearing loss.
It is tempting to speculate that defects in
the diffusion of second messengers across gap
junctions may offer a mechanistic explana-
tion in other connexin-based disorders. In
fact, a defective transfer of cyclic AMP had
already been suggested15, albeit on the basis
of indirect evidence, as the basis of the X-
linked form of Charcot-Marie-Tooth, which
is caused by mutations in Cx32.
1. Gerido, D. A. & White, T. W. Biochim. Biophys. Acta
1662, 159–170 (2004).
2. Beltramello, M. et al. Nature Cell Biol. 7, 63–69
(2005).
3. Petit, C., Levilliers, J. & Hardelin, J. P. Annu. Rev.Genet.
35, 589–646 (2001).
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(1998).
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Biol. Chem. 273, 2808–2816 (1998).
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Wnt muscles up to CREB a

Injected

The role of Wnt proteins in development runs far and wide. Despite these
many contexts in which to study the Wnt pathway, what lies between Wnt
and specific gene expression is still not clear. Most factors in the canonical
Wnt/β-catenin pathway have already been implicated in cell-fate-specific
gene activation, but lately it is non-canonical Wnt targets that have taken
centre stage. Chen et al. (Nature, DOI:10.1038/nature03126; 2004) now
report for the first time a link between Wnt and PKA-dependent CREB
signalling during myogenesis.
Using antibodies that recognize the active, phosphorylated form of
CREB on mouse embryonic sections, the authors observed that phos-
pho-CREB and the myogenic transcription factors Pax3, MyoD and
Myf5 are expressed in overlapping patterns. This prompted them to
examine whether CREB-null mutants have defects in muscle develop-
ment. In situ staining revealed that in CREB-null embryos, the levels
of each of these transcription factors is reduced compared with wild-
type. Coincidently, the authors also found that two other CREB-family
members, ATF-1 and CREM, were upregulated enough to at least par-
tially compensate for the CREB-null phenotype. They confirmed this
by injecting an adenoviral inhibitor (Ad–ACREB) that targets all CREB
family members: the myogenic defects were now much more severe, thus
reinforcing a role for CREB in myogenesis.
The phenotype seen when CREB was lost was highly reminiscent of
that previously observed for mutants lacking Wnt1 or Wnt3a; thus, Chen
et al. tested whether CREB might form part of the Wnt signalling path-
way. Similarly to previous reports, both Wnt proteins induced expression
of Pax3, MyoD and Myf5 in a co-culture assay. Importantly, Ad–ACREB
infection reduced expression of all three myogenic factors, without
affecting expression of the canonical Wnt/β-catenin pathway targets.
Consistent with this, elevated phospho-CREB levels were observed in
response to expression of both Wnt1 and Wnt3a.
So can Wnt regulate CREB-induced transcription directly? Chen et al.
see that both Wnt1 and Wnt7a potently induce transcription from the
cyclic AMP-responsive element (CRE) promoter. By testing a barrage
of inhibitors and dominant-negative mutants, they conclude that it is
non-canonical Wnt signalling — specifically the adenylyl cyclase (AC)
Uninjected
b
Uninjected
Injected
Interfering with CREB family function in vivo inhibits myogenesis. Control (a) or
ACREB-expressing (b) adenoviruses were microinjected into E9.75 somites between
the forelimb and hindlimb on one side of mouse embryos. Pax3 expression (purple)
was analysed by in situ hybridization. Reproduced, with permission, from Nature,
DOI:10.1038/nature03126 © (2004) Macmillan Magazines Ltd.
cascade — that activates CREB-mediated transcription. They also show
that the AC-dependent activation of CREB is physiologically relevant
for Wnt-induced myogenesis. Increasing AC activity (or elevating cAMP
levels) specifically enhanced expression of MyoD and Myf5 induced by
Wnt1, whereas blocking AC or PKA activity inhibited both Wnt1- and
Wnt7a-mediated myogenesis, both in cultures and in vivo.
Thus, the findings by Chen et al. suggest the existence of a novel Wnt-
mediated myogenic pathway involving PKA, AC and CREB. Although
it is unclear whether CREB directly activates expression of any of the
myogenic genes examined, it constitutes an important addition to the
vast array of Wnt effectors. The question now is whether this pathway
extends its range to other developmental programmes, not least of
which, neurogenesis.
MYRTO RAFTOPOULOU

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