Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
The orientation of cell division plays a central role in embryonic development, determining the asymmetric expression patterns of developmental fate regulators and tissue morphology. Although well studied in Caenorhabditis elegans and Drosophila melanogaster, little is known about oriented cell division in vertebrates. Gong et al. have now analyzed mitotic divisions during zebrafish gastrulation to show that cells present in dorsal tissues preferentially divide along the animal-vegetal (AV) axis of the embryo under the control of non-canonical Wnt signaling.
The dorsal epiblast, which gives rise to the neural ectoderm, consists of two to three layers of cells that divide twice during gastrulation. The authors monitored these mitotic divisions throughout the depth of the epiblast using four-dimensional confocal microscopy. Analysis of wild-type zebrafish gastrulae showed that cell divisions in all layers of the dorsal epiblast oriented along the AV axis. Previously, geometric constraints were thought to align the mitotic spindle with the cell's long axis ('Hertwig's rule'). However, this does not apply in zebrafish gastrulation, as most epiblast cells divided at right angles to their long axis. This suggests that the mitotic spindle is actively oriented in the dorsal epiblast.
Dishevelled
(Dsh) has been implicated in polarized cell division in both Drosophila
and C. elegans. Injection of a mutant form of Xenopus
Dsh (Xdd1), which blocks axis elongation, severely disrupted the
AV alignment in all layers of the epiblast in zebrafish embryos.
Xdd1 also disrupted the convergence and extension of the dorsal
tissue. Mosaic analysis demonstrated that Dsh exerts a direct,
cell-autonomous effect on division orientation.
Dsh is involved in multiple Wnt pathways, including the canonical
Wnt/β-catenin
pathway and the non-canonical pathway related to the Drosophila
planar cell polarity (PCP) pathway. To distinguish which pathway
is involved in oriented cell division, the authors used three
different Dsh deletion constructs. While each exerted a different
effect on canonical Wnt signaling, all inhibited division alignment.
Inhibition of the Wnt canonical pathway with specific antagonists
had no effect on the AV alignment of divisions, confirming that
the canonical Wnt pathway is not directly involved in the control
of oriented cell division.
The authors found that loss-of-function of the PCP factor wnt11
gave rise to less AV polarity and that Wnt5a
appears to act in parallel with Wnt11.
Disrupting Strabismus
(Stbm), which modulates PCP but does not lie in a linear cascade
with Wnt/Dsh, also resulted in misaligned mitotic division. Oriented
division accounted for a significant amount of zebrafish gastrula
AV axis extension, and the authors propose that the PCP pathway
regulates both oriented cell division and cell intercalation through
the polarization of the cell. Together with previous findings
in C. elegans and Drosophila, this work confirms
that the PCP pathway has an evolutionarily conserved role in vertebrate
development, and raises the possibility that non-canonical Wnt
signaling regulates oriented cell division universally.
Brenda Riley, Assistant Editor Signaling Gateway
article
Ying Gong, Chunhui Mo & Scott E. Fraser Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation Nature, 430, 689 - 693 (05 August 2004); doi:10.1038/nature02796
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Cytokines are part of a large family of secreted proteins that regulate a range of cellular functions — including proliferation, survival and maturation. Cytokines are essential mediators of the immune response and are implicated in a diverse set of diseases. Over 60 cytokines have been described so far, and now Dillon et al. report the discovery of yet another: interleukin 31 (IL-31), which is implicated in the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.
Infiltrating T cells that persist in the skin, and the cytokines they produce, are thought to be involved in the development of pruritis and allergic and non-allergic conditions, including psoriasis and both atopic and non-atopic dermatitis (inflamed skin conditions). Cytokines mediate their functions through ligand-induced oligomerization of a dimeric receptor complex. A subset of cytokine receptors, classified as type 1, shows common structural features within the extracellular domain. Further classification shows a subfamily of type 1 receptors composed of gp130-related chains, which includes the oncostatin M receptor (OSMR) and the orphan receptor GLMR.
By screening translated human genomic sequences for homologies to known cytokine receptor sequences, Dillon et al. identified four splice variants of a gp130-like type 1 cytokine receptor, IL-31RAv1 to IL-31RAv4, one of which is highly homologous to GLMR. Using a functional cloning approach, the cognate cytokine for at least two of the receptor splice variants, IL-31, was identified. IL-31 was secreted rapidly after TH2 cell activation, and the authors showed that it signals through a heterodimeric receptor composed of IL-31RA and OSMR that is expressed on both epithelial cells and keratinocytes. These cells responded to IL-31 stimulation and are likely to be involved in the dermatitis and pruritis observed in transgenic mice that overexpress IL-31. Similar pathologies were observed after systemic administration of IL-31 to wild-type mice, and mice deficient of IL-31RA are phenotypically normal and refractile to IL-31 administration.
IL-31 stimulation induced a variety of chemokines, indicating that IL-31 may function in the recruitment of polymorphonuclear cells, monocytes and T cells to sites of skin inflammation in vivo. Furthermore, transgenic mice overexpressing IL-31 developed piloerection, followed by mild-to-severe alopecia (hair loss), and were also highly pruritic (itchy). Finally, Dillon et al. showed that IL-31 receptors were upregulated in lung epithelium and bronchoalveolar lavage cells derived from an animal model of airway hypersensitivity.
The characterization of IL-31 by Dillon et al. suggests that this T cell-derived cytokine may be involved in promoting skin disorders and in regulating other allergenic diseases, such as asthma, adding this cytokine to the long list of known mediators of these conditions. Targeting IL-31 may prove to be an effective approach in the treatment of these diseases.
Jon Reynolds, Copy Editor Nature Cell Biology
article
Stacey R. Dillon, Cindy Sprecher, Angela Hammond, Janine Bilsborough, Maryland Rosenfeld-Franklin, Scott R. Presnell, Harald S. Haugen, Mark Maurer, Brandon Harder, Janet Johnston, Susan Bort, Sherri Mudri, Joseph L. Kuijper, Tom Bukowski, Pamela Shea, Dennis L. Dong, Maria Dasovich, Francis J. Grant, Luann Lockwood, Steven D. Levin, Cosette LeCiel, Kim Waggie, Heather Day, Stavros Topouzis, Janet Kramer, Rolf Kuestner, Zhi Chen, Don Foster, Julia Parrish-Novak & Jane A. Gross Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice Nature Immunology, 5, 752 - 760 (July 2004); doi:10.1038/ni1084
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Lysophosphatidic acid: Signaling pain
Peripheral nerve injury induces neuropathic pain, characterized
by abnormal pain sensation from non-noxious stimuli (allodynia),
and an increased pain response to normally painful stimuli (hyperalgesia).
The small phospholipid lysophosphatidic acid (LPA) is a signaling
molecule potentially involved in neuropathic pain. LPA produced
by activated platelets is released into serum. Serum leakage at
sites of tissue injury can lead to the exposure of neural cells
to micromolar concentrations of this bioactive lipid. LPA is known
to signal via G-protein-coupled LPA1
receptors (also known as EDG2) in peripheral nociceptor nerve
endings. LPA signaling activates RhoA
and consequently Rho
kinase (ROCK) through Gα12/13.
Inoue et al. now report that this LPA signaling pathway
is required for neuropathic pain sensation.
In mice, a single injection of LPA resulted in thermal hyperalgesia and mechanical allodynia, which was blocked by pharmacological inhibition of the Rho-ROCK pathway. The authors confirmed that LPA1 receptors are the main LPA receptors present in neurons and the Schwann cells of the dorsal root ganglion (DRG). LPA-induced allodynia was blocked in mice treated with LPA1 antisense oligonucleotide (LPA1 AS-ODN) and in LPA1-null mice.
Injected LPA1 AS-ODN administered pre-injury significantly inhibited allodynia and hyperalgesia, although post injury injections did not. Pharmacological inhibition of Rho and ROCK around the time of partial sciatic nerve ligation completely prevented the development of allodynia and hypergesia. This effect was not seen upon Rho inhibition 6 hours post-injury, indicating that Rho-ROCK signaling is important during the early phase after nerve injury.
Aberrant myelination is a common indicator of neuropathic pain.
Demyelination of the dorsal root, similar to that caused by partial
sciatic nerve injury, occurred 24 hours after the injection of
LPA. Injury-induced dorsal root demyelination was blocked by Rho-ROCK
inhibition. Increased expression of protein
kinase Cγ (PKCγ)
and the α2δ1 subunit of the voltage-gated calcium
channel (Caα2δ1) correlate with neuropathic pain.
LPA administration induced an increase in both PKCγ and
Caα2δ1 expression, which was susceptible to Rho-ROCK
inhibitors.
Together, these results show that LPA functions as an important signaling molecule in the induction of neuropathic pain and inhibitors of this pathway carry promise as analgesics for this type of pain.
Brenda Riley, Assistant Editor Signaling Gateway
article
Makoto Inoue, Md Harunor Rashid, Ryousuke Fujita, James J A Contos, Jerold Chun & Hiroshi Ueda Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling Nature Medicine, 10, 712 - 718 (July 2004); doi:10.1038/nm1060
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Cell cycle: It's a Smad world
During the G1 phase of the cell cycle, cells encounter a myriad
of external signals. These signals are integrated with basic cell
cycle machinery and ultimately trigger cell proliferation or growth
arrest. One such signal is TGF-β,
a potent inhibitor of cell cycle progression. TGF-β activates
Smads, which induce the transcription of growth inhibitory cell
cycle regulators such as p15 or p21,
and suppress the expression of growth stimulatory factors such as
c-myc. At the center of cell cycle regulation are the cyclin-dependent
kinases (CDKs), including CDK4
and CDK2
which act during early-mid G1 and G1/S, respectively. Fang Liu and
colleagues have now made an important new link between these CDKs
and Smad signaling.
Smads contain putative CDK phosphorylation sites and Matsuura et
al. now confirm that CDK4 and CDK2 can phosphorylate Smad2
and Smad3in vitro. Cell lysate analysis shows that Smad3 phosphorylation
occurs during G1. The in vitro and in vivo CDK
phosphorylation sites were mapped to Thr 8, Thr 178 and Ser 212
in Smad3.
Mutation of these sites altered the biological response to Smads:
a mutant Smad3 protein unable to undergo phosphorylation stimulated
transcription from the p15 promoter to a greater extent than wild-type
protein. Interference of CDK2 and CDK4 activity also increased TGF-β-induced
p15 reporter gene activity. In addition, mutant Smad3 was more effective
at down regulating c-myc.
Together, these data suggest that CDK phosphorylation reduces Smad3
activity.
Cells lacking Smad3 proliferate faster and are largely resistant
to TGF-β-induced growth arrest. When the authors re-expressed
Smad3 in these cells it reduced G1/S progression, accompanied by
increased p15 expression and reduced c-myc levels. Interestingly,
mutant Smad3 augmented these effects.
These observations suggest that Smad3 and CDKs play antagonistic
roles in G1 progression and that in the absence of a strong TGF-β
signal, CDK phosphorylation of Smad3 can block its activity to allow
cell cycle progression. As cancer cells often exhibit deregulated
G1 signaling pathways that increase CDK activity, it is tempting
to speculate that enhanced Smad phosphorylation in tumor cells may
allow them to escape TGF-β-induced arrest.
Barbara Marte, Editor Signaling Gateway
article
Isao Matsuura, Natalia G. Denissova, Guannan Wang, Dongming He,
Jianyin Long & Fang Liu Cyclin-dependent kinases regulate the antiproliferative function
of Smads Nature, 430, 226 - 231 (8 July 2004); doi:10.1038/nature02650
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Putting c-Fyn on a pedestal
Enteropathogenic Escherichia coli (EPEC), the pathogen that causes diarrhoeal disease, subverts the actin polymerization machinery of host cells to form actin pedestals during infection. A key step in this process is phosphorylation of the EPEC receptor Tir (translocated intimin receptor), and in the July issue of Nature Cell Biology, Vassilis Koronakis and colleagues identify c-Fyn as the long-sought kinase responsible for this critical event.
During infection, EPEC delivers Tir to the host cell membrane, which following phosphorylation at Tyr474, recruits the adaptor protein Nck and the actin polymerization machinery of the host cell to drive the formation of actin pedestals and bacterial adherence. To help them in the task of identifying the relevant kinase, Phillips et al. first teased apart the events controlling binding and activation of Tir, so that Tir phosphorylation could be readily detected. An important clue to which kinase might be responsible came from the observation that the Tir phosphorylation site Tyr474 lies in a region closely resembling a c-Src target motif. So they first tested a battery of kinase inhibitors, and indeed found that blocking Src family kinases prevented Tir phosphorylation at Tyr474, as well as Nck recruitment to the receptor.
Three SFKs are expressed in mammalian cells — c-Src, c-Fyn and c-Yes. So which of the three is the culprit? To address this, Phillips et al. went through a process of elimination by testing different mutant combinations and found that the key kinase for Tir phosphorylation and pedestal formation was c-Fyn. In support of the knockout phenotype, they showed that Tir is also a c-Fyn target in vitro and that knocking down c-Fyn expression using small-interfering RNAs largely prevented Tir phosphorylation and pedestal formation.
One surprise is that although c-Fyn appears to be critical for Tir phosphorylation, the kinase itself cannot be detected in actin pedestals, although Nck and the actin polymerization machinery are consistently present. One interpretation of this is that the liason between c-Fyn and Tir is brief, and this may be necessary to allow the subsequent interaction between Tir and Nck. Nonetheless, what is clear is that c-Fyn is crucial for Tir phosphorylation and without it, pedestal formation and bacterial adherence are compromised. In addition to revealing a key step in the pathogenesis of EPEC, this also adds to a growing list of Src family kinases that are subverted by pathogens to regulate the actin cytoskeleton. How c-Fyn is activated and targeted towards Tir in this context, and whether other virulence factors of EPEC participate in this activation, remains to be seen.
Alison Schuldt, Senior Editor Nature Cell Biology
article
Phillips, N., Hayward, R. D. & Koronakis, V Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation. Nature Cell Biology, 6, 618 - 625 (July 2004); doi:10.1038/ncb1148
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