Immune signaling: Fungus makes cells Syk

Following fungal infection, Syk signaling activates Nlrp3-containing inflammasomes to produce the interleukin IL-1β.

Different types of infection trigger the assembly of distinct inflammasomes — multi-protein complexes containing caspases, adaptor proteins and NLR (nucleotide-binding domain and leucine-rich-repeat-containing) receptors. Interleukin-1β (IL-1β) is a cytokine with an important role in the innate immune response. The fungus Candida albicans is known to produce IL-1β via a two-step mechanism involving NF-κB-mediated transcription of pro-IL-1β and caspase-1-mediated proteolysis to mature IL-1β at inflammasomes. In Nature, Jürgen Ruland and colleagues now reveal that the tyrosine kinase Syk has a dual role in inducing both IL-1β transcription and proteolysis following fungal infection.

Syk and the associated adaptor Card9 are known to be required for cytokine production downstream of fungal recognition. In this study, pharmacologic inhibition or genetic deletion of Syk blocked both accumulation of pro-IL-1β and caspase-1 activation. Intriguingly, Card9 was necessary for Syk-mediated NF-κB transcriptional activity but dispensable for activation of the inflammasome, indicating that Syk has a separable role in IL-1β transcription and inflammasome activation.

Intermediary physiological insults such as reactive oxygen species (ROS) or lysosomal damage stimulate the inflammasome by activating NLR proteins. Syk, but not Card9, was required for the production of ROS. Inhibition of ROS attenuated IL-1β production induced by C. albicans, suggesting that Syk activates the inflammasome by generating ROS.

Previous studies have linked ROS production to the activation of inflammasome complexes containing the NLR family member Nlrp3. Indeed, depletion of Nlrp3 blocked caspase-1 activation and IL-1β production following fungal infection. Furthermore, Nlrp3^-/- mice displayed impaired IL-1β production in response to C. albicans.

Thus, Syk has a dual effect on IL-1β production as a defense mechanism to fungal infection. First, Syk–Card9 signaling promotes NF-κB-mediated transcription of pro-IL-1β. Second, Syk promotes the production of ROS, activating Nlrp3-containing inflammasomes to stimulate caspase-1-mediated cleavage of pro-IL-1β. Syk kinase inhibitors have been shown to be beneficial in certain inflammatory disorders, and it will be interesting to determine if inflammasome inhibition contributes to this effect.

Emily J. Chenette
Signaling Gateway

Reference:
Gross, O., Poeck, H. et al.
Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence
Nature 459, 433-436 (2009)
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Immune signaling: Playing TAG in the endosomes

TAG, a splice variant of the Toll-like receptor (TLR) adaptor protein TRAM, negatively regulates TLR4 signaling in late endosomes.

In response to lipopolysaccharide (LPS) and other pro-inflammatory signals, Toll-like receptor 4 (TLR4) signals through the adaptor proteins MyD88, TRIF and TRAM to induce cytokine production. TLR4-MyD88 signaling stimulates the NF-κB transcriptional program, whereas the activated TLR4-TRAM complex recruits TRIF, which activates IRF3 to stimulate transcription of interferon-responsive genes. The TLR4-TRAM-TRIF complex continues to signal as it is internalized to endosomes. In Nature Immunology, Luke O'Neill and colleagues now show that a splice variant of TRAM called TAG inhibits TRIF-mediated activation of the IRF3 transcriptional program by binding to TRAM in late endosomes.

Bioinformatic analyses predicted the existence of TAG, which was detected in a variety of human, but not murine tissues. Unlike TRAM, TAG contains a Golgi dynamics (GOLD) domain thought to be involved in targeting proteins to vesicles, but lacks the myristoylation site and protein kinase C (PKC)-ε phosphorylation site present in TRAM. TRAM and TAG did not co-localize in resting cells, but LPS treatment promoted the co-localization of both proteins in late endosomes. In contrast to TRAM, TRIF or MyD88, however, TAG appears to antagonize TLR4 signaling, as short interfering (si)RNA-mediated knockdown of TAG increased TLR4 levels.

Overexpression of TAG inhibited interferon-responsive reporter gene expression but had no effect on NF-κB reporter gene expression. Furthermore, siRNA against TAG enhanced TLR4-mediated stimulation of interferon-responsive, but not NF-κB-responsive genes when exposed to LPS, indicating a selective effect on the IRF3 transcriptional pathway. As the interaction between TRAM and TRIF is required for IRF3 activity, does TAG interfere with binding? Indeed, co-immunoprecipitation experiments revealed that TRAM and TAG interact in vitro, and TAG disrupted the TRAM-TRIF complex. In addition, siRNA-mediated depletion of TAG resulted in a stable complex between TRIF and TRAM, explaining the enhanced transcription of IRF3-regulated genes in the absence of TAG.

Thus, TAG attenuates TLR4-TRAM-TRIF signaling by disassembling the TRAM-TRIF complex in late endosomes. As depletion of TAG increases TLR4 levels, it is possible that TAG is also involved in TLR4 trafficking and degradation. Spatial regulation is emerging as an important aspect of TLR signaling, and it will be interesting to uncover how other TLR family members are regulated at discrete cellular subdomains.

Emily J. Chenette
Signaling Gateway

Reference:
Palsson-McDermott, E. M., Doyle, S. L., McGettrick, A. F. et al.
TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway
Nature Immunology 10, 579-586 (2009)
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Hematopoietic stem cells: Home to marrow

The heterotrimeric G protein Gα_s subunit is required for the ability of primitive hematopoietic cells to home to and engraft the bone marrow.

Understanding the process by which hematopoietic stem and progenitor cells (HSPCs) home to, engraft and are retained in bone-marrow niches has important implications for clinical medicine. In Nature, David Scadden and colleagues now show that the heterotrimeric G protein stimulatory α subunit (Gα_s) is required for homing and engraftment of HSPCs in mice.

Gα_s is essential for development; therefore, to investigate the importance of Gα_s in HSPC biology, chimeric mice were established by injecting Gα_s^-/- embryonic stem cells into wild-type blastocysts. Although Gα_s^-/- cells were present at normal levels in the liver, they were not found in the bone marrow of the chimeric mice. Competitive repopulation experiments in which irradiated mice received equal numbers of both wild-type and Gα_s-deficient HSPCs revealed that Gα_s^-/- cells were absent from bone marrow following transplantation. Furthermore, Gα_s^-/- cells, when transplanted alone, were not capable of significant engraftment, resulting in the eventual death of recipient mice. Thus, Gα_s appears to play an important role in HSPC homing and engraftment.

Although Gα_s deficiency did not affect chemotaxis or general motility, Gα_s^-/- cells did not efficiently interact with the bone-marrow endothelium. In addition, conditional deletion of the gene encoding Gα_s in established bone marrow populations resulted in impaired trafficking out of the marrow, but did not affect retention in the marrow. Conversely, cholera toxin, which inhibits GTP hydrolysis and activates Gα_s signaling, enhanced the ability of wild-type, but not Gα_s-deficient cells, to home to and engraft bone marrow. These data also suggest that that HSPC homing, engraftment and retention in the bone marrow are separable biological events.

The finding that pharmacological activation of Gα_s signaling increases homing supports a potential therapeutic application for Gα_s stimulation in bone marrow transplantation. However, the proteins and regulatory pathways that lie upstream and downstream of Gα_s during homing and engraftment remain to be elucidated. It is possible that prostaglandin receptors might play a role in this process, as PGE2 has also been shown to enhance stem cell transplantation in mice.

Emily J. Chenette
Signaling Gateway

Reference:
Adams, G. B. et al.
Haematopoietic stem cells depend on Gα_s-mediated signalling to engraft bone marrow
Nature 459, 103-107 (2009)
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Prostate cancer: A new cooperative link emERGes

Misregulated expression of the transcription factor ERG cooperates with loss of the PI(3)K pathway inhibitor PTEN to promote prostate tumorigenesis.

Fusion of the ETS transcription factor gene ERG to the androgen-responsive TMPRSS2 gene promoter (TMPRSS2-ERG) occurs in about half of human prostate cancers. Previous studies have suggested that this genetic rearrangement is an early event in tumorigenesis, but the role of TMPRSS2-ERG expression in prostate cancer is still not fully understood. Two studies in Nature Genetics by King et al. and Carver et al. now show that overexpression of ERG enhances cell migration and cooperates with activation of the phosphoinositide 3-kinase (PI(3)K) pathway to promote tumor progression.

To understand the role of ERG overexpression in prostate cancer, King et al. developed mice with targeted expression of TMPRSS2-ERG in the prostate. Overexpression of TMPRSS2-ERG alone was insufficient to induce tumorigenesis, but prostates from TMPRSS2-ERG mice that also lacked a copy of PTEN (Pten^+/-;TMPRSS2-ERG) showed evidence of prostate intraepithelial neoplasia (PIN) – a pre-malignant lesion that can progress to adenocarcinoma. Furthermore, TMPRSS2-ERG mice that overexpressed AKT developed a more florid, hyperplastic form of PIN, suggesting that overexpression of ERG together with PI(3)K pathway activation facilitates PIN.

Meanwhile, Carver et al. also found evidence for ERG overexpression and concomitant PTEN loss in human prostate cancer specimens and analyzed the extent of cooperation between these two proteins in initiating prostate cancer in a mouse model. Mice lacking a copy of PTEN and overexpressing ERG in the prostate (Pten^+/-;PB-ERG) developed high-grade PIN within two months, which rapidly progressed to invasive adenocarcinoma. In contrast, Pten^+/- mice developed high-grade PIN that did not progress to adenocarcinoma. Furthermore, in agreement with the findings of King et al., Carver et al. observed that ERG overexpression had no overt phenotype.

However, cell culture experiments revealed that PTEN haploinsufficiency or AKT overexpression conferred a proliferative advantage in prostate cancer cell lines, whereas ERG expression increased migration. Two proteins with known functions in cell migration – the chemokine receptor CXCR4 and the metalloproteinase ADAMTS1 – were identified as direct targets of ERG. Small interfering (si)RNA-mediated knockdown of CXCR4 blocked migration in prostate cancer cell lines overexpressing ERG. These data suggest that the ERG-mediated increase in migration cooperates with PI(3)K pathway-driven proliferation to promote rapid tumorigenesis. It will be important to uncover other transcriptional targets of ERG, and to determine if inhibiting ERG or one of its targets can block the initiation of adenocarcinoma in high-grade PINs.

Emily J. Chenette
Signaling Gateway

References:
King, J. C. et al.
Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis
Nature Genetics 41, 524-526 (2009)
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Carver, B. S. et al.
Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate
Nature Genetics 41, 619-624 (2009)
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Development: Abrupt inhibition of cell migration

The protein Abrupt is a point of convergence for the ecdysone and JAK-STAT pathways during border cell migration in the developing Drosophila oocyte.

During Drosophila ovarian development, border cells migrate to the anterior border of the oocyte. JAK-STAT activity is required for the specification and spatial patterning of border cells, and the hormone ecdysone regulates the timing of border cell migration. However, the mechanism that integrates JAK-STAT and ecdysone signaling during migration is not yet understood. In Nature Cell Biology, Denise Montell and colleagues now show that the protein Abrupt is the point of convergence for the ecdysone and JAK-STAT pathways, providing insight into how spatial and temporal cues are integrated during border cell migration.

Although ecdysone was known to be synthesized during Drosophila developmental stages 9 and 10, it was not known how ecdysone spatial patterning is achieved. Abrupt was identified in a screen for genes that, when over-expressed, interfered with expression of an ecdysone receptor (EcR)-responsive element (EcRE-lacZ) and caused border cell migration defects. In contrast to ecdysone, Abrupt expression decreased throughout stage 9 in migrating border cell nuclei. Interestingly, Abrupt levels increased in border cells expressing dominant-negative EcR, whereas hypomorphic Abrupt mutants increased EcRE-lacZ expression, suggesting a mutually antagonistic relationship between Abrupt and ecdysone signaling.

Indeed, co-immunoprecipitation assays revealed a direct interaction between Abrupt and the EcR co-activator Taiman (Tai). Abrupt inhibited EcRE-lacZ expression when co-expressed with full-length Tai, but not with a Tai mutant that could not interact with Abrupt (TaiΔB). Expression of TaiΔB caused precocious and robust expression of EcRE-lacZ; surprisingly, early ecdysone expression completely blocked cell migration. Premature cell migration could be induced by co-expression of TaiΔB and constitutively active JAK, indicating that JAK-STAT and ecdysone are both required for the onset of cell migration.

But how is the timing of border cell migration regulated? STAT expression inversely correlated with nuclear Abrupt levels in border cells, although the mechanism by which JAK-STAT downregulates Abrupt is not known. Thus, Abrupt is negatively regulated by both the JAK-STAT and ecdysone pathways, suggesting that it functions as a nodal point between them. The authors suggest that this regulation generates a temporal gradient of ecdysone signaling comparable to morphogen gradients in spatial patterning. One benefit of such tightly orchestrated spatial and temporal control is that it would prevent a rapid increase in ecdysone, as high levels of the hormone — occurring either as a consequence of TaiΔB expression or naturally at stage 10 — appear to impede cell migration.

Emily J. Chenette
Signaling Gateway

Reference:
Jang, A. C.-C., Chang, Y.-C., Bai, J. & Montell, D.
Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt
Nature Cell Biology 11, 569-579 (2009)
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