Small-molecule inhibitors: Another one bites the Dusp

The use of a novel small-molecule inhibitor of Dusp6 reveals the importance of tightly regulated FGF signaling during cardiac development.

Dual-specificity phosphatases (Dusps) negatively regulate mitogen-activated protein kinase (MAPK) signaling by dephosphorylating components of the MAPK cascade. In zebrafish, Dusp6 is expressed in cardiac progenitor tissue and attenuates fibroblast growth factor (FGF)-ERK signaling by dephosphorylating ERK1/2. However, the precise function of Dusp6 in cardiac development has remained unknown. In Nature Chemical Biology, Molina et al. describe a novel Dusp6 small-molecule inhibitor and show that unrestrained FGF signaling stimulates cardiac progenitor cell proliferation during zebrafish development.

A chemical screen in zebrafish identified the small molecule BCI, which enhanced ligand-dependent FGF signaling and target gene transcription by blocking Dusp6- and Dusp1-mediated dephosphorylation of activated ERK. BCI reversibly inhibited Dusp1 and Dusp6 by locking them in a low-activity form. As these phosphatases are directly activated by ERK binding, BCI likely interferes with their ERK-mediated activation. Indeed, BCI had no effect on Dusp6 basal activity, but strongly suppressed ERK2-stimulated activity towards an exogenous substrate. Thus, BCI appears to be an allosteric inhibitor of Dusp1 and Dusp6.

Armed with this novel small-molecule inhibitor, the authors were able to investigate the function of Dusp6 in heart development. Transient, BCI-mediated inhibition of Dusp6 in zebrafish embryos increased FGF signaling and expanded the pool of cardiac progenitor cells at the expense of hematopoietic or endothelial cell lineages. This increase in progenitor cells caused enlarged hearts at subsequent developmental stages. BCI treatment at a later stage of embryogenesis also increased heart size, albeit less efficiently.

In this study, the use of BCI allowed the investigation of Dusp6 function during narrow developmental windows and revealed the importance of fine-tuned FGF signaling during cardiac development. It will be interesting to elucidate the function of Dusp6 or Dusp1 in other tissues during development and homeostasis. Intriguingly, loss of the Dusp6 gene in mice has also been shown to cause enlarged hearts, suggesting that FGF signaling might be an evolutionarily conserved mechanism that regulates cardiac progenitor cell proliferation.

Emily J. Chenette
Signaling Gateway

Reference:
Molina, G. et al.
Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages
Nature Chemical Biology advance online publication, 5 July 2009 (DOI 10.1038/nchembio.190)
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FGF signaling: It's a wrap

Fibroblast growth factor signaling regulates the switch from migration to differentiation in Drosophila glial cells.

Glial cells migrate from the perineurium to the eye disc to wrap and insulate photoreceptor neurons in the eye. Migrating glia begin to differentiate into wrapping glia once they reach the photoreceptor axons, but the signals that mediate the switch between migration and differentiation have remained unknown. In Nature, Christian Klämbt and colleagues now report that fibroblast growth factor (FGF) signaling is important for Drosophila glial cell migration and differentiation, providing clues about how this developmental switch is regulated.

The FGF receptor heartless (Htl) was detected at high levels in Drosophila glial cells but was absent from photoreceptor neurons. RNA-mediated interference (RNAi) against htl impaired glial cell migration and differentiation, resulting in a 40% decrease in glial cell number. Conversely, constitutively active Htl increased the number of glial cells but diminished migration, pointing to a role for FGF signaling in regulating migration and differentiation.

The FGF8-like ligand Pyramus (Pyr) is expressed in both glia and eye discs, whereas a second FGF8-like ligand, Thisbe (Ths), is restricted to photoreceptor neurons. Depletion of glial Pyr reduced glial cell number, whereas Pyr overexpression increased glial cell motility and proliferation. Depletion of neuronal Ths blocked glial cell wrapping, and Ths overexpression increased wrapping – this effect was even more pronounced when constitutively active Htl was concomitantly expressed in the wrapping glia. However, Pyr could compensate for Ths and induce wrapping when expressed ectopically in neurons.

Given that Pyr and Ths can elicit the same biological responses, what mediates the switch between migration and differentiation? Effectors downstream of Htl might regulate this process. Loss of the adaptor protein Dof, which is expressed only in glia and the eye disc, impaired cell division and wrapping. Depletion of the Ras family member Rap1 blocked proliferation but did not affect differentiation, whereas the ETS-family transcription factor Pointed controlled differentiation but not proliferation. Furthermore, the MAPK negative regulator Sprouty was selectively expressed in glial cells following contact with axons, and its overexpression blocked differentiation. Thus, the authors propose that differential expression and activation of core MAPK components might underlie the switch from migration to differentiation.

These data support a role for FGF signaling in both migration and differentiation, and suggest that an interaction between glial cells and neurons activates MAPK pathway components to inhibit glial migration and induce differentiation. It will be interesting to establish whether Ths signals in a paracrine manner when glia reach neurons, as the combination of Pyr and Ths could potentially upregulate Htl signaling to trigger differentiation.

Emily J. Chenette
Signaling Gateway

Reference:
Franzdóttir, S. R., Engelen, D., Yuva-Aydemir, Y., Schmidt, I., Aho, A. and Klämbt, C.
Switch in FGF signalling initiates glial differentiation in the Drosophila eye
Nature advance online publication, 13 July 2009 (DOI 10.1038/nature08167)
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Sphingosine 1-phosphate signaling: T_reg or not T_reg, that is the question

S1P₁ activates the mTOR–Akt pathway to suppress T_reg-cell differentiation and activity.

Regulatory T (T_reg) cells help maintain immunological tolerance by antagonizing conventional T (T_conv)-cell responses. Their suppressive effects must be halted to permit adaptive immune responses, but unrestrained T_reg-cell activity can lead to impaired immunity. The mechanism that regulates T_reg cells has remained largely uncharacterized. In Nature Immunology, Hongbo Chi and colleagues now report that sphingosine 1-phosphate receptor 1 (S1P₁) signals through the mTOR–Akt pathway to repress T_reg-cell differentiation and activity.

S1P₁ was previously shown to regulate T-cell trafficking, but its importance in T_reg cells had not been elucidated. Targeted deletion of S1P₁ in T cells upregulated FoxP3 expression — a transcription factor involved in the differentiation of CD4⁺CD25⁺FoxP3^- precursor cells into mature T_reg cells — and increased the thymic T_reg-cell population. Overexpression of S1P₁ shrank the T_reg-cell population and blocked precursor cells from differentiating into mature T_reg cells, indicating that S1P₁ antagonizes T_reg-cell differentiation.

Moreover, S1P₁ also impairs T_reg-cell function. When S1P₁-knockout or wild-type T_reg cells were mixed with T_conv cells, the knockout cells were better able to suppress T_conv-cell proliferation and IL-2 secretion. Transgenic expression of S1P₁ elicited the opposite effect. Indeed, as a result of the defective T_reg-cell function, S1P₁-transgenic mice developed autoimmune reactions in vivo that were associated with enhanced activation of T_conv cells.

Expression of IL-2 and activation of the T-cell antigen receptor (TCR) both modulate the differentiation and function of T_reg cells by activating the Akt, Erk and STAT5 signaling pathways. Overexpression of S1P₁ significantly upregulated the activity of Akt, but not Erk or STAT5. Intriguingly, blocking mTOR–Akt pathway signaling in cells overexpressing S1P₁ restored their ability to suppress T_conv-cell proliferation. Furthermore, loss of S1P₁ attenuated Akt activation in mature T_reg cells following treatment with IL-2. Thus, S1P₁-mediated activation of the mTOR–Akt pathway inhibits T_reg-cell differentiation and activity.

S1P₁–mTOR–Akt signaling in T_reg cells mediates immune tolerance by antagonizing their differentiation and function. Remarkably, S1P₁ mRNA levels declined rapidly in T_conv cells following TCR or IL-2 induction, and S1P₁ was not required for cell proliferation or Akt activation in these cells. In contrast, the amount of S1P₁ mRNA declined gradually in T_reg cells. Expression of KLF2, a transcription factor essential for S1P₁ expression in thymocytes, exhibited a similar expression pattern to that of S1P₁. Thus, KLF2 may contribute to the differential regulation of S1P₁ in T_reg and T_conv cells. It will be important to determine the extent to which KLF2 is required for maintaining immunological tolerance and to elucidate the signals that modulate KLF2 expression.

Emily J. Chenette
Signaling Gateway

Reference:
Liu, G. et al.
The receptor S1P₁ overrides regulatory T cell-mediated immune suppression through Akt–mTOR
Nature Immunology 10, 769-777 (2009)
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Further reading:
Ohkura, N. and Sakaguchi, S.
A novel modifier of regulatory T cells
Nature Immunology 10, 685-686 (2009)
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Ubiquitin ligases: Long live the worm

A specific E2/E3 ubiquitin ligase complex is essential for dietary-restriction-induced longevity in worms.

Caloric restriction has been shown to increase lifespan in many organisms. However, the molecular mechanisms linking dietary restriction to longevity have not yet been clearly defined. In Nature, Carrano et al. now report that the UBC-18 E2 and WWP-1 E3 ubiquitin ligases have important roles in mediating dietary-restriction-induced longevity in C. elegans.

In mammals, the WWP family of E3 ubiquitin ligases comprises diverse members involved in many cellular functions. C. elegans contains a single WWP ortholog, WWP-1, which was previously shown to be essential for embryonic development. Carrano et al. found that a mutant allele of wwp-1 exhibiting partial penetrance decreased stress resistance in adult worms, suggesting a potential role for wwp-1 in longevity. Indeed, overexpression of wwp-1, but not other homologous E3 ligases, significantly increased lifespan.

Intriguingly, WWP-1 was also required for the increase in longevity observed in response to dietary restriction. This effect was dependent on PHA-4 — a FOXA transcription factor required for dietary-restriction-induced longevity. However, WWP-1 was not necessary for lifespan extension in worms with reduced insulin/IGF-1 signaling or defects in mitochondrial function. A mutation in wwp-1 that abolished ubiquitination activity also blocked the increase in lifespan, suggesting that ubiquitin ligase activity is essential for dietary-restriction-induced longevity.

Ubiquitin is transferred from an E1 to an E2 to an E3 ligase, which then ubiquitinates target proteins. The E2 ubiquitin ligase UBC-18 associated with WWP-1 and was required for WWP-1 ubiquitination activity in vitro. Although overexpression of ubc-18 alone was not sufficient to extend lifespan, depletion of ubc-18 abolished the increase in longevity observed following dietary restriction or wwp-1 overexpression. Depletion of other UBC-18-associated E3 ligases had no effect on diet-induced longevity, suggesting that the UBC-18–WWP-1 complex has the unique ability to regulate lifespan in response to dietary restriction.

These data reveal an important role for ubiquitin ligases in diet-induced longevity. It will be important to determine the targets of UBC-18–WWP-1, as the authors' preliminary data suggests that PHA-4 is not ubiquitinated by this complex. In addition, future studies aimed at uncovering whether mammalian orthologs of UBC-18–WWP-1 have the same function as their worm counterparts may help explain the mechanism of fasting-associated longevity in other organisms, as well as elucidate the extent to which this pathway is conserved across species.

Emily J. Chenette
Signaling Gateway

Reference:
Carrano, A. C., Liu, Z., Dillin, A. and Hunter, T.
A conserved ubiquitination pathway determines longevity in response to diet restriction
Nature advance online publication, 24 June 2009 (DOI 10.1038/nature08130)
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TGF-β signaling: A new piece of the puzzle

TGF-β signaling promotes the nuclear accumulation of CLIC4, which mediates the transcriptional response to TGF-β by protecting phospho-Smad2 and 3 from dephosphorylation.

Chloride intracellular channel 4 (CLIC4) has an important role in a number of processes, including cell-cycle control and apoptosis. Stressors such as DNA damage induce CLIC4 nuclear translocation; furthermore, CLIC4 is excluded from the nucleus in some cancers. The nuclear function of CLIC4, and the mechanisms regulating its translocation, have not yet been clearly defined. In Nature Cell Biology, Stuart Yuspa and colleagues now report that TGF-β signaling promotes the accumulation of CLIC4 in the nucleus, where it mediates the transcriptional response to TGF-β by preventing dephosphorylation of phospho-Smad2 and 3.

Previous studies had uncovered a link between TGF-β signaling and CLIC4 expression in fibroblasts. In a screen for novel CLIC4 interacting proteins, Yuspa and colleagues identified the TGF-β pathway protein Schnurri-2. TGF-β1 treatment increased the expression of and interaction between CLIC4 and Schnurri-2. Exogenous Schnurri-2 promoted CLIC4 nuclear translocation and upregulated TGF-β-dependent transcriptional activity and growth inhibition, whereas depletion of Schnurri-2 blocked these effects. However, nuclear targeting of CLIC4 circumvented the requirement for Schnurri-2 in stimulating TGF-β transcriptional activity, indicating that Schnurri-2 regulates CLIC4 nuclear translocation but not its activity.

Intriguingly, nuclear targeting of CLIC4 inhibited DNA synthesis and caused a marked increase in TGF-β transcriptional responses, such as downregulation of c-Myc and upregulation of p21, even in the absence of TGF-β1. Indeed, nuclear CLIC4 enhanced the nuclear accumulation of phosphorylated Smad2 and 3 — the transcription factors that are required for the transcriptional response to TGF-β signaling — and small hairpin (sh)RNA against CLIC4 decreased phospho-Smad2/3 levels independently of TGF-β1. CLIC4 was found to stabilize phospho-Smad levels by directly interacting with phospho-Smad2/3 and preventing their association with the Smad phosphatase PPM1a.

These data suggest that TGF-β1 stimulates the interaction between CLIC4 and Schnurri-2, which increases CLIC4 nuclear translocation. Nuclear CLIC4 then binds phospho-Smad2/3, preventing their dephosphorylation and stimulating the TGF-β transcriptional program. Thus, CLIC4 represents a new essential component of TGF-β signaling, which is itself induced by TGF-β signaling. It will be important to explore the regulation of this pathway and the mechanism by which CLIC4 is retained in the cytoplasm in cancer cells in future studies.

Emily J. Chenette
Signaling Gateway

Reference:
Shukla, A. et al.
TGF-β signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3
Nature Cell Biology 11, 777-784 (2009)
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