Absence Of TGF-β Signaling Research Articles

TGF-β promotes stem-like T cells via enforcing their lymphoid tissue retention.

Stem-like CD8+ T cells sustain the antigen-specific CD8+ T cell response during chronic antigen exposure. However, the signals that control the maintenance and differentiation of these cells are largely unknown. Here, we demonstrated that TGF-β was essential for the optimal maintenance of these cells and inhibited their differentiation into migratory effectors during chronic viral infection. Mechanistically, stem-like CD8+ T cells carried a unique expression pattern of α4 integrins (i.e., α4β1hi and α4β7lo) controlled by TGF-β. In the absence of TGF-β signaling, greatly enhanced expression of migration-related markers, including altered expression of α4 integrins, led to enhanced egress of stem-like CD8+ T cells into circulation accompanied by further differentiation into transitional states. Blocking α4 integrin significantly promoted their lymphoid tissue retention and therefore partially rescued the defective maintenance of Tcf-1+ subset in the absence of TGF-β signaling. Thus, TGF-β promotes the maintenance and inhibits the further differentiation of stem-like T cells at least partially via enforcing their lymphoid tissue residency.

Pericyte-myofibroblast transition in the human lung

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease that includes fibroblastic foci (FF). It has been increasingly appreciated that the origin of collagen-overproducing cells such as pathological myofibroblasts in FF is pericytes. However, neither pericytes derived from the lung nor FF in the IPF lung have not been fully characterized. Human lung pericytes (HuL-P) examined in this study expressed two representative pericyte markers; platelet-derived growth factor receptor β (PDGFRB) and chondroitin sulfate proteoglycan 4 (CSPG4), and were able to migrate and cover endothelial tubes in 3D conditions, indicating that they retain characteristics of pericytes. Moreover HuL-P cells transitioned to myofibroblast-like cells in the presence of transforming growth factor (TGF)-β signaling or to pericyte-like cells in the absence of TGF-β signaling (pericyte-myofibroblast transition). On the other hand, the FF detected in this study were invariably localized between peripheral lung epithelia and capillary endothelia, the basement membranes of which are physiologically fused. The localization is highly specific in that the only cells that exist between the gap are pericytes. As expected, FF were immunohistochemically positive for PDGFRB and CSPG4, suggesting that pericytes are activated to form FF. We also found that HuL-P cells were difficult to eradicate by dual silencing of Bcl-xL plus MCL1. It would be more sensible to suppress pericyte-myofibroblast transition than to kill activated myofibroblasts for the treatment of IPF.

TGF-β signaling controls Foxp3 methylation and T reg cell differentiation by modulating Uhrf1 activity.

Regulatory T (T reg) cells are required for the maintenance of immune homeostasis. Both TGF-β signaling and epigenetic modifications are important for Foxp3 induction, but how TGF-β signaling participates in the epigenetic regulation of Foxp3 remains largely unknown. Here we showed that T cell-specific ablation of Uhrf1 resulted in T reg-biased differentiation in TCR-stimulated naive T cells in the absence of TGF-β signaling, and these Foxp3+ T cells had a suppressive function. Adoptive transfer of Uhrf1 -/- naive T cells could significantly suppress colitis due to increased iT reg cell generation. Mechanistically, Uhrf1 was induced upon TCR stimulation and participated in the maintenance of DNA methylation patterns of T reg cell-specific genes during cell division, while it was phosphorylated upon TGF-β stimulation and sequestered outside the nucleus, and ultimately underwent proteasome-dependent degradation. Collectively, our study reveals a novel epigenetic mechanism of TGF-β-mediated iT reg cell differentiation by modulating Uhrf1 activity and suggests that Uhrf1 may be a potential therapeutic target in inflammatory diseases for generating stable iT reg cells.

TGFβ signaling in germinal center B cells promotes the transition from light zone to dark zone.

B cells in germinal centers (GCs) cycle between light zone (LZ) and dark zone (DZ). The cues in the GC microenvironment that regulate the transition from LZ to DZ have not been well characterized. In Peyer's patches (PPs), transforming growth factor-β (TGFβ) promotes IgA induction in activated B cells that can then differentiate into GC B cells. We show here that TGFβ signaling occurs in B cells in GCs and is distinct from signaling that occurs in activated B cells in PPs. Whereas in activated B cells TGFβ signaling is required for IgA induction, in the GC it was instead required for the transition from LZ to DZ. In the absence of TGFβ signaling, there was an accumulation of LZ GC B cells and reduced antibody affinity maturation likely due to reduced activation of Foxo1. This work identifies TGFβ as a microenvironmental cue that is critical for GC homeostasis and function.

SKI and SMAD4 are essential for IL-21-induced Th17 differentiation

Th17 cells are essential for the pathogenesis of inflammatory and autoimmune diseases. In the presence of TGF-β, the differentiation of Th17 cells can be induced by inflammatory cytokines, especially IL-6, which is mainly produced by antigen presenting cells (APCs); or IL-21, which is derived from T cells. IL-21 is required for IL-6-induced Th17 cell differentiation. However, the key regulators and underlying mechanisms for IL-21-induced Th17 differentiation is still elusive. Here we show that SMAD4 is a key regulator in IL-21-induced Th17 differentiation. SMAD4 deficient naïve T cells can differentiate into Th17 cells in the absence of TGF-β signaling, and these Th17 cells are pathogenic during EAE. SMAD4 represses Rorc mRNA transcription to constrain IL-21-induced Th17 differentiation in the absence of TGF-β signaling. While in the presence of TGF-β, SMAD4 losses its suppressive ability due to the degradation of SKI. Mutation of Y429A or A432E on SMAD4 disrupts the interaction of SKI from SMAD4 and eliminates SMAD4 mediated suppression of Th17 differentiation. SMAD4 is indispensable for SKI binding to Rorc promoter region to regulate Th17 differentiation. Moreover, activin can induce Th17 differentiation in combination with IL-21, and the process is also subjected to the control of SKI and SMAD4. This study therefore elucidates critical mechanism for IL-21-induced Th17 differentiation to indicate SKI and SMAD4 as potential therapeutic targets for treating autoimmune diseases.

IL-4 together with IL-1\u03b2 induces antitumor Th9 cell differentiation in the absence of TGF-\u03b2 signaling

IL-9-producing CD4+ (Th9) cells are a subset of CD4+ T-helper cells that are endowed with powerful antitumor capacity. Both IL-4 and TGF-β have been reported to be indispensable for Th9 cell-priming and differentiation. Here we show, by contrast, that Th9 cell development can occur in the absence of TGF-β signaling. When TGF-β was replaced by IL-1β, the combination of IL-1β and IL-4 efficiently promoted IL-9-producing T cells (Th9IL-4+IL-1β). Th9IL-4+ IL-1β cells are phenotypically distinct T cells compared to classic Th9 cells (Th9IL-4+TGF-β) and other Th cells, and are enriched for IL-1 and NF-κB gene signatures. Inhibition of NF-κB but not TGF-β-signaling negates IL-9 production by Th9IL-4+IL-1β cells. Furthermore, when compared with classic Th9IL-4+TGF-β cells, Th9IL-4+IL-1β cells are less exhausted, exhibit cytotoxic T effector gene signature and tumor killing function, and exert a superior antitumor response in a mouse melanoma model. Our study thus describes an alternative pathway for Th9 cell differentiation and provides a potential avenue for antitumor therapies.

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

Th17 cells are critically involved in host defense, inflammation, and autoimmunity1–5. TGF-β is instrumental in Th17 differentiation by cooperating with IL-66,7. Yet, the mechanism of how TGF-β enables Th17 differentiation remains elusive. Here we reveal that TGF-β licenses Th17 differentiation by releasing Ski-Smad4-complex suppressed RORγt expression. We found serendipitously that, unlike wild-type T cells, Smad4-deficient T cells differentiated into Th17 cells in the absence of TGF-β signaling in a RORγt-dependent manner. Ectopic Smad4 expression suppressed the RORγt expression and Th17 differentiation of Smad4-deficient T cells. Unexpectedly however, TGF-β neutralized Smad4 mediated suppression without affecting Smad4 binding to Rorc locus. Proteomic analysis revealed that Smad4 interacted with Ski, a transcriptional repressor degraded upon TGF-β stimulation. Ski controlled the histone acetylation/de-acetylation of Rorc locus and Th17 differentiation via Smad4 because ectopic Ski expression inhibited H3K9Ac of Rorc locus, Rorc expression and Th17 differentiation in a Smad4-dependent manner. Therefore, TGF-β-induced disruption of Ski releases Ski-Smad4 complex imposed suppression of RORγt to license Th17 differentiation. This study reveals a critical mechanism by which TGF-β controls Th17 differentiation and uncovers Ski-Smad4 axis as a potential therapeutic target for treating Th17 related diseases.

BLIMP-1/BLMP-1 and Metastasis-Associated Protein Regulate Stress Resistant Development in Caenorhabditis elegans.

Environmental stress triggers multilevel adaptations in animal development that depend in part on epigenetic mechanisms. In response to harsh environmental conditions and pheromone signals, Caenorhabditis elegans larvae become the highly stress-resistant and long-lived dauer. Despite extensive studies of dauer formation pathways that integrate specific environmental cues and appear to depend on transcriptional reprogramming, the role of epigenetic regulation in dauer development has remained unclear. Here we report that BLMP-1, the BLIMP-1 ortholog, regulates dauer formation via epigenetic pathways; in the absence of TGF-β signaling (in daf-7 mutants), lack of blmp-1 caused lethality. Using this phenotype, we screened 283 epigenetic factors, and identified lin-40, a homolog of metastasis-associate protein 1 (MTA1) as an interactor of BLMP-1 The interaction between LIN-40 and BLMP-1 is conserved because mammalian homologs for both MTA1 and BLIMP-1 could also interact. From microarray studies, we identified several downstream target genes of blmp-1: npr-3, nhr-23, ptr-4, and sams-1 Among them S-adenosyl methionine synthase (SAMS-1), is the key enzyme for production of SAM used in histone methylation. Indeed, blmp-1 is necessary for controlling histone methylation level in daf-7 mutants, suggesting BLMP-1 regulates the expression of SAMS-1, which in turn may regulate histone methylation and dauer formation. Our results reveal a new interaction between BLMP-1/BLIMP-1 and LIN-40/MTA1, as well as potential epigenetic downstream pathways, whereby these proteins cooperate to regulate stress-specific developmental adaptations.

TGF-β and NFAT control T cell receptor restimulation-induced cell death of CD4+CD25+Foxp3+ regulatory cells

Abstract One of the key questions in the area of immunomodulation is how CD4+CD25+Foxp3+ regulatory cells (Tregs) are controlled. Here we studied how their survival is regulated in the context of T cell Receptor re-stimulation apoptosis. We show that both mouse and human Tregs and iTregs exhibit increased resistance to T cell Receptor restimulation-induced cell death (RICD) compared to conventional CD4+ cells. The enhanced survival was dependent on TGF-β signaling that results in reduced nuclear translocation of nuclear factor of activated T-cells (NFAT), as well as up-regulation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) in Tregs. Tregs also showed significantly decreased susceptibility to α-CD3 antibody mediated apoptosis compared to the effector cells in vivo. While ionomycin increased Treg apoptosis, NFAT activation inhibitors reduced the apoptosis levels of effector cells almost to Treg levels, demonstrating that reduced NFAT activity contributes to decreased RICD in Tregs. Finally, RICD resistance in Tregs manifests itself as increased protection of these cells from cell death during mouse oropharyngeal candidiasis (OPC) infection and inflammatory bowel disease (IBD). Protection from cell death was abrogated in the absence of TGF-β signaling in Tregs. Taken together, our data unravel the previously unrecognized role of TGF-β in promoting Treg survival during TCR restimulation, through regulation of multiple pathways involving NFAT and cFLIP in Tregs. This novel mechanism that offers a selective survival advantage to Tregs may have broader ramifications in immune homeostasis during inflammation.

The TGF-β-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-β are well established, we wondered whether TGF-β could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-β promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-β up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-β type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-β signaling.

TGF-β Signalling Is Required for CD4+ T Cell Homeostasis But Dispensable for Regulatory T Cell Function

Author SummaryTGF-β is a cytokine thought to be critical for the maintenance and function of tolerance in the immune system. In many studies the disruption of TGF-β signalling in CD4+ T cells (a type of white blood cell that coordinates immune responses) has resulted in autoimmune syndromes. We show here that the induced removal of this cytokine's receptor from these specialised blood cells results in an astonishingly mild outcome. Contrary to expectations, the number of regulatory T cells is actually increased, and we find that these cells are not dependent on TGF-β signalling. We also show that removal of the receptor from mature CD4+ T cells does not lead to lethal autoinflammation; only when we removed the receptor during development of the cells did we see the characteristic lethal multi-organ inflammation reported previously in constitutive models of TGF-β receptor ablation. In summary, our findings indicate that although TGF-β regulates maintenance of mature CD4+ T cells, its signals are dispensable for immune tolerance within this cell population.

Truncated form of TGF-βRII, but not its absence, induces memory CD8+ T cell expansion and lymphoproliferative disorder in mice.

Inflammatory and anti-inflammatory cytokines play an important role in the generation of effector and memory CD8(+) T cells. We used two different models, transgenic expression of truncated (dominant negative) form of TGF-βRII (dnTGFβRII) and Cre-mediated deletion of the floxed TGF-βRII to examine the role of TGF-β signaling in the formation, function, and homeostatic proliferation of memory CD8(+) T cells. Blocking TGF-β signaling in effector CD8(+) T cells using both of these models demonstrated a role for TGF-β in regulating the number of short-lived effector cells but did not alter memory CD8(+) T cell formation and their function upon Listeria monocytogenes infection in mice. Interestingly, however, a massive lymphoproliferative disorder and cellular transformation were observed in Ag-experienced and homeostatically generated memory CD8(+) T cells only in cells that express the dnTGFβRII and not in cells with a complete deletion of TGF-βRII. Furthermore, the development of transformed memory CD8(+) T cells expressing dnTGFβRII was IL-7- and IL-15-independent, and MHC class I was not required for their proliferation. We show that transgenic expression of the dnTGFβRII, rather than the absence of TGF-βRII-mediated signaling, is responsible for dysregulated expansion of memory CD8(+) T cells. This study uncovers a previously unrecognized dominant function of the dnTGFβRII in CD8(+) T cell proliferation and cellular transformation, which is caused by a mechanism that is different from the absence of TGF-β signaling. These results should be considered during both basic and translational studies where there is a desire to block TGF-β signaling in CD8(+) T cells.

Suppressing autoimmunity by TGF-β: not just through Treg cells

Transforming growth factor-β (TGF-β) is a pleiotropic factor. It regulates many aspects of T-cell biology including thymocyte development, T cell differentiation and effector T cell function.1 Previous work has shown that the loss of TGF-β signaling in the T-cell lineage leads to severe lymphoproliferative autoimmunity.2,3 Until now, it has been unclear to what extent the autoimmunity observed in those mice is driven by TGF–unresponsive conventional T cells, as TGF- is also crucial for the maintenance and function of regulatory T cells (Treg). A recent study published in Nature Immunology revealed a novel role of TGF-β signaling in non-regulatory naive T cells. It was found that TGF-β controls lymphopenia-driven T-cell proliferation in neonatal mice, thereby preventing the development of autoimmunity.4

TGF-β is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy

A major gap in our understanding of infant immunity is why natural killer (NK) cellresponses are deficient, making infants more prone to viral infection. Here we demonstrate that transforming growth factor-β (TGF-β) was responsible for NK cell immaturity during infancy. Higher numbers of fully mature NK cells were found in CD11cdnR mice, whose NK cells lack TGF-βR signaling. Importantly, ontogenic maturation of NK cells progressed faster in the absence of TGF-β signaling, resulting in the formation of mature NK cell pool early in life. As a consequence, infant CD11cdnR mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-β in ontogeny that can explain why NK cell responses are deficient early in life.

Cutting Edge: In the Absence of TGF-β Signaling in T Cells, Fewer CD103+ Regulatory T Cells Develop, but Exuberant IFN-γ Production Renders Mice More Susceptible to Helminth Infection

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk

IntroductionTransforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.MethodsFluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.ResultsIntravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.ConclusionFibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

Th17 cells generated in the absence of TGF-β induce experimental allergic encephalitis upon adoptive transfer

Evaluation of: Ghoreschi K, Laurence A, Yang XP et al. Generation of pathogenic T(h)17 cells in the absence of TGF-β signalling. Nature 467(7318), 967–971 (2010).The discovery of IL-17-producing helper T cells (Th17) has led to new concepts of T-cell differentiation and immunity. Importantly, Th17 cells are thought to be important drivers of autoimmunity. TGF-β and IL-6 in combination were shown to induce differentiation of murine naive CD4 T cells into IL-17-producing cells in vitro. By contrast, human Th17 differentiation was shown to be independent of TGF-β and could be induced by IL-6 in conjunction with IL-21 or with IL-1. Ghoreschi et al. have elegantly demonstrated that mouse Th17 cell differentiation can also occur in the absence of TGF-β. A combination of IL-23, IL-1 and IL-6 can give rise to IL-17-producing cells, which are characterized by the expression of T-bet. Intriguingly, the adoptive transfer of such in vitro differentiated Th17 cells into lymphocyte-deficient mice resulted in the induction of experimental allergic encephalitis, which was more severe than in mice receiving Th17 cells differentiated in the presence of TGF-β. Collectively, the results suggest that a subpopulation of Th17 cells differentiated in the absence of TGF-β, expressing T-bet and RORγt, occur in vivo and may be responsible for autoimmunity.

TGFβ signaling and congenital heart disease: Insights from mouse studies.

Transforming growth factor β (TGFβ) regulates one of the major signaling pathways that control tissue morphogenesis. In vitro experiments using heart explants indicated the importance of this signaling pathway for the generation of cushion mesenchymal cells, which ultimately contribute to the valves and septa of the mature heart. Recent advances in mouse genetics have enabled in vivo investigation into the roles of individual ligands, receptors, and coreceptors of this pathway, including investigation of the tissue specificity of these roles in heart development. This work has revealed that (1) cushion mesenchyme can form in the absence of TGFβ signaling, although mesenchymal cell numbers may be misregulated; (2) TGFβ signaling is essential for correct remodeling of the cushions, particularly those of the outflow tract; (3) TGFβ signaling also has a role in ensuring accurate remodeling of the pharyngeal arch arteries to form the mature aortic arch; and (4) mesenchymal cells derived from the epicardium require TGFβ signaling to promote their differentiation to vascular smooth muscle cells to support the coronary arteries. In addition, a mouse genetics approach has also been used to investigate the disease pathogenesis of Loeys-Dietz syndrome, a familial autosomal dominant human disorder characterized by a dilated aortic root, and associated with mutations in the two TGFβ signaling receptor genes, TGFBR1 and TGFBR2. Further important insights are likely as this exciting work progresses.

The differentiation of pathogenic Th17 cells is negatively regulated by the transcription factor ELF4 (152.6)

Abstract T helper 17 (Th17) cells are potent inducers of tissue inflammation involved in several autoimmune diseases such as multiple sclerosis (MS). Although the importance of environmental factors such as IL-6, IL-21, and IL-23 has been well established, the transcriptional network regulating lineage specification and acquisition of effector function in Th17 cells is not entirely understood. We found that loss of the transcription factor ELF4 led to increased in vitro Th17 differentiation without significant alteration in Th1, Th2, and Treg polarization. The increased differentiation of Elf4-null Th17 cells was also observed in the absence of TGFβ signaling, known to abrogate pathogenicity of Th17 cells, suggesting that polarized Elf4-null CD4+ T cells may be more pathogenic. By using experimental allergic encephalomyelitis (EAE), a mouse model for human MS, we found that Elf4-null mice exhibited an earlier onset and increased severity of disease compared to wild-type mice, supporting a negative role of ELF4 in pathogenicity of Th17 cells in vivo. To understand how ELF4 modulates Th17 differentiation, we investigated cytokine signaling in Elf4-null CD4+ T cells. Increased phosphorylation of STAT3 was observed in Elf4-null CD4+ T cells after 5-min and 30-min IL-6 stimulation. Collectively, our study identified a novel intrinsic regulator that may inhibit Th17 differentiation by dampening cytokine signaling through STAT3 inhibition.

TGF-β Promotion of Gli2-Induced Expression of Parathyroid Hormone-Related Protein, an Important Osteolytic Factor in Bone Metastasis, Is Independent of Canonical Hedgehog Signaling

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.