Matthew B. Greenblatt

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)—a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)—is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

Calcineurin regulates innate antifungal immunity in neutrophils

Patients taking immunosuppressive drugs, like cyclosporine A (CsA), that inhibit calcineurin are highly susceptible to disseminated fungal infections, although it is unclear how these drugs suppress resistance to these opportunistic pathogens. We show that in a mouse model of disseminated Candida albicans infection, CsA-induced susceptibility to fungal infection maps to the innate immune system. To further define the cell types targeted by CsA, we generated mice with a conditional deletion of calcineurin B (CnB) in neutrophils. These mice displayed markedly decreased resistance to infection with C. albicans, and both CnB-deficient and CsA-treated neutrophils showed a defect in the ex vivo killing of C. albicans. In response to the fungal-derived pathogen-associated molecular pattern zymosan, neutrophils lacking CnB displayed impaired up-regulation of genes (IL-10, Cox2, Egr1, and Egr2) regulated by nuclear factor of activated T cells, the best characterized CnB substrate. This activity was Myd88 independent and was reproduced by stimulation with the β(1,3) glucan curdlan, indicating that dectin-1, rather than toll-like receptors, is the upstream activator of calcineurin. Our results suggest that disseminated fungal infections seen in CsA-treated patients are not just a general consequence of systemic suppression of adaptive immunity but are, rather, a result of the specific blockade of evolutionarily conserved innate pathways for fungal resistance.

Mitogen-Activated Protein Kinase Pathways in Osteoblasts

Mitogen-activated protein kinases (MAPKs) are ancient signal transducers well characterized as mediators of inflammation and neoplastic transformation. Recent work has expanded our understanding of their developmental functions, particularly in the regulation of bone mass via control of osteoblast differentiation. Here, we review the functions of MAPK pathways in osteoblasts, including a consideration of MAPK substrates. In particular, MAPKs function to regulate the key transcriptional mediators of osteoblast differentiation, with ERK and p38 MAPKs phosphorylating RUNX2, the master regulator of osteoblast differentiation. ERK also activates RSK2, which in turn phosphorylates ATF4, a transcriptional regulator of late-stage osteoblast synthetic functions. The MAP3Ks and MAP2Ks upstream of MAPKs have also been investigated, and significant differences have been found in the wiring of MAPK pathways in osteoblasts relative to other tissues. Thus, the investigation of MAPKs in osteoblasts has both revealed critical mechanisms for the maintenance of bone mass and added to our understanding of how the individual components of MAPK pathways function in concert in a complex in vivo system.

TAK1 is an Essential Regulator of BMP Signalling in Cartilage

TGFβ activated kinase 1 (TAK1), a member of the MAPKKK family, controls diverse functions ranging from innate and adaptive immune system activation to vascular development and apoptosis. To analyse the in vivo function of TAK1 in cartilage, we generated mice with a conditional deletion of Tak1 driven by the collagen 2 promoter. Tak1col2 mice displayed severe chondrodysplasia with runting, impaired formation of secondary centres of ossification, and joint abnormalities including elbow dislocation and tarsal fusion. This phenotype resembled that of bone morphogenetic protein receptor (BMPR)1 and Gdf5‐deficient mice. BMPR signalling was markedly impaired in TAK1‐deficient chondrocytes as evidenced by reduced expression of known BMP target genes as well as reduced phosphorylation of Smad1/5/8 and p38/Jnk/Erk MAP kinases. TAK1 mediates Smad1 phosphorylation at C‐terminal serine residues. These findings provide the first in vivo evidence in a mammalian system that TAK1 is required for BMP signalling and functions as an upstream activating kinase for Smad1/5/8 in addition to its known role in regulating MAP kinase pathways. Our experiments reveal an essential role for TAK1 in the morphogenesis, growth, and maintenance of cartilage.

Graft versus Host Disease in the Bone Marrow, Liver and Thymus Humanized Mouse Model

Mice bearing a “humanized” immune system are valuable tools to experimentally manipulate human cells in vivo and facilitate disease models not normally possible in laboratory animals. Here we describe a form of GVHD that develops in NOD/SCID mice reconstituted with human fetal bone marrow, liver and thymus (NS BLT mice). The skin, lungs, gastrointestinal tract and parotid glands are affected with progressive inflammation and sclerosis. Although all mice showed involvement of at least one organ site, the incidence of overt clinical disease was approximately 35% by 22 weeks after reconstitution. The use of hosts lacking the IL2 common gamma chain (NOD/SCID/γc−/−) delayed the onset of disease, but ultimately did not affect incidence. Genetic analysis revealed that particular donor HLA class I alleles influenced the risk for the development of GVHD. At a cellular level, GVHD is associated with the infiltration of human CD4+ T cells into the skin and a shift towards Th1 cytokine production. GVHD also induced a mixed M1/M2 polarization phenotype in a dermal murine CD11b+, MHC class II+ macrophage population. The presence of xenogenic GVHD in BLT mice both presents a major obstacle in the use of humanized mice and an opportunity to conduct preclinical studies on GVHD in a humanized model.

Interspecies Comparison of Human and Murine Scleroderma Reveals IL-13 and CCL2 as Disease Subset-Specific Targets

Development of personalized treatment regimens is hampered by lack of insight into how individual animal models reflect subsets of human disease, and autoimmune and inflammatory conditions have proven resistant to such efforts. Scleroderma is a lethal autoimmune disease characterized by fibrosis, with no effective therapy. Comparative gene expression profiling showed that murine sclerodermatous graft-versus-host disease (sclGVHD) approximates an inflammatory subset of scleroderma estimated at 17% to 36% of patients analyzed with diffuse, 28% with limited, and 100% with localized scleroderma. Both sclGVHD and the inflammatory subset demonstrated IL-13 cytokine pathway activation. Host dermal myeloid cells and graft T cells were identified as sources of IL-13 in the model, and genetic deficiency of either IL-13 or IL-4Rα, an IL-13 signal transducer, protected the host from disease. To identify therapeutic targets, we explored the intersection of genes coordinately up-regulated in sclGVHD, the human inflammatory subset, and IL-13-treated fibroblasts; we identified chemokine CCL2 as a potential target. Treatment with anti-CCL2 antibodies prevented sclGVHD. Last, we showed that IL-13 pathway activation in scleroderma patients correlated with clinical skin scores, a marker of disease severity. Thus, an inflammatory subset of scleroderma is driven by IL-13 and may benefit from IL-13 or CCL2 blockade. This approach serves as a model for personalized translational medicine, in which well-characterized animal models are matched to molecularly stratified patient subsets.

MLK3 regulates bone development downstream of the faciogenital dysplasia protein FGD1 in mice

Mutations in human FYVE, RhoGEF, and PH domain-containing 1 (FGD1) cause faciogenital dysplasia (FGDY; also known as Aarskog syndrome), an X-linked disorder that affects multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase CDC42. However, the mechanisms by which mutations in FGD1 affect skeletal development are unknown. Here, we describe what we believe to be a novel signaling pathway in osteoblasts initiated by FGD1 that involves the MAP3K mixed-lineage kinase 3 (MLK3). We observed that MLK3 functions downstream of FGD1 to regulate ERK and p38 MAPK, which in turn phosphorylate and activate the master regulator of osteoblast differentiation, Runx2. Mutations in FGD1 found in individuals with FGDY ablated its ability to activate MLK3. Consistent with our description of this pathway and the phenotype of patients with FGD1 mutations, mice with a targeted deletion of Mlk3 displayed multiple skeletal defects, including dental abnormalities, deficient calvarial mineralization, and reduced bone mass. Furthermore, mice with knockin of a mutant Mlk3 allele that is resistant to activation by FGD1/CDC42 displayed similar skeletal defects, demonstrating that activation of MLK3 specifically by FGD1/CDC42 is important for skeletal mineralization. Thus, our results provide a putative biochemical mechanism for the skeletal defects in human FGDY and suggest that modulating MAPK signaling may benefit these patients.

NFATc1 and NFATc2 repress spontaneous osteoarthritis

Significance Currently, little is understood about how the transcriptional regulation of cartilage breakdown contributes to pathogenesis of osteoarthritis (OA). Here, we report that, within cartilage, the transcription factor Nuclear factor of activated T cells c1 (NFATc1) displays selective expression in superficial articular chondrocytes. Accordingly, mice lacking both NFATc1 and NFATc2 in cartilage were generated and found to develop a severe, spontaneous and early-onset OA. These findings establish NFATc1 as a key transcriptional repressor of cartilage breakdown and OA. Additionally, these findings provide a unique model of OA that is an attractive platform for the preclinical development of treatments to alter the course of OA. Osteoarthritis (OA) was once viewed originally as a mechanical disease of “wear and tear,” but advances made during the past two decades suggest that abnormal biomechanics contribute to active dysregulation of chondrocyte biology, leading to catabolism of the cartilage matrix. A number of signaling and transcriptional mechanisms have been studied in relation to the regulation of this catabolic program, but how they specifically regulate the initiation or progression of the disease is poorly understood. Here, we demonstrate that cartilage-specific ablation of Nuclear factor of activated T cells c1 (Nfatc1) in Nfatc2−/− mice leads to early onset, aggressive OA affecting multiple joints. This model recapitulates features of human OA, including loss of proteoglycans, collagen and aggrecan degradation, osteophyte formation, changes to subchondral bone architecture, and eventual progression to cartilage effacement and joint instability. Consistent with the notion that NFATC1 is an OA-suppressor gene, NFATC1 expression was significantly down-regulated in paired lesional vs. macroscopically normal cartilage samples from OA patients. The highly penetrant, early onset, and severe nature of this model make it an attractive platform for the preclinical development of treatments to alter the course of OA. Furthermore, these findings indicate that NFATs are key suppressors of OA, and regulating NFATs or their transcriptional targets in chondrocytes may lead to novel disease-modifying OA therapies.

S6K1 Negatively Regulates TAK1 Activity in the Toll-Like Receptor Signaling Pathway

ABSTRACT Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1−/− and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1−/− mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.