T cells Use Focal Adhesions to Pull Themselves Through Confined Environments.

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical composition. Their migration has classically been defined as amoeboid under the assumption that it is integrin-independent. Here we show that activated primary Th1 T cells require both confinement and extracellular matrix protein to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cell preferentially follows tracks of other T cells, suggesting that these adhesions are modifying the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated adhesions play a key role in T cell motility.

The CD28 Transmembrane Domain Contains an Essential Dimerization Motif.

CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.

Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning.

Due to the overlapping emission spectra of fluorophores, fluorescence microscopy images often have bleed-through problems, leading to a false positive detection. This problem is almost unavoidable when the samples are labeled with three or more fluorophores, and the situation is complicated even further when imaged under a multiphoton microscope. Several methods have been developed and commonly used by biologists for fluorescence microscopy spectral unmixing, such as linear unmixing, non-negative matrix factorization, deconvolution, and principal component analysis. However, they either require pre-knowledge of emission spectra or restrict the number of fluorophores to be the same as detection channels, which highly limits the real-world applications of those spectral unmixing methods. In this paper, we developed a robust and flexible spectral unmixing method: Learning Unsupervised Means of Spectra (LUMoS), which uses an unsupervised machine learning clustering method to learn individual fluorophores' spectral signatures from mixed images, and blindly separate channels without restrictions on the number of fluorophores that can be imaged. This method highly expands the hardware capability of two-photon microscopy to simultaneously image more fluorophores than is possible with instrumentation alone. Experimental and simulated results demonstrated the robustness of LUMoS in multi-channel separations of two-photon microscopy images. We also extended the application of this method to background/autofluorescence removal and colocalization analysis. Lastly, we integrated this tool into ImageJ to offer an easy to use spectral unmixing tool for fluorescence imaging. LUMoS allows us to gain a higher spectral resolution and obtain a cleaner image without the need to upgrade the imaging hardware capabilities.

PKK deletion in basal keratinocytes promotes tumorigenesis after chemical carcinogenesis.

Squamous cell carcinoma (SCC) of the skin is a keratinocyte malignancy characterized by tumors presenting on sun-exposed areas with surgery being the mainstay treatment. Despite advances in targeted therapy in other skin cancers, such as basal cell carcinoma and melanoma, there have been no such advances in the treatment of SCC. This is partly due to an incomplete knowledge of the pathogenesis of SCC. We have recently identified a protein kinase C-associated kinase (PKK) as a potential tumor suppressor in SCC. We now describe a novel conditional PKK knockout mouse model, which demonstrates that PKK deficiency promotes SCC formation during chemically induced tumorigenesis. Our results further support that PKK functions as a tumor suppressor in skin keratinocytes and is important in the pathogenesis of SCC of the skin. We further define the interactions of keratinocyte PKK with TP63 and NF-κB signaling, highlighting the importance of this protein as a tumor suppressor in SCC development.

A critical role for the protein kinase PKK in the maintenance of recirculating mature B cells and the development of B1 cells.

Protein kinase C associated kinase (PKK) regulates NF-κB activation and is required for the survival of certain lymphoma cells. Mice lacking PKK die soon after birth, and previous studies suggest that the role of PKK in B cell development might be context dependent. We have generated a mouse strain harboring conditional null alleles for PKK and a Cre-recombinase transgene under the control of the endogenous CD19 promoter. In the present study, we show that knockout of PKK in B cells results in the reduction of long-lived recirculating mature B cell population in lymph nodes and bone marrow as well as a decrease in peritoneal B1 cells, while PKK deficiency has no apparent effect on early B cell development in bone marrow or the development of follicular and marginal zone B cells in the spleen. In addition, we demonstrate that PKK-deficient B cells display defective proliferation and survival responses to stimulation of B cell receptor (BCR), which may underlie the reduction of recirculating mature B cells in PKK mutant mice. Consistently, BCR-mediated NF-κB activation, known to be required for the survival of activated but not resting B cells, is attenuated in PKK-deficient B cells. Thus, our results reveal a critical role of PKK in the maintenance of recirculating mature B cells as well as the development of B1 cells in mice.

Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

PKK suppresses tumor growth and is decreased in squamous cell carcinoma of the skin.

Non-melanoma skin cancer represents the most common cancer in the United States. Squamous cell carcinoma (SCC) of the skin is a subtype of NMSC that shows a greater potential for invasion and metastasis. The current study identifies the protein kinase C-associated kinase (PKK), which is also known as the receptor-interacting protein kinase 4, as a suppressor of tumor growth in SCC of the skin. We show that expression of PKK is decreased in human SCC of the skin compared with normal skin. Further, suppression of PKK in human keratinocytes leads to increased cell proliferation. The use of RNA interference to reduce PKK expression in keratinocytes leads to an increase in S phase and in proteins that promote cell cycle progression. Consistent with the results obtained from cell culture, there is a marked increased tumorigenesis after PKK knockdown in a xenotransplant model and in soft agar assays. The loss of tumor suppression involves the NF-κB and p63 pathways. NF-κB is inhibited through inhibition of inhibitor of NF-κB kinase function and there is increased nuclear TP63 activity after PKK knockdown. This study opens new avenues both in the discovery of disease pathogenesis and for potential treatments.

Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKß kinase activity. How IKKα and IKKß are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKß in mammalian cells and induces activation of both IKKα and IKKß via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKß as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKß in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

Protein kinase Cß is required for lupus development in Sle mice.

OBJECTIVE: To evaluate the requirement for protein kinase Cß (PKCß) in the development of lupus in mice, and to explore the potential of targeting PKCß as a therapeutic strategy in lupus. METHODS: Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which represent different stages of severity in the development of lupus, were crossed with PKCß-deficient mice. The effect of PKCß deficiency in lupus development was analyzed. In addition, the effects of the PKCß-specific inhibitor enzastaurin on the survival of B cells from mice with lupus and human 9G4-positive B cells as well as the in vivo effect of enzastaurin treatment on the development of lupus in Sle mice were investigated. RESULTS: In Sle mice, PKCß deficiency abrogated lupus-associated phenotypes, including high autoantibody levels, proteinuria, and histologic features of lupus nephritis. Significant decreases in spleen size and in the peritoneal B-1 cell population, reduced numbers of activated CD4 T cells, and normalized CD4:CD8 ratios were observed. PKCß deficiency induced an anergic B cell phenotype and preferentially inhibited autoreactive plasma cells and autoantibodies in mice with lupus. Inhibition of PKCß enhanced apoptosis of both B cells from Sle mice and human autoreactive B cells (9G4 positive). Treatment of Sle mice with the PKCß-specific inhibitor enzastaurin prevented the development of lupus. CONCLUSION: This study identifies PKCß as a central mediator of lupus pathogenesis, suggesting that PKCß represents a promising therapeutic target for the treatment of systemic lupus erythematosus. Moreover, the results indicate the feasibility of using a PKCß inhibitor for the treatment of lupus.

Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.

Protein kinase C-associated kinase is required for NF-kappaB signaling and survival in diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and the most common type of non-Hodgkin lymphoma. Despite recent advances in treatment, less than 50% of the patients are cured with current multiagent chemotherapy. Abnormal NF-kappaB activity not only contributes to tumor development but also renders cancer cells resistant to chemotherapeutic agents. Identifying and targeting signaling molecules that control NF-kappaB activation in cancer cells may thus yield more effective therapy for DLBCL. Here, we show that while overexpression of protein kinase C-associated kinase (PKK) activates NF-kappaB signaling in DLBCL cells, suppression of PKK expression inhibits NF-kappaB activity in these cells. In addition, we show that NF-kappaB activation induced by B cell-activating factor of tumor necrosis factor family (BAFF) in DLBCL cells requires PKK. Importantly, we show that knockdown of PKK impairs the survival of DLBCL cells in vitro and inhibits tumor growth of xenografted DLBCL cells in mice. Suppression of PKK expression also sensitizes DLBCL cells to treatment with chemotherapeutic agents. Together, these results indicate that PKK plays a pivotal role in the survival of human DLBCL cells and represents a potential target for DLBCL therapy.

Tight regulation of SpSoxB factors is required for patterning and morphogenesis in sea urchin embryos.

Previous studies in sea urchin embryos have demonstrated that nuclearization of beta-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by beta-catenin-dependent gene products that include the transcription repressor SpKrl. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of beta-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-beta-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow beta-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation.