Overlapping Functions Research Articles

Comparative Insight into Microglia/Macrophages-Associated Pathways in Glioblastoma and Alzheimer's Disease.

Microglia and macrophages are pivotal to the brain's innate immune response and have garnered considerable attention in the context of glioblastoma (GBM) and Alzheimer's disease (AD) research. This review delineates the complex roles of these cells within the neuropathological landscape, focusing on a range of signaling pathways-namely, NF-κB, microRNAs (miRNAs), and TREM2-that regulate the behavior of tumor-associated macrophages (TAMs) in GBM and disease-associated microglia (DAMs) in AD. These pathways are critical to the processes of neuroinflammation, angiogenesis, and apoptosis, which are hallmarks of GBM and AD. We concentrate on the multifaceted regulation of TAMs by NF-κB signaling in GBM, the influence of TREM2 on DAMs' responses to amyloid-beta deposition, and the modulation of both TAMs and DAMs by GBM- and AD-related miRNAs. Incorporating recent advancements in molecular biology, immunology, and AI techniques, through a detailed exploration of these molecular mechanisms, we aim to shed light on their distinct and overlapping regulatory functions in GBM and AD. The review culminates with a discussion on how insights into NF-κB, miRNAs, and TREM2 signaling may inform novel therapeutic approaches targeting microglia and macrophages in these neurodegenerative and neoplastic conditions. This comparative analysis underscores the potential for new, targeted treatments, offering a roadmap for future research aimed at mitigating the progression of these complex diseases.

Abstract 1646: IK-930, a paralog-selective TEAD inhibitor for treating YAP/TAZ-TEAD dependent cancers

Abstract The Hippo signaling cascade regulates cell proliferation and survival, as well as overall tissue homeostasis. These functions are mediated by the TEAD family of transcription factors which, when bound to the co-activators YAP or TAZ, induce expression of pro-growth and anti-apoptotic genes. This pathway is frequently dysregulated across tumor types, with genetic alterations along with other mechanisms driving hyper-active YAP/TAZ-TEAD. In addition, Hippo signaling mediates resistance to therapies targeting key oncogenic pathways such as EGFR and RAS. As a result, TEAD transcription factors are promising therapeutic targets. The TEAD family comprises four paralogs (TEAD1-4) that have both over-lapping and non-redundant functions. Given the essential roles of the Hippo pathway in normal physiology, it may be beneficial to target a subset of TEADs to minimize potential on-target toxicity while maintaining anti-tumor efficacy. Although highly homologous, the lipid-binding pocket of TEADs harbor some sequence divergence, highlighting an opportunity to design paralog-specific compounds. We developed IK-930 as a novel, selective inhibitor that potently blocks TEAD transcriptional activity by disrupting the auto-palmitoylation required for its interaction with YAP/TAZ. To demonstrate the selectivity profile of IK-930, a suite of biochemical assays was employed, all of which showed preferential compound binding to an individual paralog. A novel NanoBRETTM system that enabled quantitative, high-throughput measurements of cellular TEAD engagement also documented selective inhibitor interaction with the same family member. To assess how differences in selectivity affect therapeutic index, the in vivo efficacy and toxicity of IK-930 was compared to a pan-TEAD inhibitor. In several Hippo-mutated xenograft models, both compounds displayed potent anti-tumor activity. Moreover, IK-930 exhibited synergy with targeted agents, including EGFR inhibitors, indicating that its selectivity profile could drive robust efficacy in diverse tumor types. Previous studies in genetically engineered mouse models reported that perturbation of YAP/TAZ results in kidney toxicity. Treatment with a pan-TEAD inhibitor in rats and non-human primates led to substantial proteinuria and kidney pathology at exposures similar to those needed for antitumor activity in mouse models. In contrast, IK-930 demonstrated limited kidney toxicity in rats above efficacious doses and no signs of renal problems in non-human primates. Collectively, these results suggest that paralog selectivity with IK-930 broadens the therapeutic window of this novel compound class. With its distinct TEAD inhibitory profile, IK-930 offers unique therapeutic index advantages, further supporting its development as a first-in-class paralog-selective TEAD inhibitor. Citation Format: Nathan Young, George Punkosdy, Jill Cavanaugh, Collin Bantle, Alex Constan, Bin Li, James Conley, Marta Sanchez-Martin, Lan Xu, Karen McGovern, Alfredo Castro, Michelle Zhang, Jeffrey Ecsedy. IK-930, a paralog-selective TEAD inhibitor for treating YAP/TAZ-TEAD dependent cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1646.

The proximal intestinal Fatty Acid-Binding Proteins liver FABP (LFABP) and intestinal FABP (IFABP) differentially modulate whole body energy homeostasis but are not centrally involved in net dietary lipid absorption: Studies of the LFABP/IFABP double knockout mouse

Proximal intestinal enterocytes expresses both intestinal-fatty acid binding protein (IFABP; FABP2) and liver-FABP (LFABP; FABP1). These FABPs are thought to be important in the net uptake of dietary lipid from the intestinal lumen, however their specific and potentially unique functions in the enterocyte remain incompletely understood. We previously showed markedly divergent phenotypes in LFABP−/− vs. IFABP−/− mice fed high-fat diets, with the former becoming obese and the latter remaining lean relative to wild-type (WT) mice, supporting different functional roles for each protein. Interestingly, neither mouse model displayed increased fecal lipid concentration, raising the question of whether the presence of one FABP was sufficient to compensate for absence of the other. Here, we generated an LFABP and IFABP double knockout mouse (DKO) to determine whether simultaneous ablation would lead to fat malabsorption, and to further interrogate the individual vs. overlapping functions of these proteins. Male WT, IFABP−/−, LFABP−/−, and DKO mice were fed a low-fat (10 % kcal) or high-fat (45 % kcal) diet for 12 weeks. The body weights and fat mass of the DKO mice integrated those of the LFABP−/− and IFABP−/− single knockouts, supporting the notion that IFABP and LFABP have distinct functions in intestinal lipid assimilation that result in downstream alterations in systemic energy metabolism. Remarkably, no differences in fecal fat concentrations were found in the DKO compared to WT, revealing that the FABPs are not required for net intestinal uptake of dietary lipid.

Regulation and metabolic functions of mTORC1 and mTORC2

Cells metabolize nutrients for biosynthetic and bioenergetic needs to fuel growth and proliferation. The uptake of nutrients from the environment and their intracellular metabolism is a highly controlled process that involves cross talk between growth signaling and metabolic pathways. Despite constant fluctuations in nutrient availability and environmental signals, normal cells restore metabolic homeostasis to maintain cellular functions and prevent disease. A central signaling molecule that integrates growth with metabolism is the mechanistic target of rapamycin (mTOR). mTOR is a protein kinase that responds to levels of nutrients and growth signals. mTOR forms two protein complexes, mTORC1, which is sensitive to rapamycin, and mTORC2, which is not directly inhibited by this drug. Rapamycin has facilitated the discovery of the various functions of mTORC1 in metabolism. Genetic models that disrupt either mTORC1 or mTORC2 have expanded our knowledge of their cellular, tissue, as well as systemic functions in metabolism. Nevertheless, our knowledge of the regulation and functions of mTORC2, particularly in metabolism, has lagged behind. Since mTOR is an important target for cancer, aging, and other metabolism-related pathologies, understanding the distinct and overlapping regulation and functions of the two mTOR complexes is vital for the development of more effective therapeutic strategies. This review discusses the key discoveries and recent findings on the regulation and metabolic functions of the mTOR complexes. We highlight findings from cancer models but also discuss other examples of the mTOR-mediated metabolic reprogramming occurring in stem and immune cells, type 2 diabetes/obesity, neurodegenerative disorders, and aging.

Constructions of <i>n</i>-Dimensional Overlap Functions Based on Bivariate Overlap Functions

In this paper, we firstly introduce some new results on overlap functions and n-dimensional overlap functions. On the other hand, in a previous study, Gómez et al. presented some open problems. One of these open problems is “to search the construction of n-dimensional overlapping functions based on bi-dimensional overlapping functions”. To answer this open problem, in this paper, we mainly introduce one construction method of n-dimensional overlap functions based on bivariate overlap functions. We mainly use the conjunction operator ∧ to construct n-dimensional overlap functions based on bivariate overlap functions and study their basic properties.

Genetic interactions between the <i>CUP-SHAPED COTYLEDON</i> and the <i>BELLRINGER</i> genes indicate their overlapping functions in carpel boundary development in <i>Arabidopsis thaliana </i>

Genetic interactions between the <i>CUP-SHAPED COTYLEDON</i> and the <i>BELLRINGER</i> genes indicate their overlapping functions in carpel boundary development in <i>Arabidopsis thaliana </i>

Isoform-specific functions of an evolutionarily conserved 3\xa0bp micro-exon alternatively spliced from another exon in Drosophila homothorax gene

Micro-exons are exons of very small size (usually 3–30 nts). Some micro-exons are alternatively spliced. Their functions, regulation and evolution are largely unknown. Here, we present an example of an alternatively spliced 3 bp micro-exon (micro-Ex8) in the homothorax (hth) gene in Drosophila. Hth is involved in many developmental processes. It contains a MH domain and a TALE-class homeodomain (HD). It binds to another homeodomain Exd via its MH domain to promote the nuclear import of the Hth-Exd complex and serve as a cofactor for Hox proteins. The MH and HD domains in Hth as well as the HTh-Exd interaction are highly conserved in evolution. The alternatively spliced micro-exon lies between the exons encoding the MH and HD domains. We provide clear proof that the micro-Ex8 is produced by alternative splicing from a 48 bp full-length exon 8 (FL-Ex8) and the micro-Ex8 is the first three nt is FL-Ex8. We found that the micro-Ex8 is the ancient form and the 3 + 48 organization of alternatively spliced overlapping exons only emerged in the Schizophora group of Diptera and is absolutely conserved in this group. We then used several strategies to test the in vivo function of the two types of isoforms and found that the micro-Ex8 and FL-Ex8 isoforms have largely overlapping functions but also have non-redundant functions that are tissue-specific, which supports their strong evolutionary conservation. Since the different combinations of protein interaction of Hth with Exd and/or Hox can have different DNA target specificity, our finding of alternatively spliced isoforms adds to the spectrum of structural and functional diversity under developmental regulation.

β-adrenergic modulation of discrimination learning and memory in the auditory cortex.

Despite vast literature on catecholaminergic neuromodulation of auditory cortex functioning in general, knowledge about its role for long‐term memory formation is scarce. Our previous pharmacological studies on cortex‐dependent frequency‐modulated tone‐sweep discrimination learning of Mongolian gerbils showed that auditory‐cortical D1/5‐dopamine receptor activity facilitates memory consolidation and anterograde memory formation. Considering overlapping functions of D1/5‐dopamine receptors and β‐adrenoceptors, we hypothesised a role of β‐adrenergic signalling in the auditory cortex for sweep discrimination learning and memory. Supporting this hypothesis, the β1/2‐adrenoceptor antagonist propranolol bilaterally applied to the gerbil auditory cortex after task acquisition prevented the discrimination increment that was normally monitored 1 day later. The increment in the total number of hurdle crossings performed in response to the sweeps per se was normal. Propranolol infusion after the seventh training session suppressed the previously established sweep discrimination. The suppressive effect required antagonist injection in a narrow post‐session time window. When applied to the auditory cortex 1 day before initial conditioning, β1‐adrenoceptor‐antagonising and β1‐adrenoceptor‐stimulating agents retarded and facilitated, respectively, sweep discrimination learning, whereas β2‐selective drugs were ineffective. In contrast, single‐sweep detection learning was normal after propranolol infusion. By immunohistochemistry, β1‐ and β2‐adrenoceptors were identified on the neuropil and somata of pyramidal and non‐pyramidal neurons of the gerbil auditory cortex. The present findings suggest that β‐adrenergic signalling in the auditory cortex has task‐related importance for discrimination learning of complex sounds: as previously shown for D1/5‐dopamine receptor signalling, β‐adrenoceptor activity supports long‐term memory consolidation and reconsolidation; additionally, tonic input through β1‐adrenoceptors may control mechanisms permissive for memory acquisition.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

A Study on the Improvement of Philippine Maritime Traffic Environment and Safety Management System

The paper presents the Philippine maritime condition, safety systems and navigational aids. It aims to uplift the maritime safety systems and infrastructures by studying the different strata of the industry, thereby emphasizing on the structures, weaknesses and opportunities. It presents the different maritime agencies involved and each responsibilities. Data from PPA demonstrate the statistics of increase in the cargo throughput, passenger, shipcalls and cargo-container handled in the year 2014. It also highlights the insufficiency of different navigational aids, VTMS, MBS and TSS placed in the whole archipelago. It gathers data of maritime accidents in 10-year period, 2006-2015, showing the most occurred accidents, casualties and areas these accidents happened. It includes the weather phenomena which contribute to the maritime movements and accidents incidence. The results show that systems and infrastructures that focus on the lack of improvement in technology and over-lapping functions of different agencies of government require development and upgrading.

Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.

Resequencing Reveals Different Domestication Rate for BADH1 and BADH2 in Rice (Oryza sativa).

BADH1 and BADH2 are two homologous genes, encoding betaine aldehyde dehydrogenase in rice. In the present study, we scanned BADHs sequences of 295 rice cultivars, and 10 wild rice accessions to determine the polymorphisms, gene functions and domestication of these two genes. A total of 16 alleles for BADH1 and 10 alleles for BADH2 were detected in transcribed region of cultivars and wild species. Association study showed that BADH1 has significant correlation with salt tolerance in rice during germination stage, the SNP (T/A) in exon 4 is highly correlated with salt tolerance index (STI) (P<10−4). While, BADH2 was only responsible for rice fragrance, of which two BADH2 alleles (8 bp deletion in exon 7 and C/T SNP in exon 13) explain 97% of aroma variation in our germplasm. Theses indicate that there are no overlapping functions between the two homologous genes. In addition, a large LD block was detected in BADH2 region, however, there was no large LD blocks in a 4-Mb region of BADH1. We found that BADH2 region only showed significant bias in Tajima’s D value from the balance. Extended haplotype homozygosity study revealed fragrant accessions had a large LD block that extended around the mutation site (8 bp deletion in exon 7) of BADH2, while both of the BADH1 alleles (T/A in exon 4) did not show large extended LD block. All these results suggested that BADH2 was domesticated during rice evolution, while BADH1 was not selected by human beings.

The UPR branch IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to the only UPR pathway in yeast.

The unfolded protein response (UPR) signaling network encompasses two pathways in plants, one mediated by inositol-requiring protein-1 (IRE1)-bZIP60 mRNA and the other by site-1/site-2 proteases (S1P/S2P)-bZIP17/bZIP28. As the major sensor of UPR in eukaryotes, IRE1, in response to endoplasmic reticulum (ER) stress, catalyzes the unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans. Recent studies suggest that IRE1p and HAC1 mRNA, the only UPR pathway found in yeast, evolves as a cognate system responsible for the robust UPR induction. However, the functional connectivity of IRE1 and its splicing target in multicellular eukaryotes as well as the degree of conservation of IRE1 downstream signaling effectors across eukaryotes remains to be established. Here, we report that IRE1 and its substrate bZIP60 function as a strictly cognate enzyme-substrate pair to control viral pathogenesis in plants. Moreover, we show that the S1P/S2P-bZIP17/bZIP28 pathway, the other known branch of UPR in plants, does not play a detectable role in virus infection, demonstrating the distinct function of the IRE1-bZIP60 pathway in plants. Furthermore, we provide evidence that bZIP60 and HAC1, products of the enzyme-substrate duet, rather than IRE1, are functionally replaceable to cope with ER stress in yeast. Taken together, we conclude that the downstream signaling of the IRE1-mediated splicing is evolutionarily conserved in yeast and plants, and that the IRE1-bZIP60 UPR pathway not only confers overlapping functions with the other UPR branch in fundamental biology but also may exert a unique role in certain biological processes such as virus-plant interactions.

Non-targeted metabolomics of Brg1/Brm double-mutant cardiomyocytes reveals a novel role for SWI/SNF complexes in metabolic homeostasis.

Mammalian SWI/SNF chromatin-remodeling complexes utilize either BRG1 or Brm as alternative catalytic subunits to alter the position of nucleosomes and regulate gene expression. Genetic studies have demonstrated that SWI/SNF complexes are required during cardiac development and also protect against cardiovascular disease and cancer. However, Brm constitutive null mutants do not exhibit a cardiomyocyte phenotype and inducible Brg1 conditional mutations in cardiomyocyte do not demonstrate differences until stressed with transverse aortic constriction, where they exhibit a reduction in cardiac hypertrophy. We recently demonstrated the overlapping functions of Brm and Brg1 in vascular endothelial cells and sought here to test if this overlapping function occurred in cardiomyocytes. Brg1/Brm double mutants died within 21 days of severe cardiac dysfunction associated with glycogen accumulation and mitochondrial defects based on histological and ultrastructural analyses. To determine the underlying defects, we performed nontargeted metabolomics analysis of cardiac tissue by GC/MS from a line of Brg1/Brm double-mutant mice, which lack both Brg1 and Brm in cardiomyocytes in an inducible manner, and two groups of controls. Metabolites contributing most significantly to the differences between Brg1/Brm double-mutant and control-group hearts were then determined using the variable importance in projection analysis. Increased cardiac linoleic acid and oleic acid suggest alterations in fatty acid utilization or intake are perturbed in Brg1/Brm double mutants. Conversely, decreased glucose-6-phosphate, fructose-6-phosphate, and myoinositol suggest that glycolysis and glycogen formation are impaired. These novel metabolomics findings provide insight into SWI/SNF-regulated metabolic pathways and will guide mechanistic studies evaluating the role of SWI/SNF complexes in homeostasis and cardiovascular disease prevention.

Plastid division control: the PDV proteins regulate DRP5B dynamin activity

Chloroplast division represents a fundamental but complex biological process involving remnants of the ancestral bacterial division machinery and proteins of eukaryotic origin. Moreover, the chloroplast division machinery is divided into stromal and cytosolic sub machineries, which coordinate and control their activities to ensure appropriate division initiation and progression. Dynamin related protein 5B (DRP5B) and plastid division protein 1 and 2 (PDV1 and PDV2) are all plant-derived proteins and represent components of the cytosolic division machinery, where DRP5B is thought to exert constrictional force during division. However, the direct relationship between PDV1, PDV2 and DRP5B, and moreover how DRP5B is regulated during plastid constriction remains unclear. In this study we show that PDV1 and PDV2 can interact with themselves and with each other through their cytosolic domains. We demonstrate that DRP5B interacts with itself and with the cytosolic region of PDV1 and that the two functional isoforms of DRP5B have highly overlapping functions. We further show that DRP5B harbors GTPase activity and moreover that PDV1 and PDV2 inhibits DRP5B-mediated GTP hydrolysis in a ratio dependent manner. Our data suggest that the PDV proteins contribute to the regulation of DRP5B activity thereby enforcing control over the division process during early constriction.

The Roles of Whole-Genome and Small-Scale Duplications in the Functional Specialization of Saccharomyces cerevisiae Genes

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD–duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.

Genetic and Pharmacologic Inhibition of PKCθ and PKCα Prevents Acute Gvhd While Sparing GVL Activity in Mice.

AbstractAbstract 3000In T effector cells (Teff), protein kinase C theta (PKCθ) is a stabilizer of the immunologic synapse with antigen presenting cells (APCs). Signal propagation through PKCθ leads to activation of inflammatory NF-kB, thus implicating this molecule as a therapeutic target for T-cell mediated disorders as well as GVHD. PKCθ deficiency impairs donor T-cell mediated alloresponse and GVHD while leaving GVL responses intact to a certain extent. Given PKCθ is known to cooperate with its alpha isoform (PKCa) to optimize T-cell signaling and function, we have studied the role of PKCθ and PKCa in T-cell mediated alloresponse after allogeneic bone marrow transplantation (BMT) by using gene-deficient mice as well as a small molecular inhibitor of PKCa and θ.T cells from PKCa, θ and a/θ deficient (−/−) donor mice were tested in their ability to induce GVHD using an allogeneic murine model of myeloablative BMT. GVHD progression was monitored with or without A20-luciferase cells to observe GVL in vivo. To target PKC pharmacologically the small molecule inhibitor R524 (Rigel, San Francisco, CA), specific for PKCa/θ, was tested on GVHD progression in twice daily oral doses of 40 mg/kg for 6 weeks. Efficacy of R524 was assessed in an MHC-mismatched BMT model with and without the addition of A20-luciferase cells and in a minor antigen-mismatched BMT model with or without the addition of C1498-luciferase-DsRed cells to track GVHD progression and tumor relapse.Using a genetic approach, PKCa−/− donor T-cell alloreactivity is comparable to wild type (WT) T cell responses. WT T cells induced robust GVHD at a dose of 1×106/mouse. The same number of PKCθ−/− and PKCa/θ−/− T cells failed to generate even mild GVHD in recipient mice. At a high T cell dose, 5×106/mouse, PKCa/θ−/− T cells failed to induce severe acute GVHD and increased survival (P < 0.0001). The ability of PKCa/θ−/− T cells to induce GVHD was further reduced from PKCθ-/- T cells as evidenced by greater splenic cell number (P < 0.05) and B220+ B cell reconstitution in PKCa/θ−/− T-cell recipients. B cell activity in PKCa/θ−/− T-cell recipients was greater on a per cell basis; while the activity of T cells isolated from these mice was greatly reduced compared to WT or PKCθ−/− T-cell recipients. Microscopic tissue damage was also reduced (P < 0.05) in GVHD target organs in PKCa/θ−/− T-cell recipients. While not absent, GVL activity was compromised in PKCa/θ−/− T-cell recipients when compared to WT or PKCθ−/− T-cell recipients.Using a pharmacologic approach, the PKCa/θ small molecule inhibitor R524 reduced (P < 0.05) CD4+ and CD8+ T-cell proliferation (by 44% and 57% respectively) 5 days post allogeneic BMT. Long-term GVHD efficacy studies in an MHC-mismatched BMT model illustrate R524 mediated weight retention (P < 0.002) and decreased mortality (P < 0.01) compared to vehicle treatment. These observations were supported by a reduction in donor T cell expansion (P < 0.05), increased splenic retention of CD4+, CD8+, and B220+ cells and fewer donor T cells found in GVHD target organs of the recipients treated with R524 (P < 0.05). Additionally, CD4+ and CD8+ T cells isolated from spleens, livers and lungs of R524-treated mice produced less CXCR3, CCR6, TNFa and IFNg than their vehicle-treated counterparts. Similar results were obtained from studies using a minor antigen-mismatched BMT model, in which R524 increased recipient weight retention (P < 0.01) and reduced mortality by 50% (P < 0.002). Crucially, GVL activity was largely preserved in R524-treated mice compared to vehicle controls and mortality rates associated with tumor burden were minimal in both MHC-matched and mismatched models of BMT.In conclusion, we clearly show that PKCa and θ contribute to T cell activity with over-lapping functions in the induction of GVHD and GVL effects. Small molecule inhibition of PKCa/θ signaling with pharmacologic inhibitors presents a valid therapeutic option for GVHD prevention with simultaneous maintenance of GVL activity for clinical consideration. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Kif7 regulates Gli2 through Sufu-dependent and -independent functions during skin development and tumorigenesis

Abnormal activation of Hedgehog (Hh) signaling leads to basal cell carcinoma (BCC) of the skin, the most common human cancer. Gli2, the major transcriptional activator of Hh signaling, is essential for hair follicle development and its overexpression in epidermis induces BCC formation and maintains tumor growth. Despite its importance in skin development and tumorigenesis, little is known about the molecular regulation of Gli2. Sufu and Kif7 are two evolutionarily conserved regulators of Gli transcription factors. Here, we show that Sufu and Kif7 regulate Gli2 through distinct mechanisms in keratinocytes. Sufu restricts the activity of Gli2 through cytoplasmic sequestration. Kif7 possesses Sufu-dependent and -independent regulatory functions in Hh signaling: while it promotes Hh pathway activity through the dissociation of Sufu-Gli2 complex, it also contributes to the repression of Hh target genes in the absence of Sufu. Deletion of both Sufu and Kif7 in embryonic skin leads to complete loss of follicular fate. Importantly, although inactivation of Sufu or Kif7 alone in adult epidermis cannot promote BCC formation, their simultaneous deletion induces BCC. These studies establish Sufu and Kif7 as crucial components in the regulation of Gli2 localization and activity, and illustrate their overlapping functions in skin development and tumor suppression.

Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that act as post-transcriptional regulators of target mRNA. In this study, we sought to identify the microRNA underlying local inflammation in a murine model of chronic kidney disease (CKD). In microarray analysis of kidneys, the expression of miR-146a/b was elevated in B6.MRLc1 CKD mice that spontaneously develop renal inflammation with age. Primary-microRNA analysis found that elevated miR-146a/b expression in the kidneys of B6.MRLc1 mice was mainly derived from miR-146a rather than miR-146b, and this expression increased with the development of CKD. Histopathological scores for glomerular and interstitial lesions, mRNA expression of inflammatory mediators, and macrophage infiltration were significantly higher in B6.MRLc1 than C57BL/6 mice and were positively correlated with miR-146a expression. In situ hybridization and laser microdissection-RT-PCR showed that miR-146a expression in interstitial lesions containing inflammatory cells was higher than in the glomerulus. The increased expression of the inflammatory-associated genes RELA, IRAK1, IL1B, IL10, and CXCLs was noted in miR-146a/b-silenced human monocytes. The amount of miR-146a was higher in urine sediments of B6.MRLc1 than of C57BL/6 mice. Thus, miR-146a expression in the kidneys and its urinary excretion was specifically associated with the development of interstitial lesions and correlated with inflammatory cell infiltration.