Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production.

AIMS/HYPOTHESIS: Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. METHODS: Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 (Cre); PaskBKO) or alpha (Ppg (Cre) [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. RESULTS: Ins1 (Cre)-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. CONCLUSIONS/INTERPRETATION: The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.

The Snf1-related kinase, Hunk, is essential for mammary tumor metastasis.

We previously identified a SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for this kinase in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer in a manner consistent with the pro-metastatic function of Hunk in mice. These findings identify a direct role for Hunk kinase activity in metastasis and establish an in vivo function for this kinase.

Enabling 1536-Well High-Throughput Cell-Based Screening through the Application of Novel Centrifugal Plate Washing.

Cell-based assays have long been important within hit discovery paradigms; however, improving the disease relevance of the assay system can positively affect the translation of small-molecule drug discovery, especially if adopted in the initial hit identification assay. Consequently, there is an increasing need for disease-relevant assay systems capable of running at large scale, including the use of induced pluripotent stem cells and donor-derived primary cells. Major hurdles to adopting these assays for high-throughput screening are the cost, availability of cells, and complex protocols. Miniaturization of such assays to 1536-well format is an approach that can reduce costs and increase throughput. Adaptation of these complex cell assays to 1536-well format brings major challenges in liquid handling for high-content assays requiring washing steps and coating of plates. In addition, problematic edge effects and reduced assay quality are frequently encountered. In this study, we describe the novel application of a centrifugal plate washer to facilitate miniaturization of a range of 1536-well cell assays and techniques to reduce edge effects, all of which improved throughput and data quality. Cell assays currently limited in throughput because of cost and complex protocols may be enabled by the techniques presented in this study.

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine.

At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.

Intrarectal immunization of mice with VP6 and either LT(R192G) or CTA1-DD as adjuvant protects against fecal rotavirus shedding after EDIM challenge.

Intranasal or oral delivery of the chimeric rotavirus VP6 protein MBP::VP6 to mice elicited >90% reductions in fecal rotavirus shedding after murine rotavirus challenge. Protection depended on co-administration of adjuvants, the most effective being bacterial toxins. Because of safety and efficacy concerns following intranasal or oral toxin delivery, protective efficacy of MBP::VP6 after intrarectal delivery with toxin adjuvants was determined and compared to that induced after intranasal and oral immunization. Adult BALB/c mice were orally challenged with the murine rotavirus strain EDIM 4 weeks after their second immunization with MBP::VP6 and either LT(R192G), an attenuated Escherichia coli heat-labile toxin, or CTA1-DD, a cholera toxin derivative. Reductions in fecal rotavirus shedding were then determined relative to mock-immunized mice. Immunization with MBP::VP6 and either adjuvant by any route (except oral immunization with CTA1-DD) significantly (P<0.0001) reduced rotavirus shedding. As was previously found after oral and intranasal immunization, intrarectal immunization with MBP::VP6 and adjuvant was associated with T cell responses (IFNgamma and IL-17) but not B cell (antibody) responses.

Improved design and intranasal delivery of an M2e-based human influenza A vaccine.

M2 is the third integral membrane protein of influenza A. M2e, the extracellular, 23 amino acid residues of M2, has been remarkably conserved in all human influenza A strains. This prompted us to evaluate the use of M2e as a potential broad-spectrum immunogen in a mouse model for influenza infection. Genetic fusion of the M2e and hepatitis B virus core (HBc) coding sequences allowed us to obtain highly immunogenic virus-like particles. This M2e-HBc vaccine induced complete protection in mice against a lethal influenza challenge. Protective immunity was obtained regardless of the position of M2e in the M2e-HBc chimera at the amino-terminus or inserted in the immuno-dominant loop of the HBc protein. Increasing the copy number of M2e inserted at the N-terminus from one to three per monomer (240-720 per particle) significantly enhanced the immune response and reduced the number of vaccinations required for complete protection against a lethal challenge with influenza A virus. A series of M2e-HBc constructs was subsequently combined with CTA1-DD, a recombinant cholera toxin A1 derived mucosal adjuvant, to test its efficacy as an intranasally delivered vaccine. All hybrid VLPs tested with CTA1-DD completely protected mice from a potentially lethal infection and, in addition, significantly reduced morbidity. Overall, increased resistance to influenza challenge in the mice correlated with an enhanced Th1-type M2e-specific antibody response induced by vaccination. These results show that M2e is a valid and versatile vaccine candidate to protect against any strain of human influenza A.

The universal influenza vaccine M2e-HBc administered intranasally in combination with the adjuvant CTA1-DD provides complete protection.

Mucosal vaccination requires effective and safe adjuvants. We have evaluated the non-toxic adjuvant CTA1-DD for mucosal vaccination against influenza. CTA1-DD contains the enzymatically active CTA1 subunit of cholera toxin (CT) genetically fused to a gene encoding a dimer of the D-fragment from Staphylococcus aureus protein A. CTA1-DD only binds to Ig-receptor carrying cells of the immune system. Nasal administration of the universal influenza vaccine M2e-HBc in combination with CTA1-DD completely protected mice from a potentially lethal infection and significantly reduced morbidity. Sera of mice immunized with M2e-HBc + CTA1-DD revealed IgG subclass profiles consistent with an enhanced Th1-type immunity. When the vaccine was administered intraperitoneally, the adjuvant improved the M2e antibody titer in circulation, but did not significantly reduce the morbidity.

Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance.

We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae. We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances. Hog1 concentrates in the nucleus in oxidative stress. Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells. We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen. We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2. Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents. In a fraction of untreated pbs2 cells, Yap2 is nuclear. Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2. These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.

Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6.

In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ1-3 and SNO1-3. In media lacking vitamin B(6), SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B(6) is essential for growth, copies 2 and 3 seem more related with B(1) biosynthesis during the exponential phase.