James M. Trevillyan

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

T cell activation induces a noncoding RNA transcript sensitive to inhibition by immunosuppressant drugs and encoded by the proto-oncogene, BIC

In a search for novel early T cell activation transcripts, we identified expressed sequence tags (ESTs) more abundantly expressed in normal human CD4(+) T lymphocytes fully activated by a 5 h exposure to CD3 plus CD28 mAbs, compared to the same cells stimulated with either CD3 mAb or CD28 mAb alone. An EST was identified that hybridized with a 1.7 kb transcript expressed in activated T cells but was undetectable by Northern blot analysis in resting T cells or other normal tissues. The T cell transcript was maximally induced within 6 h and remained elevated for at least 47 h. Induction of the transcript was blocked by cyclosporin A, FK506, and dexamethasone but not by rapamycin. The transcript was polyadenylated but lacked an open reading. A BLAST search of the NCBI database revealed that the transcript shared identity with the recently reported human BIC proto-oncogene that encodes a noncoding mRNA (W. Tam, Gene 274 (2001) 157). Our data demonstrate that transcriptional activation of the BIC proto-oncogene is an early and sustained T cell activation event and suggest an important role for noncoding mRNA in T cell function.

Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line

Protein tyrosine phosphatase 1B (PTP1B) has recently been implicated in the regulation of body weight. A surprising phenotype of PTP1B-deficient mice is their resistance to diet-induced obesity. Since leptin is one of the primary hormones involved in the regulation of body weight and energy homeostasis, we investigated whether PTP1B affects leptin receptor (lepR) signaling directly. A mouse hypothalamic cell line, GT1-7, was established as a suitable cell model for the study of leptin signaling. Stimulation of GT1-7 cells by leptin caused tyrosine phosphorylation of endogenous STAT3 and activation of a STAT-dependent luciferase reporter gene. Over-expression of PTP1B in GT1-7 cells resulted in a dose-dependent decrease in endogenous JAK2 and STAT3 tyrosine phosphorylation compared with cells transfected with lepR alone. Consistent with inhibition of JAK-STAT signaling, PTP1B over-expression caused a dose-dependent decrease in leptin-induced, STAT-dependent luciferase reporter gene activation in GT1-7 cells. Furthermore, over-expression of PTP1B led to a decrease in mRNA accumulation of suppressor-of-cytokine-signalling-3 (SOCS3) and c-fos, genes that are acutely induced by leptin. Using gene microarray analysis, we confirmed that PTP1B reduces the level of gene expression of SOCS3 and showed that the expression level of other leptin-regulated genes was affected. Genes up-regulated by leptin were decreased in cells over-expressing PTP1B. Conversely, the expression of genes down-regulated by leptin was enhanced by PTP1B over-expression in GT1-7 cells. Our findings indicate that PTP1B is a negative regulator of leptin signaling and suggest that PTP1B inhibitors might be efficacious in the treatment of obesity by increasing leptin sensitivity.

Potent Inhibition of NFAT Activation and T Cell Cytokine Production by Novel Low Molecular Weight Pyrazole Compounds

NFAT (nuclearfactor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca2+/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-κB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaNin vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.

Zotarolimus, a novel sirolimus analogue with potent anti-proliferative activity on coronary smooth muscle cells and reduced potential for systemic immunosuppression

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2 values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

Liver-specific Knockdown of JNK1 Up-regulates Proliferator-activated Receptor γ Coactivator 1β and Increases Plasma Triglyceride despite Reduced Glucose and Insulin Levels in Diet-induced Obese Mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor γ coactivator 1β, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Potent, Selective Inhibitors of Protein Tyrosine Phosphatase 1B

We have previously reported a novel series of oxalyl-aryl-amino benzoic acid-based, catalytic site-directed, competitive, reversible protein tyrosine phosphatase 1B (PTP1B) inhibitors. With readily access to key intermediates, we utilized a solution phase parallel synthesis approach and rapidly identified a highly potent PTP1B inhibitor (19, K(i)=76 nM) with moderate selectivity (5-fold) over T-cell PTPase (TCPTP) through interacting with a second phosphotyrosine binding site (site 2) in the close proximity to the catalytic site.

PTP1B antisense-treated mice show regulation of genes involved in lipogenesis in liver and fat

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.

Reduction of protein-tyrosine phosphatase-1B increases insulin signaling in FAO hepatoma cells

Protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. PTP1B dephosphorylates the insulin receptor and insulin receptor substrates (IRS-1/2), inhibiting the insulin-signaling pathway. PTP1B has been reported to be elevated in diabetes and insulin-resistant states. Conversely, PTP1B null mice have increased insulin sensitivity. To further investigate the effect of PTP1B reduction on insulin signaling, FAO rat hepatoma cells were transfected, by electroporation, with a specific PTP1B antisense oligonucleotide (ASO), or a control oligonucleotide. The PTP1B ASO caused a 50-70% reduction in PTP1B protein expression as measured by Western blot analysis. Upon insulin stimulation, an increase in the phosphorylation of the insulin receptor and insulin receptor substrates was observed, without any change in protein expression levels. Reduction of PTP1B expression in FAO cells also caused an increase in insulin-stimulated phosphorylation of PKB and GSK3, without any change in protein expression. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor.

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of immunomodulators.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.