Insulin and insulin analogs stimulate proliferation of human mammary epithelial cells. We identified and analyzed the signaling pathways related to cell proliferation induced by regular insulin and by four insulin analogs presently approved for therapeutical use. Benign and malignant mammary cell lines showing different insulin receptor (IR) and IGF-I receptor (IGF-IR) expression patterns were studied. Cell proliferation was studied by crystal violet staining (BrdU-FACS analysis). Activation of insulin and IGF signaling pathways was studied by analysis of the phosphorylation status of IGF-IR and of key signaling proteins of the phosphoinositide 3-kinase (PI3K)/Akt and MAP kinase pathways, by the use of specific PI3K and MAP kinase inhibitors, and by silencing of IR and IGF-IR. Lantus stimulated the growth of MCF7 cells, which show high IGF-IR/IR ratio, significantly at 0.3 nmol/l, while regular insulin (Actrapid and bovine insulin) and other insulin analogs (Novorapid, Humalog, and Levemir) stimulated cell growth at 1.5-15 nmol/l concentrations. No difference between Lantus and the other insulin analogs was observed regarding growth stimulation of MCF10A cells showing low IGF-IR/IR ratio. Growth stimulation of MCF7 cells by Lantus was mainly due to strong activation of the IGF-IR and the MAP kinase pathway. Regular insulin and other insulin analogs tested activated mainly the IR and the PI3K/Akt pathway. We conclude that unlike regular insulin and other insulin analogs, Lantus strongly activates the IGF-IR and the MAP kinase pathway in MCF7 cells and is a strong mitogen for cells characterized by a high-IGF-IR/IR ratio.

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Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wildtype JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.T he Janus kinases (JAK1-3, TYK2) are a family of nonreceptor tyrosine kinases with essential functions in the regulation of hematopoiesis, the immune system, and cellular metabolism. JAKs interact specifically with various cytokine receptors and couple cytokine binding to cytoplasmic signaling cascades, including the signal transducers and activators of transcription (STAT) pathway. JAKs consist of an N-terminal FERM domain, an SH2-like (Src homology 2) domain, a pseudokinase domain (JAK homology 2, JH2), and the C-terminal tyrosine kinase domain (JH1). JH2 mediates critical regulatory functions in JAKs and primarily serves to inhibit basal JH1 activity. Experimental deletion of JH2 increases JH1 activity in full-length JAK in the absence of stimulation (1-3), and in recombinant systems addition of JH2 suppresses JH1 activity (4-6). JH2 is, however, also required for ligand-induced activation of full-length JAKs in cell (1-3, 6, 7). The regulatory functions of JH2 are corroborated by the multitude of human disease mutations identified in the domain. The most common JAK2 mutation, V617F, leads to cytokine-independent signaling through the exclusively JAK2-dependent homotypic receptors for erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and thrombopoietin (8). The V617F mutation is found in ∼95% of patients with polycythemia vera (PV) (9...

Major progress has been recently made in understanding the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in one of four genes-JAK2, MPL, CALR, and CSF3R-can be found in the vast majority of patients with MPN and represent driver mutations that can induce the MPN phenotype. Hyperactive JAK/STAT signaling appears to be the common denominator of MPN, even in patients with CALR mutations and the so-called "triple-negative" MPN, where the driver gene mutation is still unknown. Mutations in epigenetic regulators, transcription factors, and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. The central role of JAK2 in MPN allowed development of small molecular inhibitors that are in clinical use and are active in almost all patients with MPN. Advances in understanding the mechanism of JAK2 activation open new perspectives of developing the next generation of inhibitors that will be selective for the mutated forms of JAK2.

Key Points• JAK2-V617F cells show a competitive advantage over wild-type cells in BM transplantation assays.• A preclinical mouse model allows the examination of the effects of therapeutic agents on blood parameters and JAK2-V617F mutant allele burden.To establish a preclinical animal model for testing drugs with potential effects on myeloproliferative neoplasms (MPNs), we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2-knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelet or granulocyte levels. In a transplantation assay, JAK2-V617F ؉ BM cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea (HU). HU led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized RBC and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced the phospho-Stat5 levels, whereas HU strongly increased phospho-Stat5, most likely because of the elevated erythropoietin levels in response to the HU-induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of HU and represents an adverse effect that should be avoided. (Blood. 2013;121(7):1188-1199) IntroductionMyeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell diseases characterized by increased proliferation of the erythroid, megakaryocytic, or myeloid lineages. An acquired somatic mutation in the JAK2 gene resulting in a valine to phenylalanine substitution at position 617 (JAK2-V617F) is present in the majority of patients with MPNs. [1][2][3][4] Several mouse models expressing JAK2-V617F have been established that mimic the MPN phenotypes observed in patients (for review, see Van Etten et al 5 and Li et al 6 ). We described previously a Cre-loxP-inducible transgenic mouse model that expresses human JAK2-V617F under the endogenous JAK2 promoter and displays a phenotype resembling polycythemia vera (PV) or essential thrombocythemia (ET). 7 The phenotype was correlated with the ratio between the BM mRNA levels for the mutant human JAK2-V617F and the wildtype mouse Jak2 with a PV phenotype present at ratios of 1:1 (V617F: wild-type), whereas ET was observed at ratios of approximately 1:2. In the present study, we corroborated this correlation by studying the effects of decreased expression of the wild-type mouse Jak2 by crossing JAK2-V617F-transgenic mice with an inducible Jak2-knockout strain. Using a competitive transplantation assay, we established a preclinical mouse model that allows monitoring drug effects on the mutant JAK2-V617F allele burden. We used this mouse mod...

Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1β, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1β, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.

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Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.