Publications by authors named "Kay-Uwe Wagner"

99 Publications

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity.

Mol Cells 2021 03;44(3):168-178

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor 101 (), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14348/molcells.2021.0027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019596PMC
March 2021

The transcription factor Sox10 is an essential determinant of branching morphogenesis and involution in the mouse mammary gland.

Sci Rep 2020 10 20;10(1):17807. Epub 2020 Oct 20.

Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054, Erlangen, Germany.

The high mobility group-domain containing transcription factor Sox10 is an essential regulator of developmental processes and homeostasis in the neural crest, several neural crest-derived lineages and myelinating glia. Recent studies have also implicated Sox10 as an important factor in mammary stem and precursor cells. Here we employ a series of mouse mutants with constitutive and conditional Sox10 deficiencies to show that Sox10 has multiple functions in the developing mammary gland. While there is no indication for a requirement of Sox10 in the specification of the mammary placode or descending mammary bud, it is essential for both the prenatal hormone-independent as well as the pubertal hormone-dependent branching of the mammary epithelium and for proper alveologenesis during pregnancy. It furthermore acts in a dosage-dependent manner. Sox10 also plays a role during the involution process at the end of the lactation period. Whereas its effect on epithelial branching and alveologenesis are likely causally related to its function in mammary stem and precursor cells, this is not the case for its function during involution where Sox10 seems to work at least in part through regulation of the miR-424(322)/503 cluster.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-74664-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575560PMC
October 2020

Spatiotemporally controlled overexpression of cyclin D1 triggers generation of supernumerary cells in the postnatal mouse inner ear.

Hear Res 2020 05 19;390:107951. Epub 2020 Mar 19.

Creighton University School of Dentistry, Dept. of Oral Biology, Omaha, NE, 68178, USA. Electronic address:

The retinoblastoma family of pocket proteins (pRBs), composed of Rb1, p107, and p130 are negative regulators of cell-cycle progression. The deletion of any individual pRB in the auditory system triggers hair cells' (HCs) and supporting cells' (SCs) proliferation to different extents. Nevertheless, accessing their combined role in the inner ear through conditional or complete knockout methods is limited by the early mortality of the triple knockout. In quiescent cells, hyperphosphorylation and inactivation of the pRBs are maintained through the activity of the Cyclin-D1-cdk4/6 complex. Cyclin D1 (CycD1) is expressed in the embryonic and neonatal inner ear. In the mature organ of Corti (OC), CycD1 expression is significantly downregulated, paralleling the OC mitotic quiescence. Earlier studies showed that CycD1 overexpression leads to cell-cycle reactivation in cultures of inner ear explants. Here, we characterize a Cre-activated, Doxycycline (Dox)-controlled, conditional CycD1 overexpression model, which when bred to a tetracycline-controlled transcriptional activator and the Atoh1-cre mouse lines, allow for transient CycD1 overexpression and pRBs' downregulation in the inner ear in a reversible fashion. Analyses of postnatal mice's inner ears at various time points revealed the presence of supernumerary cells throughout the length of the cochlea and in the vestibular end-organs. Notably, most supernumerary cells were observed in the inner hair cells' (IHCs) region, expressed myosin VIIa (M7a), and showed no signs of apoptosis at any of the time points analyzed. Auditory and vestibular phenotypes were similar between the different genotypes and treatment groups. The fact that no significant differences were observed in auditory and vestibular function supports the notion that the supernumerary cells detected in the adult mice cochlea and macular end-organs may not impair auditory functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.heares.2020.107951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204889PMC
May 2020

Regulation and New Treatment Strategies in Breast Cancer.

J Life Sci (Westlake Village) 2019 Dec;1(3):23-38

Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, MI 48202.

Breast cancer classifications are based on the presence or absence of estrogen receptor and progesterone receptor along with the overexpression or amplification of the Her2 receptor. Although the overall 5-year survival rate of breast cancer patients has increased due to the use of targeted therapies, a subset of patients can acquire resistance over time or are unresponsive when presented in the clinic. Novel therapies focusing on molecular pathways and cell cycle regulation currently being used in the clinic may lead to increased response in this subset of patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039658PMC
December 2019

The Multifaceted Roles of the Tumor Susceptibility Gene 101 (TSG101) in Normal Development and Disease.

Cancers (Basel) 2020 Feb 14;12(2). Epub 2020 Feb 14.

Department of Oncology, Wayne State University School of Medicine and Tumor Biology Program, Barbara Ann Karmanos Cancer Institute, 4100 John R, EL01TM, Detroit, MI 48201, USA.

The multidomain protein encoded by the () is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12020450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073217PMC
February 2020

Efficient tissue-type specific expression of target genes in a tetracycline-controlled manner from the ubiquitously active Eef1a1 locus.

Sci Rep 2020 01 14;10(1):207. Epub 2020 Jan 14.

Department of Oncology, Wayne State University School of Medicine and Tumor Biology Program, Barbara Ann Karmanos Cancer Institute, 4100 John R, EL01TM, Detroit, MI, 48201, USA.

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-57052-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959320PMC
January 2020

Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells.

Am J Transl Res 2019 15;11(2):624-640. Epub 2019 Feb 15.

The Center for Biomedical Research, Ministry of Education and Ministry of Health, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430030, China.

Chronic rejection acts as the most formidable obstacle for organ transplantation in clinical settings. Herein we demonstrated in a cardiac transplantation model that blockade of Janus kinase 2 (Jak2) provides protection for cardiac allografts against chronic rejection. Specifically, loss of almost completely abolished the production of IFN-γ Th1 cells, while the percentage of Foxp3 regulatory T cells (Tregs) was significantly increased. As a result, loss of significantly prolonged allograft survival (58 ± 30.6 days vs. 7 ± 0.3 days). Particularly, 4 out of 13 deficient recipients (30%) showed long-term acceptance of allografts as manifested by the graft survival time > 100 days. Cellular studies revealed that deficiency did not impact the intrinsic proliferative capability for CD4 T cells in response to nonspecific polyclonal and allogenic stimulation. Mechanistic studies documented that the impaired Th1 development was caused by the attenuated IFN-γ/STAT1 and IL-12/STAT4 signaling along with repressed expression of Th1 transcription factors T-bet, Hlx and Runx3. However, the IL-2/STAT5 signaling remained intact, which ensured normal Treg development in naïve CD4 T cells. Together, our data support that blockade of Jak2 may have therapeutic potential for prevention and treatment of allograft rejection in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413256PMC
February 2019

Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

FASEB J 2019 06 18;33(6):7451-7466. Epub 2019 Mar 18.

Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.

Development of physiologic cardiac hypertrophy has primarily been ascribed to the IGF-1 and its receptor, IGF-1 receptor (IGF-1R), and subsequent activation of the protein kinase B (Akt) pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiologic hypertrophy has not been investigated. In a physiologic hypertrophy model of treadmill-exercised mice, we observed that levels of tumor susceptibility gene 101 (Tsg101), a key member of the endosomal sorting complex required for transport, were dramatically elevated in the heart compared with sedentary controls. To determine the role of Tsg101 on physiologic hypertrophy, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101. These TG mice exhibited a physiologic-like cardiac hypertrophy phenotype at 8 wk evidenced by: ) the absence of cardiac fibrosis, ) significant improvement of cardiac function, and ) increased total and plasma membrane levels of IGF-1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with family-interacting protein 3 (FIP3) and IGF-1R, thereby stabilizing FIP3 and enhancing recycling of IGF-1R. , adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of monensin, an inhibitor of endosome-mediated recycling, and by small interfering RNA-mediated knockdown (KD) of FIP3. Furthermore, cardiac-specific KD of Tsg101 showed a significant reduction in levels of endosomal recycling compartment members (Rab11a and FIP3), IGF-1R, and Akt phosphorylation. Most interestingly, Tsg101-KD mice failed to develop cardiac hypertrophy after intense treadmill training. Taken together, our data identify Tsg101 as a novel positive regulator of physiologic cardiac hypertrophy through facilitating the FIP3-mediated endosomal recycling of IGF-1R.-Essandoh, K., Deng, S., Wang, X., Jiang, M., Mu, X., Peng, J., Li, Y., Peng, T., Wagner, K.-U., Rubinstein, J., Fan, G.-C. Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201802338RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529337PMC
June 2019

Janus Kinase 1 Plays a Critical Role in Mammary Cancer Progression.

Cell Rep 2018 11;25(8):2192-2207.e5

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA; Tumor Biology Program, Barbara Ann Karmanos Cancer Institute, 4100 John R Street, EL01TM, Detroit, MI 48201, USA. Electronic address:

Janus kinases (JAKs) and their downstream STAT proteins play key roles in cytokine signaling, tissue homeostasis, and cancer development. Using a breast cancer model that conditionally lacks Janus kinase 1, we show here that JAK1 is essential for IL-6-class inflammatory cytokine signaling and plays a critical role in metastatic cancer progression. JAK1 is indispensable for the oncogenic activation of STAT1, STAT3, and STAT6 in ERBB2-expressing cancer cells, suggesting that ERBB2 receptor tyrosine kinase complexes do not directly activate these STAT proteins in vivo. A genome-wide gene expression analysis revealed that JAK1 signaling has pleiotropic effects on several pathways associated with cancer progression. We established that FOS and MAP3K8 are targets of JAK1/STAT3 signaling, which promotes tumorsphere formation and cell migration. The results highlight the significance of JAK1 as a rational therapeutic target to block IL-6-class cytokines, which are master regulators of cancer-associated inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.10.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431084PMC
November 2018

AKT3 drives adenoid cystic carcinoma development in salivary glands.

Cancer Med 2018 02 28;7(2):445-453. Epub 2017 Dec 28.

Department of Physiology, Center of Physiology and Pharmacology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only ~0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant. Treatment of ACC patients is afflicted by high recurrence rates, the high potential of the tumors to metastasize, as well as the poor response of ACC to chemotherapy. A prerequisite for the development of targeted therapies is insightful genetic information for driver core cancer pathways. Here, we developed a transgenic mouse model toward establishment of a preclinical model. There is currently no available mouse model for adenoid cystic carcinomas as a rare disease entity to serve as a test system to block salivary gland tumors with targeted therapy. Based on tumor genomic data of ACC patients, a key role for the activation of the PI3K-AKT-mTOR pathway was suggested in tumors of secretory glands. Therefore, we investigated the role of Akt3 expression in tumorigenesis and report that Akt3 overexpression results in ACC of salivary glands with 100% penetrance, while abrogation of transgenic Akt3 expression could revert the phenotype. In summary, our findings validate a novel mouse model to study ACC and highlight the druggable potential of AKT3 in the treatment of salivary gland patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cam4.1293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806106PMC
February 2018

Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation.

Sci Rep 2017 08 9;7(1):7653. Epub 2017 Aug 9.

Toronto General Hospital Research Institute, University Health Network, Toronto, M5G 1L7, Canada.

During obesity, macrophages can infiltrate metabolic tissues, and contribute to chronic low-grade inflammation, and mediate insulin resistance and diabetes. Recent studies have elucidated the metabolic role of JAK2, a key mediator downstream of various cytokines and growth factors. Our study addresses the essential role of macrophage JAK2 in the pathogenesis to obesity-associated inflammation and insulin resistance. During high-fat diet (HFD) feeding, macrophage-specific JAK2 knockout (M-JAK2) mice gained less body weight compared to wildtype littermate control (M-JAK2) mice and were protected from HFD-induced systemic insulin resistance. Histological analysis revealed smaller adipocytes and qPCR analysis showed upregulated expression of some adipogenesis markers in visceral adipose tissue (VAT) of HFD-fed M-JAK2 mice. There were decreased crown-like structures in VAT along with reduced mRNA expression of some macrophage markers and chemokines in liver and VAT of HFD-fed M-JAK2 mice. Peritoneal macrophages from M-JAK2 mice and Jak2 knockdown in macrophage cell line RAW 264.7 also showed lower levels of chemokine expression and reduced phosphorylated STAT3. However, leptin-dependent effects on augmenting chemokine expression in RAW 264.7 cells did not require JAK2. Collectively, our findings show that macrophage JAK2 deficiency improves systemic insulin sensitivity and reduces inflammation in VAT and liver in response to metabolic stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-07923-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550513PMC
August 2017

Hepatic JAK2 protects against atherosclerosis through circulating IGF-1.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada.

Atherosclerosis is considered both a metabolic and inflammatory disease; however, the specific tissue and signaling molecules that instigate and propagate this disease remain unclear. The liver is a central site of inflammation and lipid metabolism that is critical for atherosclerosis, and JAK2 is a key mediator of inflammation and, more recently, of hepatic lipid metabolism. However, precise effects of hepatic Jak2 on atherosclerosis remain unknown. We show here that hepatic Jak2 deficiency in atherosclerosis-prone mouse models exhibited accelerated atherosclerosis with increased plaque macrophages and decreased plaque smooth muscle cell content. JAK2's essential role in growth hormone signalling in liver that resulted in reduced IGF-1 with hepatic Jak2 deficiency played a causal role in exacerbating atherosclerosis. As such, restoring IGF-1 either pharmacologically or genetically attenuated atherosclerotic burden. Together, our data show hepatic Jak2 to play a protective role in atherogenesis through actions mediated by circulating IGF-1 and, to our knowledge, provide a novel liver-centric mechanism in atheroprotection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.93735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518558PMC
July 2017

Crosstalk between STAT5 activation and PI3K/AKT functions in normal and transformed mammary epithelial cells.

Mol Cell Endocrinol 2017 08 8;451:31-39. Epub 2017 May 8.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA; Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA. Electronic address:

Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to function downstream of several peptide hormones and cytokines that are required for postnatal development and secretory function of the mammary gland. As part of an extended network, these signal transducers can engage in crosstalk with other pathways to facilitate synergistic, and sometimes antagonistic, actions of different growth factors. Specifically, signaling through the JAK2/STAT5 cascade has been demonstrated to be indispensable for the specification, proliferation, differentiation, and survival of secretory mammary epithelial cells. Following a concise description of major cellular programs in mammary gland development and the role of growth factors that rely on JAK/STAT signaling to orchestrate these programs, this review highlights the significance of active STAT5 and its crosstalk with the PI3 kinase and AKT1 for mediating the proliferation of alveolar progenitors and survival of their functionally differentiated descendants in the mammary gland. Based on its ability to provide self-sufficiency in growth signals that are also capable of overriding intrinsic cell death programs, persistently active STAT5 can serve as a potent oncoprotein that contributes to the genesis of breast cancer. Recent experimental evidence demonstrated that, similar to normal developmental programs, oncogenic functions of STAT5 rely on molecular crosstalk with PI3K/AKT signaling for the initiation, and in some instances the progression, of breast cancer. The multitude by which STATs can interact with individual mediators of the PI3K/AKT signaling cascade may provide novel avenues for targeting signaling nodes within molecular networks that are crucial for the survival of cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2017.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515553PMC
August 2017

Autocrine IGF1 Signaling Mediates Pancreatic Tumor Cell Dormancy in the Absence of Oncogenic Drivers.

Cell Rep 2017 02;18(9):2243-2255

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA. Electronic address:

Mutant KRAS and c-MYC are oncogenic drivers and rational therapeutic targets for the treatment of pancreatic cancer. Although tumor growth and homeostasis are largely dependent on these oncogenes, a few residual cancer cells are able to survive the ablation of mutant KRAS and c-MYC. By performing a genome-wide gene expression analysis of in vivo-derived bulk tumor cells and residual cancer cells lacking the expression of mutant KRAS or c-MYC, we have identified an increase in autocrine IGF1/AKT signaling as a common survival mechanism in dormant cancer cells. The pharmacological inhibition of IGF-1R reduces residual disease burden and cancer recurrence, suggesting that this molecular pathway is crucial for the survival of cancer cells in the absence of the primary oncogenic drivers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369772PMC
February 2017

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Blood 2017 03 10;129(13):1823-1830. Epub 2017 Feb 10.

Department of Internal Medicine VI (Infectious Diseases, Immunology, Rheumatology and Pneumology), Medical University of Innsbruck, Innsbruck, Austria.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2016-09-740092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693322PMC
March 2017

Janus Kinase 2 (JAK2) Dissociates Hepatosteatosis from Hepatocellular Carcinoma in Mice.

J Biol Chem 2017 03 18;292(9):3789-3799. Epub 2017 Jan 18.

From the Toronto General Hospital Research Institute, Toronto, Ontario M5G 2C4, Canada,

Hepatocellular carcinoma is an end-stage complication of non-alcoholic fatty liver disease (NAFLD). Inflammation plays a critical role in the progression of non-alcoholic fatty liver disease and the development of hepatocellular carcinoma. However, whether steatosis promotes liver cancer, and the molecular mechanisms that control the progression in this disease spectrum remain largely elusive. The Janus kinase signal transducers and activators of transcription (JAK-STAT) pathway mediates signal transduction by numerous cytokines that regulate inflammation and may contribute to hepatocarcinogenesis. Mice with hepatocyte-specific deletion of JAK2 (L-JAK2 KO) develop extensive fatty liver spontaneously. We show here that this simple steatosis was insufficient to drive carcinogenesis. In fact, L-JAK2 KO mice were markedly protected from chemically induced tumor formation. Using the methionine choline-deficient dietary model to induce steatohepatitis, we found that steatohepatitis development was completely arrested in L-JAK2 KO mice despite the presence of steatosis, suggesting that JAK2 is the critical factor required for inflammatory progression in the liver. In line with this, L-JAK2 KO mice exhibited attenuated inflammation after chemical carcinogen challenge. This was associated with increased hepatocyte apoptosis without elevated compensatory proliferation, thus thwarting expansion of transformed hepatocytes. Taken together, our findings identify an indispensable role of JAK2 in hepatocarcinogenesis through regulating critical inflammatory pathways. Targeting the JAK-STAT pathway may provide a novel therapeutic option for the treatment of hepatocellular carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.752519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339761PMC
March 2017

Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation.

Proc Natl Acad Sci U S A 2016 12 5;113(51):E8228-E8237. Epub 2016 Dec 5.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198;

Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b-null, Cbl-c-null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1615677113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187741PMC
December 2016

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

Biol Cell 2016 Nov 23;108(11):324-337. Epub 2016 Aug 23.

McLaughlin Research Institute, Great Falls, MT, 59405, USA.

Background Information: Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture.

Results: To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change.

Conclusions: Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation.

Significance: This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/boc.201600014DOI Listing
November 2016

Generation of Janus kinase 1 (JAK1) conditional knockout mice.

Genesis 2016 Nov 19;54(11):582-588. Epub 2016 Oct 19.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska, 68198-5950, USA.

The biological functions of the Janus kinase 1 (JAK1) are suggested to be pleiotropic since this signal transducer is ubiquitously expressed and coupled to a variety of cytokine receptors. Consequently, mice that are deficient in this tyrosine kinase were reported to die shortly after birth. To facilitate studies that address the biological and molecular functions of JAK1 during postnatal development, we performed gene targeting in embryonic stem cells and generated a Cre/lox-based conditional knockout mouse model. Expression of Cre recombinase in the germline converted the Jak1 conditional knockout allele (Jak1 ) into a null allele (Jak1 ) that when subsequently crossed into homozygosity led to a complete absence of the JAK1 protein in developing embryos. JAK1 deficient embryos were visibly smaller starting at E15.5. Newborn pups exhibited signs of apnea and died within hours after birth. The examination of fibroblasts from conditional knockout embryos and their littermate wildtype controls expressing JAK1 showed that lack of this Janus kinase resulted in an impaired tyrosine phosphorylation and activation of the downstream Signal Transducers and Activators of Transcription (STATs) 1, 3, and 6. JAK1 conditional knockout mice will be an invaluable tool to study cytokine signaling during normal development and disease progression in adult animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvg.22982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988131PMC
November 2016

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice.

Sci Rep 2016 10 7;6:34719. Epub 2016 Oct 7.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2) to GH transgenic mice (GH) and compared them to GHSTAT5 mice. Similar to GHSTAT5 mice, JAK2 deficiency resulted in severe steatosis in the GH background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHSTAT5 mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep34719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054456PMC
October 2016

Janus Kinase 1 Is Essential for Inflammatory Cytokine Signaling and Mammary Gland Remodeling.

Mol Cell Biol 2016 06 16;36(11):1673-90. Epub 2016 May 16.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA

Despite a wealth of knowledge about the significance of individual signal transducers and activators of transcription (STATs), essential functions of their upstream Janus kinases (JAKs) during postnatal development are less well defined. Using a novel mammary gland-specific JAK1 knockout model, we demonstrate here that this tyrosine kinase is essential for the activation of STAT1, STAT3, and STAT6 in the mammary epithelium. The loss of JAK1 uncouples interleukin-6-class ligands from their downstream effector, STAT3, which leads to the decreased expression of STAT3 target genes that are associated with the acute-phase response, inflammation, and wound healing. Consequently, JAK1-deficient mice exhibit impaired apoptosis and a significant delay in mammary gland remodeling. Using RNA sequencing, we identified several new JAK1 target genes that are upregulated during involution. These include Bmf and Bim, which are known regulators of programmed cell death. Using a BMF/BIM-double-knockout epithelial transplant model, we further validated that the synergistic action of these proapoptotic JAK1 targets is obligatory for the remodeling of the mammary epithelium. The collective results of this study suggest that JAK1 has nonredundant roles in the activation of particular STAT proteins and this tyrosine kinase is essential for coupling inflammatory cytokine signals to the cell death machinery in the differentiated mammary epithelium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00999-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959311PMC
June 2016

Myocardial Hypertrophic Remodeling and Impaired Left Ventricular Function in Mice with a Cardiac-Specific Deletion of Janus Kinase 2.

Am J Pathol 2015 Dec;185(12):3202-10

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada. Electronic address:

The Janus kinase (JAK) system is involved in numerous cell signaling processes and is highly expressed in cardiac tissue. The JAK isoform JAK2 is activated by numerous factors known to influence cardiac function and pathologic conditions. However, although abundant, the role of JAK2 in the regulation or maintenance of cardiac homeostasis remains poorly understood. Using the Cre-loxP system, we generated a cardiac-specific deletion of Jak2 in the mouse to assess the effect on cardiac function with animals followed up for a 4-month period after birth. These animals had marked mortality during this period, although at 4 months mortality in male mice (47%) was substantially higher compared with female mice (30%). Both male and female cardiac Jak2-deleted mice had hypertrophy, dilated cardiomyopathy, and severe left ventricular dysfunction, including a marked reduction in ejection fractions as assessed by serial echocardiography, although the responses in females were somewhat less severe. Defective cardiac function was associated with altered protein levels of sarcoplasmic reticulum calcium-regulatory proteins particularly in hearts from male mice that had depressed levels of SERCA2 and phosphorylated phospholamban. In contrast, SERCA2 was unchanged in hearts of female mice, whereas phosphorylated phospholamban was increased. Our findings suggest that cardiac JAK2 is critical for maintaining normal heart function, and its ablation produces a severe pathologic phenotype composed of myocardial remodeling, heart failure, and pronounced mortality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2015.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729232PMC
December 2015

Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis.

Gut 2017 01 17;66(1):145-155. Epub 2015 Sep 17.

Department of Internal Medicine I, University of Bonn, Bonn, Germany.

Objective: Angiotensin II (AngII) activates via angiotensin-II-type-I receptor (AT1R) Janus-kinase-2 (JAK2)/Arhgef1 pathway and subsequently RHOA/Rho-kinase (ROCK), which induces experimental and probably human liver fibrosis. This study investigated the relationship of JAK2 to experimental and human portal hypertension.

Design: The mRNA and protein levels of JAK2/ARHGEF1 signalling components were analysed in 49 human liver samples and correlated with clinical parameters of portal hypertension in these patients. Correspondingly, liver fibrosis (bile duct ligation (BDL), carbon tetrachloride (CCl)) was induced in floxed-Jak2 knock-out mice with SM22-promotor (SM22-Jak2). Transcription and contraction of primary myofibroblasts from healthy and fibrotic mice and rats were analysed. In two different cirrhosis models (BDL, CCl) in rats, the acute haemodynamic effect of the JAK2 inhibitor AG490 was assessed using microsphere technique and isolated liver perfusion experiments.

Results: Hepatic transcription of JAK2/ARHGEF1 pathway components was upregulated in liver cirrhosis dependent on aetiology, severity and complications of human liver cirrhosis (Model for End-stage Liver disease (MELD) score, Child score as well as ascites, high-risk varices, spontaneous bacterial peritonitis). SM22- Jak2 mice lacking Jak2 developed less fibrosis and lower portal pressure (PP) than SM22-Jak2 upon fibrosis induction. Myofibroblasts from SM22-Jak2 mice expressed less collagen and profibrotic markers upon activation. AG490 relaxed activated hepatic stellate cells in vitro. In cirrhotic rats, AG490 decreased hepatic vascular resistance and consequently the PP in vivo and in situ.

Conclusions: Hepatic JAK2/ARHGEF1/ROCK expression is associated with portal hypertension and decompensation in human cirrhosis. The deletion of Jak2 in myofibroblasts attenuated experimental fibrosis and acute inhibition of JAK2 decreased PP. Thus, JAK2 inhibitors, already in clinical use for other indications, might be a new approach to treat cirrhosis with portal hypertension.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2015-309600DOI Listing
January 2017

Cyr61 as mediator of Src signaling in triple negative breast cancer cells.

Oncotarget 2015 May;6(15):13520-38

Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Madrid 28029, Spain.

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537031PMC
http://dx.doi.org/10.18632/oncotarget.3760DOI Listing
May 2015

Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.

Nat Commun 2015 Apr 9;6:6744. Epub 2015 Apr 9.

1] Simmons Cancer Center, UT Southwestern Medical Center, Dallas, Texas 75390, USA [2] Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75390, USA.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms7744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403382PMC
April 2015

Carboxyl-terminal domain of MUC16 imparts tumorigenic and metastatic functions through nuclear translocation of JAK2 to pancreatic cancer cells.

Oncotarget 2015 Mar;6(8):5772-87

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.

MUC16 (CA125) is a type-I transmembrane glycoprotein that is up-regulated in multiple cancers including pancreatic cancer (PC). However, the existence and role of carboxyl-terminal MUC16 generated following its cleavage in PC is unknown. Our previous study using a systematic dual-epitope tagged domain deletion approach of carboxyl-terminal MUC16 has demonstrated the generation of a 17-kDa cleaved MUC16 (MUC16-Cter). Here, we demonstrate the functional significance of MUC16-Cter in PC using the dual-epitope tagged version (N-terminal FLAG- and C-terminal HA-tag) of 114 carboxyl-terminal residues of MUC16 (F114HA). In vitro analyses using F114HA transfected MiaPaCa-2 and T3M4 cells showed enhanced proliferation, motility and increased accumulation of cells in the G2/M phase with apoptosis resistance, a feature associated with cancer stem cells (CSCs). This was supported by enrichment of ALDH+ CSCs along with enhanced drug-resistance. Mechanistically, we demonstrate a novel function of MUC16-Cter that promotes nuclear translocation of JAK2 resulting in phosphorylation of Histone-3 up-regulating stemness-specific genes LMO2 and NANOG. Jak2 dependence was demonstrated using Jak2+/+ and Jak2-/- cells. Using eGFP-Luciferase labeled cells, we demonstrate enhanced tumorigenic and metastatic potential of MUC16-Cter in vivo. Taken together, we demonstrate that MUC16-Cter mediated enrichment of CSCs is partly responsible for tumorigenic, metastatic and drug-resistant properties of PC cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467401PMC
http://dx.doi.org/10.18632/oncotarget.3308DOI Listing
March 2015

Mouse models of breast cancer.

Methods Mol Biol 2015 ;1267:47-71

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE, 68198-5950, USA,

Breast cancer is the most common cause of cancer death in women worldwide. This malignancy is a complex disease, which is defined by an intrinsic heterogeneity on the histopathological and molecular level as well as response to therapy and outcome. In addition to classical histopathological features, breast cancer can be categorized into at least five major subtypes based on comprehensive gene expression profiling: luminal A, luminal B, basal-like, ERBB2-positive, and normal-like breast cancer. Genetically engineered mouse models can serve as tools to study the molecular underpinnings for this disease. Given the genetic complexity that drives the initiation and progression of individual breast cancer subtypes, it is evident that certain models can reflect only particular aspects of this malignancy. In this book chapter, we will primarily focus on advances in modeling breast cancer at defined stages of carcinogenesis using genetically engineered mice. We will discuss the ability as well as shortcomings of these models to faithfully recapitulate the spectrum of human breast cancer subtypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-2297-0_3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492279PMC
September 2015

Genetically engineered mucin mouse models for inflammation and cancer.

Cancer Metastasis Rev 2015 Dec;34(4):593-609

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Mucins are heavily O-glycosylated proteins primarily produced by glandular and ductal epithelial cells, either in membrane-tethered or secretory forms, for providing lubrication and protection from various exogenous and endogenous insults. However, recent studies have linked their aberrant overexpression with infection, inflammation, and cancer that underscores their importance in tissue homeostasis. In this review, we present current status of the existing mouse models that have been developed to gain insights into the functional role(s) of mucins under physiological and pathological conditions. Knockout mouse models for membrane-associated (Muc1 and Muc16) and secretory mucins (Muc2) have helped us to elucidate the role of mucins in providing effective and protective barrier functions against pathological threats, participation in disease progression, and improved our understanding of mucin interaction with biotic and abiotic environmental components. Emphasis is also given to available transgenic mouse models (MUC1 and MUC7), which has been exploited to understand the context-dependent regulation and therapeutic potential of human mucins during inflammation and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10555-015-9549-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520780PMC
December 2015

Generation of conditional knockout mice.

Methods Mol Biol 2014 ;1194:21-35

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, DRCII, Rm. 5031, 985950 Nebraska Medical Center, Omaha, NE, 68198-5950, USA,

Conditional knockout mouse models are powerful tools to examine the biological and molecular function(s) of genes in specific tissues. The general procedure to generate such genetically engineered mouse models consists of three main steps. The first step is to find the appropriate genomic clone of the gene of interest and to design the cloning and Southern blot strategies. The second step is the cloning of the gene-targeting vector with all its essential components including positive and negative selection cassettes and the insertion of LoxP sites. Although conventional methods are still being widely used for DNA cloning, we describe in this book chapter the use of λ Red phage-based homologous recombination in Escherichia coli to capture the genomic DNA of the gene of interest and to assemble the gene-targeting vector. This new method provides several advantages as it does not require the presence of restriction sites within the gene of interest to insert LoxP-flanked DNA fragments. In the final step, the gene-targeting vector is transferred into embryonic stem (ES) cells, and successfully targeted ES cell clones are injected into mouse blastocysts to generate conditional knockout mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1215-5_2DOI Listing
March 2015

Critical role of Jak2 in the maintenance and function of adult hematopoietic stem cells.

Stem Cells 2014 Jul;32(7):1878-89

Department of Pharmacology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York, USA.

Jak2, a member of the Janus kinase family of nonreceptor protein tyrosine kinases, is activated in response to a variety of cytokines, and functions in survival and proliferation of cells. An activating JAK2V617F mutation has been found in most patients with myeloproliferative neoplasms, and patients treated with Jak2 inhibitors show significant hematopoietic toxicities. However, the role of Jak2 in adult hematopoietic stem cells (HSCs) has not been clearly elucidated. Using a conditional Jak2 knockout allele, we have found that Jak2 deletion results in rapid loss of HSCs/progenitors leading to bone marrow failure and early lethality in adult mice. Jak2 deficiency causes marked impairment in HSC function, and the mutant HSCs are severely defective in reconstituting hematopoiesis in recipient animals. Jak2 deficiency also causes significant apoptosis and loss of quiescence in HSC-enriched LSK (Lin(-)Sca-1(+)c-Kit(+)) cells. Jak2-deficient LSK cells exhibit elevated reactive oxygen species levels and enhanced p38 MAPK activation. Mutant LSK cells also show defective Stat5, Erk, and Akt activation in response to thrombopoietin and stem cell factor. Gene expression analysis reveals significant downregulation of genes related to HSC quiescence and self-renewal in Jak2-deficient LSK cells. These data suggest that Jak2 plays a critical role in the maintenance and function of adult HSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.1711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063883PMC
July 2014