Browsing by Subject "Cells, Cultured"
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Item Acute Parathyroid Hormone Injection Increases C-Terminal but Not Intact Fibroblast Growth Factor 23 Levels(Endocrine Society, 2017-05-01) Knab, Vanessa M.; Corbin, Braden; Andrukhova, Olena; Hum, Julia M.; Ni, Pu; Rabadi, Seham; Maeda, Akira; White, Kenneth E.; Erben, Reinhold G.; Jüppner, Harald; Christov, Marta; Medical and Molecular Genetics, School of MedicineThe acute effects of parathyroid hormone (PTH) on fibroblast growth factor 23 (FGF23) in vivo are not well understood. After a single subcutaneous PTH (1-34) injection (50 nmol/kg) in mice, FGF23 levels were assessed in plasma using assays that measure either intact alone (iFGF23) or intact/C-terminal FGF23 (cFGF23). Furthermore, FGF23 messenger RNA (mRNA) and protein levels were assessed in bone. In addition, we examined the effects of PTH treatment on FGF23 production in vitro using differentiated calvarial osteocyte-like cells. cFGF23 levels increased by three- to fivefold within 2 hours following PTH injection, which returned to baseline by 4 hours. In contrast, iFGF23 levels remained unchanged for the first 2 hours, yet declined to ∼60% by 6 hours and remained suppressed before returning to baseline after 24 hours. Using homozygous mice for an autosomal dominant hypophosphatemic rickets-FGF23 mutation or animals treated with a furin inhibitor, we showed that cFGF23 and iFGF23 levels increased equivalently after PTH injection. These findings are consistent with increased FGF23 production in bone, yet rapid cleavage of the secreted intact protein. Using primary osteocyte-like cell cultures, we showed that PTH increased FGF23 mRNA expression through cyclic adenosine monophosphate/protein kinase A, but not inositol triphosphate/protein kinase C signaling; PTH also increased furin protein levels. In conclusion, PTH injection rapidly increases FGF23 production in bone in vivo and in vitro. However, iFGF23 is rapidly degraded. At later time points through an unidentified mechanism, a sustained decrease in FGF23 production occurs.Item ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells(Oxford, 2014-10-29) Plotnik, Joshua P.; Budka, Justin A.; Ferris, Mary W.; Hollenhorst, Peter C.; Medicine, School of MedicineThe RAS/ERK pathway is commonly activated in carcinomas and promotes oncogenesis by altering transcriptional programs. However, the array of cis-regulatory elements and trans-acting factors that mediate these transcriptional changes is still unclear. Our genome-wide analysis determined that a sequence consisting of neighboring ETS and AP-1 transcription factor binding sites is enriched near cell migration genes activated by RAS/ERK signaling in epithelial cells. In vivo screening of candidate ETS proteins revealed that ETS1 is specifically required for migration of RAS/ERK activated cells. Furthermore, both migration and transcriptional activation through ETS/AP-1 required ERK phosphorylation of ETS1. Genome-wide mapping of multiple ETS proteins demonstrated that ETS1 binds specifically to enhancer ETS/AP-1 sequences. ETS1 occupancy, and its role in cell migration, was conserved in epithelial cells derived from multiple tissues, consistent with a chromatin organization common to epithelial cell lines. Genome-wide expression analysis showed that ETS1 was required for activation of RAS-regulated cell migration genes, but also identified a surprising role for ETS1 in the repression of genes such as DUSP4, DUSP6 and SPRY4 that provide negative feedback to the RAS/ERK pathway. Consistently, ETS1 was required for robust RAS/ERK pathway activation. Therefore, ETS1 has dual roles in mediating epithelial-specific RAS/ERK transcriptional functions.Item Identification of TMEM230 mutations in familial Parkinson's disease(Nature Research, 2016-07) Deng, Han-Xiang; Shi, Yong; Yang, Yi; Ahmeti, Kreshnik B.; Miller, Nimrod; Huang, Cao; Cheng, Lijun; Zhai, Hong; Deng, Sheng; Nuytemans, Karen; Corbett, Nicola J.; Kim, Myung Jong; Deng, Hao; Tang, Baisha; Yang, Ziquang; Xu, Yanming; Chen, Piao; Huang, Bo; Gao, Xiao-Ping; Song, Zhi; Liu, Zhenhua; Fecto, Faisal; Siddique, Nailah; Foroud, Tatiana; Jankovic, Joseph; Ghetti, Bernardino; Nicholson, Daniel A.; Krainc, Dimitri; Melen, Onur; Vance, Jeffery M.; Pericak-Vance, Margaret A.; Ma, Yong-Chao; Rajput, Ali H.; Siddique, Teepu; Medical and Molecular Genetics, School of MedicineParkinson's disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson's disease or parkinsonian disorders. The pathogenesis of Parkinson's disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body-confirmed Parkinson's disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson's disease, with implications for understanding the pathogenic mechanism of Parkinson's disease and for developing rational therapies.Item IGF-1R modulation of acute GH-induced STAT5 signaling: role of protein tyrosine phosphatase activity(Endocrine Society, 2013-11) Gan, Yujun; Zhang, Yue; Buckels, Ashiya; Paterson, Andrew J.; Jiang, Jing; Clemens, Thomas L.; Zhang, Zhong-Yin; Du, Keyong; Chang, Yingzi; Frank, Stuart J.; Biochemistry & Molecular Biology, School of MedicineGH is a potent anabolic and metabolic factor that binds its cell surface receptor (GHR), activating the GHR-associated tyrosine kinase, Janus kinase 2, which phosphorylates and activates the latent transcription factor, signal transducer and activator of transcription 5 (STAT5). Some GH actions are mediated by the elaboration of IGF-1, which exerts effects by binding and activating the heterotetrameric tyrosine kinase growth factor receptor, IGF-1R. In addition to this GH-GHR-IGF-1-IGF-1R scheme, we have demonstrated in primary osteoblasts and in islet β-cells that then deletion or silencing of IGF-1R results in diminished GH-induced STAT5 phosphorylation, suggesting that the presence of IGF-1R may facilitate GH signaling. In this study, we explore potential roles for protein tyrosine phosphatase activity in modulating GH-induced signaling, comparing conditions in which IGF-1R is present or diminished. We confirm that in mouse primary osteoblasts harboring loxP sites flanking the IGF-1R gene, infection with an adenovirus that expresses the Cre recombinase results in IGF-1R deletion and diminished acute GH-induced STAT5 phosphorylation. Furthermore, we present a new model of IGF-1R silencing, in which expression of short hairpin RNA directed at IGF-1R greatly reduces IGF-1R abundance in LNCaP human prostate cancer cells. In both models, treatment with a chemical inhibitor of protein tyrosine phosphatase-1B (PTP-1B), but not one of src homology region 2 domain-containing phosphotase-1 (SHP-1) and SHP-2, reverses the loss of GH-induced STAT5 phosphorylation in cells lacking IGF-1R but has no effect in cells with intact IGF-1R. Furthermore, expression of either a dominant-negative PTP-1B or the PTP-1B-interacting inhibitory protein, constitutive photomorphogenesis 1, also rescues acute GH-induced STAT5 signaling in IGF-1R-deficient cells but has no effect in IGF-1R replete cells. By expressing a substrate-trapping mutant PTP-1B, we demonstrate that tyrosine phosphorylated Janus kinase-2 is a PTP-1B substrate only in cells lacking IGF-1R. Collectively, our data suggest that IGF-1R positively regulates acute GH signaling by preventing access of PTP-1B activity to Janus kinase 2 and thereby preventing PTP-1B-mediated suppression of GH-induced STAT5 activation.Item Inhibition of 12/15-Lipoxygenase Protects Against β-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes(American Diabetes Association, 2017-11) Hernandez-Perez, Marimar; Chopra, Gaurav; Fine, Jonathan; Conteh, Abass M.; Anderson, Ryan M.; Linnemann, Amelia K.; Benjamin, Chanelle; Nelson, Jennifer B.; Benninger, Kara S.; Nadler, Jerry L.; Maloney, David J.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicineIslet β-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in β-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate β-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced β-cell oxidative stress, and preservation of β-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced β-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of β-cell dysfunction and glycemic deterioration in models of T1D.Item Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain(Lippincott, Williams & Wilkins, 2018-10) François-Moutal, Liberty; Dustrude, Erik T.; Wang, Yue; Brustovetsky, Tatiana; Dorame, Angie; Ju, Weina; Moutal, Aubin; Perez-Miller, Samantha; Brustovetsky, Nickolay; Gokhale, Vijay; Khanna, May; Khanna, Rajesh; Pharmacology and Toxicology, School of MedicineWe previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.Item Monitoring focal adhesion kinase phosphorylation dynamics in live cells(Royal Society of Chemistry, 2017-07-24) Damayanti, Nur P.; Buno, Kevin; Narayanan, Nagarajan; Harbin, Sherry L Voytik; Deng, Meng; Irudayaraj, Joseph M.K.; Medicine, School of MedicineFocal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.Item Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination(American Society for Microbiology (ASM), 2014-06) Byrd, Daniel; Shepherd, Nicole; Lan, Jie; Hu, Ningjie; Amet, Tohti; Yang, Kai; Desai, Mona; Yu, Qigui; Department of Microbiology & Immunology, IU School of MedicineHuman monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1β treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.Item Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture(Society for Neuroscience, 1994-08) Vasko, MR; Campbell, WB; Waite, KJ; Pharmacology and Toxicology, School of MedicineProstaglandins are known to enhance the inflammatory and nociceptive actions of other chemical mediators of inflammation such as bradykinin. One possible mechanism for this sensitizing action is that prostanoids augment the release of neuroactive substances from sensory neurons. To initially test this hypothesis, we examined whether selected prostaglandins could enhance the resting or bradykinin-evoked release of immunoreactive substance P (iSP) and/or immunoreactive calcitonin gene-related peptide (iCGRP) from sensory neurons in culture. Bradykinin alone causes a concentration-dependent increase in the release of iSP and iCGRP from isolated sensory neurons, and this action is abolished in the absence of extracellular calcium. Pretreating the neurons with PGE2 (10 nM to 1 microM) potentiates the bradykinin-evoked release of both iSP and iCGRP by approximately two-to fourfold. At these concentrations, PGE2 alone did not significantly alter peptide release. Exposing the cultures to 1 microM PGF2 alpha is ineffective in altering either resting or bradykinin-evoked peptide release. Sensory neurons in culture contain cyclooxygenase-like immunoreactivity suggesting that the enzyme that converts arachidonic acid to prostaglandins is present. In addition, pretreating cultures with 14C-arachidonic acid yields radiolabeled eicosanoids that cochromatograph with known prostaglandin standards. Preexposing cultures to indomethacin abolishes the production of prostaglandins and attenuates the bradykinin-stimulated release of iSP and iCGRP. This implies that the synthesis of prostaglandins contributes to the bradykinin-evoked release of peptides. The augmentation of bradykinin-induced release of iSP and iCGRP by PGE2 may be one mechanism to account for the inflammatory and hyperalgesic actions of this eicosanoid.Item The protective role of DOT1L in UV-induced melanomagenesis(Nature Publishing Group, 2018-01-17) Zhu, Bo; Chen, Shuyang; Wang, Hongshen; Yin, Chengqian; Han, Changpeng; Peng, Cong; Liu, Zhaoqian; Wan, Lixin; Zhang, Zhang; Zhang, Jie; Lian, Christine G.; Ma, Peilin; Xu, Zhi-xiang; Prince, Sharon; Wang, Tao; Gao, Xiumei; Shi, Yujiang; Liu, Dali; Liu, Min; Wei, Wenyi; Wei, Zhi; Pan, Jingxuan; Wang, Yongjun; Xuan, Zhenyu; Hess, Jay L.; Hayward, Nicholas K.; Goding, Colin R.; Chen, Xiang; Zhou, Jun; Cui, Rutao; Pathology and Laboratory Medicine, School of MedicineThe DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.