Browsing by Author "Lee, Daniel D."

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    Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 simultaneously binds Glutamyl-Prolyl-tRNA synthetase and scaffold protein aminoacyl tRNA synthetase complex interacting multifunctional protein 3 of the multi-tRNA synthetase complex
    (Elsevier, 2018-06) Schwarz, Margaret A.; Lee, Daniel D.; Bartlett, Seamus; IU School of Medicine
    Higher eukaryotes have developed extensive compartmentalization of amino acid (aa) - tRNA coupling through the formation of a multi-synthetase complex (MSC) that is composed of eight aa-tRNA synthetases (ARS) and three scaffold proteins: aminoacyl tRNA synthetase complex interacting multifunctional proteins (AIMP1, 2 and 3). Lower eukaryotes have a much smaller complex while yeast MSC consists of only two ARS (MetRS and GluRS) and one ARS cofactor 1 protein, Arc1p (Simos et al., 1996), the homolog of the mammalian AIMP1. Arc1p is reported to form a tripartite complex with GluRS and MetRS through association of the N-terminus GST-like domains (GST-L) of the three proteins (Koehler et al., 2013). Mammalian AIMP1 has no GST-L domain corresponding to Arc1p N-terminus. Instead, AIMP3, another scaffold protein of 18 kDa composed entirely of a GST-L domain, interacts with Methionyl-tRNA synthetase (MARS) (Quevillon et al., 1999) and Glutamyl-Prolyl-tRNA Synthetase (EPRS) (Cho et al., 2015). Here we report two new interactions between MSC members: AIMP1 binds to EPRS and AIMP1 binds to AIMP3. Interestingly, the interaction between AIMP1 and AIMP3 complex makes it the functional equivalent of a single Arc1p polypeptide in yeast. This interaction is not mapped to AIMP1 N-terminal coiled-coil domain, but rather requires an intact tertiary structure of the entire protein. Since AIMP1 also interacts with AIMP2, all three proteins appear to compose a core docking structure for the eight ARS in the MSC complex.
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    Cell-Cell Communication Breakdown and Endothelial Dysfunction
    (Elsevier, 2020-04) Lee, Daniel D.; Schwarz, Margaret A.; Medicine, School of Medicine
    Guided by organ-specific signals in both development and disease response, the heterogeneous endothelial cell population is a dynamic member of the vasculature. Functioning as the gatekeeper to fluid, inflammatory cells, oxygen, and nutrients, endothelial cell communication with its local environment is critical. Impairment of endothelial cell-cell communication not only disrupts this signaling process, but also contributes to pathologic disease progression. Expanding our understanding of those processes that mediate endothelial cell-cell communication is an important step in the approach to treatment of disease processes.
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    A distinct transcriptional profile in response to endothelial monocyte activating polypeptide II is partially mediated by JAK-STAT3 in murine macrophages
    (American Physiological Society, 2019-09-01) Lee, Daniel D.; Hochstetler, Alexandra; Murphy, Christina; Lowe, Chinn-Woan; Schwarz, Margaret A.; Pediatrics, School of Medicine
    Macrophages are important responders to environmental changes such as secreted factors. Among the secreted factors in injured tissues, the highly conserved endothelial monocyte activating polypeptide II (EMAP II) has been characterized to limit vessel formation, to be locally expressed near sites of injury labeling it a “find-me” signal, and to recruit macrophages and neutrophils. The molecular mechanisms mediated by EMAP II within macrophages once they are recruited are unknown. In this study, using a model of partially activated, recruited thioglycollate-elicited peritoneal macrophages, a transient, transcription profile of key functional genes in macrophages exposed to EMAP II was characterized. We found that EMAP II-mediated changes were elicited mainly through signal transducer and activator of transcription 3 (STAT3) as evidenced by increased Y705 phosphorylation and changes in activity and upstream of it, Janus associated kinase (JAK)1/2 upstream. Both inhibition of JAK1/2 and knockdown of Stat3 abrogated a subset of genes that are upregulated by EMAP II. Our results identify a rapid EMAP II-mediated STAT3 activation that coincides with altered pro- and anti-inflammatory gene expression in macrophages.
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    Influence of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 on epithelial differentiation and organization during lung development
    (American Physiological Society, 2020-08) Lee, Daniel D.; Hochstetler, Alexandra; Sah, Eric; Xu, Haiming; Lowe, Chinn-Woan; Santiaguel, Sara; Thornton, Janet Lea; Pajakowski, Adam; Schwarz, Margaret A.; Anatomy and Cell Biology, School of Medicine
    Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1−/− mutant mice. Mating of Aimp1+/− produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1−/− pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1−/− lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1−/− mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.
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    Mechanistic regulation of SPHK1 expression and translocation by EMAP II in pulmonary smooth muscle cells
    (Elsevier, 2020-12) Ranasinghe, A. Dushani C.U.; Lee, Daniel D.; Schwarz, Margaret A.; Pediatrics, School of Medicine
    Phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) produces the bioactive sphingolipid sphingosine-1-phosphate (S1P), a microvascular and immuno-modulator associated with vascular remodeling in pulmonary arterial hypertension (PAH). The low intracellular concentration of S1P is under tight spatial-temporal control. Molecular mechanisms that mediate S1P burden and S1P regulation of vascular remodeling are poorly understood. Similarities between two early response pro-inflammatory cytokine gene transcript activation profiles, S1P and Endothelial Monocyte Activating Polypeptide II (EMAP II), suggested a strategic link between their signaling pathways. We determined that EMAP II triggers a bimodal phosphorylation, transcriptional regulation and membrane translocation of SPHK1 through a common upstream process in both macrophages and pulmonary artery smooth muscle cells (PASMCs). EMAP II initiates a dual function of ERK1/2: phosphorylation of SPHK1 and regulation of the transcription factor EGR1 that induces expression of SPHK1. Activated ERK1/2 induces a bimodal phosphorylation of SPHK1 which reciprocally increases S1P levels. This identified common upstream signaling mechanism between a protein and a bioactive lipid initiates cell specific downstream signaling representing a multifactorial mechanism that contributes to inflammation and PASMC proliferation which are cardinal histopathological phenotypes of PAH.
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    Pancreatic ductal adenocarcinoma cell secreted extracellular vesicles containing ceramide-1-phosphate promote pancreatic cancer stem cell motility
    (Elsevier, 2018-10) Kuc, Norbert; Doermann, Allison; Shirey, Carolyn; Lee, Daniel D.; Lowe, Chinn-Woan; Awasthi, Niranjan; Schwarz, Roderich E.; Stahelin, Robert V.; Schwarz, Margaret A.; Cellular and Integrative Physiology, School of Medicine
    The high mortality rate associated with pancreatic ductal adenocarcinoma (PDAC) is in part due to lack of effective therapy for this highly chemoresistant tumor. Cancer stem cells, a subset of cancer cells responsible for tumor initiation and metastasis, are not targeted by conventional cytotoxic agents, which renders the identification of factors that facilitate cancer stem cell activation useful in defining targetable mechanisms. We determined that bioactive sphingolipid induced migration of pancreatic cancer stem cells (PCSC) and signaling was specific to ceramide-1-phosphate (C1P). Furthermore, PDAC cells were identified as a rich source of C1P. Importantly, PDAC cells express the C1P converting enzyme ceramide kinase (CerK), secrete C1P-containing extracellular vesicles that mediate PCSC migration, and when co-injected with PCSC reduce animal survival in a PDAC peritoneal dissemination model. Our findings suggest that PDAC secrete C1P-containing extracellular vesicles as a means of recruiting PCSC to sustain tumor growth therefore making C1P release a mechanism that could facilitate tumor progression.
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    A shift from glycolytic and fatty acid derivatives toward one-carbon metabolites in the developing lung during transitions of the early postnatal period
    (American Physiological Society, 2021) Lee, Daniel D.; Park, Sang Jun; Zborek, Kirsten L.; Schwarz, Margaret A.; Pediatrics, School of Medicine
    During postnatal lung development, metabolic changes that coincide with stages of alveolar formation are poorly understood. Responding to developmental and environmental factors, metabolic changes can be rapidly and adaptively altered. The objective of the present study was to determine biological and technical determinants of metabolic changes during postnatal lung development. Over 118 metabolic features were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS, Sciex QTRAP 5500 Triple Quadrupole). Biological determinants of metabolic changes were the transition from the postnatal saccular to alveolar stages and exposure to 85% hyperoxia, an environmental insult. Technical determinants of metabolic identification were brevity and temperature of harvesting, both of which improved metabolic preservation within samples. Multivariate statistical analyses revealed the transition between stages of lung development as the period of major metabolic alteration. Of three distinctive groups that clustered by age, the saccular stage was identified by its enrichment of both glycolytic and fatty acid derivatives. The critical transition between stages of development were denoted by changes in amino acid derivatives. Of the amino acid derivatives that significantly changed, a majority were linked to metabolites of the one-carbon metabolic pathway. The enrichment of one-carbon metabolites was independent of age and environmental insult. Temperature was also found to significantly influence the metabolic levels within the postmortem sampled lung, which underscored the importance of methodology. Collectively, these data support not only distinctive stages of metabolic change but also highlight amino acid metabolism, in particular one-carbon metabolites as metabolic signatures of the early postnatal lung.
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    Targeted FGFR/VEGFR/PDGFR inhibition with dovitinib enhances the effects of nab-paclitaxel in preclinical gastric cancer models
    (Taylor & Francis, 2021) Crawford, Kate; Bontrager, Erin; Schwarz, Margaret A.; Chaturvedi, Apurva; Lee, Daniel D.; Sazzad, Hassan Md; von Holzen, Urs; Zhang, Changhua; Schwarz, Roderich E.; Awasthi, Niranjan; Surgery, School of Medicine
    Standard chemotherapy regimens for gastric adenocarcinoma (GAC) have limited efficacy and considerable toxicity profiles. Nab-paclitaxel has shown promising antitumor benefits in previous GAC preclinical studies. Dovitinib inhibits members of the receptor tyrosine kinase family including FGFR, VEGFR and PDGFR, and has exhibited antitumor effects in many solid tumors including GAC. Based on the antimitotic, antistromal and EPR effects of nab-paclitaxel, we investigated augmentation of nab-paclitaxel response by dovitinib in multiple GAC preclinical models. In MKN-45 subcutaneous xenografts, inhibition in tumor growth by nab-paclitaxel and dovitinib was 75% and 76%, respectively. Dovitinib plus nab-paclitaxel had an additive effect on tumor growth inhibition and resulted in tumor regression (85% of its original value). Dovitinib monotherapy resulted in minimal improvement in animal survival (25 days) compared to control (23 days), while nab-paclitaxel monotherapy or dovitinib plus nab-paclitaxel combination therapy led to a clinically significant lifespan extension of 83% (42 days) and 187% (66 days), respectively. IHC analysis of subcutaneous tumors exhibited reduced tumor cell proliferation and tumor vasculature by dovitinib. In vitro studies demonstrated that dovitinib and nab-paclitaxel individually reduced tumor cell proliferation, with an additive effect from combination therapy. Immunoblot analyses of MKN-45 and KATO-III cells revealed that dovitinib decreased phospho-FGFR, phospho-AKT, phospho-ERK, phospho-p70S6K, phospho-4EBP1, Bcl-2 and increased cleaved PARP-1, cleaved-caspase-3, p27, Bax, Bim, with an additive effect from combination therapy. These results demonstrate that the FGFR/VEGFR/PDGFR inhibitor, dovitinib, has the potential to augment the antitumor effects of nab-paclitaxel, with implications for use in the advancement of clinical GAC therapy.
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    α5β1 integrin mediates pulmonary epithelial cyst formation
    (Wiley, 2017-06) Legan, Susan K.; Lee, Daniel D.; Schwarz, Margaret A.; Pediatrics, School of Medicine
    BACKGROUND: Formation of the epithelial cyst involves the establishment of apical-basolateral polarity through a series of cellular interactions that are in part mediated by the extracellular matrix (ECM). We report that in a three-dimensional multi-cellular self-assembly model of lung development, α5 integrin regulates epithelial cyst formation through organization of soluble fibronectin matrix into insoluble fibrils through a process called fibrillogenesis. RESULTS: Dissociated murine embryonic lung cells self-assemble into three-dimensional pulmonary bodies that are dependent on α5β1 integrin mediated fibrillogenesis for cell-cell mediated self-assembly: compaction and epithelial cyst formation. Knockdown of α5 integrin resulted in a significant increase in another mediator of fibrillogenesis, αV integrin. Compensatory increased expression of another mediator of fibrillogenesis, αV integrin, was not sufficient to normalize epithelial cyst formation. Loss of α5 integrin-mediated fibrillogenesis perturbed the ability of clustered epithelial cells to establish clear polarity, loss of epithelial cell pyramidal shape, and disrupted apical F-actin-rich deposition. Lack of rich central epithelial localization of F-actin cytoskeleton and Podocalyxin suggests that loss of α5 integrin-mediated fibrillogenesis interferes with the normal cytoskeleton organization that facilitates epithelial cysts polarization. CONCLUSIONS: We conclude that lung epithelial cyst formation in development is mediated in part by α5β1 integrin dependent fibrillogenesis