Immortalized Cell Model Research Articles

Piezo2 interacts with E-cadherin in specialized gastrointestinal epithelial mechanoreceptors.

Piezo2 is a mechanically gated ion channel most commonly expressed by specialized mechanoreceptors, such as the enteroendocrine cells (EECs) of the gastrointestinal epithelium. A subpopulation of EECs expresses Piezo2 and functionally resembles the skin's touch sensors, called Merkel cells. Low-magnitude mechanical stimuli delivered to the mucosal layer are primarily sensed by mechanosensitive EECs in a process we term "gut touch." Piezo2 transduces cellular forces into ionic currents, a process that is sensitive to bilayer tension and cytoskeletal depolymerization. E-cadherin is a widely expressed protein that mediates cell-cell adhesion in epithelia and interacts with scaffold proteins that anchor it to actin fibers. E-cadherin was shown to interact with Piezo2 in immortalized cell models. We hypothesized that the Piezo2-E-cadherin interaction is important for EEC mechanosensitivity. To test this, we used super-resolution imaging, co-immunoprecipitation, and functional assays in primary tissues from mice and gut organoids. In tissue EECs and intestinal organoids, we observed multiple Piezo2 cellular pools, including one that overlaps with actin and E-cadherin at the cells' lateral walls. Further, E-cadherin co-immunoprecipitated with Piezo2 in the primary colonic epithelium. We found that E-cadherin knockdown decreases mechanosensitive calcium responses in mechanically stimulated primary EECs. In all, our results demonstrate that Piezo2 localizes to the lateral wall of EECs, where it physically interacts with E-cadherin and actin. They suggest that the Piezo2-E-cadherin-actin interaction is important for mechanosensitivity in the gut epithelium and possibly in tissues where E-cadherin and Piezo2 are co-expressed in epithelial mechanoreceptors, such as skin, lung, and bladder.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/− male to generate Shmt2+/+ (wild-type) or Shmt2+/− (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/− MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

The MAPK/ERK Pathway and the Role of DUSP1 in JCPyV Infection of Primary Astrocytes.

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

Kidney organoids—a new tool for kidney therapeutic development

Kidney organoids, derived from human pluripotent stem cells, have the potential to greatly facilitate drug development. Boreström etal. have used CRISPR/Cas9 to create kidney fluorescent lineage markers for SIX2 and NPHS1 to monitor the differentiation process to tubular and glomerular structures and optimize maturity. The convergence of "personalized" kidney organoids with genome editing and single-cell sequencing technology hold great promise to result in better insight to disease, better human cell disease models, more predictive toxicology, and potentially "clinical trials in a dish."

Assessing the degradation of tau in primary neurons: The role of autophagy.

Tau is a neuronal cytosolic, highly regulated protein. Although first identified as a protein that binds and stabilizes microtubules, it is now clear that tau plays numerous other roles in neurons. In addition to its key physiological roles in neuronal structure and function, tau is also involved in the pathogenesis of Alzheimer's disease and numerous other neurodegenerative disorders. In all tauopathies, there are pathogenic accumulations of tau. Given that tau homeostasis requires a balance of synthesis and degradation, understanding the pathways that mediate tau clearance and regulate this process in the disease state is of fundamental importance. In neurons, macroautophagy (referred to as autophagy in this chapter) plays a pivotal role in clearing damaged or misfolded proteins under normal conditions. However, in the disease state autophagy is impaired and tau may not be efficiently targeted for degradation which contributes to the increases in pathological tau species. Therefore, establishing model systems that allow for the analysis of tau clearance by autophagy and quantitative assessment of interventions that increase autophagy and tau clearance are needed. Of particular importance is the use of primary neurons as a model system, as they are more reflective of the relevant in vivo autophagy pathway than clonal or immortalized cell models. In this chapter we present detailed methods for the preparation of neurons, immunoblotting and imaging analyses, genetic and pharmacological manipulation of autophagy with analyses, and methods to quantitatively measure changes in tau and phospho-tau levels.

Abstract A16: Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions

Abstract Introduction: Drug testing of Glioblastoma multiforme (GBM) has revealed that many promising preclinical therapies fail when translated to the clinic. Serious limitations in preclinical models (often monolayer, serum-treated immortalized cell models) likely contribute to this translational failure. Although patient-derived xenografts (PDX), in which patient tumors are serially passaged in immunocompromised mice, are potentially superior model systems of human disease, high throughput drug studies in GBM PDX tumors are not feasible due to time, cost and throughput. To overcome these challenges, we have developed a GBM PDX MicroTumor system in which GBM PDX cells are grown in a novel 3D extracellular matrix material (HuBiogel™, Vivo Biosciences Inc., Birmingham, AL). We hypothesized that this 3D MicroTumor system would better emulate in vivo PDX tumor growth while providing a high throughput assay system for drug combination studies that can be performed in a more rapid timeframe. Methods: Six GBM PDX tumor “xenolines” were selected from the UAB Brain Tumor Animal Model Core to represent the 4 known molecular subtypes of GBM: Classical (JX10, X1016, X1046); Proneural (XD456); Neural (JX10) and Mesenchymal (JX22P). Tumor cells were prepared as single cell suspensions from subcutaneously passaged tumors and embedded in HuBiogel™ beads to form MicroTumors using Neuro-basal media devoid of serum. Single and combinatorial drug cytotoxicity studies were performed using MTT mitochondrial viability assays and Calcein AM imaging. Small molecule inhibitors selected for drug testing were: 1) selumetinib-targeting MEK1/2; 2) crizotinib-targeting c-MET and ALK; 3) cediranib-targeting VEGFR, FLT-1, FLT-4, c-KIT, and PDGFR; and 4) WP1066-targeting JAK2/STAT3. Combinatorial interactions were determined by Chou-Talalay combination index (CI) calculation using Calcusyn software. Global kinase signaling (kinomic profiling) was assessed using paired total cell lysates of GBM-PDX from orthotopic tumors grown in athymic nu/nu mice and as 3D MicroTumors. Testing was performed on the PamStation12 high content peptide microarray platform within the UAB Kinome Core. Results: Following optimization of model system conditions, 4 drugs were tested at multiple concentrations to identify effective dose ranges. While crizotinib, cediranib and WP1066 demonstrated significant cytotoxicity in at least 1 of the GBM-PDX MicroTumors tested, selumetinib was ineffective in producing cytotoxicity as a monotherapy. However, in combinatorial drug testing, selumetinib was effective in promoting synergistic activity (CI<1.0) when combined with each of the other drugs in several of the GBM-PDX tested. Conversely, WP1066 demonstrated some antagonistic interactions with the other drugs in certain tumors. Overall, cediranib and crizotinib demonstrated the most consistent level of efficacy both as monotherapies and combinatorial therapies across the GBM-PDX MicroTumors. Kinomic profiles were compared between GBM-PDX MicroTumors and matched GBM-PDX tumor xenolines grown as orthotopic intracranial tumors to confirm kinase signaling fidelity between the model systems. Paired t-test of kinomic phosphopeptide probes revealed only 20 of 246 peptides significantly differed (FDR<0.05) between the model systems suggesting a high level of signaling preservation within the MicroTumors. Conclusions: The GBM-PDX 3D MicroTumor provides a relatively high throughput drug screening model system that displays similar kinase signaling pathway activation as compared to their in vivo intracranial counterpart. Specific synergistic drug combinations were identified through MicroTumor testing. Ongoing drug combination testing of intracranially implanted GBM-PDX will determine how robustly the MicroTumor system predicts in vivo efficacy. Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Christine W. Duarte, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A16.

Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons

BackgroundOvernutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood.MethodsHere we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis.ResultsImportantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor-β-activated kinase 1 binding protein (TAB1) interaction as identified in the periphery.ConclusionsTaken together, GPR120 is functionally active in the hypothalamic neuronal line, rHypoE-7, wherein it mediates the anti-inflammatory actions of DHA to reduce the inflammatory response to TNFα.

Rhodacyanine Derivative Selectively Targets Cancer Cells and Overcomes Tamoxifen Resistance

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease

Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88(Tg737Rpw)) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na(+) movement in cilium-deficient ("mutant") cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent ("rescued") monolayers. To examine NHE activity, we measured intracellular pH (pH(i)) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na(+)-dependent acid-base transporter activity in the nominal absence of CO(2)/HCO(3)(-). However, only the mutant cells displayed appreciable apical Na(+)-induced pH(i) recoveries from NH(4)(+) prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pH(i) dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pH(i) 6.23-6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD.

Titration of Synaptotagmin I Expression Differentially Regulates Release of Neuropeptide Y and Norepinephrine

Synaptotagmin (syt) I is a primary regulator of Ca2+-dependent vesicle secretion. Pheochromocytoma (PC12) cells are used as an immortalized cell model system for neurons to study regulated vesicle release. In this study, we stably transfected multiple PC12 cell lines with a single plasmid that contains a syt I-targeting short hairpin RNA to knockdown expression of syt I. Stable cells were selected, expanded, and tested for stable incorporation of the plasmid with PCR and for specific targeting of syt I with immunoblot analysis. As previously reported (Cahill et al., 2007), individual stable cell lines derived from incorporation of a single short hairpin RNA expressed varying levels of syt I knockdown. This variability offers the advantages of studying the functional effects of graded levels of syt I protein expression in regulated release of transmitter. We measured Ca2+-stimulated release of two transmitters, neuropeptide Y (NPY) and norepinephrine (NE). NPY was measured by an enzyme-immunoassay after depolarizing with 50 mM K+ solution. Stable cell lines that expressed 50-60% of control levels of syt I exhibited NPY release that were similar to control cells. NPY release was reduced to about 18% of control cell release when expression of syt I was reduced to ∼20%, and NPY release was abolished when syt I expression was abolished. Stimulated NE release from single vesicles was measured by carbon-fiber amperometry. Unlike NPY release, NE release ranged from 50-100% compared to control release, but was not abolished even for the cells that did not express syt I. These results show that syt I is required for NPY release, but NE release is only partially dependent on syt I expression.

The oncogenic kinase Pim-1 is modulated by K-Ras signaling and mediates transformed growth and radioresistance in human pancreatic ductal adenocarcinoma cells

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons

Significant information on reproductive function has been generated based on the rat model, including many seminal discoveries. Yet little is known about the molecular and cellular events involved in control of reproductive function, mainly due to the pervasive lack of cell models from rat. We have therefore generated a wide array of cell lines using primary cell culture from the rat hypothalamus. Immortalization of the primary cells was achieved through retroviral transfer of T-antigen, followed by selection with geneticin. The mixed cell populations were subcloned and each clonal cell line was analyzed for expression of specific cellular markers. Each line has a distinct phenotypic profile, with expression of key neuroendocrine markers. We have functionally analyzed two clonal cell lines, rHypoE-7 and rHypoE-8, for hormones implicated in the control of gonadotropin-releasing hormone neuronal function through melatonin, specifically kisspeptin (KISS) and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). We detected functional melatonin receptor activity, as each cell line exhibited inhibition of forskolin-stimulated 3′-5′-cyclic adenosine monophosphate (cAMP) accumulation. Upon treatment with 10 nM melatonin, we found that KISS gene expression was decreased in the rHypoE-8 cell line, while RFRP-3 was increased in the rHypoE-7 cell line. These results are in accordance with the differential regulatory functions of these two peptides, particularly on GnRH neuronal control. These cell lines will serve as novel tools for the analysis of the cellular and molecular mechanisms involved in hypothalamic control of a number of physiological processes described in the rat animal model.