scholarly journals Moderate Autophagy Inhibits Vascular Smooth Muscle Cell Senescence to Stabilize Progressed Atherosclerotic Plaque via the mTORC1/ULK1/ATG13 Signal Pathway

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Zhenli Luo ◽  
Wenhuan Xu ◽  
Sai Ma ◽  
Hongyu Qiao ◽  
Lei Gao ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Foam Cell ◽  
Cell Model ◽  
P53 Protein ◽  
Atherosclerosis Progression

In order to investigate the effects of autophagy induced by rapamycin in the development of atherosclerosis plaque we established murine atherosclerosis model which was induced in ApoE−/− mice by high fat and cholesterol diet (HFD) for 16 weeks. Rapamycin and 3-Methyladenine (MA) were used as autophagy inducer and inhibitor respectively. The plaque areas in aortic artery were detected with HE and Oil Red O staining. Immunohistochemical staining were applied to investigate content of plaque respectively. In contrast to control and 3-MA groups, rapamycin could inhibit atherosclerosis progression. Rapamycin was able to increase collagen content and a-SMA distribution relatively, as well as decrease necrotic core area. Then we used MOVAS and culture with ox-LDL for 72 h to induce smooth muscle-derived foam cell model in vitro. Rapamycin and 3-MA were cultured together respectively. Flow cytometry assay and SA-β-Gal staining experiments were performed to detect survival and senescence of VSMCs. Western blot analysis were utilized to analyze the levels of protein expression. We found that rapamycin could promote ox-LDL-induced VSMCs autophagy survival and alleviate cellular senescence, in comparison to control and 3-MA groups. Western blot analysis showed that rapamycin could upregulate ULK1, ATG13 and downregulate mTORC1 and p53 protein expression.

Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Spencer Barnhill ◽  
Prakash Arumugam ◽  
John Matsuura ◽  
Scott Berceli ◽  
Katie Carroll ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Western Blot ◽  
Vascular Smooth Muscle Cells ◽  
Blot Analysis ◽  
Intimal Hyperplasia ◽  
Western Blot Analysis ◽  
Interleukin 2 ◽  
And Migration ◽  
Proliferation And Migration

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

scholarly journals FGF21 Ameliorates Hepatic Fibrosis by Multiple Mechanisms

2021 ◽  
Author(s):  
Fanrui Meng ◽  
Mir Hassan Khoso ◽  
Kai Kang ◽  
Qi He ◽  
Yukai Cao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Model ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Abstract Previous study reports that FGF21 could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. CCL4 and DMN were respectively used to induce hepatic fibrosis animal models. Our results demonstrated that liver index and liver function were deteriorated in both models. HE and Masson’s staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1β, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. RT-PCR and Western blot analysis showed that mRNA and protein expression of collagenI, α-SMA and TGF-β were significantly decreased by treatment with FGF21. PDGF-BB stimulant was used to establish the experimental cell model in HSCs. RT-PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expression of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 could significantly reduce leptin expression in a dose dependent manner and these effects were reversed in siRNA (against β-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-β were significantly downregulated by FGF21 treatment in a dose dependent manner. The expression of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs.

scholarly journals Estrogen Induces Metastatic Potential of Papillary Thyroid Cancer Cells through Estrogen Receptorαandβ

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Wenwu Dong ◽  
Hao Zhang ◽  
Jing Li ◽  
Haixia Guan ◽  
Liang He ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Metastatic Potential ◽  
Migration And Invasion ◽  
Selective Agonist ◽  
E Cadherin

Estradiol (E2) promotes metastatic propensity. However, the detailed mechanism remains largely unknown. E-cadherin, vimentin, and MMP-9 play a dominant role in the metastatic process. We aimed to investigate the effects of E2 on metastatic potential of PTC cell line BCPAP and on E-cadherin, vimentin, and MMP-9 protein expression. PTC cell line BCPAP was evaluated for the presence of estrogen receptor (ER) by western blot analysis. The effects of E2, PPT (a potent ERα-selective agonist), and DPN (a potent ERβ-selective agonist) on modulation of metastatic phenotype were determined by usingin vitroscratch wound assay and invasion assay. In addition, the effects on E-cadherin, vimentin, and matrix metalloproteinase-9 (MMP-9) protein expression were evaluated by Western blot analysis. We found that BCPAP cells expressed ERαand ERβ. E2 and PPT enhanced, but DPN inhibited, the migration and invasion of BCPAP cells in anin vitroexperimental model system that is modulated by E-cadherin, vimentin, and MMP-9. These findings indicate that E2 induces the metastatic potential of BCPAP cells through ERαand ERβ. The two ER subtypes play differential roles in modulation of BCPAP cell metastasis and the related molecule expressions including E-cadherin, vimentin, and MMP-9.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney

2021 ◽  
Author(s):  
Koji Eguchi ◽  
Yuichiro Izumi ◽  
Yukiko Yasuoka ◽  
Terumasa Nakagawa ◽  
Makoto Ono ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Membrane Fraction ◽  
Potassium Level ◽  
Collecting Duct ◽  
Subcutaneous Administration ◽  
Extracellular Potassium ◽  
Membrane Expression

Rh C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and Gӧ6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Abstract WP433: Targeting Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) Expression at the Blood-brain Barrier: Implications for Treatment of Ischemic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Wazir Abdullahi ◽  
Hrvoje Brzica ◽  
Patrick Ronaldson
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Organic Anion ◽  
Stroke Treatment ◽  
Brain Microvessels ◽  
Cns Delivery

Introduction: Stroke is a leading cause of mortality and morbidity. Several drugs with neuroprotective properties have been proposed for stroke treatment but many have failed in clinical trials. These failures may be due to limited drug permeability across the blood-brain barrier (BBB). Targeting endogenous BBB uptake transporters (i.e., organic anion transporting polypeptides (Oatps)) is a novel approach that can improve CNS delivery of drugs relevant to stroke therapy (i.e., statins). Optimal CNS drug delivery via Oatp in the setting of stroke requires characterization of regulatory pathways such as transforming growth factor (TGF)-β signaling. The goal of the present study was to investigate, in vivo , involvement of TGF-β signaling via the activin-like kinase (ALK)-1 receptor on Oatp1a4 expression at the BBB. Methods: Female Sprague-Dawley rats (200-250 g) were administered bone morphogenic protein-9 (BMP-9; 0-5 μg/kg, i.p.), an ALK1 agonist, or vehicle (i.e., 0.9% saline). Inhibition experiments were performed using LDN193189 (0-5 mg/kg, i.p.), an ALK1 antagonist. Western blot analysis and fluorescence microscopy of isolated brain microvessels were used to determine protein expression and localization in rat brain microvessels respectively. Results: Fluorescence staining demonstrated localization of Oatp1a4 and ALK1 in rat brain microvessels. Western blot analysis showed a dose dependent increase in Oatp1a4 protein expression in brain microvessels isolated from BMP-9 treated rats as compared to controls. Treatment with 0.5 μg/kg and 5 μg/kg BMP-9 resulted in a 55% and 116% increase in Oatp1a4 protein expression, implying that activation of ALK1 signaling can up-regulate Oatp1a4 at the brain microvasculature. In contrast, 6 h treatment with LDN193189 did not alter Oatp1a4 expression across a dose range of 0-5 mg/kg, suggesting that ALK1 inhibition does not modulate basal Oatp1a4 expression at the BBB. Conclusions: Taken together, our data implies that TGF-β/ALK1 signaling may play a role in altering Oatp1a4 protein expression at the BBB. Studies are currently being undertaken in our laboratory to fully characterize the role of TGF-β/ALK1 signaling in determining CNS delivery of drugs relevant to stroke treatment (i.e., statins).

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Nathamon Yimpring ◽  
Sittiruk Roytrakul ◽  
Janthima Jaresitthikunchai ◽  
Narumon Phaonakrop ◽  
Sucheewin Krobthong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Profiles ◽  
Tumor Necrosis Factor Receptor ◽  
Principal Component ◽  
Peptide Mass ◽  
Different Ages

Abstract Background Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry. Results A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. Conclusions The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

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