Heme-Regulated eIF2α Kinase (HRI) Inhibition Decreases PKR Activation in HUDEP2 Cells

Abstract Introduction: Sickle Cell Disease (SCD) is a group of inherited disorders caused by mutations in the β-globin gene which encodes the hemoglobin subunit β in erythrocytes [1]. Hemoglobin containing the mutant β-globin polymerizes and causes sickling of erythrocytes, which subsequently leads to vaso-occlusion, hemolysis, and activation of the immune system by release of free heme [2]. Heme-regulated eIF2α kinase, also known as heme-regulated inhibitor (HRI/EIF2AK1), the dsRNA-activated kinase Protein Kinase R (PKR/EIF2AK2), PKR-like endoplasmic reticulum kinase (PERK/EIF2AK3) and General Control Non-Depressible 2 (GCN2/EIF2AK4) are four kinases belonging to the eIF2α kinases family and play key functions in the Integrated Stress Response-ATF4 pathway, which is critical for translational control in response to various stress conditions [3]. These kinases are activated respectively by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, and they phosphorylate eukaryotic initiation factor-2α (eIF2α). Recently, it has been shown that HRI inhibition induces fetal hemoglobin in HUDEP-2 cells and CD34+ hematopoietic progenitor stem cells and prevents sickling, suggesting HRI as a potential therapeutic target for SCD [4, 5]. Moreover, it has been well documented that kinase inhibition can activate compensatory loops (bypass signaling) to circumvent the inhibited target, in particular by overexpression and activation of kinases having the same substrate [6, 7]. Thus, in this study, we investigated if the inhibition of HRI in HUDEP-2 cells lead to compensatory mechanisms by modulation of the expression and activation of the other eIF2α kinases. Methods: To inhibit HRI, we generated HUDEP-2 [8] HRI Knock-Out cells (HRI-KO) and HUDEP-2 HRI Kinase Dead cells (K196R) (clones HRI-KD1 and HRI-KD2) by using CRISPR-Cas9 gene editing technology [9]. We confirmed the induction of fetal hemoglobin for each clone by flow cytometry. To evaluate a potential compensatory mechanism, we measured the effects of HRI inhibition on the expression and activation of the other eIF2α kinases by western blot (WB) and the regulation at the transcriptomic level by qPCR. Based on the results of preliminary studies, we generated HUDEP-2 PKR Knock-Out cells (PKR-KO) by CRISPR-Cas9. We differentiated them for 7 days and we quantified the level of fetal hemoglobin by flow cytometry, AlphaLISA® and WB. Results: HRI-KO, HRI-KD1 and HRI-KD2 clones expressed fetal hemoglobin after 7-day of differentiation consistent with published data [4]. HRI inhibition did not result in any modulation of PKR protein expression, but the activation of PKR, measured by phosphorylation at its residue Threonine 446, was decreased in HRI-KD1, HRI-KD2, and in HRI-KO cells at day 0 and day 7 of differentiation. As PKR and HRI have the same downstream target eIF2α and HRI inhibition induces fetal hemoglobin through eIF2α-ATF4-Bcl11a axis, we verified if fetal hemoglobin induction is due to HRI inhibition exclusively and is not a consequence of the decrease in PKR activation when HRI is inhibited. We measured the protein expression level of fetal hemoglobin in PKR-KO cells and results obtained by flow cytometry, western blot and AlphaLISA® did not show any regulation in fetal hemoglobin in PKR-KO cells after 7 days of differentiation. Finally, HRI inhibition did not result in any regulation of kinases PERK and GCN2 activation and expression, at the RNA and protein level. The expression of these two eIF2α kinases was low compared to HRI and PKR. Conclusion: HRI inhibition does not cause any modulation in the expression and activation of GCN2 and PERK in HUDEP-2 cells but results in a decrease in PKR activation. This outcome could be explained by a possible increase in the expression of proteins that inhibit PKR, such as TRBP or Hsp40, and are induced by HRI silencing (Hsp40) [10-12]. Nonetheless PKR inhibition does not induce fetal hemoglobin in HUDEP-2 cells after 7 days of differentiation. Overall, this study provides evidence that fetal hemoglobin induction by HRI inhibition in HUDEP-2 cells is independent from the other eIF2α kinases and supports HRI as a potential therapeutic target in SCD. However, the biological implications of a potential compensatory effect on PKR signaling in HRI-expressing tissues warrant further investigation. Disclosures Krishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.

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The sigma 1 receptor (S1R) is a potential therapeutic target for the treatment of Huntington's disease

10.31219/osf.io/mcefx ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Endoplasmic Reticulum ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Therapeutic Target ◽

Structural Integrity ◽

Neuronal Cell ◽

Glutamate Excitotoxicity ◽

Potential Therapeutic Target ◽

Sigma 1 Receptor ◽

Sigma 1

During the progression of Huntington's disease (HD), changes in Ca2+ signaling cause neuronal cells to lose a range of functional properties. GABAergic medium spiny neurons (MSNs) are able to prevent Ca2+ imbalance in the early stages of the illness through a number of compensatory strategies. However, as people become older, their neuroprotective potential diminishes due to a decrease in metabolic activity and the generation of Ca2+-buffering proteins. Continuing Ca2+ regulation problems exhaust the cells' compensatory abilities, resulting in a continuous surge in cytosolic Ca2+ and neuronal degeneration.The sigma 1 receptor (S1R) is a potential therapeutic target for the treatment of HD because it regulates a number of cytosolic Ca2+-dependent signaling cascades. S1R activation by selective agonists protects neurons from glutamate excitotoxicity, reduces store-operated Ca2+ entry (SOCE) hyperactivation, and maintains the structural integrity of mitochondria-associated endoplasmic reticulum membranes (MAMs), which is required for synchronizing mitochondrial and endoplasmic reticulum (ER) activity to maintain cell bioenergetics balance. Because of the stability of Ca2+ signaling in neurons, pridopidine, a highly selective S1R agonist, has been demonstrated to protect neurons in cellular and animal models of HD.The synaptoprotective effect of pridopidine is very important since it is found in both cortical and striatal neurons, indicating that pridopidine has a systemic influence on HD therapy. Because synaptic dysfunctions are one of the earliest markers of neuropathology at the cellular level, normalization of Ca2+ balance by pridopidine may prevent disease development at the molecular level at the earliest stages. In this regard, the most significant therapeutic advantage of pridopidine will almost certainly be in preventative treatment, even before the start of the first clinical indications, which will improve neuronal cell compensatory abilities and significantly reduce the progression of HD.

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Effects of Fluvastatin on the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

Blood ◽

10.1182/blood.v118.21.2253.2253 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2253-2253

Author(s):

Keiko Maruyama ◽

Eriko Morishita ◽

Hiroki Torishima ◽

Akiko Sekiya ◽

Hidesaku Asakura ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Western Blot ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Umbilical Vein ◽

The Other ◽

Mrna And Protein Expression ◽

Tissue Factor Pathway ◽

Umbilical Vein Endothelial Cells

Abstract Abstract 2253 OBJECTIVE: 3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) inhibit the production of mevalonate and other isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Statins can protect the vasculature from inflammation and atherosclerosis caused by cholesterol-dependent and cholesterol-independent mechanisms. The latest investigations show that statins modulate the expression of genes related to inflammation, blood coagulation and fibrinolysis in cultured endothelial cells. Tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. The aim of this study was to elucidate the effects of fluvastatin, lipophilic statin, on expressions of TFPI in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were incubated for 24 h in culture medium including fluvastatin (0.1, 1.0, 10.0 μM). The expression of TFPI mRNA and protein was evaluated by western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. To identify which product of statin reaction is necessary for the effect of fluvastatin, HUVECs were incubated for 24h with fluvastatin with mavalonate, FPP, or GGPP. On the other hand, it is known that fluvastatin increase nitric oxide (NO) bioavailability. To determine whether fluvastatin induced NO affects TFPI mRNA and protein expression, HUVECs were incubated for 24h with fluvastatin with NG-Nitro-L-arginine methyl ester, hydrochloride (L-NAME: specific inhibitor of NO synthase). Additionally, to determine whether fluvastatin affects p38MAPK, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) pathways, HUVECs were incubated for 24h with fluvastatin with the inhibitors of p38MAPK (SB203580), JNK (SP600125), MEK (U0126), PI3K (LY294002), and PKC (GF109203). The expression of TFPI mRNA and protein was evaluated by western blot. RESULTS: Fluvastatin increased TFPI mRNA and protein expression (1μM: p<0.01, 10μM: p<0.05; Figure 1). This fluvastatin-dependent up-regulation of TFPI was prevented by mevalonate and geranylgeranylphosphate (GG-PP). In contrast, the addition of L-NAME did not alter induction of TFPI expression by fluvastatin. Similarly, Y-27632 (Rho kinase inhibitor) and NSC23766 (Rac1 inhibitor) were ineffective. Additionally, the inhibitors of p38MAPK, PI3K, and PKC prevented fluvastatin-dependent up-regulation. On the other hand, the inhibitors of JNK and MEK were ineffective. CONCLUSIONS: This study suggests that fluvastatin significantly increases TFPI mRNA and protein expression, and this effect of fluvastatin is accompanied by the activation of p38 MAPK, PI3K, and PKC pathways. Therefore, this effect may play an important role in preventing cardiovascular events. Disclosures: No relevant conflicts of interest to declare.

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Relative quantification of protein expression in metastatic renal cell carcinoma using iTRAQ LC-MS/MS analysis reveals galectin-1 as a potential prognostic marker and therapeutic target.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.6_suppl.412 ◽

2013 ◽

Vol 31 (6_suppl) ◽

pp. 412-412

Author(s):

Nicole M.A. White ◽

Olena Masui ◽

Leroi DeSouza ◽

Olga Krakovska ◽

Ajay Matta ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Overall Survival ◽

Protein Expression ◽

Western Blot ◽

Cell Motility ◽

Cell Carcinoma ◽

Therapeutic Target ◽

Renal Cell ◽

Cellular Migration ◽

Metastatic Rcc

412 Background: Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant cancers. Identification of proteins involved in tumor progression will help gain a better understanding of the disease and will form the basis for the identification of novel therapeutic targets. Methods: Using six fresh-frozen primary and six unmatched metastatic RCC tumors, we used iTRAQ labeling and LC-MS/MS analysis to identify proteins differentially expressed in metastatic versus primary RCC. We verified protein expression by western blot and immunohistochemical analyses and the measured the effect of dysregulated protein expression on biological processes with RCC cell line models. Results: After analysis, we identified 29 proteins differentially expressed in metastatic versus primary RCC. We verified expressions of profilin-1, 14-3-3 zeta/delta, and galectin-1 (Gal-1) on two independent tissue sets by western blot (10 primary and 10 metastatic RCC tissues) and immunohistochemistry (22 primary and 23 metastatic tissues). Overexpression of Gal-1 in CAKI-1 cells lead to decreased actin, increased vimentin expression, and increased cellular migration. Additionally, when Gal-1 was decreased via siRNA, cells showed decreased cellular migration. Protein array analysis showed expression of cell motility-related proteins HSP27, JNK, and RSK, were altered after siRNA transfection. We also showed that Gal-1 expression was increased in response to HIF-1alpha. Furthermore, we analyzed the expression of Gal-1 mRNA in 404 RCC patients using the Cancer Genome Anatomy Project, and found that patients who had higher Gal-1 expression in the primary RCC had significantly decreased overall survival (41 vs. 78 months; p < 0.01). Conclusions: Gal-1 is increased in metastatic RCC and can effect cell migration by targeting proteins involved in cell motility. This may be a downstream effect of HIF-1α dysregulation. Decreased Gal-1 significantly decreased cellular migration suggesting Gal-1 may serve as a potential therapeutic target. Additionally, we showed that increased Gal-1 expression was associated with decreased overall survival.

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EFFECT OF EDEM1 OVEREXPRESSION ON THE GENERATION AND ASSEMBLY OF MAJOR HISTOCOMPATIBILITY COMPLEXES

Romanian Journal of Rheumatology ◽

10.37897/rjr.2016.4.2 ◽

2016 ◽

Vol 25 (4) ◽

pp. 181-186

Author(s):

Alexandra Circiumaru ◽

◽

Gabriela Chiritoiu ◽

Livia Sima ◽

Mihai Bojinca ◽

...

Keyword(s):

Flow Cytometry ◽

Endoplasmic Reticulum ◽

Western Blot ◽

Autoimmune Diseases ◽

Cell Line ◽

Hek293t Cell ◽

Western Blot Assay ◽

Mhc I ◽

Transfected Cells ◽

Hek293t Cell Line

Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target.

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Rituximab-Induced CD20-Mediated Signals and Suppression of PI3K-AKT Pathway Cooperates to Inhibit B-Cell Lymphoma Growth By Down-Regulation of Myc.

Blood ◽

10.1182/blood.v128.22.5312.5312 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5312-5312 ◽

Cited By ~ 2

Author(s):

Tsuyoshi Nakamaki ◽

Yuta Baba ◽

Maasa Abe ◽

Sou Murai ◽

Megumi Watanuki ◽

...

Keyword(s):

Protein Expression ◽

Western Blot ◽

B Cell ◽

Cell Line ◽

Therapeutic Target ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Akt Pathway ◽

Down Regulation ◽

Rb Protein

Abstract Rituximab, anti-CD20 monoclonal antibody, shows anti-lymphoma effects by either activating host immune mechanism against lymphoma cells such as ADCC and CDC or suppressing signal(s) important for growth of B-cell lymphoma. Accumulated evidence shows de-regulated PI3K-AKT pathway is important therapeutic target in B-cell lymphoma. Although studies suggested suppression of PI3K-AKT pathway is possibly critical in rituximab-induced anti-lymphoma signals, details of molecular mechanisms covering CD20-mediated signals and PI3K-AKT pathway are still unknown. We analyzed signals in B-cell lymphoma cell line, SD07, which had bi-allelic deletions of both CD20(MS4A1) and PTEN gene. Briefly, SD07 was established from a patient with diffuse large B-cell lymphoma(DLBCL)who showed refractory CD20-negative relapse(Eur.J. Haematol. 2012). We transiently restored protein expression of either or both of CD20 and PTEN by electroporation of expression plasmid having cDNA of those genes in SD07. In this experiment, expression of both proteins were clearly detected by western blot until 72 hours after transfection. CD20 protein was also detectable on cell surface of SD07 cells by flow cytometric(FCM) analysis at comparable levels of those of Daudi cells after transfection on CD20 cDNA. Our previous study showed expression of both c-myc RNA and protein, as judged by FCM, was induced by transient transfection of CD20 cDNA in SD07 cells. Western blot analysis also showed significant increase of myc protein 24hrs after transfection (vector=13.7±0.2, CD20=14.5±0.4, P<0.05, arbitrary density unit) and the increase was not affected by the addition of rituximab(20µg/ml)in culture. In contrast transfection of PTEN cDNA produced significant decrease of myc protein (PTEN=11.2±0.1, P<0.01). Myc protein levels was comparable between cells transfected with CD20 plus PTEN and PTEN alone. However in the presence of rituximab, myc protein expression was markedly downregulated in SD07 cells at both 24 hours(vector=13.7±0.2,CD20+PTEN=8.3±0.1,P=0.0008)and 48hours(vector=12.4±0.3 vs CD20+PTEN=11.2±0.1,P=0.04). 24 hours after transfection, de-phosphorylation and down-regulation of retinoblastoma(RB) protein expression was evident only in SD07 cells transfected with both CD20 and PTEN, but not with either alone. Rituximab also augmented down-regulation of RB protein in SD07 cells transfected with both CD20 and PTEN. After transfection, in vitro cell growth of SD07, as evaluated by MTT, was as follows;vector=4.6±0.3, CD20=4.8±0.3(P=NS), PTEN=4.1±0.2(P<0.05), CD20+PTEN=3.6±0.2(P<0.01), CD20+PTEN+rituximab=2.7±0.2(P<0.01),OD570nm, days3) 72 hours after transfection, by FCM, SD07 transfected with both CD20 and PTEN showed significant increase of subG1 DNA content potion compared with cells transfected with either alone(vector=2.5±0.2%,CD20=5.3±0.2%,PTEN=8.8 ±0.5%,CD20+PTEN= 16.1±0.6, P<0.01). Furthermore, addition of rituximab significantly increase of cells in subG1 in SD07 with transfected with both CD20 and PTEN(18.6±0.5%,P<0.01), suggesting suppression of PI3K-AKT is important for mediateing rituximab-induced apoptotis. Accordance with proliferative effect of CD20 in SD07, transfection of CD20 alone produced significant increase of phosphorylation of AktSer473(vector=11.2±2.3, CD20=13.3±2.3,pAkt/total Akt,P<0.05). Co-transfection of PTEN with CD20 suppress the CD20-induced phosphorylation of Akt in SD07 cells. (CD20+PTEN=12.8±2.3,P=NS). In summary, although data is limited in a cell line, it clearly shows rituximab-induced CD20-mediated signals and suppression of PI3K-AKT pathway by tumor suppressor PTEN cooperatively inhibits growth and induce apoptosis of DLBCL cell line, SD07. Myc protein is critical molecules in this particular cooperative pathways. The present result partly agree studies which showed that inhibition of PI3K-AKT signaling and down-regulation of myc is important in PTEN-induced cytotoxicity in GCB(germinal center B cell)type DLBCL. In addition, we show here myc is not only for PTEN, but also involved in rituximab-induced anti-lymphoma effect. The present result provide a rationale for combination of rituximab and PI3K inhibitor in the treatment of DLBCL. In this context, myc is a candidate of the therapeutic target in rituximab-based immunochemotherapy for B cell lymphoma. Disclosures No relevant conflicts of interest to declare.

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Abstract 4266: Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer

10.1158/1538-7445.am2019-4266 ◽

2019 ◽

Author(s):

Ashish Tyagi ◽

Balaji chandrasekaran ◽

Venkatesh Kolluru ◽

Becca V. Baby ◽

Alatassi Houda ◽

...

Keyword(s):

Colorectal Cancer ◽

Endoplasmic Reticulum ◽

Therapeutic Target ◽

Mutant P53 ◽

Potential Therapeutic Target

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Abstract 4266: Endoplasmic reticulum (ER), a potential therapeutic target for mutant p53 colorectal cancer

10.1158/1538-7445.sabcs18-4266 ◽

2019 ◽

Author(s):

Ashish Tyagi ◽

Balaji chandrasekaran ◽

Venkatesh Kolluru ◽

Becca V. Baby ◽

Alatassi Houda ◽

...

Keyword(s):

Colorectal Cancer ◽

Endoplasmic Reticulum ◽

Therapeutic Target ◽

Mutant P53 ◽

Potential Therapeutic Target

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Mir-21 Knock Out Creates a Radiosensitive Phenotype and is Therefore a Potential Therapeutic Target

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/j.ijrobp.2014.05.151 ◽

2014 ◽

Vol 90 (1) ◽

pp. S36-S37

Author(s):

C.M. Wright ◽

T. Dan ◽

L. Liotta ◽

L. Jin ◽

R.G. Pestell ◽

...

Keyword(s):

Therapeutic Target ◽

Potential Therapeutic Target ◽

Knock Out

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PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C fromPeucedanum praeruptorumDunn

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/156574 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Ling Huang ◽

Qian Wu ◽

Yu-Hua Li ◽

Yi-Tao Wang ◽

Hui-Chang Bi

Keyword(s):

Gene Expression ◽

Catalytic Activity ◽

Protein Expression ◽

Western Blot ◽

Blot Analysis ◽

Pregnane X Receptor ◽

Transient Transfection ◽

The Other ◽

Primary Hepatocytes ◽

Cyp3a4 Substrate

We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on theCYP3Agene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level ofCYP3a11gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs.

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