Deregulation of oncogene‐induced senescence and p53 translational control in X‐linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X‐linked dyskeratosis congenita (X‐DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene‐induced senescence (OIS). Here, we show that during OIS, a switch between cap‐ and internal ribosome entry site (IRES)‐dependent translation occurs. During this switch, an IRES element positioned in the 5′untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1^m cells, p53 IRES‐dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X‐DC human patient cells in which similar impairments in p53 IRES‐dependent translation are observed. Importantly, re‐introduction of wild‐type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.     

Regulation of autophagy and chloroquine sensitivity by oncogenic RAS in vitro is context-dependent

Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.     

Regulation of autophagy and chloroquine sensitivity by oncogenic RAS in vitro is context-dependent

Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.     

A novel SOS1 mutation in Costello/CFC syndrome affects signaling in both RAS and PI3K pathways

Abstract

Context: Pathological upregulation of the RAS/MAPK pathway causes Costello, Noonan and cardio–facio–cutaneous (CFC) syndrome; however, little is known about PI3K/AKT signal transduction in these syndromes. Previously, we found a novel mutation of the SOS1 gene (T158A) in a patient with Costello/CFC overlapping phenotype. Objective: The aim of this study was to investigate how this mutation affects RAS/MAPK as well as PI3K/AKT pathway signal transduction.

Materials and methods: Wild-type and mutant (T158A) Son of Sevenless 1 (SOS1) were transfected into 293T cells. The levels of phospho- and total ERK1/2, AKT, p70S6K and pS6 were examined under epidermal growth factor (EGF) stimulation. Results: After EGF stimulation, the ratio of phospho-ERK1/2 to total ERK1/2 was highest at 5?min in mutant (T158A) SOS1 cells, and at 15?min in wild-type SOS1 cells. Phospho-AKT was less abundant at 60?min in mutant than in wild-type SOS1 cells. Phosphorylation at various sites in p70S6K differed between wild-type and mutant cells. Eighteen hours after activation by EGF, the ratio of phospho-ERK1/2 to total ERK1/2 remained significantly higher in mutant than in wild-type SOS1 cells, but that of phospho-AKT to total AKT was unchanged. Discussion: T158A is located in the histone-like domain, which may have a role in auto-inhibition of RAS exchanger activity of SOS1. T158A may disrupt auto-inhibition and enhance RAS signaling. T158A also affects PI3K/AKT signaling, probably via negative feedback via phospho-p70S6K. Conclusion: The SOS1 T158A mutation altered the phosphorylation of gene products involved in both RAS/MAPK and PI3K/AKT pathways.     

Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2–5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.     

Valsartan chronotherapy reverts the non-dipper pattern and improves blood pressure control through mediation of circadian rhythms of the renin-angiotensin system in spontaneous hypertension rats

ABSTRACT

Background: Numerous clinical studies have evaluated valsartan and found more efficacious control of blood pressure (BP) variability when administered before sleep. The treatment leads to improved outcomes when compared to administration upon awakening. The mechanism underlying this etiology is not fully understood. The present study investigates the safety and efficacy of asleep administration of valsartan in spontaneously hypertensive rats (SHR) with a non-dipping blood pressure pattern compared to SHRs receiving administration during awake time. Materials and Methods: 84 Male SHRs and 28 male Wistar-Kyoto rats (WKY) were kept under a strict alternating 12-h light/dark cycle. WKYs were utilized as a non-disease control. Meanwhile, SHRs were randomly divided into three groups: untreated, Valsartan asleep administration (VSA) and Valsartan awake administration (VWA) respectively. The VSA group was treated with valsartan (30 mg/kg/d) after the light onset, while the VWA group was treated with valsartan (30 mg/kg/d) after light offset. Both groups were treated for 6 weeks. Tail artery blood pressure was measured every 4 h via a noninvasive tail cuff blood pressure measurement method. HE and Masson staining were used to evaluate any damage within the target organs. ELISA was used to determine the 24-h plasma renin-angiotensin system (RAS) concentration at 4-h intervals. Results: Based on our findings, VSA significantly reduced 24-h and evening mean BP and restored the abnormal circadian rhythm compared to VWA, which attenuated injuries in the majority of target organs except for the kidneys. Furthermore, VSA was found to activate RAS during the light cycle and inhibit it during the dark cycle. Conversely, VWA was found to deactivate RAS throughout the day which may be related to the circadian BP rhythm. Conclusion: VSA may be more efficacious than VWA in controlling BP, circadian BP rhythm and blood RAS rhythm. Recent cardiovascular outcome investigations substantiate that chronotherapy treatment might be a novel therapeutic strategy for hypertension therapy.

Abbreviations: Angiotensin-converting enzyme (ACE); Angiotensin converting enzyme inhibitors (ACEIs); Angiotensin II (ANG II); Analysis of variance (ANOVA); Angiotensin receptor blockers (ARBs); Blood Pressure (BP); Calcium Antagonists Calcium Channel Blockers (CCB); Chronic kidney diseases (CKD); Sodium carboxyl methyl cellulose (CMC-Na); Cardiac mass index (CMI); Cardiovascular diseases (CVD); Diastolic blood pressure (DBP); Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E); Kidney mass index (KMI); Liver mass index (LMI); Mean arterial blood pressure (MAP); Plasma renin concentration (PRC); Renin-angiotensin system (RAS); Rennin (REN); Systolic blood pressure (SBP); Student-Newman-Keuls q test (SNK-q test); Spontaneous hypertension rats (SHR); Valsartan asleep Administration (VSA); Valsartan awake Administration (VWA); Wistar-Kyoto (WKY); Mesor (M); Amplitude (A); Phase (φ).     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain (∼310 amino acids), a single transmembrane domain (∼20 amino acids) and an intracellular domain (∼19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain (∼30 amino acids), the IC domain is also involved in assembly of V₀ portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 Å (maltose-free) and 2.15 Å (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP–PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR-IC domain in protein oligomerization.     

Understanding the dual mechanism of bioactive peptides targeting the enzymes involved in Renin Angiotensin System (RAS): An in-silico approach

Abstract

Understanding the dual inhibition mechanism of food derivative peptides targeting the enzymes (Renin and Angiotensin Converting enzyme) in the Renin Angiotensin System. Two peptides RALP and WYT were reported to possess antihypertensive activity targeting both renin and ACE, and we have used molecular docking and molecular dynamics simulation, in order to understand the underlying mechanism. The selected peptides (RALP and WYT) from the series of peptides reported were docked to renin and ACE and two binding modes were selected based on the binding energy, interaction pattern and clusters of docking simulation. The enzyme-peptide complexes for renin and ACE (Renin/RALP_1,2; ACE/RALP_1,2; Renin/WYT_1,2 and ACE/WYT_1,2) were subjected to molecular dynamics simulation. Our results identified that the peptides inhibiting renin, tends to move out of the binding pockets (S1’ S2’) which is critical for potent binding and occupies the less important pockets (S4 and S3). This could possibly be the reason for its low potency. Whereas, the same peptides targeting ACE, tends to be intact in the pocket because of the metal ion coordination and there is an ample room to improve on its efficacy. Our results further pave way for the biochemist, medicinal chemist to design dual peptides targeting the RAS effectively.

Communicated by Ramaswamy H. Sarma     

Altered dipsogenic responses and expression of angiotensin receptors in the offspring exposed to prenatal high sucrose

The present study determined water and salt intake as well as expression of AT₁ and AT₂ receptors in the brain and kidney in the adult offspring rats prenatally exposed to high sucrose. Following the exposure during pregnancy, water intake and salt intake at baseline levels were not changed in the adult offspring. However, after 24 h water deprivation, consumption of water and salt was significantly increased compared to that of the control. Plasma sodium and osmolality levels remained the same between the offspring in the control and the exposed groups, while hematocrit was higher in the offspring exposed to prenatal high sucrose immediately following water deprivation. Density of renal AT₁ receptor protein was the same between the control and the exposed group, while AT₂ receptor protein in the kidney was significantly increased in the offspring exposed to prenatal high sucrose in association of thicker basal membrane of glomerular. In the forebrain, both AT₁ and AT₂ receptor levels were significantly increased in the offspring with history of prenatal high sucrose. In addition, water deprivation induced more c-fos expression in the central dipsogenic areas, including the paraventricular and supraoptic nuclei in the offspring exposed to prenatal high sucrose. The results suggested that prenatal high intake of sucrose may affect development of pathways in regulation of dipsogenic behavior in face of dehydration, which was associated with altered expression of AT₁ or/and AT₂ receptors in the kidney and brain.