Inhibition of microRNA-103 attenuates inflammation and endoplasmic reticulum stress in atherosclerosis through disrupting the PTEN-mediated MAPK signaling

Atherosclerosis (AS), a chronic disorder of large arteries, is the underlying pathological process of heart disease and stroke. Former researchers have found that microRNAs (miRs) are involved in the several key processes of AS. Apolipoprotein E knockout (ApoE^−/−) mice fed a high-fat-diet (HFD) to establish AS model. The expression of miR-103 was characterized in the mice model. The effects of miR-103 on inflammation and endoplasmic reticulum stress (ERS) were analyzed when the expression of miR-103 was inhibited in ApoE ^−/− mice fed an HFD and human aortic endothelial cells (HAECs) exposed to oxidized low-density lipoprotein (ox-LDL). The relationship between miR-103 and phosphatase and tensin homolog (PTEN) was identified by luciferase activity detection and real-time quantitative polymerase chain reaction (RT-qPCR). Gain- and loss-function approaches were further applied for investigating the regulatory effects of miR-103 and PTEN on ERS. Role of MAPK signaling was then analyzed using PD98059 to block this pathway. miR-103 was highly expressed in the ApoEApoE ^−/− mice fed an HFD. Downregulation of miR-103 suppressed inflammation and ERS in endothelial cells isolated from ApoE ^−/− mice fed a HFD and ox-LDL-exposed HAECs. In addition, miR-103 can target PTEN and downregulate its expression. Overexpression of PTEN reversed the miR-103-induced activation of MAPK signaling. Moreover, PTEN upregulation or MAPK signaling inhibition ease miR-103-induced inflammation and ERS in vivo and in vitro. Thus, miR-103 depletion restrains the progression of AS through blocking PTEN-mediated MAPK signaling.     

Stimulation of σ1-receptor restores abnormal mitochondrial Ca mobilization and ATP production following cardiac hypertrophy

Background

We previously reported that the σ₁-receptor (σ₁R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ₁R stimulation with the selective σ₁R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ₁R localized in the sarcoplasmic reticulum (SR).

Methods

We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ₁R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ₁R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).

Results

σ₁R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca^2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca^2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.

Conclusions

σ₁R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca^2 + mobilization and ATP production via σ₁R stimulation.

General significance

Our observations suggest that σ₁R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or,a novel flavoheme reductase

NADH cytochrome b₅ oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Bax Inhibitor-1-mediated Ca leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca²⁺ levels. Recently, we elucidated BI-1's Ca²⁺-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca²⁺-channel pore-dead mutant BI-1 (BI-1^D213R) was developed. We determined whether BI-1 behaves as a bona fide H⁺/Ca²⁺ antiporter or as an ER Ca²⁺-leak channel by investigating the effect of pH on unidirectional Ca²⁺-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1^−/− cells increased the ER Ca²⁺-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1^D231R expression in BI-1^−/−, despite its ER localization, did not increase the ER Ca²⁺-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca²⁺ leak was blocked. Finally, a peptide representing the Ca²⁺-channel pore of BI-1 promoting Ca²⁺ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca²⁺ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca²⁺-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.