Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204.

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

Evidence for a protective role of Protein Disulfide Isomerase-A1 against aortic dissection.

BACKGROUND AND AIMS: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection. METHODS: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days. RESULTS: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation. CONCLUSIONS: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling.

Hippocampal Endoplasmic Reticulum Stress Hastens Motor and Cognitive Decline in Adult Male Rats Sustainedly Exposed to High-Sucrose Diet.

Metabolic dysfunctions, such as hyperglycemia and insulin resistance, have been associated to cognitive impairment and dementia regardless of advanced age, although the underlying mechanisms are still elusive. Thus, this study investigates the deleterious effects of metabolic syndrome (MetS) induced by long-term exposure to a high-sucrose diet on motor and cognitive functions of male adult rats and its relationship with hippocampal endoplasmic reticulum (ER) stress. Weaned Wistar male rats were fed a high-sucrose diet until adulthood (HSD; 6 months old) and compared to both age-matched (CTR; 6 months old) and middle-aged chow-fed rats (OLD; 20 months old). MetS development, serum redox profile, behavioral, motor, and cognitive functions, and hippocampal gene/protein expressions for ER stress pro-adaptive and pro-apoptotic pathways, as well as senescence markers were assessed. Prolonged exposure to HSD induced MetS hallmarked by body weight gain associated to central obesity, hypertriglyceridemia, insulin resistance, and oxidative stress. Furthermore, HSD rats showed motor and cognitive decline similar to that in OLD animals. Noteworthy, HSD rats presented marked hippocampal ER stress characterized by failure of pro-adaptive signaling and increased expression of Chop, p21, and Parp-1 cleavage, markers of cell death and aging. This panorama resembles that found in OLD rats. In toto, our data showed that early and sustained exposure to a high-sucrose diet induced MetS, which subsequently led to hippocampus homeostasis disruption and premature impairment of motor and cognitive functions in adult rats.

Analysis of splice variants of the human protein disulfide isomerase (P4HB) gene.

BACKGROUND: Protein Disulfide Isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily with crucial roles in endoplasmic reticulum proteostasis, implicated in many diseases. The family prototype PDIA1 is also involved in vascular redox cell signaling. PDIA1 is coded by the P4HB gene. While forced changes in P4HB gene expression promote physiological effects, little is known about endogenous P4HB gene regulation and, in particular, gene modulation by alternative splicing. This study addressed the P4HB splice variant landscape. RESULTS: Ten protein coding sequences (Ensembl) of the P4HB gene originating from alternative splicing were characterized. Structural features suggest that except for P4HB-021, other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. These indicated widespread expression but significant variability in the degree of isoform expression among distinct tissues and even among distinct locations of the same cell, e.g., vascular smooth muscle cells from different origins. P4HB-02, P4HB-027 and P4HB-021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. Expression of such variants was validated by qRT-PCR in some cell types. The most consistently expressed splice variant was P4HB-021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation. CONCLUSIONS: Our study details the splice variant landscape of the P4HB gene, indicating their potential role to diversify the functional reach of this crucial gene. P4HB-021 splice variant deserves further investigation in vascular smooth muscle cells.

Evidence for sub-functionalization of tandemly duplicated XPB nucleotide excision repair genes in Arabidopsis thaliana.

Plants are continuously exposed to agents that can generate DNA lesions. Nucleotide Excision Repair (NER) is one of the repair pathways employed by plants to protect their genome, including from sunlight. The Xeroderma Pigmentosum type B (XPB) protein is a DNA helicase shown to be involved in NER and is also an essential subunitof the Transcription Factor IIH (TFIIH) complex. XPB was found to be a single copy gene in eukaryotes, but found as a tandem duplication in the plant Arabidopsis thaliana, AtXPB1 and AtXPB2. We aimed to investigate whether the XPB in tandem duplication was common within members of the Brassicaceae. We analyzed genomic DNA of species from different tribes of the family and the results indicate that the tandem duplication occurred in Camelineae tribe ancestor, of which A. thaliana belongs, at approximately 8 million years ago. Further experiments were devised to study possible functional roles for the A. thaliana AtXPB paralogs. A non-coincident expression profile of the paralogs was observed in various plant organs, developmental and cell cycle stages. AtXPB2 expression was observed in proliferating cells and clustered with the transcription of other components of the TFIIH such as p44, p52 and XPD/UVH6 along the cell cycle. AtXPB1 gene transcription, on the other hand, was enhanced specifically after UV-B irradiation in leaf trichomes. Altogether, our results reported herein suggest a functional specialization for the AtXPB paralogs: while the AtXPB2 paralog may have a role in cell proliferation and repair as XPB of other eukaryotes, the AtXPB1 paralog is most likely implicated in repair functions in highly specialized A. thaliana cells.

Long-term exposure to high-sucrose diet down-regulates hepatic endoplasmic reticulum-stress adaptive pathways and potentiates de novo lipogenesis in weaned male mice.

Childhood consumption of added sugars, such as sucrose, has been associated to increased risk of metabolic syndrome (MetS) and nonalcoholic fatty liver disease (NAFLD). Although the mechanisms underlying NAFLD onset are incompletely defined, recent evidence has proposed a role for the endoplasmic reticulum (ER) stress. Thus, the present study sought to investigate the metabolic outcomes of high-sucrose intake on weaned Swiss mice fed a 25% sucrose diet for 30, 60 and 90 days in comparison to regular chow-fed controls. High-sucrose feeding promoted progressive metabolic and oxidative disturbances, starting from fasting and fed hyperglycemia, hyperinsulinemia, glucose intolerance and increased adiposity at 30-days; passing by insulin resistance, hypertriglyceridemia and NAFLD onset at 60 days; until late hepatic oxidative damage at 90 days. In parallel, assessment of transcriptional and/or translational levels of de novo lipogenesis (DNL) and ER stress markers showed up-regulation of both fatty acid synthesis (ChREBP and SCD1) and oxidation (PPARα and CPT-1α), as well as overexpression of unfolded protein response sensors (IRE1α, PERK and ATF6), chaperones (GRP78 and PDIA1) and antioxidant defense (NRF2) genes at 30 days. At 60 days, fatty acid oxidation genes were down-regulated, and ER stress switched over toward a proapoptotic pattern via up-regulation of BAK protein and CHOP gene levels. Finally, down-regulation of both NRF2 and CPT-1α protein levels led to late up-regulation of SREBP-1c and exponential raise of fatty acids synthesis. In conclusion, our study originally demonstrates a temporal relationship between DNL and ER stress pathways toward MetS and NAFLD development on weaned rats fed a high-sucrose diet.

Conserved Gene Microsynteny Unveils Functional Interaction Between Protein Disulfide Isomerase and Rho Guanine-Dissociation Inhibitor Families.

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.

Conserved gene microsynteny unveils functional interaction between protein disulfide isomerase and rho guanine-dissociation inhibitor families

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIa were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.

Early and sustained exposure to high-sucrose diet triggers hippocampal ER stress in young rats.

Early-life environmental insults have been shown to promote long-term development of chronic non-communicable diseases, including metabolic disturbances and mental illnesses. As such, premature consumption of high-sugar foods has been associated to early onset of detrimental outcomes, whereas underlying mechanisms are still poorly understood. In the present study, we sought to investigate whether early and sustained exposure to high-sucrose diet promotes metabolic disturbances that ultimately might anticipate neurological injuries. At postnatal day 21, weaned male rats started to be fed a standard chow (10 % sucrose, CTR) or a high-sucrose diet (25 % sucrose, HSD) for 9 weeks prior to euthanasia at postnatal day 90. HSD did not alter weight gain and feed efficiency between groups, but increased visceral, non-visceral and brown adipose tissue accumulation. HSD rats demonstrated elevated blood glucose levels in both fasting and fed states, which were associated to impaired glucose tolerance. Peripheral insulin sensitivity did not change, whereas hepatic insulin resistance was supported by increased serum triglyceride levels, as well as higher TyG index values. Assessment of hippocampal gene expression showed endoplasmic reticulum (ER) stress pathways were activated in HSD rats, as compared to CTR. HSD rats had overexpression of unfolded protein response sensors, PERK and ATF6; ER chaperone, PDIA2 and apoptosis-related genes, CHOP and Caspase 3; but decreased expression of chaperone GRP78. Finally, HSD rats demonstrated impaired neuromuscular function and anxious behavior, but preserved cognitive parameters. In conclusion, our data indicate that early exposure to HSD promote metabolic disturbances, which disrupt hippocampus homeostasis and might precociously affect its neurobehavioral functions.

Altered gene expression profiles of wheat genotypes against Fusarium head blight.

Fusarium graminearum is responsible for Fusarium head blight (FHB), which is a destructive disease of wheat that makes its quality unsuitable for end use. To understand the temporal molecular response against this pathogen, microarray gene expression analysis was carried out at two time points on three wheat genotypes, the spikes of which were infected by Fusarium graminearum. The greatest number of genes was upregulated in Nobeokabouzu-komugi followed by Sumai 3, whereas the minimum expression in Gamenya was at three days after inoculation (dai). In Nobeokabouzu-komugi, high expression of detoxification genes, such as multidrug-resistant protein, multidrug resistance-associated protein, UDP-glycosyltransferase and ABC transporters, in addition to systemic defense-related genes, were identified at the early stage of infection. This early response of the highly-resistant genotype implies a different resistance response from the other resistant genotype, Sumai 3, primarily containing local defense-related genes, such as cell wall defense genes. In Gamenya, the expression of all three functional groups was minimal. The differences in these molecular responses with respect to the time points confirmed the variation in the genotypes. For the first time, we report the nature of gene expression in the FHB-highly resistant cv. Nobeokabouzu-komugi during the disease establishment stage and the possible underlying molecular response.

Functional characterization of sugarcane mustang domesticated transposases and comparative diversity in sugarcane, rice, maize and sorghum.

Transposable elements (TEs) account for a large portion of plant genomes, particularly in grasses, in which they correspond to 50%-80% of the genomic content. TEs have recently been shown to be a source of new genes and new regulatory networks. The most striking contribution of TEs is referred as "molecular domestication", by which the element coding sequence loses its movement capacity and acquires cellular function. Recently, domesticated transposases known as mustang and derived from the Mutator element have been described in sugarcane. In order to improve our understanding of the function of these proteins, we identified mustang genes from Sorghum bicolor and Zea mays and performed a phenetic analysis to assess the diversity and evolutionary history of this gene family. This analysis identified orthologous groups and showed that mustang genes are highly conserved in grass genomes. We also explored the transcriptional activity of sugarcane mustang genes in heterologous and homologous systems. These genes were found to be ubiquitously transcribed, with shoot apical meristem having the highest expression levels, and were downregulated by phytohormones. Together, these findings suggest the possible involvement of mustang proteins in the maintenance of hormonal homeostasis.

Transcriptionally active transposable elements in recent hybrid sugarcane.

Transposable elements (TEs) are considered to be important components of the maintenance and diversification of genomes. The recent increase in genome sequence data has created an opportunity to evaluate the impact of these active mobile elements on the evolution of plant genomes. Analysis of the sugarcane transcriptome identified 267 clones with significant similarity to previously described plant TEs. After full cDNA sequencing, 68 sugarcane TE clones were assigned to 11 families according to their best sequence alignment against a fully characterized element. Expression was further investigated through a combined study utilizing electronic Northerns, macroarray, transient and stable sugarcane transformation. Newly synthesized cDNA probes from flower, leaf roll, apical meristem and callus tissues confirm previous results. Callus was identified as the tissue with the highest number of TEs being expressed, revealing that tissue culture drastically induced the expression of different elements. No tissue-specific family was identified. Different representatives within a TE family displayed differential expression patterns, showing that each family presented expression in almost every tissue. Transformation experiments demonstrated that most Hopscotch clone-derived U3 regions are, indeed, active promoters, although under a strong transcriptional regulation. This is a large-scale study about the expression pattern of TEs and indicates that mobile genetic elements are transcriptionally active in the highly polyploid and complex sugarcane genome.