Redox biology of the intestine

Abstract

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Role of high-mobility group box-1 protein in disruption of vascular barriers and regulation of leukocyte–endothelial interactions

Abstract

High-mobility group box-1 protein (HMGB1) is a highly conserved non-histone DNA-binding protein present in the nuclei and cytoplasm of nearly all cell types. The results from recent research provide evidence that HMGB1 is secreted into the extracellular milieu and acts as a pro-inflammatory cytokine and exhibits angiogenic effects to fire the immunological response against the pathological effects. Recently, a great deal of evidence has indicated the critical importance of HMGB1 in mediating vascular barriers dysfunction by modulating the expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular cell adhesion protein 1 and E-selectin on the surface of endothelial cells. Such process promotes the adhesion and migration of leukocytes across the endothelium, leading to breakdown of vascular barriers (blood–brain barrier and blood–retinal barrier) via modulating the expression, content, phosphorylation, and distribution of tight junction proteins. Therefore, here we give an abridged review to understand the mechanistic link between HMGB1 and vascular barriers dysfunction, including interaction with cell-surface receptors and intracellular signaling pathways.     

The phosphatidylinositol‐transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling

Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)‐stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)‐transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C‐terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF‐stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA‐binding capability and PI‐transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.     

Wnk kinases are positive regulators of canonical Wnt/β-catenin signalling

Wnt/β-catenin signalling is central to development and its regulation is essential in preventing cancer. Using phosphorylation of Dishevelled as readout of pathway activation, we identified Drosophila Wnk kinase as a new regulator of canonical Wnt/β-catenin signalling. WNK kinases are known for regulating ion co-transporters associated with hypertension disorders. We demonstrate that wnk loss-of-function phenotypes resemble canonical Wnt pathway mutants, while Wnk overexpression causes gain-of-function canonical Wnt-signalling phenotypes. Importantly, knockdown of human WNK1 and WNK2 also results in decreased Wnt signalling in mammalian cell culture, suggesting that Wnk kinases have a conserved function in ensuring peak levels of canonical Wnt signalling.     

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway

The DNA‐binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/β‐catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, β‐catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced β‐catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/β‐catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.     

Wnt and planar cell polarity signaling in cystic renal disease

Cystic kidney diseases can cause end stage renal disease, affecting millions of individuals worldwide. They may arise early or later in life, are characterized by a spectrum of symptoms and can be caused by diverse genetic defects. The primary cilium, a microtubule-based organelle that can serve as a signaling antenna, has been demonstrated to have a significant role in ensuring correct kidney development and function. In the kidney, one of the signaling pathways that requires the cilium for normal development is Wnt signaling. In this review, the roles of primary cilia in relation to canonical and non-canonical Wnt/PCP signaling in cystic renal disease are described. The evidence of the associations between cilia, Wnt signaling and cystic renal disease is discussed and the significance of planar cell polarity-related mechanisms in cystic kidney disease is presented. Although defective Wnt signaling is not the only cause of renal disease, research is increasingly highlighting its importance, encouraging the development of Wnt-associated diagnostic and prognostic tools for cystic renal disease.     

Fabrication,Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.     

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2′-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2′-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2′-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.