Adipose tissue responds to stress-induced immunosuppression affecting immune response partially by miR-145-5p/S1PR1 pathway.

Stress-induced immunosuppression (SIIS) is one of the most common problems in intensive poultry production, which can cause immunized chickens to still develop diseases and bring huge losses to production. Recently, adipose tissue, as an immunomodulatory organ, has become a hot topic of attention. However, the function and mechanism of adipose tissue involved in SIIS and its influence on the immune response are still unclear. In this study, we dynamically analyzed the correlations between the T cells migration and change of sphingosine-1-phosphate receptor 1(S1PR1) gene in adipose tissue using chicken models with different immune states, and further explored the regulatory mechanisms and application. The results showed that SIIS could significantly change the expressions of lymphocytes migration related S1PR1 gene, and SIIS could inhibit the Newcastle disease virus (NDV) immune response partially by affecting the migration and proliferation of TCRα+ T cells in adipose tissue. Moreover, the miR-145-5p/S1PR1 pathway was a potential key mechanism to regulate T cells migration in adipose tissue, and circulating miR-145-5p had potential value as a molecular marker. This research can provide innovative reference for in-depth studying the immunoregulatory function and mechanism of adipose tissue.

Fluorescence detection of adenosine triphosphate based on dimeric G-quadruplex.

Adenosine triphosphate (ATP) is a major chemical energy carrier in organisms and is involved in numerous biological processes. ATP levels are associated with many diseases, cell viability, and food freshness. Thus, it has become an important biomarker. Many strategies have been used to detect ATP. However, the problems of difficult-to-prepare materials, too much dependence on instruments, and complicated processes restrict the application of these methods. In this study, we proposed a novel ATP detection sensor. The method is based on the fluorescence enhancement effect of dimeric G-quadruplex (Di-G4) on thioflavin T (ThT). First, the cleavage of Di-G4 by S1 nuclease decreases system fluorescence. However, it can be recovered by increases in ATP concentrations, which act as an inhibitor of S1 nuclease. Under the optimized conditions, a good linear relationship was observed between fluorescence intensity and ATP concentrations within the range of 0.5-120 µM. The detection limit was 245 nM. The method was utilized to measure the ATP content in apples and compared with ATP assay kits, resulting in satisfactory results.

C-Reactive Protein-to-Albumin Ratio as a Predictive Indicator for Evaluating Tolerability in S-1 Adjuvant Chemotherapy after Curative Surgery for Pancreatic Cancer: An External Validation Cohort Study.

BACKGROUND: S-1 in adjuvant chemotherapy (AC) administration after pancreatic cancer (PC) surgery has been standardized in Japan. The Ehime study confirmed that a postoperative higher C-reactive protein-to-albumin ratio (CAR) value predicted the risk of adverse event (AE)-related S-1 non-completion as an AC in patients with PC after curative surgery. This study aimed to investigate the index to predict S-1 tolerance among patients who underwent curative surgery for PC (the Dokkyo study). METHODS: This retrospective validation cohort study included 172 patients at the Department of Hepato-Biliary Pancreatic Surgery, Dokkyo Medical University, Japan, from January 2010 to December 2022. All patients underwent nutritional screening using the postoperative CAR. S-1 completion status and its effect on prognosis were systematically followed up and investigated. We conducted a statistical analysis of predictive markers to investigate their association with S-1 completion. RESULTS: Patients were categorized into the S-1 completion (N = 91) and non-completion (N = 81) groups. The S-1 completion group demonstrated a significantly lower CAR than the S1 non-completion group. Moreover, the current study revealed a significant difference in the S-1 completion rate, applying the CAR cutoff value of 0.05 established in the Ehime study. Additionally, univariate and multivariate analyses confirmed that a CAR of <0.05 was significantly associated with S-1 completion. CONCLUSIONS: The Dokkyo study confirmed the results observed in the Ehime study. Consequently, an increased postoperative CAR value appeared as a universal applicable marker for the risk factor of AE-related S-1 non-completion after curative surgery for patients with PC.

S1PR1 mediates Th17 cell migration from the thymus to the skin in health and disease.

Th17 cells play crucial roles in host defense and the pathogenesis of autoimmune diseases in the skin. While their differentiation mechanisms have been extensively studied, the origin of skin Th17 cells remains unclear. In this study, we analyzed single-cell RNA-sequencing data and identify the presence of Th17 cells in the human thymus. Thymic Th17 cells were characterized by high expression levels of Sphingosine-1-Phosphate Receptor 1 (S1PR1), a receptor crucial for T cell egress from lymphoid tissues. In mice, Th17 cell-specific knockout of S1pr1 resulted in the accumulation of Th17 cells in the thymus and a corresponding decrease in their numbers in the skin. Th17 cells that accumulated in the thymus exhibited a lower IL-17A production capacity compared to those in the skin, indicating that the local environment in the skin is important for maintaining the Th17 cell phenotype. Additionally, using a murine psoriasis model, we demonstrated that Th17 cell-specific knockout of S1pr1 reduced their migration to the inflamed skin, thereby ameliorating disease progression. Collectively, our data suggest that S1PR1 mediates Th17 cell migration from the thymus to the skin, thereby modulating their functional engagement in both homeostatic and inflammatory conditions.

Phase II Trial of Adjuvant S-1 Following Neoadjuvant Chemotherapy and Surgery in Patients with Locally Advanced Esophageal Squamous Cell Carcinoma: The PIECE Trial.

BACKGROUND: Neoadjuvant chemotherapy followed by surgery (NAC-S) is the standard therapy for locally advanced esophageal squamous cell carcinoma (ESCC) in Japan. OBJECTIVE: The aim of this phase II trial was to assess the efficacy and safety of the addition of adjuvant S-1 after R0 resection in ESCC patients who received NAC-S. PATIENTS AND METHODS: Key eligibility criteria included clinical stage IB-III (without T4 disease) ESCC, age 20-75 years, and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients received adjuvant therapy with four cycles of S-1 (80 mg/m2/day) administered orally for 4 weeks of 6-week cycles. The primary endpoint was 3 year relapse-free survival (RFS). If the lower confidence limit for 3 year RFS was >50%, we judged that the primary endpoint of this study was met. RESULTS: A total of 52 patients were enrolled between January 2016 and January 2019. Two patients were excluded from analysis; five patients were determined to have R1 or R2 resection, and seven patients did not receive adjuvant S-1. The 3-year RFS and overall survival rates in the intention-to-treat population were 72.3% (90% confidence interval [CI] 59.9-81.5) and 85.0% (90% CI 73.9-91.6), indicating that the primary endpoint was met. Grade ≥3 adverse events with an incidence ≥10% included neutropenia (13.2%), anorexia (13.2%), and diarrhea (10.5%). There were no treatment-related deaths. CONCLUSION: Adjuvant S-1 after NAC-S showed promising efficacy with a manageable safety profile for patients with resectable ESCC and warrants further evaluation in larger studies.

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Safety evaluation of the food enzyme endonuclease from the non-genetically modified Penicillium citrinum strain NP 11-15.

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non-genetically modified Penicillium citrinum strain NP 11-15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knockout, and LacZ knockin mice.

FAM19A5 is a novel secretory protein expressed primarily in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function through sphingosine-1-phosphate receptor 2 (S1PR2). In our study, we investigated FAM19A5 transcript and protein levels in peripheral tissues, including adipose tissues, from wild-type, FAM19A5 knockout, and FAM19A5 LacZ knockin mice. We found that the FAM19A5 transcript levels in the central nervous system were much greater than those in any of the peripheral tissues, including adipose tissues. Furthermore, the FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis and ELISA. Additionally, we found that the FAM19A5 protein did not interact with S1PR2 in terms of G protein-mediated signal transduction, ß-arrestin recruitment, or ligand-mediated internalization. Taken together, our findings revealed basal levels of FAM19A5 transcripts and proteins in peripheral tissues, confirming its primary expression in the central nervous system and lack of significant interaction with S1PR2.

Sphingosine 1-phosphate receptor subtype 1 (S1P1) activity in the course of Alzheimer's disease.

Some specific lipid molecules in the brain act as signaling molecules, neurotransmitters, or neuromodulators, by binding to specific G protein-coupled receptors (GPCR) for neurolipids. One such receptor, sphingosine 1-phosphate receptor subtype 1 (S1P1), is coupled to Gi/o proteins and is involved in cell proliferation, growth, and neuroprotection. S1P1 constitutes an interesting target for neurodegenerative diseases like multiple sclerosis and Alzheimer's disease (AD), in which changes in the sphingolipid metabolism have been observed. This study analyzes S1P1 receptor-mediated activity in healthy brains and during AD progression using postmortem samples from controls and patients at different Braak's stages. Additionally, the distribution of S1P1 receptor activity in human brains is compared to that in commonly used rodent models, rats and mice, through functional autoradiography, measuring [35S]GTPγS binding stimulated by the S1P1 receptor selective agonist CYM-5442 to obtain the distribution of functional activity of S1P1 receptors. S1P1 receptor-mediated activity, along with that of the cannabinoid CB1 receptor, is one of the highest recorded for any GPCR in many gray matter areas of the brain, reaching maximum values in the cerebellar cortex, specific areas of the hippocampus and the basal forebrain. S1P1 signaling is crucial in areas that regulate learning, memory, motor control, and nociception, such as the basal forebrain and basal ganglia. In AD, S1P1 receptor activity is increased in the inner layers of the frontal cortex and underlying cortical white matter at early stages, but decreases in the hippocampus in advanced stages, indicating ongoing brain impairment. Importantly, we identified significant correlations between S1P1 receptor activity and Braak stages, suggesting that S1P1 receptor dysfunction is associated to disease progression, particularly in memory-related regions. The S1P signaling via S1P1 receptor is a promising neurological target due to its role in key neurophysiological functions and its potential to modify the progression of neurodegenerative diseases. Finally, rats are suggested as a preferred experimental model for studying S1P1 receptor-mediated responses in the human brain.

Exploring the antioxidant, antidiabetic, and antibacterial potential of postbiotic compounds derived from Lactiplantibacillus plantarum O7S1.

Probiotic bacteria are distinguished by their ability to produce various functional postbiotic metabolites. Therefore, this study aimed to explore the in vitro antioxidant, antidiabetic, and antibacterial properties of two postbiotics generated by Lactiplantibacillus plantarum O7S1 (Lpb. plantarum O7S1) during the fermentation process: cell-free supernatant (CFS) and exopolysaccharides (EPS). The antioxidant potential of these postbiotics was assessed using various radical scavenging assays and ferric-reducing antioxidant potential. The antidiabetic activity was evaluated through α-amylase inhibitory assays, while antibacterial activity was determined using agar well diffusion assays. The results of the present study revealed that CFS exhibited significant antioxidant and antidiabetic efficacy in contrast to EPS (P < 0.001). Specifically, CFS displayed remarkable scavenging ability against DPPH, hydroxyl, and superoxide radicals, with inhibition rates of 88.78, 78.91, and 34.85%, respectively, while EPS showed comparatively lower inhibition rates. Additionally, CFS demonstrated higher reducing activity (0.30 optical density units at 700 nm) and potent α-amylase inhibitory activity (95.87%) compared to EPS (67.17%) (P < 0.001). The agar well diffusion assay reported that CFS showed significant antimicrobial activity against both Gram-positive and Gram-negative pathogens, while no activity was observed with EPS. Furthermore, carbohydrate fermentation analysis indicated the strain's ability to metabolize various carbohydrates and their derivatives, potentially enhancing digestive health. These findings suggest that both CFS and EPS exhibit promising hypoglycemic, antioxidant, and antibacterial properties, making them potential candidates for incorporation into functional foods and pharmaceuticals aimed at preventing oxidative damage, diabetes, and pathogenic bacterial infections.

SARS-CoV-2 spike S1 protein induces microglial NLRP3-dependent neuroinflammation and cognitive impairment in mice.

Cognitive impairment is often found at the acute stages and sequelae of coronavirus disease 2019 (COVID-19), and the underlying mechanisms remain unclear. The S1 protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be a cause of cognitive impairment associated with COVID-19. The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome and neuroinflammation play important roles in Alzheimer's disease (AD) with cognitive impairment. However, their roles remain unknown in COVID-19 with cognitive impairment. We stimulated BV2 cells with S1 protein in vitro and injected the hippocampi of wild-type (WT) mice, NLRP3 knockout (KO), and microglia NLRP3 KO mice in vivo with S1 protein to induce cognitive impairment. We assessed exploratory behavior as associative memory using novel object recognition and Morris water maze tests. Neuroinflammation was analyzed using immunofluorescence and western blotting to detect inflammatory markers. Co-localized NLRP3 and S1 proteins were investigated using confocal microscopy. We found that S1 protein injection led to cognitive impairment, neuronal loss, and neuroinflammation by activating NLRP3 inflammation, and this was reduced by global NLRP3 KO and microglia NLRP3 KO. Furthermore, TAK 242, a specific inhibitor of Toll-like receptor-4, resulted in a significant reduction in NLRP3 and pro-IL-1ß in BV2 cells with S1 protein stimulation. These results reveal a distinct mechanism through which the SARS-CoV-2 spike S1 protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Early-Life Milk αS1-Casein Allergy Induces the Activation of Astrocytes in Mice and Leads to Stress Vulnerability in Adulthood.

In recent years, the incidence of food allergies in children has been increasing annually, significantly affecting the quality of life for patients and their families. It has long been suspected that childhood allergies might potentially lead to behavioral and psychological issues in adulthood, but the specific connection remains unclear. In this study, we established a model of young mice allergic to milk αS1-casein, conducted behavioral tests, and employed transcriptomics, immunohistochemistry, Golgi staining, and fecal microbiota transplantation to explore the link between early life allergies and adult psychological problems. The results showed that early life milk protein allergy significantly increased intestinal epithelial permeability in mice, leading to the translocation of gut microbiota metabolites. This process subsequently activated astrocyte lysosomes via SLC15a3, making astrocytes more susceptible. This susceptibility caused mice with early life milk protein allergy to have more activated astrocytes and excessive dendritic spine phagocytosis (normal group: 5.4 ± 1.26 spines/10 µm, allergy group: 3.2 ± 0.92 spines/10 µm) under acute stress in adulthood, leading to anxiety and depressive behaviors.

Optimized aptamer-based next generation biosensor for the ultra-sensitive determination of SARS-CoV-2 S1 protein in saliva samples.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, which rapidly spread worldwide and resulted in a pandemic. Efficient and sensitive detection techniques have been devised since the onset of the epidemic and continue to be improved at present. Due to the crucial role of the SARS-CoV-2 S1 protein in facilitating the virus's entry into cells, efforts in detection and treatment have primarily centered upon this protein. In this study, a rapid, ultrasensitive, disposable, easy-to-use, cost-effective next generation biosensor based on optimized aptamer (Optimer, OPT) was developed by using a disposable pencil graphite electrode (PGE) and applied for the impedimetric determination of SARS-CoV-2 S1 protein. The S1 protein interacted with the OPT in the solution phase and then immobilized onto the PGE surface. Subsequently, measurements using electrochemical impedance spectroscopy (EIS) were conducted in a solution containing a redox probe of 1 mM [Fe(CN)6]3-/4-. Under optimum conditions, the limit of detection (LOD) for the S1 protein in buffer medium at concentrations ranging from 101 to 106 ag/mL was calculated as 8.80 ag/mL (0.11 aM). The selectivity of the developed biosensor was studied against MERS-CoV-S1 protein (MERS) and Influenza Hemagglutinin antigen (HA). Furthermore, the application of the biosensor in artificial saliva medium is demonstrated. The LOD was also calculated in artificial saliva medium in the concentration range of 101-105 ag/mL and calculated as 2.01 ag/mL (0.025 aM). This medium was also used to assess the selectivity of optimized-aptamer based biosensor.

One-Selector-One-Resistor Integrated Memory Cells Based on Two-Dimensional Heterojunction Memory Selectors.

Selectors are critical components for reducing the sneak path leakage currents in emerging resistive random-access memory (RRAM) arrays. Two-dimensional (2D) materials provide a rich choice of materials with van der Waals stacking to form the heterostructure selectors with controllable energy barriers. Here, we experimentally demonstrate 2D-material-based heterostructure selectors with exponential current-voltage (I-V) relationships and integrate them with hafnium oxide (HfOx)-based RRAMs, forming one-selector-one-resistor (1S1R) cells. The multilayer graphene (MG)/tungsten disulfide (WS2)/platinum (Pt) selector contains two asymmetric heterojunctions with different Schottky barriers, which lead to highly nonlinear and asymmetric I-V characteristics. The 2D selectors in 1S1R cells can successfully drive RRAMs, reduce sneak path leakage current by more than 100 times, and provide the set compliance current. The 1S1R cells are further modeled and integrated into both planar and 3D memory arrays, with circuit-level simulations demonstrating that the presence of 2D selectors in large memory arrays can reduce the power consumption by up to 86%, improve the read/write margin by up to 31%, and avoid write failure. Such a platform holds high potential for constructing 3D high-density memories and performing in-memory computing.

Lysophospholipid receptors in neurodegeneration and neuroprotection.

The central nervous system (CNS) is one of the most complex physiological systems, and treatment of CNS disorders represents an area of major medical need. One critical aspect of the CNS is its lack of regeneration, such that damage is often permanent. The damage often leads to neurodegeneration, and so strategies for neuroprotection could lead to major medical advances. The G protein-coupled receptor (GPCR) family is one of the major receptor classes, and they have been successfully targeted clinically. One class of GPCRs is those activated by bioactive lysophospholipids as ligands, especially sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA). Research has been increasingly demonstrating the important roles that S1P and LPA, and their receptors, play in physiology and disease. In this review, I describe the role of S1P and LPA receptors in neurodegeneration and potential roles in neuroprotection. Much of our understanding of the role of S1P receptors has been through pharmacological tools. One such tool, fingolimod (also known as FTY720), which is a S1P receptor agonist but a functional antagonist in the immune system, is clinically efficacious in multiple sclerosis by producing a lymphopenia to reduce autoimmune attacks; however, there is evidence that fingolimod is also neuroprotective. Furthermore, fingolimod is neuroprotective in many other neuropathologies, including stroke, Parkinson's disease, Huntington's disease, Rett syndrome, Alzheimer's disease, and others that are discussed here. LPA receptors also appear to be involved, being upregulated in a variety of neuropathologies. Antagonists or mutations of LPA receptors, especially LPA1, are neuroprotective in a variety of conditions, including cortical development, traumatic brain injury, spinal cord injury, stroke and others discussed here. Finally, LPA receptors may interact with other receptors, including a functional interaction with plasticity related genes.

Deciphering the role of sphingosine 1-phosphate in central nervous system myelination and repair.

Sphingosine 1-phosphate (S1P) is a bioactive lipid of the sphingolipid family and plays a pivotal role in the mammalian nervous system. Indeed, S1P is a therapeutic target for treating demyelinating diseases such as multiple sclerosis. Being part of an interconnected sphingolipid metabolic network, the amount of S1P available for signalling is equilibrated between its synthetic (sphingosine kinases 1 and 2) and degradative (sphingosine 1-phosphate lyase) enzymes. Once produced, S1P exerts its biological roles via signalling to a family of five G protein-coupled S1P receptors 1-5 (S1PR1-5). Despite significant progress, the precise roles that S1P metabolism and downstream signalling play in regulating myelin formation and repair remain largely opaque and somewhat controversial. Genetic or pharmacological studies adopting various model systems identify that stimulating S1P-S1PR signalling protects myelin-forming oligodendrocytes after central nervous system (CNS) injury and attenuates demyelination in vivo. However, evidence to support its role in remyelination of the mammalian CNS is limited, although blocking S1P synthesis sheds light on the role of endogenous S1P in promoting CNS remyelination. This review focuses on summarising the current understanding of S1P in CNS myelin formation and repair, discussing the complexity of S1P-S1PR interaction and the underlying mechanism by which S1P biosynthesis and signalling regulates oligodendrocyte myelination in the healthy and injured mammalian CNS, raising new questions for future investigation.

L5-S1 facet joint pathology in pelvic ring injuries.

BACKGROUND: The authors believe that the L5-S1 facet joint injury in the setting of pelvic fractures is underappreciated by orthopedic traumatologists. The purpose of this study was to draw attention to the L5/S1 facet joint in the setting of pelvic ring injuries. METHODS: This was a retrospective comparative study of all patients greater than or equal to 18 years of age with an acute pelvic ring injury (AO/OTA 62 B to C) presenting to a single level I trauma center. The primary objective was to determine demographic and injury characteristics associated with L5-S1 facet joint injuries in patients with pelvic ring injuries. The secondary objective was to determine the proportion of L5-S1 facet joint injuries that were missed on initial radiographic workup. RESULTS: There were 476 patients included in the analysis, 53 (11.1%) of whom had an L5-S1 facet joint injury. Patients with an L5-S1 injury were more likely to be younger (44.1 vs. 53.2 years, p = 0.001) and experience a high energy mechanism of injury (95.0% vs. 78.0%, p = 0.002). Certain injury patterns were associated with L5-S1 facet joint injuries: any sacral fracture (96.2% vs. 73.8%, p < 0.001), Denis zone 2 fractures (43.4% vs. 20.1%, p < 0.001), Denis zone 3 fractures (34.0% vs. 4.7%, p < 0.001), bilateral displaced sacral fractures (18.9% vs. 3.5%, p < 0.001), and L5 transverse process fractures (64.2% vs. 18.0%, p < 0.001). Only 16.0% of radiology reports identified an L5-S1 injury. CONCLUSIONS: Orthopedic traumatologists should scrutinize imaging for L5-S1 facet joint injuries in the presence of pelvic ring injuries, especially in patients with certain sacral fracture patterns.

miR-495 promotes intestinal epithelial cell apoptosis through downregulation of Sphingosine-1-phosphate.

Many pathological conditions lead to defects in intestinal epithelial integrity and loss of barrier function; Sphingosine-1-phosphate (S1P) has been shown to augment intestinal barrier integrity, though the exact mechanisms are not completely understood. We have previously shown that overexpression of Sphingosine Kinase 1 (SphK1), the rate limiting enzyme for S1P synthesis, significantly increased S1P production and cell proliferation. Here we show that microRNA 495 (miR-495) upregulation led to decreased levels of SphK1 resultant from a direct effect at the SphK1 mRNA. Increasing expression of miR-495 in intestinal epithelial cells resulted in decreased proliferation and increased susceptibility to apoptosis. Transgenic expression of miR-495 inhibited mucosal growth, as well as decreased proliferation in the crypts. The intestinal villi also expressed decreased levels of barrier proteins and exaggerated damage upon exposure to cecal ligation-puncture. These results implicate miR-495 as a critical negative regulator of intestinal epithelial protection and proliferation through direct regulation of SphK1, the rate limiting enzyme critical for production of S1P.

Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells.

BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.