MicroRNA-30c attenuates fibrosis progression and vascular dysfunction in systemic sclerosis model mice

Molecular Biology Reports - Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that...     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.     

Mechanism of p38 MAPK–induced EGFR endocytosis and its crosstalk with ligand-induced pathways

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.     

Identification of a Metabolizing Enzyme in Human Kidney by Proteomic Correlation Profiling

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein''s correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.Molecular identification for an enzyme reaction or biologically active substance in an organism is challenging, although molecular biological methodologies such as expression cloning (1), recombinant protein panel (2) and RNAi screening (3) have been introduced recently as alternative approaches. Conventional biochemical purification has provided a number of successes and thus still remains a powerful, though labor-intensive strategy.In the traditional protein purification, it had been necessary to purify an individual protein nearly to homogeneity at a microgram amount so that the purified protein could be analyzed by N-terminal amino acid sequencing. Protein identification by mass spectrometry subsequently revolutionized this technology by enabling identification of proteins at much lower abundances: individual proteins could then be associated with specific activities as soon as a band in SDS-PAGE could be observed, even when the purified protein was far from homogeneity (4–6). Although this streamlined the workflow by reducing the required starting materials as well as the separation steps for protein purification, a faster and more generalized approach from smaller starting material has still been desired because some proteins are physiochemically difficult for example in solubilization and stability. To solve these problems, we devised a proteomic correlation profiling methodology (7).The basic concept of proteomic correlation profiling was originally developed by Andersen et al. (8). They quantitatively profiled hundreds of proteins across several centrifugation fractions by mass spectrometry and identified centrosomal proteins by calculating the correlation of these protein expression profiles with already known centrosomal proteins. In the following study, Foster et al. applied this strategy to map more than 1400 proteins to ten subcellular locations (9). Although these studies used centrifugation as a separation method and a known marker profile as a standard for correlation, we extended this concept to use chromatography as a separation method and kinase activity as a basis for comparison; our approach successfully identified a kinase responsible for phosphorylation of peptide substrates just after one step chromatography, and was termed proteomic correlation profiling (7). Independently, Kuromitsu et al. reported identification of an active substance in the serum response element-dependent luciferase assay from interstitial cystitis urine after three-step chromatography by a similar concept (10). In theory, this general proteomic correlation profiling strategy can be adapted to any kind of separation method and activity profile but no other example has been reported thus far, therefore, actual examples where the method can be applied to other enzyme classes are required to prove its generality.Multiple sclerosis is the most common autoimmune disorder of the central nerve system in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring (11, 12). Until recently, the standard treatments for multiple sclerosis such as interferon beta, glatiramer acetate, mitoxantrone, and natalizumab would often cause severe adverse events (13, 14), providing an opportunity for development of less dangerous treatments for this disease. However, in 2010, Food and Drug Administration approved fingolimod (Gilenya; chemical structure in Fig. 1) as the first oral medicine, and recommended this as a first-line treatment for relapsing-remitting multiple sclerosis, opening up a new therapeutic approach to the disease (15).Open in a separate windowFig. 1.The chemical structures of CS-0777, fingolimod and their phosphorylated derivatives.Sphingosine 1-phosphate receptor 1 (S1P1)¹ modulators are emerging as a new class of drugs with potential therapeutic application in multiple sclerosis (15), and fingolimod is a nonselective sphingosine 1-phosphate (S1P) receptor modulator (16–18, 21, 22). Given its structural similarity to sphingosine, fingolimod is phosphorylated in vivo by sphingosine kinase, in particular sphingosine kinase 2 (SPHK2) (19, 20), and the fingolimod-phosphate (fingolimod-P, Fig. 1) binds to and activates four G protein-coupled S1P receptors (21, 22). By this mechanism, fingolimod-P induces internalization of S1P1 on lymphocytes, blocking the ability of the receptor to support lymphocyte egress and recirculation through secondary lymphoid organs. This suppresses immune responses and is presumably the main immunomodulatory mode of action of fingolimod.CS-0777 (Fig. 1) is a novel selective S1P1 modulator (23). Although the immunomodulatory effects are supposed to be mainly mediated by S1P1, some lines of evidence suggest that the agonist activity on S1P receptor 3 (S1P3) could cause acute toxicity and cardiovascular deregulation, including bradycardia in rodents (24, 25). Thus, CS-0777 was designed to have more selectivity on S1P1 over S1P3 in contrast to fingolimod-P which has potent agonistic activity for S1P3, S1P4, and S1P5 in vitro (22). Like fingolimod, CS-0777 is also a prodrug phosphorylated in vivo, and the phosphorylated CS-0777 (CS-0777-P, Fig. 1) agonizes S1P1 with more than 300-fold selectivity relative to S1P3 whereas CS-0777-P has weaker effects on S1P5 and no activity on S1P2 (23). CS-0777 showed immunosuppressive activity in mouse and rat models of experimental autoimmune encephalitis, animal models for multiple sclerosis. In healthy volunteers, single oral doses of CS-0777 caused marked, dose-dependent decreases in numbers of circulating lymphocytes, including marked and reversible decreases in circulating T and B cells (26). Furthermore, in multiple sclerosis patients, single oral doses of CS-0777 caused dose-dependent decreases in circulating lymphocytes, with a slightly greater suppression of CD4+ versus CD8+ T cells. Therefore, CS-0777 would alter immune responses solely through activation of S1P1 without S1P3 modulation in humans, which could circumvent a bradycardia adverse effect, although the relationships associating selectivity of S1P1 to S1P3 with bradycardia in humans are not fully understood (12).Orally administrated CS-0777 is phosphorylated and rapidly reaches equilibrium with CS-0777-P as in the case of fingolimod (22), suggesting that the high kinase activity in blood is balanced by phosphatases. Therefore, identification of a phosphatase, the inactivating enzyme of an active metabolite, as well as identification of a kinase, the activating enzyme of a prodrug, are critical to fully understand the mechanism of action at the molecular level for both CS-0777 and fingolimod. Sphingosine kinase 2 (SPHK2) was identified as the major kinase of fingolimod (21, 28, 29) and lipid phosphate phosphatase 3 (LPP3) was reported to be a phosphatase for fingolimod-P dephosphorylation (30), although contribution of LPP3 in vivo has not been fully studied. In our previous work, we have identified CS-0777 kinases in human blood as fructosamine 3-kinase-related protein (FN3K-RP) and fructosamine 3-kinase (FN3K) (6), whereas the phosphatase of CS-0777-P had not been identified thus far.In this study, we have successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as the major CS-0777-P phosphatase candidate in the human kidney by proteomic correlation profiling. According to available information, this is the first report applying proteomic correlation profiling to enzyme classes other than kinases; similarly, we believe this to be first application of proteomic correlation profiling to human tissue extract, which therefore has opened up wide usage of proteomic correlation profiling for all types of enzyme identification.     

Molecular Markers for Granulovacuolar Degeneration Are Present in Rimmed Vacuoles

Background

Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.

Methods

Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.

Results

GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.

Conclusions

These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.     

Glucagon-Like Peptide-1 Secretory Function as an Independent Determinant of Blood Pressure: Analysis in the Tanno-Sobetsu Study

Aims

Roles of glucagon-like peptide-1 (GLP-1) in extra-pancreatic tissues remain unclear. The aim of this study was to examine determinants of GLP-1 secretory function and possible contribution of GLP-1 to blood pressure (BP) regulation.

Methods and Results

We recruited 128 subjects who received annual examinations and 75g-oral glucose tolerance tests (OGTT) in the Tanno-Sobetsu cohort. Subjects on regular medications for cardiovascular and/or metabolic diseases were excluded, and data for the remaining 103 subjects were used for the univariate and multivariate analyses. Age, plasma glucose (PG), hemoglobin A1c (HbA1c), plasma insulin, and serum lipids were not selected as independent determinants of fasting GLP-1 level by multiple linear regression analysis. However, age and female sex were selected as independent positive determinants of the area under the curve of GLP-1 level during OGTT (AUC_GLP-1), an index of GLP-1 secretory function. Multiple linear regression analysis indicated that AUC_GLP-1 was an independent negative predictor of systolic BP (SBP), while AUC_GLP-1 was not correlated with fasting PG or HbA1c level. In subgroup analyses using the median of AUC_GLP-1 to divide the study subjects into high and low GLP-1 response groups, AUC_GLP-1 was significantly correlated with both SBP and diastolic BP (r = 0.40 and 0.28, respectively) in the low GLP-1 response group but not in the high GLP-1 response group.

Conclusions

The results of the present study suggest that GLP-1 secretory function is involved in prevention of BP elevation and that the GLP-1 response to oral glucose rather increases with aging perhaps as an adaptive phenomenon.