Insights into Activation Mechanisms of Store-Operated TRPC1 Channels in Vascular Smooth Muscle.

In vascular smooth muscle cells (VMSCs), the stimulation of store-operated channels (SOCs) mediate Ca2+ influx pathways which regulate important cellular functions including contraction, proliferation, migration, and growth that are associated with the development of vascular diseases. It is therefore important that we understand the biophysical, molecular composition, activation pathways, and physiological significance of SOCs in VSMCs as these maybe future therapeutic targets for conditions such as hypertension and atherosclerosis. Archetypal SOCs called calcium release-activated channels (CRACs) are composed of Orai1 proteins and are stimulated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) following store depletion. In contrast, this review focuses on proposals that canonical transient receptor potential (TRPC) channels composed of a heteromeric TRPC1/C5 molecular template, with TRPC1 conferring activation by store depletion, mediate SOCs in native contractile VSMCs. In particular, it summarizes our recent findings which describe a novel activation pathway of these TRPC1-based SOCs, in which protein kinase C (PKC)-dependent TRPC1 phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) are obligatory for channel opening. This PKC- and PIP2-mediated gating mechanism is regulated by the PIP2-binding protein myristoylated alanine-rich C kinase (MARCKS) and is coupled to store depletion by TRPC1-STIM1 interactions which induce Gq/PLCβ1 activity. Interestingly, the biophysical properties and activation mechanisms of TRPC1-based SOCs in native contractile VSMCs are unlikely to involve Orai1

PPM1A Controls Diabetic Gene Programming through Directly Dephosphorylating PPAR?? at Ser273

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a master regulator of adipose tissue biology. In obesity, phosphorylation of PPAR gamma at Ser273 (pSer273) by cyclin-dependent kinase 5 (CDK5)/extracellular signal-regulated kinase (ERK) orchestrates diabetic gene reprogramming via dysregulation of specific gene expression. Although many recent studies have focused on the development of non-classical agonist drugs that inhibit the phosphorylation of PPAR gamma at Ser273, the molecular mechanism of PPAR gamma dephosphorylation at Ser273 is not well characterized. Here, we report that protein phosphatase Mg2+/Mn2+-dependent 1A (PPM1A) is a novel PPAR gamma phosphatase that directly dephosphorylates Ser273 and restores diabetic gene expression which is dysregulated by pSer273. The expression of PPM1A significantly decreases in two models of insulin resistance: diet-induced obese (DIO) mice and db/db mice, in which it negatively correlates with pSer273. Transcriptomic analysis using microarray and genotype-tissue expression (GTEx) data in humans shows positive correlations between PPM1A and most of the genes that are dysregulated by pSer273. These findings suggest that PPM1A dephosphorylates PPAR gamma at Ser273 and represents a potential target for the treatment of obesity-linked metabolic disorders

Transcriptional Regulator TonEBP Mediates Oxidative Damages in Ischemic Kidney Injury

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient mice subjected to bilateral renal ischemia followed by reperfusion (I/R). In the TonEBP haplo-deficient animals, induction of TonEBP, oxidative stress, inflammation, cell death, and functional injury in the kidney in response to I/R were all reduced. Analyses of renal transcriptome revealed that genes in several cellular pathways including peroxisome and mitochondrial inner membrane were suppressed in response to I/R, and the suppression was relieved in the TonEBP deficiency. Production of reactive oxygen species (ROS) and the cellular injury was reproduced in a renal epithelial cell line in response to hypoxia, ATP depletion, or hydrogen peroxide. The knockdown of TonEBP reduced ROS production and cellular injury in correlation with increased expression of the suppressed genes. The cellular injury was also blocked by inhibitors of necrosis. These results demonstrate that ischemic insult suppresses many genes involved in cellular metabolism leading to local oxidative stress by way of TonEBP induction. Thus, TonEBP is a promising target to prevent AKI

Phenotypic characterization of human prostatic stromal cells in primary cultures derived from human tissue samples

Emerging evidence has shown that the tumor microenvironment plays a crucial role in prostate cancer (PCa) development and progression. However, the mechanism(s) through which stromal cells regulate epithelial cells and the differences among prostatic stromal cells of different histological/pathological origin in PCa progression remain unclear. Therefore, it is necessary to characterize the stromal cell populations present in benign prostatic hyperplasia (BPH) and PCa. To this end, we used cultures from stromal cells obtained from BPH-derived (15 cases) and PCa-derived (30 cases) primary cultures. In culture, stromal cells are a mixture of fibroblasts, myofibroblasts (MFs) and muscle cells. Fibroblasts are characterized for the expression of vimentin, MFs for the co-expression of α-smooth muscle actin (α-SMA) and vimentin, whereas muscle cells for the expression of α-SMA and desmin. Fibroblasts were present in large amounts in the BPH-compared to the PCa-derived cultures, whereas MFs were more representative of PCa-as opposed to BPH-derived cultures. Some α-SMA-positive cells retained the expression of basal cytokeratin K14. This population was defined as myoepithelial cells and was associated with senescent cultures. The percentage of MFs was higher in high-grade compared to moderate-and low-grade PCa-derived cultures, whereas the number of myoepithelial cells was lower in high-grade compared to moderate-and low-grade PCa-derived cultures. In addition, we analyzed the expression of p75NTR, as well as the expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of MMPs (TIMPs). p75NTR expression was elevated in the stromal cultures derived from PCa compared to those derived from BPH and in cultures derived from cases with Gleason scores.7 compared to those derived from cases with Gleason scores <7, as well as in cultures with a high concentration of MFs compared to those with a high concentration of fibroblasts. MMP-2 was secreted by all primary cultures, whereas MMP-9 secretion was observed only in some PCa-derived stromal cells, when the percentage of MFs was significantly higher compared to BPH-derived cultures. TIMP1, TIMP2 and TIMP3 were secreted in elevated amounts in the BPH-compared to the PCa-derived stromal cultures, suggesting the differential regulation of extracellular matrix (ECM) degradation. When we used 22rv1 and PC3 PCa xenograft models for the isolation and characterization of murine cancer-associated fibroblasts (CAFs) we noted that the angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, vimentin, tenascin, calponin, desmin and Masson's trichrome. In conclusion, MF stromal cells from PCa participate in the progression and metastasis of PCa, modualting inflammation, angiogenesis and epithelial cancer cell proliferation

MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs; qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs

Mucosal Mast Cells Response in the Jejunum of Ascaridia Galli-Infected Laying Hens

Intestinal defense mechanism against helminthes parasitic nematode to be associated with mucosal mast cells reaction. The aim of this research was to examine the effect of infection by Ascaridia galli parasite to trigger mucosal defense based on mucosal mast cells response in laying hens. Amount of ten head laying hens 12-wk old were divided into two groups containing five chickens of each. The first group, chickens were left as un-infected controls. The second group, chickens were infected orally with 1,000 embryonated eggs of A. galli. Mucosal mast cell responses were assayed by in situ jejunal mast cell counts in stained serial histological sections with Alcian blue (pH 0.3) and Safranin-O (pH 0.1) of the jejunum. Mucosal mast cells response were observed and counted on days 14 post infection. The result showed that A. galli infection was able to increase significantly (P<0.05) mast cells response. This research concluded that the A. galli infection can trigger the involment of mucosal mast cells response in jejunal defense of laying hens against parasitic diseases caused by A. galli

Impact of sitagliptin on endometrial mesenchymal stem-like progenitor cells : a randomised, double-blind placebo-controlled feasibility trial

Background: Recurrent pregnancy loss (RPL) is associated with the loss of endometrial mesenchymal stem-like progenitor cells (eMSC). DPP4 inhibitors may increase homing and engraftment of bone marrow-derived cells to sites of tissue injury. Here, we evaluated the effect of the DPP4 inhibitor sitagliptin on eMSC in women with RPL, determined the impact on endometrial decidualization, and assessed the feasibility of a full-scale clinical trial. Methods: A double-blind, randomised, placebo-controlled feasibility trial on women aged 18 to 42 years with a history of 3 or more miscarriages, regular menstrual cycles, and no contraindications to sitagliptin. Thirty-eight subjects were randomised to either 100 mg sitagliptin daily for 3 consecutive cycles or identical placebo capsules. Computer generated, permuted block randomisation was used to allocate treatment packs. Colony forming unit (CFU) assays were used to quantify eMSC in midluteal endometrial biopsies. The primary outcome measure was CFU counts. Secondary outcome measures were endometrial thickness, study acceptability, and first pregnancy outcome within 12 months following the study. Tissue samples were subjected to explorative investigations. Findings: CFU counts following sitagliptin were higher compared to placebo only when adjusted for baseline CFU counts and age (RR: 1.52, 95% CI: 1.32–1.75, P<0.01). The change in CFU count was 1.68 in the sitagliptin group and 1.08 in the placebo group. Trial recruitment, acceptability, and drug compliance were high. There were no serious adverse events. Explorative investigations showed that sitagliptin inhibits the expression of DIO2, a marker gene of senescent decidual cells. Interpretation: Sitagliptin increases eMSCs and decreases decidual senescence. A large-scale clinical trial evaluating the impact of preconception sitagliptin treatment on pregnancy outcome in RPL is feasible and warranted. Funding: Tommy's Baby Charity. Clinical trial registration: EU Clinical Trials Register no. 2016-001120-54

Cells and cacti

Let $(W,S)$ be a Coxeter system, let $\varphi$ be a weight function on $S$ and let ${\mathrm{Cact}}\_W$ denote the associated {\it cactus group}. Following an idea of I. Losev, we construct an action of ${\mathrm{Cact}}\_W \times {\mathrm{Cact}}\_W$ on $W$ which has nice properties with respect to the partition of $W$ into left, right or two-sided cells (under some hypothesis, which hold for instance if $\varphi$ is constant or if $W$ is finite of rank \textless{} 5). It must be noticed that the action depends heavily on $\varphi$.Comment: 23 pages. This new version extends the scope of validity of the main result by removing an hypothesis. For this purpose, we have slightly extended some results of arXiv:1502.0166