Cell Physiol Biochem Research Articles

Cdc42 Rho GTPase Activity Sustains Epithelial Barrier Function in the Cystic Fibrosis Intestine

Increased paracellular permeability is a common manifestation of cystic fibrosis (CF) intestinal disease in people with CF and in CF mouse models. Increased paracellular permeability has been found in CF airway epithelium cultured in the absence of inflammatory factors, suggesting an inherent defect in CF epithelial barrier function (Weiser et al., Cell Physiol Biochem. 2011;28(2):289-96). CF transmembrane conductance regulator knockout ( Cftr KO) mouse intestinal crypts demonstrate an increase in epithelial cell proliferation, which correlates with an alkaline intracellular pH (pHi), a pHi-dependent increase in lateral cell membrane localization of the Wnt transducer Disheveled (Dvl), and increased Wnt/β-catenin signaling (Strubberg et al., Cell Mol Gastroenterol Hepatol. 2017 Dec 7;5(3):253-271). We hypothesized that the alkaline pHi of Cftr KO crypts also facilitates Dvl-dependent noncanonical Wnt signaling to increase Rho GTPase activity and thereby tight junction remodeling during increased proliferation. In Dvl2-eGFP rescue mice, Dvl2-eGFP intensity near the apical membrane of Cftr KO enteroid crypts was greater versus wild-type (WT) at both the crypt base and the transit-amplifying zone. Immunofluorescence studies of both freshly isolated crypts and early passage enteroids also found increased accumulation of the Rho GTPase Cdc42 at the tight junctions of Cftr KO versus WT crypts. Live imaging revealed a small increase in leak pathway permeability (3kD dextran) in Cftr KO as compared to WT murine enteroids. Increased leak permeability and Cdc42 tight junction localization were normalized in Cftr KO enteroids by suppressing proliferation through inhibition of Wnt/β-catenin signaling. There was no difference in Cdc42 expression in Cftr KO fresh crypts or enteroids, but initial studies show increased Cdc42 GTP-loaded activity in Cftr KO enteroids relative to WT. Similarly, CFTR KO human intestinal epithelial Caco-2 cells, which also show an alkaline pHi, increased Wnt/β-catenin signaling and a hyperproliferative phenotype, demonstrated a significant increase in GTP-loaded Cdc42 activity compared to WT cells. Inhibition of Cdc42 activity with ML141 (R&D Systems) resulted in a significant disruption of the Cftr KO epithelial barrier, whereas WT enteroid permeability was not affected by Cdc42 inhibition, Wnt inhibition or Wnt3a-induced proliferation. We conclude that increased Cdc42 Rho GTPase activity is cell-autonomous and is necessary for maintaining barrier integrity of the Cftr KO crypt epithelium. This work was supported by the Cystic Fibrosis Foundation grant CLARKE20G0 and the Crohn’s and Colitis Foundation grant #693938. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MO610: A Graphene-Based Bioactive Compound Reduces Adipogenesis and Increases Lipolysis Via Integrin-Beta1 Interaction: A New Therapeutical Approach Against Obesity

Abstract BACKGROUND AND AIMS Type 2 diabetes and obesity are metabolic disorders related to kidney risk and associated with white adipose tissue (WAT) hypertrophy, which is based in a pro-adipogenic transdifferentiation of the adipocytes and their precursors’ stem cells present in WAT. Several transcription factors are key regulators during this transdifferentiation. The consequence is that adipocytes’ size increases due to the excessive storage of triglycerides (TG) inside the cells. Therapeutic strategies for reducing WAT size may be based in the capacity of increasing lipolysis and reducing adipogenesis. We have previously demonstrated the implication of the extracellular matrix (ECM) mediator integrin beta 1 (INTB1) and intracellular mediation of actin cytoskeleton during obesity (Hatem-Vaquero. J Endocrinol 2017; Cell Physiol Biochem 2020). Here, we demonstrate for the first time the antiobesogenic properties of a new highly biocompatible and bioactive graphene-based product named BioGraph (Llorens-Gámez M. Int J Biol Macromol 2020; Salesa B. Biomedicines 2021), which physicochemical interaction with INTB1 give a great potential as a diagnostic and bioactive therapeutic tool. METHOD Pluripotent c3h10t1/2 cells were differentiated into adipocytes using a specific culture medium. After 7 days of differentiation, the adipocytes were treated for 24 h with a suspension of the graphene derivative BioGraph, developed, registered and patented by Graphenano Medical Care S.L. To determine viability and toxicity on adipocytes, trypan blue and MTT labeling techniques were used. The levels of intracellular TG contained in the lipid droplets were determined by fluorescent labeling with AdipoRed. PPARgamma, CEBP and INTB1 mRNA levels were determined by RT-qPCR. Actin polymerization in the cytoskeleton was quantified by fluorescent phalloidin staining. Active INTB1 quantification (phosphorylated isoform at thr788/9) and total and active lipase HSL (phosphorylated isoform at ser660) were performed by western blot. RESULTS BioGraph is safe and non-toxic on adipocytes in a two-order concentration range. The TG content is reduced in a dose-dependent manner. Adipogenesis markers PPARgamma and CEBP and actin polymerization were reduced after treatment with an intermediate dose. The expression of INTB1 was not modified, but its phosphorylation was increased rapidly after treatment. Lipolysis-main enzyme HSL expression and activity were increased 24 h after treatment. CONCLUSION BioGraph is biocompatible and bioactive on adipocytes. A rapid modulation of INTB1 produces the depolymerization of the cytoskeleton, the reduction of intracellular TG content and the expression of adipogenesis markers, while HSL-mediated lipolysis is increased. We demonstrate for the first time the mechanism followed by a graphene-based material modulating the adipocyte phenotype and behavior. BioGraph may be considered a new anti-obesogenic agent.

Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

MicroRNA-26a-5p: multiple functions, multiple possibilities – a mini-review

1. Xie T, Huang M, Wang Y, et al. MicroRNAs as Regulators, Biomarkers and Therapeutic Targets in the Drug Resistance of Colorectal Cancer. Cell Physiol Biochem. 2016; 40(1–2): 62–76. https://doi.org/10.1159/000452.... CrossRef Google Scholar

P0992PHARMACOLOGICAL UPREGULATION OF INTEGRIN-LINKED KINASE (ILK) REDUCES VISCERAL ADIPOSITY IN A METABOLIC SYNDROME MICE MODEL

Abstract Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

P0980IMPLICATIONS OF ADIPOSE EARLY DOWNREGULATION OF INTEGRIN-LINKED KINASE (ILK) IN THE DEVELOPMENT OF METABOLIC SYNDROME IN MICE

Abstract Background and Aims Insulin resistance (IR)-related metabolic disorders, such as diabetes type 2 and obesity, are linked to renal and cardiovascular risk. Unfortunately, they lack a better prognosis before their full establishment. An important mechanism associated to the IR development is the dysregulation of lipolysis in white adipose tissue (WAT), which is driven by the excessive lipid accumulation, together with inflammation and the excessive deposition of ECM components such as collagens and fibronectin, known as fibrosis. The intracellular lipid adjustment is affected by changes in the expression and activity of the hormone-sensitive lipase (HSL) and the intracellular and circulating balance of the lipolysis products glycerol and free fatty acids. We published that transgenic depletion of the scaffold intracellular protein Integrin-linked kinase (ILK), one of the adhesome translators of the ECM signals, modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017]. We also published that ILK downregulation in WAT is an early event in a short-term high fat diet (HFD) mice model that correlates with a faster insulin sensitivity loss, accompanied with an early increase of body weight and the erratic expression of WAT adipokines and metabolites transporters. [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Here we used in vivo and in vitro approaches to decode the role of ILK downregulation during WAT expansion and altered lipolysis. Method Adult mice with global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. We determined the weight changes on body and WAT depots, epidydimal (eWAT) and subcutaneous (scWAT), and the circulating lypolisis product glycerol. The expression of ECM component fibronectin (FN), fibrosis marker TGF-beta and the glycerol transporter AQP3 were determined in eWAT by RT-qPCR or Western blot. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). We determined the expression of ILK, total and active HSL isoforms (HSL, p-HSL s660) by RT-qPCR or Western blot and the intracellular fat droplet content (by adipo-red dye) Results HFD increased progressively CT eWAT, scWAT and whole body weight gains. HFD-fed cKD-ILK have an earlier increase of body and eWAT weights. However, the scWAT weight ratio against eWAT was inverted in the same cKD-ILK animals. This early depot-specific response to HFD coincide with increase in IR [Wajchenberg BL. Endocr Rev 2000]. HFD-fed cKD-ILK WAT early expressed altered levels of fibrotic markers (TGF-beta and Fibronectin). HFD-fed cKD-ILK shown a temporary increase of circulating glycerol and its WAT transporter AQP3. In cultured adipocytes with downregulated expression of ILK, the intracellular fat droplet was increased and the HSL contain and its lipolysis activity (measured as levels of p-HSL isoform) were reduced. Conclusion We suggest ILK as a modulator during the obesity establishment. Considering the observed relation between ILK expression with the altered lipolysis and fibrosis patterns observed in visceral WAT, ILK downregulation may be a predictive value and probably its pharmacological upregulation may be a novel therapeutic strategy against obesity and IR.

Abstract B087: PARK2 expression reduces migration and invasion and deregulates metabolic pathways in ccRCC

Abstract PARK2 is an E3-ubiquitin-ligase, which plays an important role in mitophagy, a macroautophagy process that selectively degrades defective mitochondria. The significant downregulation of mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) were shown to be associated with aggressive disease and poor clinical outcome (Toma et al., 2013). The aim of our study was to investigate the role of of PARK2 in ccRCC. First, we transduced the ccRCC cell line 786-O with a PLVX-EF1a-PARK2-IRES-mcherry construct to overexpress PARK2. Subsequently, we performed functional assays, analyzed the mitochondria and mitochondrial respiration, and RNA-Sequencing. While apoptosis, cell cycle and cell proliferation remained unaffected, we found a marked reduction of migration and invasion in PARK2 overexpressing cells compared to wt controls. PARK2 is involved in the degradation of damaged mitochondria during mitophagy, therefore, we investigated the amount of active and total mitochondria and found more active mitochondria in the PARK2 clones. The overall amount of mitochondria was unchanged. To verify the results and get a further insight into the functionality of the mitochondria mitochondrial respiration was analyzed. As expected, the mitochondrial respiration of the PARK2 clones was upregulated. In addition, we observed a general shift towards metabolic upregulation in 786-O cells expressing PARK2. To find specific pathways affected by the expression of PARK2, we performed RNA Sequencing. Several metabolic pathways were deregulated in the cells expressing PARK2 compared to wt controls. We were able to identify several genes being downregulated in transgenic wild type cells that play a role in the epithelial-to-mesenchymal transition underlining our phenotypic findings of invasion and migration. In conclusion, PARK2 has an impact on the aggressiveness of the disease, the health of the mitochondria and effects a deregulation of metabolic pathways. Lately, several groups reported on the connection between migration/invasion and metabolic changes (Kenny et al., 2019; Yizhak et al., 2014; Gaude and Frezza; 2016, Yao et al., 2018) suggesting a potential link between these two events. Our study adds additional proof towards that link, nevertheless, further research needs to be conducted to identify if these two events are indeed interconnected and dependent on each other. Literature Gaude E and Frezza C. Tissue-specific and convergent metabolic transformation of cancer correlates with metastatic potential and patient survival. Nat Commun. 7:13041. 2016. Kenny TC et al. Mitohormesis Primes Tumor Invasion and Metastasis. Cell Rep. 27(8):2292-2303. 2019. Toma MI et al. PARK2 and PACRG are commonly downregulated in clear-cell renal cell carcinoma and are associated with aggressive disease and poor clinical outcome. Genes Chromosomes Cancer. 52(3):265-73. 2013. Yao L, Guo X, Gui Y. Acetyl-CoA Synthetase 2 Promotes Cell Migration and Invasion of Renal Cell Carcinoma by Upregulating Lysosomal Associated Membrane Protein 1 Expression. Cell Physiol Biochem. 45(3):984-992. 2018. Yizhak K et al. A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration. Mol Syst Biol. 10:744. 2014. Citation Format: Laura K Eßer, Lena Arévalo, Glen Kristiansen, Hubert Schorle, Marieta I Toma. PARK2 expression reduces migration and invasion and deregulates metabolic pathways in ccRCC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B087. doi:10.1158/1535-7163.TARG-19-B087

Cytosolic Internalization of the Na/K ATPase Ouabain Receptor Complex (NORC)

In early (1960–80) classic work, digitalis (ouabain) binding to the Na/K ATPase (NKA) was considered mainly a disruption of the cellular ionic steady state and in terms of ligand dependency. During the past 2–3 decades, the role of NKA as signal transducer involving protein/protein interactions with subsequent protein phosphorylation and gene transcriptional upregulation was elucidated regarding cell survival and apoptosis. Recently re‐established, the significance of a cytosolic internalization of the NKA‐ouabain receptor‐complex (here called NORC) is not yet understood in terms of details of participating proteins and organelles, and its potential for gene transcriptional upregulation. The fate of NORC was followed by immunocytochemistry, and enhanced dark field hyperspectral microscopy (EDHM) in two different lens epithelial cells (LECs) (FHL124 and B3) with the aim to characterize some of the protein partners of the hypothetical membrane‐to‐cytosol escalator involved in this process. Partners of interest were the Bcl‐2 protein BclXL, proposed by us to interact with NKA's N‐terminal domain (Lauf et al., Cell Physiol Biochem 31:257–276, 2013) and detected by co‐immunoprecipitation (Lauf et al., Am J Physiol Cell Physiol. 308, C51–60, 2015), α‐tubulin (Zampar et al. Biochem J. 422:129,2009), caveolin (Cui & Zie, Molecules 22:990, 2017), and the endosomal and lysosomal proteins EEA1 and LAMP1 (Cheriavsky et al., JBC 289:1049, 2014). The presence of these protein partners was also studied in B3 LECs and verified by PAGEL and Western blots. Protein interactions were tested with Bodipy‐green fluorescent ouabain (BFO) and green‐ or red‐fluorescent labeled antibodies against all protein components, verified immuno‐cytochemically by overlapping yellow/orange fluorescence and further refined by MATLAB photo‐editing for background noise. Results: Based on immuno‐colocalization of BFO with Cy‐3 Red‐stained anti‐α1 NKA isoform antibody, NORC transgressed the plasmalemma into cytosol 45 min after exposure to μM BFO. This was further supported by finding of intracellular BFO labeling using EDHM in FHL124 cells. Higher ouabain concentrations affected cell size and NKA distribution. However, interaction of NKA with Bcl‐XS, a shorter variant of BclXL lacking the B1 motif, and caveolin‐1 in B3 LECs was independent of ouabain concentrations, and not seen in all cells. Increase in ouabain concentration enhanced the interaction of NKA with α‐tubulin, which was attenuated by preventing its deacetylation with Trichostatin A, a histone deacetylase inhibitor. Colocalization of NKA with EEA1 was not affected by ouabain, which appeared to increase NKA/Lamp1 immuno‐colocalization. A model is proposed by which NORC is translocated across the plasma membranes within minutes after its generation. In some B3 cells, by interaction with BclXS, caveolin and α‐tubulin, NORC may be guided to endosomes and, if not recycled back to the plasma membrane, subsequently to lysosomes. The future fate of NORC, especially of a putative nuclear receptor capture of lysosome‐processed ouabain to mediate gene transcriptional upregulation of various pro‐apoptotic and pro‐inflammatory proteins is under study and reported elsewhere at this meeting.Support or Funding InformationWright State University Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long‐term Ouabain treatment promotes ATP1A1‐dependent IL‐6 expression in a fetal human lens epithelial cell line (FHL124)

The cardiac glycoside ouabain (OUA) selectively inhibits Na+/K+‐ATPase (NKA) governing the steady state maintenance of the electro‐chemical gradient of Na+ and K+ across the cell membranes. OUA, following binding to the NAK, activates several growth‐related signaling pathways: the EGFR/Src–Ras–ERK pathway and the PI3K1A–PDK–Akt pathway (Xie and Askari, Eur J Biochem 269(10):2434–2439, 2002). OUA also influences pro‐ and anti‐inflammation (Cavalcante‐Silva et al., Frontiers in Physiology, 8:895, 2017) and cell proliferation depending on OUA concentration and incubation time, and cell type (Kulikov et al., Biochim et Biophys Acta 1768: 1691–1702, 2007 and Nguyen et al. JASN 18 (1): 46–57, 2007). Our group proposed and showed the direct interaction between NKA and BcL2 family proteins, which promote or inhibit apoptosis in human fetal lens epithelial cell line (FHL124) (Lauf et al., Cell Physiol Biochem 31:257–276, 2013; Lauf et al., Am J Physiol. Cell Physiol. 308, C51‐60, 2015). Here, we aimed to study the time dependence of OUA action at a concentration of 10−7M on cell viability and transcriptional gene regulation of various pro‐apoptotic and pro‐inflammatory related genes in FHL124 cells. Cells were incubated with either 1–100 μM OUA in 7 μM DMSO or 7 μM DMSO alone (solvent control) relative to an untreated control for 1, 24 and 48 h. The OUA‐treated cells cultured for 24 h revealed a slightly decreased cell viability, however, 48 h incubation dramatically decreased the number of cells by 75–80% while DMSO alone reduced cell viability by 40–50% compared to untreated cells. Like all epithelial cells, FHL124 cells possess α1 (atp1a1) isoform of NAK. The 5′‐untranslated region (5′UTR) of the atp1a1 gene contains two p53 and three NF‐kB (transcription factors nuclear factor‐kappa‐B) response elements. To test for the functional expression of the p53 and NFKB target genes, we selected mdm2, which negatively regulates the p53 gene and IL‐6 (interleukin‐6), which is induced by NF‐KB. Quantitative polymerase chain reaction (qPCR) method, using TaqMan probes, was employed to measure comparative gene expression of the atp1a1, mdm2, and IL‐6 genes. Cells OUA‐treated for 48 h promoted an increase of the IL‐6 expression by ~eight fold and the atp1a1 expression by two fold, but not the mdm2 expression. These findings suggest that OUA, in addition to utilizing existing signaling pathways mentioned above, may act either in complex with cytosolically internalized NKA (see Stricker et al., this FASEB meeting 2018) or derived therefrom through to be determined gene regulation factors proximal to the NF‐kB response elements. Further studies are aimed to confirm NKA‐dependent IL‐6 expression after long‐treatment with OUA.Support or Funding InformationSupported by the P&T Graduate program and WSU Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Receptor Potential Melastatin 8 (TRPM8) Induced Relaxation of Pulmonary Artery is Mediated Through Inhibition of Store‐Operated Ca2+ Entry in Normoxic and Chronic Hypoxic Rats

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Members of multiple transient receptor potential subfamilies (TRPC, TRPV, and TRPM) have been identified in pulmonary vascular tissue. Our previous studies showed that the expression and functions of the store‐operated TRPC1, receptor‐operated TRPC6, and the mechanosensitive TRPV4 channels are augmented in PASMCs during PH, and their upregulation contribute to PH development. In contrast, TRPM8 is down‐regulated in PASMCs of rats PH models, and activation of TRPM8 with agonists causes relaxation of pulmonary arteries (PAs)(Liu et al., Cell Physiol Biochem 2013, 31:892–904). However, the mechanism of TRPM8‐induced PA relaxation has not been determined. In this study, we examined the possible interactions of TRPM8 activation and store‐operated Ca2+ entry (SOCE) in PAs and PASMCs of normoxic and chronic hypoxic pulmonary hypertensive (CHPH) rats. We found that TRPM8 expression was down‐regulated in PAs and the TRPM8‐mediated cation entry was reduced significantly in the PASMCs of rats after 3 weeks of hypoxia (10% O2) exposure. Activation of TRPM8 with icilin (0.1–100 μM) caused concentration‐dependent relaxation of cyclopiazonic acid (CPA) and ET‐1 pre‐contracted endothelium‐denuded PAs in the presence of nifedipine. The vasorelaxant effect of icilin was abolished in the presence of the SOCE antagonist Gd3+ at submicromolar concentration. Icilin suppressed the CPA‐induced cation entry in PASMCs determined by the Mn2+ quenching technique and by Ca2+ transient measurement. The inhibitory effect of icilin on SOCE‐induced PA relaxation and Ca2+ entry were abolished by the TRPM8 antagonist AMTB, indicating that the effect was mediated specifically by TRPM8 channels. Moreover, TRPM8‐mediated inhibitory effects on CPA‐induced contraction in PAs and SOCE in PASMCs were significantly augmented in CHPH rats, probably related to the enhanced SOCE caused by PH. These results demonstrate for the first time that TRPM8 activation can cause relaxation of PA through inhibition of SOCE. Since SOCE plays many important roles in pulmonary vascular functions and is crucial for the vascular remodeling and the enhanced vasoconstriction in PH, the downregulation of TRPM8 expression and activity in PASMCs during PH may minimized the TRPM8‐dependent inhibition of SOCE, and allow unimpeded SOCE activity for PH development.Support or Funding InformationSupported by grants NSFC31571179, NSFC31371165, NSF of Fujian Province 2015J01313, and Fujian Province Hundred Experts Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pig Renal Na/K ATPase and Bcl‐2 Proteins: A Co‐Immuno‐Precipitation Study

The Na/K ATPase (NKA) has at least two Bcl‐2 like sequences (Cell Physiol. Biochem. Cell Physiol Biochem 31: 257–276, 2013) and, in human lens epithelial cells, co‐immuno‐precipitates with the Bcl‐2 proteins BclXL and Bak (Am J. Physiol. Cell Physiol 2015: 308: C51–C60). Here we searched for immunochemical evidence of the presence of BH3 motif‐containing Bak co‐immuno‐precipitated with the well‐characterized pig renal NKA (PRNKA) (Klodos et al., Kidney Int. 62:2097–2100, 2002).ResultsPRNKA was prepared by the Klodos procedure omitting the final SDS purification step to preserve NKA activity at 0.45 μmol in 5.54 mg/ml. Mouse (ms) anti‐NKA (α6F) antibody (ab) showed the α1 subunit of ~110 kDa in immunoblots (IB) of the PRNKA input, in the protein‐G coated metal bead (PGCMB) ‐ α6F immuno‐pulldown, as well as in PGCMB‐ms and rabbit (rb) IgG pulldowns. A rb polyclonal anti‐human Bak BH3 motif ab (Cell Signaling #3814) together with a secondary (2nd) goat anti‐Rb Fc’ heavy (H) chain ab detected a low level staining of a ~27 kDa peptide in the PRNKA input and a prominent band in the α6F immuno‐pull down suggesting the presence of Bak in the enzyme preparation. IBs with the 2nd anti‐Fc’ H‐chain ab alone, however, not only detected IgG H chains in α6F ab‐, and ms‐ and rb‐IgG immuno‐pull downs, but unexpectedly also in PRNKA input. These findings suggest the presence of 1) Bak, and 2) NKA in PRNKA inputs and in immune and “non‐immune” pulldowns, with an absence of both proteins in ms and rb IgG input only; and 3) an apparent IgG contamination in PRNKA. Because of the latter, our efforts are now directed to discriminate further between Bak and L chains both of very close molecular mass. Whether the triple occurrence of NKA, Bak and IgG in all pull downs is IgG‐molecular specific but not species‐specific or coincidental due to non‐specific adsorption to the protein G beads, or both, is currently under study.Support or Funding InformationSupported by Wright State University Foundation to the Cell Biophysics Laboratory, by the Pharmacology and Toxicology Program to C.K.M., and by NIH to JH.

Downregulation of BRCA1 Protein in BCR-ABL1–positive Cells Depends on Tiar-Mediated Repression of BRCA1 mRNA Translation

Expression of the BCR-ABL1 kinase causes chronic myeloid leukemia in chronic phase (CML). BCR-ABL1 modulates DNA repair mechanisms leading to genomic instability causing resistance to the tyrosine kinase inhibitors and/or disease transition from chronic phase to more malignant stages. BCR-ABL1 –mediated downregulation of breast cancer type 1 susceptibility (BRCA1) protein, a key element in homologous recombination repair (HRR) of DNA double-strand breaks (DSB) was first observed by Deutsch et al, Blood 2003 and later confirmed in our laboratories (Wolanin et al, Mol Cancer Ther 2010; Cramer-Morales et al, Blood 2013). In addition to HRR, BRCA1 protein has been implicated in a broad range of cellular processes, including cell cycle control, cell division and gene transcription (Wu et al, Protein Cell 2010; Moiola et al, Cell Physiol Biochem 2012). We previously reported that downregulation of BRCA1 protein affected the function of spindle assembly checkpoint and postmitotic checkpoint leading to aneuploidy and resistance to spindle poisons in CML cells (Wolanin et al). Recently we also showed that BRCA1-dependent defects in DSB repair in CML cells can sensitize them to synthetic lethality induced by RAD52 inhibitor (Cramer-Morales et al).The mechanism of BRCA1 downregulation in CML cells remained unknown. We observed that it was not due to decreased BRCA1 mRNA expression or shortened protein half-life (Piwocka et al, InTech Press 2011), and it was partially reversible by inhibition of BCR-ABL1 kinase by imatinib (Deutsch et al; Wolanin et al). In addition, mutations abrogating the expression of BRCA1 protein have not been detected in CML (Friedenson, BMC Cancer 2007).Here we report that downregulation of BRCA1 protein is paradoxically associated with enhanced half-life and increased levels of BRCA1 mRNA in a BCR-ABL1 transformed cell line and in CML primary cells. Using polysome profiling and luciferase-BRCA1 3’UTR reporter system we demonstrated that downregulation of BRCA1 protein in BCR-ABL1 expressing cells is caused by inhibition of BRCA1 mRNA translation. We show that this is not accompanied by increased protein degradation. Altogether, these results implicated the involvement of mRNA binding proteins such as Hu protein R (HuR) and TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAR) proteins because of their capability to bind the AU-Rich Element (ARE) sites in 3’UTR of human BRCA1 mRNA. It has been reported that in breast cancer HuR can regulate BRCA1 mRNA stability by binding to its 3’UTR (Saunus et al, FEBS Lett 2007; Cancer Res 2008). The other ARE-site binding protein – TIAR – is a well characterized translation repressor protein activated by stress which is known to inhibit protein synthesis transiently by formation of translational silent stress granules (Anderson & Kedersha, J Cell Sci 2002). We have described that BCR-ABL1 expression causes ER stress and thus activation of Unfolded Protein Response (UPR) stress-induced pathway (Kusio-Kobialka et al, Cell Cycle 2012) which stimulates TIAR (Kedersha & Anderson, Biochem Soc Trans 2002).We observed that BCR-ABL1 kinase promoted cytosolic localization and formation of the TIAR-HuR complex, which facilitated the association with BRCA1 mRNA. We found that HuR protein positively regulated BRCA1 mRNA stability and translation. Conversely TIAR, which was localized predominantly in the cytosolic stress granules in CML cells, abrogated BRCA1 mRNA translation and downregulated BRCA1 protein level. Altogether, we postulate that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein levels in BCR-ABL1 –positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in “BRCAness” CML and BCR-ABL1 –positive ALL cells.Acknowledgments: We acknowledge Prof. Anne Willis and Dr. Lindsay Wilson for technical help and advice. Prof. Myriam Gorospe for providing plasmid with HuR cDNA. This work was supported by the National Science Centre research grant 2011/01/B/NZ3/02145 to K.P. and the Polish Ministry of Science and Higher Education grants IP2011 043071 and IP2010 032870 to P.P-B. Skorski’s lab was supported by NIH/NCI R01 CA123014. DisclosuresNo relevant conflicts of interest to declare.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

Unique Regulation of AQP1 and AQP5 Expression by HIF-1 in Nucleus Pulposus Cells

Introduction Aquaporins are a family of transmembrane water channels that aid in osmoregulation, a critical function in the proteoglycan-rich nucleus pulposus. Previous reports have demonstrated expression of aquaporins in the intervertebral disc.1,2 Hypoxia, a defining feature of the nucleus pulposus environment has been shown to regulate expression of aquaporin1 (AQP1) and aquaporin 5 (AQP5).3-6 In some cases, this regulation has been shown to occur through HIF-1α and to depend on hypoxia responsive elements (HREs). The goal of this study was to examine if hypoxia and HIF-1α play a role in regulation and function of aquaporin1 (AQP1) and aquaporin5 (AQP5) in nucleus pulposus (NP) cells of the intervertebral disc. Materials and Methods Quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry were used to measure AQP1 and AQP5 expression in nucleus pulposus (NP) and annulus fibrosus (AF) cells and tissues. Transfections were used to determine the role of HIF-1α on AQP1 and AQP5 promoter activity in normoxia and hypoxia. The JASPAR database was used to identify two putative hypoxia response elements (HREs) in the promoter of each aquaporin. HIF-1α levels were modulated with treatment by DMOG or shRNAs. Expression of aquaporins in human tissue samples was evaluated using immunohistochemistry and polymerase chain reaction. Results We found that both AQP1 and AQP5 were expressed in tissues and cells of human and rat discs. To determine whether expression of the aquaporins was regulated by hypoxia, we treated NP cells with hypoxia (1% O2) for 8 to 72 hours. Treatment had no effect on promoter activity, mRNA expression, or protein expression of either aquaporin. Using the JASPAR database, we identified two HREs in the promoter of each aquaporin. Mutation of the HREs in either AQP1 or AQO5 had no suppressive effect on the basal activity of the promoters. We then investigated whether AQP1 and AQP5 were regulated in a HIF1α-dependent manner. Accumulation of HIF-1α by DMOG did not affect expression of either aquaporin. However, suppression of HIF-1α by lentiviral delivery of shRNA significantly decreased both mRNA and protein expression of AQP1 and AQP5. In addition, we have found changes in expression of these aquaporins in human tissue samples from patients with varying degrees of intervertebral disc degeneration. Conclusion These results indicate that, under hypoxic conditions, HIF-1α maintains basal expression of both AQP1 and AQP5 in the NP; however, this regulation is independent of HIF binding to the identified HREs in their promoters. Analysis of human tissue samples suggests that aquaporin expression may be linked to health of the intervertebral disc. Disclosure of Interest None declared References Richardson SM, Knowles R, Marples D, Hoyland JA, Mobasheri A. Aquaporin expression in the human intervertebral disc. J Mol Histol 2008;39(3):303–309 Gajghate S, Hiyama A, Shah M, et al. Osmolarity and intracellular calcium regulate aquaporin2 expression through TonEBP in nucleus pulposus cells of the intervertebral disc. J Bone Miner Res 2009;24(6):992–1001 Tanaka A, Sakurai K, Kaneko K, et al. The role of the hypoxia-inducible factor 1 binding site in the induction of aquaporin-1 mRNA expression by hypoxia. DNA Cell Biol 2011;30(8):539–544 Tie L, Lu N, Pan XY, et al. Hypoxia-induced up-regulation of aquaporin-1 protein in prostate cancer cells in a p38-dependent manner. Cell Physiol Biochem 2012;29(1-2):269–280 Zhang J, Xiong Y, Lu LX, et al. AQP1 expression alterations affect morphology and water transport in Schwann cells and hypoxia-induced up-regulation of AQP1 occurs in a HIF-1α-dependent manner. Neuroscience 2013;252(252):68–79 Kawedia JD, Yang F, Sartor MA, Gozal D, Czyzyk-Krzeska M, Menon AG. Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways. PLoS ONE 2013;8(3):e57541

Novel Mechanism of Na/K Pump Inhibition by Chelerythrine (CET), a PKC Inhibitor, Uncovers Potential Early Signal Transducers of Apoptosis.

In human lens epithelial cells (HLECs), CET abolishes Na/K pump (NKP) activity, concomitantly inhibits K fluxes via the Na‐K‐2Cl isoform 1 and stimulates K channels (Lauf et al. Cell Physiol Biochem 2012). Since in HLECs neither ATP, nor phosphorylation of the α1 subunit or FXYD2 proteins were altered, nor ouabain binding to pig renal Na/K ATPase, an alternate direct action of CET on NKP was explored. CET is a BH3‐mimetic for proapoptotic Bcl‐2 proteins interacting with their BH1 domains. Thus we hypothesized that the NKP might also possess BH1 domains to which a) the BH3‐mimetic CET and b) BH3‐domain type Bcl‐2 proteins would bind and hence modify NKP activity crucial for cellular homeostasis. Indeed, MAFT sequence alignments between Bcl‐2 and BclXl revealed the presence of at least one BH1‐like domain within the NKP potentially interacting with CET or Bcl‐2 BH3 proteins. This site consists of some 13 amino acids (aa) within the N‐terminal 100 aa of the actuator (A) within the NKP (and other P‐type pumps). We propose that CET inhibits the NKP by conformational changes of its A domain thus disrupting the α1 subunit catalytic activity. Consequently, by interacting with the putative NKP CET‐binding BH1‐like site, Bcl‐2 BH3 domain proteins may be intermediary cytosolic sensors of normal NKP function thus constituting important signal transducers in the initial phases of apoptosis.

Maturing EPCs into endothelial cells: may the force be with the EPCs. Focus on “Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells”

endothelial progenitor cells (EPCs) are a unique cell type found circulating in the peripheral blood with the capacity to become mature endothelial cells. EPCs can be released from many sources including the bone marrow, adipose tissue, the vessel wall, as well as potentially the spleen, liver, and

Evidence for a Na/K pump in nematocytes isolated from sea anemones

Nematocytes, the stinging cells of the primitive marine animals jellyfish and sea anemones respond with regulatory volume decrease (RVD) to osmotic swelling in 35% diluted artificial sea water (65% ASW, Marino et al., Cell Physiol Biochem 2010). Since RVD is attained through K+ and Cl‐efflux via channels and cotransporters, an active pump mechanism must be required to maintain the cellular K+ gradient and expel Na, which was here explored. Cells were isolated from acontia of the anemone Aiptasia mutabilis (Strait of Messina, Italy) by applying 605mM NaSCN plus 0.01mM Ca2+ and then exposed to isosmotic or 65% hyposmotic ASW in the presence and absence of ouabain, the specific Na/K pump inhibitor. Ouabain present at 10‐4 M, but not at 10‐6 and 10‐8M, induced significant cell swelling in isosmotic ASW suggesting Na/K pump inhibition and failure to extrude Na causing an osmotic influx of water. This effect was attenuated by replacing Na with choline in the ASW. Ouabain applied at 10‐4M prior to 65% ASW did not affect the initial osmotic swelling phase but inhibited RVD consistent with loss of K required for this process. Results suggest that nematocytes possess Na/K pumps with low ouabain affinity that actively extrude Na and maintain cellular K steady state levels, necessary to perform RVD.

Safety of recombinant human deoxyribonuclease as a rescue treatment for persistent atelectasis in newborns

BACKGROUND AND OBJECTIVEPulmonary problems are vitally important in newborns. Increased intense and mucoid secretions may lead to atelectasis, pulmonary infections, respiratory distress, prolonged mechanical ventilation or even death. The aim of this study was to evaluate the safety of recombinant human deoxyribonuclease (rhDNase) in the management of persistent atelectasis in term and preterm newborns, unresponsive to the conventional treatment.DESIGN AND SETTINGProspective study of patients admitted to a general community setting of a neonatal intensive care unit between December 2007 and December 2009.PATIENTS AND METHODSThe study included 22 patients (12 premature and 10 term) who were admitted to the neonatal intensive care unit because of respiratory distress and developed atelectasis, and were unresponsive to conventional treatment. Nebulized rhDNase was administered to all patients at a dose of 1 mg/m2 twice daily for 3 days. In patients who did not respond to 3 days of treatment, endotracheal rhDNase was administered at a dose of 1 mg/m2. We assessed the clinical (respiratory rate and oxygen requirement) and radiologic responses (chest radiographic score), recurrence of atelectasis, the need for a repetitive treatment, and mortality rate.RESULTSA clinical and radiologic improvement of atelectasis was observed in 18 of 22 patients following 3 days of nebulized rhDNase treatment. Atelectasis relapsed in 4 patients. Following the administration of combined endotracheal and nebulized rhDNase treatment, an improvement of atelectasis was noted in all four recurrent cases. No adverse events were observed in patients because of the rhDNase treatment.CONCLUSIONSrhDNase treatment is a safe option and may be used as an effective method for the management of persistent atelectasis in newborns, which is resistant to other conventional treatment methods.

Hyperosmotic stress strongly potentiates serum response factor (SRF)‐dependent transcriptional activity in ehrlich lettré ascites cells through a mechanism involving p38 mitogen‐activated protein kinase

Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway is transiently inhibited while p38 MAPK is activated, in turn impacting on cell survival (Pedersen et al., 2007, Cell Physiol Biochem 20: 735-750). Here, we show that downstream of these kinases, two transcription factors with major roles in control of cell proliferation and death, serum response factor (SRF) and cAMP response element-binding protein (CREB) are differentially regulated in ELA cells. SRF Ser(103) phosphorylation and SRF-dependent transcriptional activity were strongly augmented 5-30 min and 24 h, respectively, after hyperosmotic stress (50% increase in extracellular ionic strength), in a p38 MAPK-dependent manner. In contrast, CREB Ser(133) was transiently dephosphorylated upon osmotic shrinkage. The ERK1/2 effector ribosomal S kinase (RSK) and the ERK1/2- and p38 MAPK effector mitogen- stress-activated protein kinase 1 (MSK1) both phosphorylate CREB at Ser(133) . RSK and MSK1 were dephosphorylated within 5 min of shrinkage. MSK1 phosphorylation recovered within 30 min in a p38-MAPK-dependent manner. CREB was transiently dephosphorylated after shrinkage in a manner exacerbated by p38 MAPK inhibition or MSK1 knockdown, but unaffected by inhibition of RSK. In conclusion, in ELA cells, hyperosmotic stress activates SRF in a p38 MAPK-dependent manner and transiently inactivates CREB, likely due to MSK1 inactivation. We suggest that these events contribute to shrinkage-induced changes in gene transcription and death/survival balance.