bluuebunny/crossref_metadata_2025 huggingface dataset:
SKP2 Inactivation Suppresses Cell Proliferation and Regulates AR/FOXA1 Expression in PCa Cells
Authors: Celada Sherly I.
Published: June 2021

Increased levels of SKP2 (S‐phase kinase‐associated protein 2), an E3 ubiquitin ligase, is frequently seen in advanced stages of prostate cancer (PCa). Correlations between SKP2 and the ubiquitin‐mediated degradation of key cell cycle regulators such as p21 and p27, as well as between SKP2 and the regulation of androgen receptor (AR) activity is frequently observed in PCa progression. Despite these associations, the molecular mechanisms responsible for the proto‐oncogenic effects of SKP2 in PCa remain elusive. The transcription factor FOXA1 (Forkhead box protein A1) is known to induce AR activity, and by doing so cause the activation of AR target genes. Deregulation of the AR/FOXA1 complex has been found to contribute to the progression of PCa and castration‐resistant prostate cancer (CRPC). We hypothesized that SKP2 impacts the function of the AR/FOXA1 complex contributing to the progression of CRPC. Using shRNA technology, we established stable SKP2 knockdown PCa cell lines to investigate the effects of SKP2 on CRPC growth. Our results demonstrate that SKP2 plays a critical role in the regulation of AR and FOXA1 expression in CRPC cell lines. SKP2 knockdown increased AR and FOXA1 levels in C4‐2B and 22RV1 cells, two CRPC cell lines. Furthermore, SKP2 knockdown significantly inhibited cellular proliferation and restored sensitivity to the AR antagonist (MDV3100) in C4‐2B and 22RV1 cells. Combined exposure to the AR antagonist (MDV3100) and a SKP2 inhibitor significantly reduced cellular proliferation, a result of cell cycle arrest and cellular senescence in C4‐2B and 22RV1. Endogenous and Myc‐tagged SKP2 as well as Flag‐tagged FOXA1 immunoprecipitation displayed a physical interaction between SKP2 and FOXA1. In vivo ubiquitination assay was performed using HEK293T cells transfected with HA‐Ub constructs demonstrating a SKP2 E3 ubiquitin ligase function for FOXA1. Our findings present a potential SKP2‐AR/FOXA1 signaling pathway that may be targeted therapeutically to inhibit CRPC malignancy.

Support or Funding Information

2U54CA163066

MVTCP CA163069

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural organization of the FtsLB complex of the bacterial divisome
Authors: Senes Alessandro , Condon Samson G.F. , Mahbuba Deena‐Al , Armstrong Claire R. , Diaz‐Vazquez Gladys , Craven Samuel J. , Cui Qiang
Published: June 2021

In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. We present a structural analysis of the FtsLB complex – performed with biophysical, computational and in vivo methods – that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co‐evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain likely terminates near the critical Constriction Control Domain, which might correspond to a structural transition. Presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ‐binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly‐rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions, and that integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

Support or Funding Information

The work was supported by National Institutes of Health Grant (NIH) R01‐GM0997522 and National Science Foundation (NSF) grant CHE‐1415910 and DMS‐1661900. S.G.F.C. and G.D.V. acknowledge support by the NLM training grant 5T15LM0073.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating host protein binding to SARS coronavirus untranslated region using immobilized RNA
Authors: Gonzalez Fabiola , Nag Anita
Published: June 2021

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) encoded nonstructural protein 1 (Nsp1) is a key factor in dampening host gene expression, also known as host shut‐off. Nsp1 binds to the 40S ribosome, stalls ribosome assembly, and results cleavage of cellular messenger RNA (mRNA). Surprisingly, SCoV mRNA is resistant to cleavage under the same condition, allowing us to investigate host protein binding by SCoV mRNA that confers resistant to Nsp1‐initiated cleavage. The untranslated region of SCoV mRNA is about 100 nucleotides long and contain a RNA stem‐loop necessary for resisting endonucleolytic cleavage. We used T7 polymerase driven transcription and T4 RNA ligase to synthesize biotinylated RNA containing untranslated region of SCoV mRNA. The RNA was then incubated with cellular proteins in the presence of Nsp1 followed by UV crosslinking. RNA‐protein complexes were isolated using streptavidin‐coated magnetic beads. Proteins were separated by gel electrophoresis followed by their identification using LC‐MS/MS. Identification of these proteins will allow us to decipher the mechanism of host shutoff by Nsp1.

Support or Funding Information

SC INBRE

Furman Advantage

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glyceraldehyde‐3‐phosphate dehydrogenase differentiates the cytotoxic mechanisms of cancer‐active electrophiles in a yeast model
Authors: Harach Janel L , Lynch Connor J , Montovano Giancarlo , Olsen Stefan H , Foley Timothy D
Published: June 2021

Diverse examples of thiol‐reactive electrophiles, ranging from trivalent arsenicals to 3‐bromopyruvate (3BP), have shown promise as anti‐cancer agents. Inhibition of the glycolytic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has been identified as a primary mechanism underlying the anti‐cancer activity of 3BP but the importance of targeting GAPDH for the cellular actions of arsenicals remains uncertain. We have studied the effects of 3BP and the representative trivalent arsenical phenylarsine oxide (PAO) on the growth, viability, metabolism, and GAPDH activity of the yeast Saccharomyces cerevisiae, which reproduces metabolic features of Warburg‐positive tumor cells. Both 3BP and PAO abolished growth and, within hours, caused substantial cell death measured by propidium uptake and fluorescence. Neither compound increased ROS levels nor inhibited protein synthesis. Indeed, lower concentrations of PAO appeared to decrease cellular ROS. Critically, exposure of intact cells to 3BP produced a rapid and nearly complete inhibition of GAPDH activity. In contrast, PAO did not inhibit the cellular activity of GAPDH despite findings, also to be presented, that GAPDH was one of many proteins from yeast lysates able to bind immobilized PAO. These results suggest that changes in the activity of GAPDH, in situ, may differentiate the cytotoxic pathways of cancer‐active electrophiles. The mechanism(s) underlying the impairment of growth and viability of S. cerevisiae by arsenicals remains under investigation.

Support or Funding Information

Funding was provided by the University of Scranton

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Calcineurin B Homologous Protein (CHP) Isoform Specific Function
Authors: Davis Shane , Provost Joseph
Published: June 2021

Mammalian cells ubiquitously express the Sodium‐Hydrogen Exchanger Isoform 1 (NHE1) a membrane transporter responsible for intracellular pHi, motility and proliferation. Calcineurin B Homologous Protein (CHP) in part regulates activation and kinetics of the NHE1. CHP has two isoforms, CHP1 and CHP2, whose physiological function remains unclear but both have a unique role in nascent tumor survival. CHP binds NHE via the N and C terminal domains. There is a considerable tertiary structure between both the N terminal domains (amino acids 1–90) and the C terminal domain (aa 111/110–195/196) with a 72% and 74% conserved homology, an intervening loop domain is unique to each CUR. This signature domain CHP unique region (CUR: CHP1 R91 to S110 and CHP2 R91 to S111) when bound to NHE has a specific and distinct structure indicating its potential role in effecting CHP‐NHE1 related functions. The purpose of this study is to investigate how the these CHP unique regions contribute to CHP isoform cell function, specifically cell proliferation and location. Using site directed mutagenesis, we have generated epitope tagged (DDK and MYC) wild type versions of both isoforms of CHP and a variant with the respective CUR domain deleted (CHP1 ΔCUR1 and CHP2ΔCUR2). To distinguish the importance of the CUR domains we have swapped CUR domains between each CHP (CHP2(CUR1) and CHP1(CUR2). These mutations will be expressed in lung fibroblasts expressing NHE1 (CCL39) and PS120 cells (null NHE1 expressing CCL39 derivatives) to determine the impact of each CUR domain on cellular location in the absence and presence of NHE1 and on cell proliferation. We hypothesize that the CUR domain is critical for CHP isoform specific function and NHE1 interactions and present the role of both CHP isoforms on NHE1 related behavior.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Vesicle Formation at the Golgi Complex by the Arf GEFs Gea1 and Gea2
Authors: Muccini Arnold , Brownfield Bryce , Gustafson Margaret , Fromme J. Christopher
Published: June 2021

In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs) Gea1, Gea2, and Sec7. Sec7 activates Arf1 at the trans‐Golgi network, and mechanisms regulating the activity and recruitment of Sec7 to the Golgi have been well characterized. For example, Sec7 is recruited to the TGN by four small GTPases including its substrate Arf1. However, Gea1 and Gea2, which activate Arf1 at earlier Golgi compartments, do not share the same regulatory mechanisms as Sec7. While initially thought to be redundant paralogs, we have found that Gea1 and Gea2 localize to separate regions of the Golgi despite sharing common regulatory elements. As such, the purpose and mechanism for their differential localization remains unclear. I aim to understand the functional relevance for their distinct localization and will present preliminary evidence suggesting that Gea recruitment is coordinated by Golgi SNARE complexes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel p53 barrier destruction mechanism through MMP‐7 mediating nucleolar matrix assembly & intra‐nucleolar cleaving p53 during cancer stem cell transformation
Authors: Yu wei‐hsuan , Wu Erxi , Li Yongqing , Yu Shuan Su C. , Huang Po Tsang
Published: June 2021

The molecular mechanism underlying the striking enlarged nucleolus instead of disperse small nucleolus in cancer stem cells remains elusive. Our novel findings that MUC‐1 C‐ter participates in nucleolus matrix assembly has further prompted the idea that the cell surface receptor MUC‐1, via MMP‐7 proteolytic cleavage of the MUC‐1 SEA domain releases cryptic bioactive fragments that orchestrate nucleolar activities. Furthermore, MUC‐1 C‐ter induces a unique porous bulky ball‐shaped, cage‐like nucleolus that functions as a nucleus molecular “garage”for potent transcription factors, such as γ‐catenin and p53. Specifically, this cage segregates the transcription factors from its target promoter. In addition, the intra nucleolus degradation of p53 by MMP‐7 to generate a 35 kDa p53 fragment suggests that the nucleolus could also facilitate the novel subnucleus compartment for proteolytic processing tumor suppressor protein, p53 in cancer stem like cell, which implicate the novel molecular mechanism for destructing the p53 barrier and inducing cancer stem cell transformation. Salinomycin, an anti‐cancer stem cell agent, can disrupt the nucleolus inducing translocation of p53 and sensitize cancer stem cell to chemotherapy drug. These data provide important new insights into the dysregulations of cancer stem cells as well as highlight targets for cancer therapeutics.

Support or Funding Information

Taiwan Ministry of Science and technology 94‐2320‐B‐002 ‐120

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CaMKIIa regulates let‐7 microRNAs in opioid tolerance
Authors: He Ying , Wang Zaijie Jim
Published: June 2021

We have previously reported that let‐7 miRNAs modulate opioid tolerance, through complex mechanisms that involve the μ opioid receptor and other signaling molecules. Chronic opioid treatment up‐regulated brain let‐7 miRNA expression, which was functionally correlated with the development of opioid tolerance. The aim of this study was to understand the mechanisms by which opioids regulate let‐7 miRNA. We determined the let‐7 transcript and found that the expression of primary let‐7 (pri‐let‐7) remained unchanged in human neuroblastoma SH‐SY5Y cells that were treated with morphine (1 mM, for 48 h). In agreement with the in vitro findings, chronic morphine treatment induced opioid tolerance, but did not alter the level of pri‐let‐7 in mouse brain. These data suggested that the robust elevation of let‐7 occurred at the post‐transcriptional level. In the presence of KN93, inhibitor of Ca2+ /calmodulin‐dependent protein kinase II (CaMKII), chronic morphine treatment failed to generate let‐7 up‐regulation in SH‐SY5Y cells. In CaMKIIaT286A point mutation mice lacking functional CaMKIIa, opioid tolerance was absent in −/− mice, but tolerance was full developed in littermate wildtype +/+ mice. Interestingly, let‐7 miRNA levels were much lower in CaMKIIaT286A mutant mice than those in wild‐type mice. Moreover, morphine failed to regulate let‐7 miRNA in CaMKIIaT286A mutant (−/−) mice. Taken together, these data suggested that CaMKIIa is required for the biogenesis and possibly regulation of let‐7 miRNA in opioid tolerance.

Support or Funding Information

R01 DA041809

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Doxorubicin Mitochondrial Dysfunction and Cytotoxicity in Human Proximal Tubular Epithelial Cells is Attenuated by Resveratrol (RES)
Authors: Valentovic Monica A , Brown Kathleen C , Dial Mason , McGuffey Rachel
Published: June 2021

Doxorubicin (DOX, Adriamycin), is a cancer chemotherapy agent used in the treatment regimen for breast, Hodgkin and Non‐Hodgkin lymphomas, small cell lung cancer and acute lymphoid leukemia. Unfortunately, doxorubicin clinical usage is associated with permanent cardiomyopathy and renal impairment. The cellular mechanisms for doxorubicin mediated renal cytotoxicity have yet to be understood. Resveratrol (RES) is a phytoalexin found in many fruits including grapes, blueberries, cranberries as well as chocolate and peanuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies in cancer patients as well as animal models. This study tested the hypothesis that RES protection for DOX renal cytotoxicity, in human noncancerous renal proximal tubular epithelial cells (HK‐2), is mediated by preserving mitochondrial function and attenuating oxidative stress. HK‐2 cells were plated and grown in cell culture equilibrated for 48 h. Cells were next pre‐incubated for 1 h with 0 (DMSO) or 5 or 7.5 uM RES followed by a 24 h co‐incubation with 0–5 uM DOX. All results were obtained from 3 independent experiments. Western analysis probed for protein carbonylation was used as an indicator of oxidative stress. Mitochondrial function was assessed using a Seahorse analyzer system. Prior to examining mitochondrial function, cell number and substrate concentrations were optimized based on the manufacturer's recommendations. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Western blot was conducted on whole cell lysate for SIRT1 and PGC1alpha. Viability was assessed using MTT leakage and tyrpan blue exclusion cell counts. RES did not alter cell viability as indicated by comparable MTT values between DMSO and RES groups (p>0.05). DOX was cytotoxic to HK‐2 cells within 24 h. Pretreatment with RES provided protection from DOX to HK‐2 cells. Mitochondrial respiration assessed as oxygen consumption rate (OCR) was diminished by DOX. DOX impaired mitochondria function within 24 h and RES provided protection. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial function. Further studies are needed to probe changes in specific mitochondrial proteins mediated by DOX.

Support or Funding Information

Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence. M.D. and R.M. supported by WV NASA Undergraduate Research Fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physical Interactions between Heme Oxygenase 1 and the Cytochromes P450 in the Endoplasmic Reticulum Membrane
Authors: Connick John Patrick , Reed James R , Backes Wayne L
Published: June 2021

Heme oxygenase 1 (HO‐1) and the cytochromes P450 (P450s) are endoplasmic reticulum (ER)‐bound enzymes that rely on NADPH‐cytochrome P450 reductase (POR) to provide the electrons that fuel their catalytic cycle. Previous work has shown that under normal conditions POR levels are limiting and P450s must compete for POR binding. Our lab and others have also shown that, in addition to simple competition, certain P450s can bind POR as multimeric P450•P450 complexes that are associated with altered P450 activity. Because HO‐1 also relies on physical interaction with POR in the ER, we hypothesized that it too would compete with the P450s for POR binding. Additionally, the prevalence of P450•P450 interactions led us to consider the possibility that HO‐1 might form analogous homomeric (HO‐1•HO‐1) or heteromeric (HO‐1•P450) complexes that could affect POR binding and enzyme activity. To gain insight into how these enzymes are physically organized, we used bioluminescence resonance energy transfer (BRET). We implemented BRET by expressing a pair of tagged proteins, one with a green fluorescent protein (GFP) tag and one with a Renilla luciferase tag, in HEK293T cells. Energy transfer only occurs if the tags are in close proximity, allowing us to detect protein complexes by observing changes in the tag's emission spectrum. We are also able to assess the relative stability of protein complexes by observing how co‐expression of a third, untagged protein affects the BRET signal of the tagged pair. Using this technique, we have determined that HO‐1 forms homomeric complexes that are more stable when POR is limiting. We have also found that HO‐1 forms heteromeric complexes with several P450s including CYP1A1 and CYP2D6. The BRET signal for the HO‐1•CYP2D6 complex was robust in both the presence and absence of co‐transfected untagged POR, showing that the complex is stable when POR is present. Interestingly, the BRET signal for the HO‐1•CYP1A1 complex was low in the absence of POR but increased significantly when cells were co‐transfected with untagged POR. Doubling the amount of POR led to an attenuation of the BRET signal. Current and future work will use BRET to determine whether HO‐1 affects the physical interaction between POR and P450s and, conversely, whether the presence of a P450 can affect the HO‐1•POR complex.

Support or Funding Information

NIH ES004344, NIH ES013648, NIH GM123253

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA
Authors: Riaz Maryam , Cushen Spencer C. , Phillips Nicole R. , Goulopoulou Styliani
Published: June 2021

Background

Vascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell‐free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell‐free mtDNA.

Hypothesis

Mitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death‐dependent mechanisms.

Material and Methods

Human umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80–90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM ‐ 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non‐apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real‐time PCR techniques.

Results

Treatment of HUVECs with rotenone increased early apoptosis (Fig. A) and late apoptosis/necrosis (Fig. B; 1‐way ANOVA followed by Sidak's post‐hoc test). Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone (Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.07; Sidak's post‐hoc test). Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p≥0.84).

Conclusions

Mitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.

Support or Funding Information

Research support: NIH (R25HL125447)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Egr‐1 contributes to the enhanced expression of Giα proteins and hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats
Authors: Polchtchikov Stephanie , Li Yuan , Anand‐Srivastava Madhu B.
Published: June 2021

Early growth response, Egr‐1, is known to activate the expression of several genes implicated in the development of vascular dysfunction. Earlier studies have shown that angiotensin II (Ang II) enhances the expression of Egr‐1 and Giα proteins in vascular smooth muscle cells (VSMC). In addition, the role of enhanced levels of endogenous Ang II has been shown to contribute to the enhanced expression of Giα proteins in spontaneously hypertensive rats (SHR). The present study was therefore undertaken to examine if VSMC from SHR also exhibit enhanced expression of Egr‐1 proteins and explore its role and underlying mechanisms in the hyperproliferation of VSMC in SHR. The levels of Egr‐1 proteins in VSMC from SHR started increasing at 2 weeks as compared to their normotensive control, WKY (Wistar‐Kyoto rats) and at 12 weeks, the levels of Egr‐1 proteins were increased significantly by 80%. The enhanced levels of Egr‐1 proteins were restored to control levels by losartan (an AT1 receptor antagonist), BQ123 (an ETA receptor antagonist), BQ788 (an ETB receptor antagonist), as well as by antioxidants, such as N‐acetyl‐L‐cysteine (NAC) and diphenyleneiodonium (DPI) but not by PD123319 (an AT2 receptor antagonist). In addition, pharmacological inhibitors of platelet‐derived growth factor (PDGF), insulin‐like growth factor (IGF) and epidermal growth factor (EGF) receptors also inhibited the overexpression of Egr‐1 proteins in VSMC from SHR. Furthermore, the enhanced expression of Egr‐1 proteins was attenuated by PD098059, an inhibitor of mitogen‐activated protein kinase (MAPK), but not by wortmannin, an inhibitor of phosphatidylinositol‐3‐kinase (PI‐3‐K). VSMC from SHR exhibit hyperproliferation which was attenuated by knocking down Egr‐1 by siRNA. In addition, siRNA of Egr‐1 also inhibited the overexpression of Giα‐2 and Giα‐3 proteins, as well as cell cycle proteins, including cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4), Cdk2 and phosphorylated retinoblastoma protein (pRb). These results suggest that VSMC from SHR exhibit an overexpression of Egr‐1 proteins, which is attributed to the enhanced levels of endogenous Ang II and ET‐1, oxidative stress, growth factor receptors and MAPK and that the overexpression of Egr‐1, through the enhanced expression of Giα and cell cycle proteins, contributes to the hyperproliferation of VSMC from SHR.

Support or Funding Information

Grant from CIHR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Lipid and Glucose Homeostasis in Mice with Liver‐Specific Deletion of Protein Kinase Cbeta
Authors: Mehta Kamal D , Hassan Faizule , Coppola Vincenzo , Shu Yaoling
Published: June 2021

It is widely recognized that a Western‐style diet increases the risk of non‐alcoholic fatty liver disease (NAFLD) development and its subsequent progression to NASH. Several mechanisms have been proposed to explain the dramatic inducing effect of dietary cholesterol on the progression of inflammation and apoptosis/necrosis of hepatocytes and nonparenchymal cells. Some of these mechanisms are related to dysregulational lipid homeostasis, mitochondrial dysfunction, and oxidative stress. However, the diet‐induced signaling events integrating with the mechanistic processes driving lipid accumulation, mitochondrial dysfunction, and inflammation in non‐alcoholic steatohepatitis (NASH) are not well understood. We recently reported that PKCβ can coordinate multiple sequential and parallel pathways that cooperatively trigger dysregulation of triglyceride, cholesterol and glucose homeostasis, mitochondrial signaling and autophagy, thus can drive critical events in the genesis and progression of NAFLD/NASH. Furthermore, PKCβ has also been reported to be involved in the activation of two main downstream pathways, nuclear factor‐κB and C‐Jun N‐terminal kinase. Consistent with the role of PKCβ in metabolism, systemic PKCβ deficiency protected mice from diet‐induced adiposity, hepatic steatosis and insulin‐resistance. The goal of this study is to define the individual contribution of hepatic PKCβ in the control of hepatic metabolism. To accomplish this, we generated mice specifically lacking the expression of PKCβ in the liver, which represents the first tissue‐specific deletion of this kinase. We compared body compositions, hepatic lipids, insulin sensitivity, glucose tolerance, and insulin signaling responses between high‐fat/high‐cholesterol‐fed control PKCβflox/flox and PKCβLiv−/− mice. We found that hepatocyte‐specific PKCβ deficiency protects from diet‐induced adiposity, with modest alterations in glucose homeostasis. Our data establish that hepatocyte‐restricted PKCβ deletion is sufficient to protect from steatosis, emphasizing this kinase's relevance as a drug target in NAFLD. Hence, hepatocyte PKCβ is a critical hub for integrating nutritional inputs with downstream metabolic events.

Support or Funding Information

NIH R01 HL138198

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genistein, daidzein, and resveratrols stimulate PGC‐1β‐mediated gene expression
Authors: Kamei Yasutomi , Uchitomi Ran , Nakai Shiho , Matsuda Rintaro , Onishi Takumi , Hatazawa Yukino
Published: June 2021

PGC‐1β is a transcriptional co‐activator of nuclear receptors such as the estrogen receptor‐related receptor (ERR). Transgenic overexpression of PGC‐1β in mice increases energy expenditure and suppresses high‐fat diet‐induced obesity. In this study, we screened various food‐derived and natural compounds using a reporter assay system to measure the transcriptional activity of PGC‐1β. Soy‐derived isoflavones, genistein and daidzein, and several resveratrols activated PGC‐1β. Genistein, daidzein, and trans‐oxyresveratrol activated ERR‐responsive element‐mediated reporter activity in the presence of PGC‐1β. Stable overexpression of PGC‐1β in C2C12 myoblasts increased the expression of medium‐chain acyl‐CoA dehydrogenase (MCAD), an important enzyme in fatty acid β‐oxidation. Genistein and daidzein increased MCAD mRNA levels and mitochondrial content in PGC‐1β‐expressing C2C12 cells. These compounds activated ERR/PGC‐1β complex‐mediated gene expression, and our findings may be a practical foundation for developing functional foods targeting obesity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Characterization of Leucine‐Rich Repeat Containing 8 A (LRRC8A) Homomeric Channel
Authors: Yamada Toshiki , Denton Jerod S. , Strange Kevin
Published: June 2021

Regulation of cell volume in response to osmotic perturbations is an essential cellular process. The volume‐regulated anion channel (VRAC) is ubiquitously expressed in vertebrate cells and mediates the swelling‐induced release of chloride and organic solutes. VRAC is encoded by LRRC8 gene family members, LRRC8A‐E. VRAC is a heteromeric channel comprising an essential LRRC8A subunit plus at least one other LRRC8 member. Several high‐resolution cryo‐EM structures of the LRRC8A homomeric channel have recently been published. However, until now, the detailed functional and regulatory properties of the LRRC8A homomeric channel have not yet been characterized. Here, we show that HCT116 cells in which all five Lrrc8 genes have been knocked out produce a small whole‐cell current (~20 pA/pF at +100 mV) when transfected with a plasmid encoding human LRRC8A. In striking contrast to the heteromeric VRAC current, the LRRC8A current is not activated by cell swelling or inhibited by cell shrinkage. However, when cells are patch clamped with an intracellular solution of low ionic strength, the current exhibits slow and modest activation, and is dramatically activated by cell swelling. The degree of swelling‐induced LRRC8A current activation is directly and dose‐dependently related to intracellular ionic strength. Consistent with the heteromeric VRAC current, swelling‐induced activation of LRRC8A current is prevented by removing intracellular ATP. We are currently evaluating the anion selectivity and pharmacological properties of LRRC8A. Our studies provide important new insights into the functional and regulatory properties of the LRRC8A homomeric channels.

Support or Funding Information

This work was funded by National Institutes of Health grant R01DK051610 to K.S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of exogenous ceramide on barrier function of human airway epithelial cells
Authors: Kalsi Kameljit Kaur , Jackson Sonya , Baines Deborah Louise
Published: June 2021

Maintaining the integrity of the airway epithelial barrier is essential in preventing increased permeability to harmful molecules and pathogens, which may lead to increased risk of developing respiratory infection and disease. We have previously shown that in human airway epithelial cells metformin increases tight junction (TJ) assembly after pathogen disruption by promoting the abundance and assembly of full length occludin, and that this process involves phosphorylation of occludin via an AMPK‐PKCζ pathway. During bacterial infection alpha toxin activates the acid sphingomyelinase/ceramide system which can cleave membrane proteins releasing ceramide to form ceramide rich domains, this signals the reorganization of receptors and activation of protein kinase C isoforms such as PKCζ. This study aimed to investigate the effect of N‐Palmitoyl‐d‐erythro‐sphingosine (C16:0 ceramide) on epithelial integrity of human airway epithelial cells (H441 cells). TJ assembly was measured after calcium switch assay by measuring the transepithelial electrical resistance (TEER) and paracellular transport of sodium‐fluorescein which was used as an index of permeability (Papp) in the presence of 0.1 μM or 1 μM ceramide. We found that after 24h there was 11% change in TEER (138 ± 15 vs 125 ± 14 Ω/cm2, P = 0.34; n=8) and 10% change in Papp (16 ± 6 vs 18 ± 7 cm/s x 10−6, P = 0.19; n=3) with 0.1 μM ceramide compared to control. No changes were observed with 1 μM ceramide. Immunofluorescence staining for occludin, claudin‐1 or claudin‐3 did not show a difference in localisation with ceramide treatment compared to control untreated cells. These results indicate so far that in airway epithelial cells activation of PKCζ by ceramide only evokes a minor effect on tight junction reassembly or permeability.

Support or Funding Information

AstraZeneca R&D

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Exosome Release and Inhibited TRPML1 Channel Activity in Podocytes from Mice with Podocyte‐Restricted Deletion of Asah1 Gene
Authors: Li Guangbi , Zhang Qinghua , Yuan Xinxu , Li Pin‐Lan
Published: June 2021

The ceramide hydrolysis by lysosomal acid ceramidase (AC) has been reported to regulate lysosome function, which determines the degradation of multivesicular bodies. In this regard, we found that urinary exosome excretion was remarkably increased in podocyte‐specific AC gene knockout (Asah1fl/fl/Podocre) mice. Given the importance of transient receptor potential mucolipin 1 (TRPML1) channel‐mediated Ca2+ release to lysosome function and its regulation by sphingolipids, the present study tested a hypothesis that podocyte‐specific Asah1 gene deletion inhibits TRPML1 channel activity, leading to lysosome dysfunction and exosome release from podocytes. It was found that exosomes released from Asah1fl/fl/Podocre podocytes were remarkably higher than WT/WT podocytes as detected by nanoparticle tracking analysis. Super‐resolution microscopy showed less fusion of multivesicular bodies and lysosomes in Asah1fl/fl/Podocre podocytes compared with WT/WT podocytes. Using GCaMP3 Ca2+ imaging, it was found that ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased Ca2+ release from lysosomes by 7.35 folds in WT/WT podocytes. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in WT/WT podocytes. In Asah1fl/fl/Podocre podocytes, however, ML‐SA1 was found to have no effects on TRPML1 channel‐mediated Ca2+ release, but sphingosine as an AC product of ceramide hydrolysis remarkably increased TRPML1 channel‐mediated Ca2+ release in both WT/WT podocytes (5.42 folds) and Asah1fl/fl/Podocre podocytes (4.52 folds). Using whole‐lysosome patch clamp recording, we detected ML‐SA1‐induced Ca2+ currents in a dose‐dependent manner through the membrane of lysosomes isolated from WT/WT podocytes. Compared to WT/WT podocytes, intact lysosomes isolated from Asah1fl/fl/Podocre podocytes had evidently blunted opening of TRPML1 channels in response to its agonist, ML‐SA1. AC associated sphingolipids, sphingomyelin, ceramide and sphingosine had different effects on TRPML1 channel activity in WT/WT podocytes, with inhibition by sphingomyelin, no effect from ceramide, but enhancement by sphingosine (2.25 folds). In Asah1fl/fl/Podocre podocytes, sphingosine remained to increase ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release (3.96 folds). These results suggest that the AC deficiency decreases TRPML1 channel activity due to reduction of sphingosine production, which may contribute to lysosome dysfunction and enhanced exosome release.

Support or Funding Information

This study was supported by NIH grants DK54927, DK120491, and HL057244.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Depolarization of mitochondrial membrane by H2O2 regulates Ca2+ signalling in Endothelial Cells in Intact Blood Vessels
Authors: Zhang Xun , Wilson Calum , Lee Matthew , McCarron John G
Published: June 2021

Hydrogen peroxide (H2O2) is a key mitochondrial derived reactive oxygen species (ROS) that acts as a signalling hub in regulating signalling transduction, vasocontraction and vasodilation. Although the physiological roles of ROS in mechano‐stress signal transduction, vascular relaxation and permeability of endothelial cells is acknowledged, there is uncertainty on the mechanisms by which how H2O2 regulates endothelial signalling. In the present study, the effects of H2O2 on Ca2+ signalling and on vasocontraction and vasodilation were investigated in the endothelium of intact rat mesenteric arteries. Endothelium‐dependent vasodilation of intact mesenteric arteries to acetylcholine (ACh) was suppressed by H2O2 (100 μM) and a greater vasoconstriction to phenylephrine occurred. To investigate the underlying endothelium‐dependent mechanisms of H2O2 on vascular response, intracellular Ca2+ signalling in endothelial cells of intact arteries was visualized and recorded with high‐speed and spatial‐resolution. H2O2 (100 nM – 1 mM) alone did not significantly alter basal Ca2+ activity in endothelial cells. However, H2O2 inhibited acetylcholine‐evoked Ca2+ signalling as revealed by decrease in the amplitude of the Ca2+ signal in each activated cell and a reduced number of cells activated. H2O2‐evoked inhibition of Ca2+ signalling was reversed by catalase (1000 U/ml). To determine if the effects of H2O2 were explained by decreased IP3 production or decreased IP3‐evoked Ca2+ release, photoactivatable caged‐inositol 1,4,5‐trisphosphate (cIP3) was used to bypass the synthesis of IP3. Ca2+ release evoked by photolysis of cIP3 supressed by 100 μM H2O2. These results suggest that H2O2 may alter the activity of IP3 receptors. One mechanism by which this may occur is by H2O2 altering mitochondrial activity. Mitochondria exert a profound control of IP3‐evoked Ca2+ release. Acetylchonline‐evoked Ca2+ release was inhibited by proton uncoupler carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) and rotenone, a mitochondrial complex inhibitor. H2O2 (100 μM) depolarized mitochondria membrane potential as measured by the mitochondrial membrane potential sensitive dye, TMRE. The depolarization of mitochondria decreased the organelle's ability to produce ATP and sequester Ca2+ from the local microdomain near IP3 receptors. A higher local concentration of Ca2+ around IP3 receptors inhibited Ca2+ release from endoplasmic reticulum. In summary, H2O2 inhibit Ca2+ release from the internal store in endothelial cells by depolarizing mitochondrial membrane potential to suppress vasodilation in intact mesenteric arteries.

Support or Funding Information

Wellcome Trust and British Heart Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fecal metabolomic response of IL‐10‐deficient mice supplemented with Goji
Authors: Wang Hongbin , Zhang Shuming , Zhu Meijun
Published: June 2021

Background

Chronic gut inflammation is an important risk factor for colorectal cancer (CRC). Our recent study suggests that dietary Goji reduces gut inflammation and increases Bifidobacterium and butyrate‐producing bacteria in interleukin (IL)‐10‐deficient mice. We hypothesized that enhanced Bifidobacterium and other probiotics associated with Goji supplementation alter gut metabolites, which exert beneficial effects to host epithelial cells.

Methods

The GC‐MS analysis was used to profile the fecal metabolomic changes due to Goji supplementation to uncover the potential mechanisms underlying the preventive effects of Goji supplementation on inflammatory bowel diseases.

Results

Thirty‐four compounds were altered due to Goji supplementation, including polyunsaturated long‐chain fatty acids (PUFAs), sugar and sugar alcohols, benzene derivatives, pyridines derivatives, dicarboxylic acids, short‐chain hydroxy acids. Pathway analysis showed that the α‐linolenic acid and linoleic acid metabolism were dramatically affected by dietary Goji intake. Four ω‐6 PUFAs including linoleic acid and arachidonic acid were reduced due to Goji supplementation. Fecal levels of most amino acids, positively correlated with gut inflammation and CRC risk, tended to decrease in the Goji supplemented group along with decreased urease activity.

Conclusion

data provides valuable metabolic clues for unravelling the beneficial effects of Goji supplementation against gut inflammation and related diseases.

Support or Funding Information

USDA‐NIFA #2018‐67017‐27517 and WSU ERI competitive grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Characterization of the Atlantic salmon, Salmo salar L., Di/Tripeptide Transporters PepT1a and PepT1b
Authors: Gomes Ana Silva , Rønnestad Ivar , Murashita Koji , Vacca Francesca , Cinquetti Raffaella , Barca Amilcare , Aksnes Anders , Bossi Elena , Verri Tiziano
Published: June 2021

Amino acids and small peptides in the form of di/tripeptides are key nutrients in animal diets and essential contributors to growth. In mammals, di/tripeptides represent a highly significant fraction of dietary protein after digestion and are transported across the brush‐border membrane of the intestinal epithelium via a single transport protein, the Peptide Transporter 1 (PepT1), a member of the Solute Carrier 15 (Slc15) family. PepT1 is a low‐affinity/high‐capacity transporter that uses an inwardly‐directed proton electrochemical gradient to drive the uptake of di/tripeptides, and peptidomimetics, across the cell membrane. However, in teleost fishes two pept1 genes, namely pept1b and pept1a, have been identified. PepT1b has been studied in many teleost species, including the Atlantic (A.) salmon (Salmo salar L.), whereas PepT1a remain unexplored. The aim of our study was to characterize, the A. salmon PepT1a vs. PepT1b in terms of tissue/organ distribution and function, and possibly extrapolate their physiological role(s).

Despite the extra (4th) whole genome duplication (WGD) in the Actinopterygian lineage, A. salmon has only two pept1 genes (namely pept1a and pept1b), like in other telosts that didn't undergo the extra WGD, e.g., the zebrafish (Danio rerio). At the amino acid level, the two paralogues share 77% similarity, 64% identity. Tissue distribution and relative abundance of A. salmon pept1a and pept1b revealed that both transcripts have a similar expression profile. A. salmon pept1a is highly expressed in the proximal intestine (pyloric caeca ≈ anterior midgut > midgut >> posterior midgut), but ~5‐fold less than pept1b, which is expressed in the same gut regions. No expression signals were detected in the anterior and posterior stomach or the anterior and posterior hindgut.

As assessed by heterologous expression in Xenopus laevis oocytes and Two‐Electrode Voltage Clamp measurements, A. salmon PepT1a is, like PepT1b, a low‐affinity/high‐capacity system, able to sustain an electrogenic, sodium‐independent and pH‐dependent transport of di/tripeptides. Transport associated currents show that the protein exhibits a K0.5 value for the reference dipeptide Gly‐Gln of 0.5 mM at pH 7.6 and of 0.07 mM at pH 6.5.

The pre‐steady state and steady‐state transport currents analysis indicate important functional differences between A. salmon PepT1a and PepT1b transporters. The difference in expression levels and a significantly dissimilar substrate specificity for certain di/tripeptides, such as lysine‐containing peptides, supports the idea of distinct roles in peptide recognition and transport for PepT1a and PepT1b.

Support or Funding Information

Funded by RFFV; 247978 SalmoFeedPlus and Cargill

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ezrin is a novel target in cyst initiation in ADPKD
Authors: Dixon Eryn E , Woodward Owen M
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive renal cystogenesis and other extrarenal manifestations, is one of the most common monogenetic diseases, and is caused by loss of function mutations in PKD1 and PKD2, which encode transmembrane proteins polycystin 1 and 2 (PC1/2). The polycystin proteins have been hypothesized to function as a complex that has been localized to the plasma membrane, cilia, endoplasmic reticulum, and junctions of epithelial cells. The downstream effects of either PC1 or PC2 function loss have not been elucidated. Previously, an investigation of smaller cysts from nephrectomized human ADPKD kidneys revealed a pattern of increased cell width and an altered apical compartment. Subsequently, we observed a decreased abundance and disrupted localization of ezrin, a master scaffold and apical compartment regulator, in similarly characterized cysts. Following this observation, we explored the role of ezrin disruption in a three‐dimensional in vitro model of cystogenesis. Acute inactivation of Pkd2 lead to significant loss of ezrin protein. We confirmed the regulatory relationship between PC2 and ezrin with immunohistochemistry in a genetically inducible in vivo short induction model of Pkd2 inactivation (Pkd2fl/fl Pax8 rtTA TetOCre) that revealed a kidney tubule wide decrease in ezrin at the apical membrane. Lipid protein overlay assays and immunoprecipitation experiments demonstrate both PC2 and ezrin interact with similar phosphoinositides, and possibly with each other, suggesting a role for PC2 in apical compartment and junctional assembly. Functionally, we found that PC2 is critical in junctional integrity, with acute inactivation of Pkd2 causing decreases in transepithelial electrical resistance in inducible primary renal epithelial cells (Pkd2fl/fl Pax8 rtTA TetOCre). Ezrin, expressed in all segments of the kidney, is a regulator of many components of the junctions and apical compartment of renal epithelial cells. The disruption of ezrin following inactivation of Pkd2 in both in vitro and in vivo models, and shared interacting lipids, suggests that PC2 may play a role in the regulation of ezrin in renal epithelial cells and that ezrin may be involved in the initiation of cystogenesis following loss of PC2.

Support or Funding Information

NIDDK F31 NRSA, PKD Foundation, NIDDK Baltimore Polycystic Kidney Disease Research and Clinical Core Center

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.