Investigating the role of retinoic acid (RA) in the non-human primate testis

Retinoic Acid (RA) is essential for germ cell development in the rodent testis. In the absence of RA, germ cell development is halted. RA triggers undifferentiated A spermatogonia to transition into differentiating A1 spermatogonia, which is commonly referred to as the A to A1 transition. This transition is marked by the co-expression of STRA8 and KIT (both RA responsive markers) and by the expression of retinoic acid receptor gamma (RARG) directly prior to this transition, but loss of expression of this receptor shortly after the transition occurs. Whether or not an equivalent of this transition exists in the primate testis and/or whether RA is involved is unknown. We attempted to stain non-human primate testis with the mouse STRA8 antibody, but no staining occurred. Next, we produced a rabbit polyclonal antibody against the full-length recombinant primate STRA8 (pSTRA8) protein. Using immunohistochemistry, we stained non-human primate testis with pSTRA8. Our preliminary data suggests that a subpopulation of primate spermatogonia and primary spermatocytes may be RA responsive as they express pSTRA8 in specific stages along the length of the seminiferous tubules. Specifically, Stage VII-IX contains a ring of pSTRA8 immuno-positive primary spermatocytes along the outside of the tubule, which are the stages where entry into meiosis occurs. Additionally, we performed an organotypic culture of fresh non-human primate explant tissue and treated with DMSO (vehicle control), BDAD (inhibitor of RA synthesis) only, and either BDAD + all-trans RA or BDAD + 13cis RA. The inhibition of RA synthesis or the addition of RA in culture with non-human primate testis tissue altered the RA degrading enzyme, Cyp26a1, expression suggesting the cells in the non-human primate testis are responding appropriately to the treatment. However, more work needs to be done to measure Stra8 expression. The expression of pSTRA8 in a subpopulation of primate spermatogonia and in primary spermatocytes implies that RA may play a role in primate spermatogonial differentiation, as in the rodent testis involving a similar A to A1 transition, as well as entry into meiosis. This data aids in our investigation of the role of RA in spermatogonial differentiation in the non-human primate testis

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection

Glutamine uptake is increased by latent KSHV infection.

<p><b>(A)</b> Intracellular glutamine levels are elevated following KSHV infection. Box and whisker plot showing relative level of intracellular glutamine determined in a metabolomics screen of Mock- (white) and KSHV-infected (grey) TIME cells at 48 and 96 hpi[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005052#ppat.1005052.ref008" target="_blank">8</a>]. The average intensity of intracellular glutamine from six biological replicates is denoted by (+) sign. <i>P</i>-value < 0.0001 at both time points. <b>(B)</b> Glutamine uptake is increased during latent KSHV-infection. Ninety-six hpi, Mock- and KSHV-infected TIME cells were incubated with [³H]-Glutamine for 10 min, followed by intracellular quantification of radioactivity, normalized to total protein. Error bars represent the SEM of three independent experiments, <i>p</i>-value < 0.05.</p

Glutamine metabolism is required for the survival of latently infected endothelial cells.

<p><b>(A)</b> At 20 hpi, mock- or KSHV-infected TIME cells were re-seeded into 24-well plates in triplicate. Cells were treated with replete (REP) or glutamine-free (-GLUT) media containing the fluorescent dye YOYO-1 to identify dead cells or SytoGreen24 to identify total cell number. Cells were imaged every 2 hours for 48 hours on the Essen BioScience IncuCyte. The line graph displays percent cell death (Average YOYO-1 positive cells/Average SytoGreen positive cells) for every time point over 48 hours of treatment, while the bar graph shows the percent cell death at t = 0, 24, and 48 hours post treatment. Data shown represents the average of three independent experiments. Error bars are SEM, and a <i>p</i>-value < 0.0001 is represented by three asterisks. <b>(B)</b> IncuCyte microscopy images identifying dead cell nuclei (YOYO-1) for Mock- and KSHV-infected cells in replete or glutamine-free media at 48 hours post treatment (72 hpi). Essen software was used to identify cell nuclei by size and fluorescent intensity, with background subtracted. YOYO-1 positive nuclei are in white. <b>(C)</b> TIME cells were Mock-, KSHV-, or KSHV-UV (UV-irradiated) infected and prepared as described in panel A. Cells were imaged every 2 hours for 48 hours on the Essen BioScience IncuCyte. Fold increase in cell death of each sample over Mock replete at t = 0 is shown. Error bars represent SEM, <i>p</i>-value < 0.0001 and “ns” is shown where the averages are not significantly different. <b>(D)</b> Primary hDMVECs were re-seeded into 24-well plates at 48 or 72 hpi and were treated with Replete (4 mM glutamine) or glutamine-free media and 48 hours post treatment were scanned on the Typhoon 9400 variable mode imager (GE Healthcare) and analyzed with ImageJ software for relative fluorescence. Data shown represent the average of three independent experiments. Error bars are SEM. A <i>p</i>-value < 0.0001 is represented by three asterisks and “ns” is shown where the averages are not significantly different.</p

Glutamine starvation leads to apoptosis of KSHV-infected endothelial cells.

<p>At 20 hpi, Mock- or KSHV-infected TIME cells were re-seeded into 24-well plates in triplicate. Cells were treated with replete (REP) or glutamine-free (-GLUT) media in the presence or absence of 20 μM QVD (pancaspase-inhibitor) and <b>(A)</b> YOYO-1 or SytoGreen24 were added to wells to quantify dead cells and total cell numbers, respectively. Each condition was examined in triplicate, using the Essen BioSciences IncuCyte. Percent cell death was calculated (YOYO-1 positive cells/SytoGreen24 positive cells). Data represents the average of three independent experiments. Error bars represent SEM. A <i>p</i>-value < 0.01 is denoted by two asterisks and “ns” is shown for averages that are not significantly different. <b>(B)</b> The Cell Event Caspase-3/7 substrate was added to all wells. Each condition was examined in triplicate at 48 hours post treatment, and scanned for relative fluorescence on the Typhoon. Relative fluorescence shown is normalized to SytoGreen24 levels (total cell count). Data represents the average of three independent experiments. Error bars represent SEM. A <i>p</i>-value < 0.0001 is denoted by three asterisks and “ns” is shown where two averages are not significantly different. <b>(C)</b> Representative microscopy images of Caspase-3/7 samples at 48 hours post treatment (72 hpi). Live cell imaging was captured on the Cellomics ArrayScan Vti.</p

MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV.

<p><b>(A)</b> TIME cells were transfected with siControl or siMondoA and then Mock- or KSHV-infected 24 hours post transfection. Whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis for MondoA, Mlx, SLC1A5 and TXNIP. KSHV infection elevates levels of all four proteins and loss of MondoA reduces the observed increase in protein expression. The standard γ-tubulin was included as a loading control. <b>(B)</b> Twenty-four hours post transfection of TIME cells with siControl or siMondoA, cells were Mock- or KSHV-infected and overlaid with replete media in the presence or absence of αKG. YOYO-1 or SytoGreen was added to the media to monitor relative fluorescence for cell death or total cells, respectively. Forty-eight hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM. A <i>p</i> < 0.05 is denoted by one asterisk and “ns” is shown where two averages are not significantly different.</p

Endothelial cells latently infected with KSHV require the glutamine transporter SLC1A5 for survival.

<p><b>(A)</b> 20 hpi, Mock- or KSHV-infected TIME cells were re-seeded into 24-well plates in triplicate. Cells were treated with replete (REP) media in the presence or absence of 5mM GPNA, a specific inhibitor of SLC1A5, in the presence or absence of 3.5 mM αKG and scanned on the Typhoon at 48 hours post treatment (72 hpi). Glutamine-deprived (-GLUT) controls were included for comparison. Data shown is the average relative fluorescence (YOYO-1 positive cells/SytoGreen24 positive cells) from three independent experiments. Error bars represent the SEM. A <i>p</i>-value < 0.01 is denoted by two asterisks. <b>(B)</b> TIME cells were transfected with siControl or siSLC1A5. siSLC1A5 treatment leads to an approximately 70% reduction in SLC1A5 expression, determined by qRT-PCR for SLC1A5. Expression was normalized to the housekeeping gene GAPDH. <b>(C)</b> Twenty-four hours post transfection of TIME cells with siControl or siSLC1A5, cells were Mock- or KSHV-infected. Upon completion of the infection, cells were treated with replete media containing YOYO-1 to identify dead cells or SytoGreen24 to identify total cell number. 48 hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM.</p

KSHV infection of endothelial cells increases protein expression of the Myc/MondoA network and downstream targets, including the glutamine transporter SLC1A5.

<p>TIME cells were Mock- or KSHV-infected and whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis using the indicated antibodies. Υ-tubulin and β-actin standards were included as loading controls.</p

Glutamine uptake is increased by latent KSHV infection.

<p><b>(A)</b> Intracellular glutamine levels are elevated following KSHV infection. Box and whisker plot showing relative level of intracellular glutamine determined in a metabolomics screen of Mock- (white) and KSHV-infected (grey) TIME cells at 48 and 96 hpi[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005052#ppat.1005052.ref008" target="_blank">8</a>]. The average intensity of intracellular glutamine from six biological replicates is denoted by (+) sign. <i>P</i>-value < 0.0001 at both time points. <b>(B)</b> Glutamine uptake is increased during latent KSHV-infection. Ninety-six hpi, Mock- and KSHV-infected TIME cells were incubated with [³H]-Glutamine for 10 min, followed by intracellular quantification of radioactivity, normalized to total protein. Error bars represent the SEM of three independent experiments, <i>p</i>-value < 0.05.</p