Browsing by Author "Prigge, Justin R."

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Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase
(2017-06) Prigge, Justin R.; Coppo, Lucia; Martin, Sebastin S.; Ogata, Fernando; Miller, Colin G.; Bruschwein, Michael D.; Orlicky, David J.; Shearn, Colin T.; Kundert, Jean A.; Lytchier, Julia; Herr, Alix E.; Mattsson, Åse; Taylor, Matthew P.; Gustafsson, Tomas; Arnér, Elias S. J.; Holmgren, Arne; Schmidt, Edward E.
Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.
Hydrodynamic Delivery of Cre Proteins to Lineage-Mark or Time-Stamp Hepatocytes In Situ
(2014-03) Sonsteng, K. M.; Prigge, Justin R.; June, Ronald K.; Schmidt, Edward E.
Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles.
Non-Invasive Quantification of Cartilage Using a Novel In Vivo Bioluminescent Reporter Mouse
(2015-07) Mailhiot, Sarah E.; Zignego, Donald L.; Prigge, Justin R.; Wardwell, Ella R.; Schmidt, Edward E.; June, Ronald K.
Mouse models are common tools for examining post-traumatic osteoarthritis (OA), which involves cartilage deterioration following injury or stress. One challenge to current mouse models is longitudinal monitoring of the cartilage deterioration in vivo in the same mouse during an experiment. The objective of this study was to assess the feasibility for using a novel transgenic mouse for non-invasive quantification of cartilage. Chondrocytes are defined by expression of the matrix protein aggrecan, and we developed a novel mouse containing a reporter luciferase cassette under the inducible control of the endogenous aggrecan promoter. We generated these mice by crossing a Cre-dependent luciferase reporter allele with an aggrecan creERT2 knockin allele. The advantage of this design is that the targeted knockin retains the intact endogenous aggrecan locus and expresses the tamoxifen-inducible CreERT2 protein from a second IRES-driven open reading frame. These mice display bioluminescence in the joints, tail, and trachea, consistent with patterns of aggrecan expression. To evaluate this mouse as a technology for non-invasive quantification of cartilage loss, we characterized the relationship between loss of bioluminescence and loss of cartilage after induction with (i) ex vivo collagenase digestion, (ii) an in vivo OA model utilizing treadmill running, and (iii) age. Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence. In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease. We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age. These results indicate that the bioluminescent signal from this mouse may be used as a non-invasive quantitative measure of cartilage. Future studies may use this reporter mouse to advance basic and preclinical studies of murine experimental OA with applications in synovial joint biology, disease pathogenesis, and drug delivery.
TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy
(2019-06) McLoughlin, Michael R.; Orlicky, David J.; Prigge, Justin R.; Krishna, Pushya; Talago, Emily A.; Cavigli, Ian R.; Eriksson, Sofi; Miller, Colin G.; Kundert, Jean A.; Sayin, Volkan I.; Sabol, Rachel A.; Heinemann, Joshua; Brandenberger, Luke O.; Iverson, Sonya V.; Bothner, Brian; Papagiannakopoulos, Thales; Shearn, Colin T.; Arner, Elias S. J.; Schmidt, Edward E.
Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsrâ€“double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.