Abstract Pancreatic cancer (PC) is currently the only major form of cancer that still has survival rates in the single digits. The limited success of cancer therapies in treating pancreatic cancer has been primarily due to the immunosuppressive tumor microenvironment. In particular, there is an increased prevalence of regulatory T cells (Tregs) (CD4+CD25+CD127−), which suppress anti-tumor immune responses in PC tumor-bearing (TB) hosts. Tregs express the Forkhead BoxP3 (FoxP3) gene, which is critical for their suppressive function. Studies have shown that Ikaros, a zinc finger transcription factor, is an important regulator of T lymphocyte development and function. The regulation of Ikaros’ expression and function is controlled by post-translational modification events. Deficiencies in Ikaros are observed in various T cell leukemias and lymphomas. However, little is known about the possible role of Ikaros in regulating immune cell development in response to solid cancers. In this study, we aim to identify the role of Ikaros in regulating Treg homeostasis and function in a pancreatic tumor microenvironment. Therefore, using our murine model of pancreatic cancer, we isolated splenocytes from TB and control mice and performed flow cytometry and magnetic activated cell sorting (MACS) to immunophenotype and enrich T cell populations for in vivo and in vitro analyses. Also, we performed quantitative real-time PCR (qRT-PCR) and western blot analyses to evaluate Ikaros and FoxP3 mRNA and protein expression in whole and enriched CD3+ T cells from TB and control splenocytes. Our results showed that effector T cell percentages (CD4+ and CD8+) were significantly lower in splenocytes from TB mice compared to control. However, our results also showed a significant expansion of Tregs in splenocytes from TB mice compared to control. In addition, enriched TB Tregs (CD4+CD25+) suppressed antigen-specific CD8+T cell immune responses in a dose-dependent manner, in vitro. Preliminary qRT-PCR results revealed no significant difference in Ikaros mRNA expression; whereas, Ikaros protein expression was reduced in TB whole splenocytes compared to control. Also, Ikaros protein expression was reduced in TB enriched CD3+ T cells compared to control. Furthermore, our results showed an increase in FoxP3 protein expression in TB CD3+ T cells compared to control. These findings suggest that the pancreatic tumor microenvironment potentially down-regulates Ikaros’ protein expression, which may contribute to the expansion of Tregs and their suppression of CD8+T cell (anti-tumor) immune responses. Citation Format: Nadine Nelson, Maya Jerald, Laura Pendleton, Karoly Szekeres, Nasreen Vohra, Shari Pilon-Thomas, Tomar Ghansah. The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2013-4975
Argininosuccinate synthase (AS) is an essential mediator of endothelial health by providing a dedicated source of arginine for nitric oxide (NO) production and promoting endothelial cell viability. Our laboratory has previously demonstrated that AS is present in endothelial caveolar fractions along with endothelial nitric oxide synthase (eNOS) and argininosuccinate lyase (AL), the core components of the citrulline‐NO cycle. To expand on those studies, we utilized immunofluorescence microscopy and showed that AS localized to the Golgi, perinuclear region and plasma membrane in conjunction with eNOS. We also demonstrated that AS co‐localizes with caveolin‐1 and HSP90, key regulators of eNOS function. Co‐immunoprecipitation studies showed that AS interacts with caveolin‐1 and HSP90. In the case of caveolin‐1, a binding motif in the AS protein sequence suggested a direct interaction. To characterize the nitric oxide metabolome from a more global perspective, we utilized co‐immunoprecipitation followed by mass spectrometry to identify several putative AS and eNOS interacting partners that are either novel or understudied in the regulation of NO production. Our work highlights the functional co‐localization and interactions of citrulline‐NO cycle components in vascular endothelial cells, which has important implications for our understanding of vascular biology.
Abstract Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for cellular nucleic acid-binding protein which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7 alpha-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feed-back regulation of hepatic HMG-CoA reductase gene expression does not occur at the level of transcription.
Nitric oxide (NO) signaling is required for vascular function and its homeostasis is altered in atherosclerosis and/or vascular complications of diabetes. The recycling of citrulline to arginine creates a distinct pool of arginine critical for NO production and is known as the citrulline‐NO cycle. Argininosuccinate synthase (AS) is the rate‐limiting enzyme for NO production, and therefore its regulation affects NO synthesis. Our laboratory has recently identified a biologically relevant AS phosphorylation site at serine 328 that responds to physiological cues in cultured bovine aortic endothelial cells (BAECs). In this report, we describe an investigation into the relevant pathways that regulate AS function through the phosphorylation of serine 328. We show by kinase inhibition, kinase knockdown, and in vitro kinase activity assays that protein kinase C α (PKCα) phosphorylates AS at serine 328 as the result of calcium dependent stimulation of NO in BAECs. In addition, we show signaling pathways that do not involve PKCα, such as insulin or VEGF signaling, decrease phosphorylation of serine 328. In conclusion, these data suggest that PKCα mediated phosphorylation of serine 328 occurs under conditions that promote NO production in response to distinct physiological cues that are dependent on calcium signaling in endothelial cells.
Livers from hypophysectomized rats had low levels of glyceraldehyde 3-phosphate dehydrogenase mRNA. Administration of L-triiodothyronine increased these levels over 20-fold. The peak response was seen 72 h after hormone administration. A half-maximal response was obtained with 5 micrograms of T3 per 100 g of body weight. Thus the expression of hepatic glyceraldehyde 3-phosphate dehydrogenase appears to be regulated by thyroid hormone.
Endothelial dysfunction associated with elevated serum levels of TNF-α observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-α (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-α with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-α suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-α may be mediated, in part, via the NF-κB signaling pathway. These findings demonstrate that TNF-α coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-α is an added insult to endothelial function because of its important role in NO production and in endothelial viability.
Ammonium, or a metabolite of ammonium, represses the expression of nitrate reductase (NR) in Chlorella vulgaris. The removal of ammonium and addition of nitrate (induction) resulted in a rapid (20 min) peaked synthesis of NR mRNA. Nitrate reductase protein and activity increased at a much lower rate, reaching their maxima by 8 h. Ammonium added to nitrate-grown cells resulted in a dramatic decrease in NR mRNA from a steady-state level to undetectable levels within 15 min of ammonium addition. Nitrate reductase activity and protein levels decreased to 20% and 40% of initial levels respectively over 8 h. The half-life for NR mRNA under these conditions was estimated to be less than 5 min, compared with 120 min for NR protein. Such rapid decreases in NR mRNA suggested a degradation and/or cessation in NR mRNA transcription. No apparent difference in NR mRNA-specific RNAase activity of crude cell extracts (NR-induced or repressed) was observed. However, a significant difference in the susceptibility to degradation of NR mRNA from long-term nitrate-grown cells compared with the NR mRNA isolated from short-term induced cells (20 min in nitrate) was observed. NR mRNA isolated from long-term-nitrate-grown cells was completely degraded by RNAases in cell extracts under conditions in which the NR mRNA isolated from short-term induced cells was resistant to degradation. These results suggest that mRNA stability may be an important factor in the metabolic regulation of assimilatory nitrate reductase in Chlorella.
