Laura C. Pendleton

Effect of thyroid hormone on hepatic cholesterol 7α hydroxylase, LDL receptor, HMG-CoA reductase, farnesyl pyrophosphate synthetase and apolipoprotein A-I mRNA levels in hypophysectomized rats

The effects of thyroid hormone on cholesterol 7 alpha hydroxylase, LDL receptor, HMG-CoA reductase, apo A-I and farnesyl pyrophosphate synthetase hepatic mRNA levels were investigated in hypophysectomized rats. Of these mRNAs cholesterol 7 alpha hydroxylase responded the most rapidly and required the lowest dose of T3. Maximal mRNA levels were reached one hr after T3 administration and required 10 micrograms/100g of body weight. These results suggest that the hypocholesterolemic effect of thyroid hormone may be mediated by a primary effect on cholesterol 7 alpha hydroxylase gene expression.

The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells

SUMMARY The enzyme endothelial nitric oxide synthase (eNOS) catalyzes the conversion of arginine, oxygen and NADPH to NO and citrulline. Previous results suggest an efficient, compartmentalized system for recycling of citrulline to arginine utilized for NO production. In support of this hypothesis, the recycling enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase (AL), have been shown to colocalize with eNOS in caveolae, a subcompartment of the plasma membrane. Under unstimulated conditions, the degree of recycling is minimal. Upon stimulation of NO production by bradykinin, however, recycling is co-stimulated to the extent that more than 80% of the citrulline produced is recycled to arginine. These results suggest an efficient caveolar recycling complex that supports the receptor-mediated stimulation of endothelial NO production. To investigate the molecular basis for the unique location and function of endothelial AS and AL, endothelial AS mRNA was compared with liver AS mRNA. No differences were found in the coding region of the mRNA species, but significant differences were found in the 5′-untranslated region (5′-UTR). The results of these studies suggest that sequence in the endothelial AS-encoding gene, represented by position -92 nt to -43 nt from the translation start site in the extended AS mRNA 5′-UTRs, plays an important role in differential and tissue-specific expression. Overall, a strong evidential case has been developed supporting the proposal that arginine availability, governed by a caveolar-localized arginine regeneration system, plays a key role in receptor-mediated endothelial NO production.

Regulation of Endothelial Argininosuccinate Synthase Expression and NO Production by an Upstream Open Reading Frame

Argininosuccinate synthase (AS) catalyzes the rate-limiting step in the recycling of citrulline to arginine, which in endothelial cells, is tightly coupled to the production of nitric oxide (NO). In previous work, we established that endothelial AS mRNA can be initiated from multiple start sites, generating co-expressed mRNA variants with different 5′-untranslated regions (5′-UTRs). One of the 5′-UTRs, the shortest form, represents greater than 90% of the total AS mRNA. Two other extended 5′-UTR forms of AS mRNA, resulting from upstream initiations, contain an out-of-frame, upstream open reading frame (uORF). In this study, the function of the extended 5′-UTRs of AS mRNA was investigated. Single base insertions to place the uORF in-frame, and mutations to extend the uORF, demonstrated functionality, both in vitro with AS constructs and in vivo with luciferase constructs. Overexpression of the uORF suppressed endothelial AS protein expression, whereas specific silencing of the uORF AS mRNAs resulted in the coordinate up-regulation of AS protein and NO production. Expression of the full-length of the uORF was necessary to mediate a trans-suppressive effect on endothelial AS expression, demonstrating that the translation product itself affects regulation. In conclusion, the uORF found in the extended, overlapping 5′-UTR AS mRNA species suppresses endothelial AS expression, providing a novel mechanism for regulating endothelial NO production by limiting the availability of arginine.

Murine pancreatic adenocarcinoma dampens SHIP-1 expression and alters MDSC homeostasis and function.

Background Pancreatic cancer is one of the most aggressive cancers, with tumor-induced myeloid-derived suppressor cells (MDSC) contributing to its pathogenesis and ineffective therapies. In response to cytokine/chemokine receptor activation, src homology 2 domain-containing inositol 5′-phosphatase-1 (SHIP-1) influences phosphatidylinositol-3-kinase (PI3K) signaling events, which regulate immunohomeostasis. We hypothesize that factors from murine pancreatic cancer cells cause the down-regulation of SHIP-1 expression, which may potentially contribute to MDSC expansion, and the suppression of CD8+ T cell immune responses. Therefore, we sought to determine the role of SHIP-1 in solid tumor progression, such as murine pancreatic cancer. Methodology and Principal Findings Immunocompetent C57BL/6 mice were inoculated with either murine Panc02 cells (tumor-bearing [TB] mice) or Phosphate Buffer Saline (PBS) (control mice). Cytometric Bead Array (CBA) analysis of supernatants of cultured Panc02 detected pro-inflammatory cytokines such as IL-6, IL-10 and MCP-1. TB mice showed a significant increase in serum levels of pro-inflammatory factors IL-6 and MCP-1 measured by CBA. qRT-PCR and Western blot analyses revealed the in vivo down-regulation of SHIP-1 expression in splenocytes from TB mice. Western blot analyses also detected reduced SHIP-1 activity, increased AKT-1 and BAD hyper-phosphorylation and up-regulation of BCL-2 expression in splenocytes from TB mice. In vitro, qRT-PCR and Western blot analyses detected reduced SHIP-1 mRNA and protein expression in control splenocytes co-cultured with Panc02 cells. Flow cytometry results showed significant expansion of MDSC in peripheral blood and splenocytes from TB mice. AutoMACS sorted TB MDSC exhibited hyper-phosphorylation of AKT-1 and over-expression of BCL-2 detected by western blot analysis. TB MDSC significantly suppressed antigen-specific CD8+ T cell immune responses in vitro. Conclusion/Significance SHIP-1 may regulate immune development that impacts MDSC expansion and function, contributing to pancreatic tumor progression. Thus, SHIP-1 can be a potential therapeutic target to help restore immunohomeostasis and improve therapeutic responses in patients with pancreatic cancer.

Thyroid hormone rapidly increases cholesterol 7α-hydroxylase mRNA levels in hypophysectomized rats

The induction of hepatic cholesterol 7 alpha-hydroxylase mRNA by triiodothyronine was investigated in hypophysectomized rats. These rats exhibited markedly decreased levels of cholesterol 7 alpha-hydroxylase mRNA compared to normal controls. An increase in this mRNA could be detected within 30 min after giving triiodothyronine. A triiodothyronine dose of 0.25 microgram per 100 g of body weight, which produces 50% occupancy of hepatic nuclear thyroid hormone receptors, caused significant increases in hydroxylase mRNA levels. The half-life of cholesterol 7 alpha-hydroxylase mRNA was about 30 min and was not affected by thyroid hormone. Administration of the protein synthesis inhibitor, cycloheximide, after triiodothyronine caused superinduction. Hepatic HMG-CoA reductase mRNA levels, which are also low in livers from hypophysectomized rats, were unaffected by these doses of hormone within the 2 h time-frame examined in these studies. The results suggest that thyroid hormone may exert a primary affect on the expression of the hepatic cholesterol 7 alpha-hydroxylase gene. This could explain, in part, the hypocholesterolemic effect of thyroid hormone.

Loss of NADPH during assays of HMG-CoA reductase: implications and approaches to minimize errors

In assays of 3-hydroxy-3-methylglutary coenzyme A (HMG-CoA) reductase activity, preincubation of isolated washed microsomes with NADPH led to a time- and protein concentration-dependent, loss of enzyme activity. This occurred despite the presence of an NADPH regenerating system. Addition of fresh NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase restored activity. Of the individual components, only NADP was effective. Errors due to loss of NADPH are most pronounced in assays using high microsomal protein, low NADPH levels and preincubation with NADPH and when glutathione rather than dithiothreitol is present. To minimize the effects of NADPH depletion, it is recommended that (i) NADP and NADPH not be present during the preincubation period; (ii) incubation periods be relatively short; (iii) microsomal protein concentrations be less than 1 mg; and (iv) NADPH concentrations be 1 to 2 mM.

Thyroid hormone increases glyceraldehyde 3-phosphate dehydrogenase gene expression in rat liver

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 μg of T3 per 100 g of body weight. Thus the expression of hepatic glyceraldehyde 3‐phosphate dehydrogenase appears to be regulated by thyroid hormone.

Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues.

Troglitazone up-regulates vascular endothelial argininosuccinate synthase.

Vascular endothelial nitric oxide (NO) production via the citrulline-NO cycle not only involves the regulation of endothelial nitric oxide synthase (eNOS), but also regulation of caveolar-localized endothelial argininosuccinate synthase (AS), which catalyzes the rate-limiting step of the cycle. In the present study, we demonstrated that exposure of endothelial cells to troglitazone coordinately induced AS expression and NO production. Western blot analysis demonstrated an increase in AS protein expression. This increased expression was due to transcriptional upregulation of AS mRNA, as determined by quantitative real time RT-PCR and inhibition by 1-d-ribofuranosylbenzimidazole (DRB), a transcriptional inhibitor. Reporter gene assays and EMSA analyses identified a distal PPARgamma response element (PPRE) (-2471 to -2458) that mediated the troglitazone increase in AS expression. Overall, this study defines a novel molecular mechanism through which a thiazolidinedione (TZD) like troglitazone supports endothelial function via the transcriptional up-regulation of AS expression.

In sire determination of the functional size of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase by radiation inactivation analysis

Radiation inactivation analysis of liver pieces yielded a target size of 210 kDa for hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) from rats fed a normal diet. Feeding a diet containing mevinolin and colestipol, which causes a marked increase in enzyme activity, resulted in a reduction of the target size to 120 kDa. These results are consistent with those obtained by radiation inactivation and immunoblotting analysis of isolated microsomes and suggest that the increase in HMG-CoA reductase activity caused by these dietary agents is accompanied by a change from a dimer to a monomer form of the enzyme.