Journal of Molecular Endocrinology current issue

Leu27IGF2 plays an opposite role to IGF1 to induce H9c2 cardiomyoblast cell apoptosis via G{alpha}q signaling

Thu, 19 Nov 2009 09:07:54 PST

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Gq using the immunoprecipitation assay, we found that IGF2R could directly interact with Gq and after treatment with Leu27IGF2 the binding ability of Gq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-β, a key downstream modulator of Gq, on serine 537. Moreover, disruption of the Gq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Gq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

Chisalita, S I, Johansson, G S, Liefvendahl, E, Back, K, Arnqvist, H J — Thu, 19 Nov 2009 09:07:54 PST

Whether insulin, at physiological concentrations, has direct effects on vascular smooth muscle cells (VSMCs) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, the effects of IGF1 and IGF2 were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analyzed by quantitative reverse transcription PCR and receptor protein by ELISA and western blot. Biological effects were studied by thymidine incorporation and glucose accumulation. In HASMC, both mRNA and protein expression of IGF1 receptors (IGF1R) were fivefold higher compared to insulin receptor (IR). IR isoform A mRNA was 13-fold more expressed than IR isoform B. IR and IGF1R co-precipitated, indicating the presence of hybrid IR/IGF1R. Phosphorylation of the IGF1R β-subunit was obtained by IGF1 10^–9–10^–8 mol/l and IGF2 10^–8 mol/l. IR β-subunit was phosphorylated by IGF1 10^–8 mol/l but not by insulin. IGF1 stimulated IR substrate-1 and AKT at 10^–8 mol/l and extracellular signal-regulated kinases 1 and 2 at 10^–9–10^–8 mol/l respectively. IGF1 and 2 at a concentration of 10^–8–10^–7 mol/l significantly stimulated ³H-thymidine incorporation, whereas insulin did not. ¹⁴C-Glucose accumulation was stimulated by IGF1 or IGF2 10^–8–10^–7 mol/l, and also by insulin 10^–7 mol/l. Our results suggest that IGF1R and hybrid IR/IGF1R are activated by physiological concentrations of IGF1 and 2 in HASMC and this propagates downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF1R and incorporation of IR into hybrid IR/IGF1R.

Downregulation of peroxisome proliferator-activated receptor {alpha} and its coactivators in liver and skeletal muscle mediates the metabolic adaptations during lactation in mice

Gutgesell, A., Ringseis, R., Schmidt, E., Brandsch, C., Stangl, G. I, Eder, K. — Thu, 19 Nov 2009 09:07:54 PST

Previous studies have shown that genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis are downregulated in liver and skeletal muscle of rats during lactation. However, biochemical mechanisms underlying these important metabolic adaptations during lactation have not yet been elucidated. As all these genes are transcriptionally regulated by peroxisome proliferator-activated receptor (Ppar), we hypothesized that their downregulation is mediated by a suppression of Ppar during lactation. In order to investigate this hypothesis, we performed an experiment with lactating and nonlactating Ppar knockout and corresponding wild-type mice. In wild-type mice, lactation led to a considerable downregulation of Ppar, Ppar coactivators Pgc1 and Pgc1β, and Ppar target genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis in liver and skeletal muscle (P<0.05). Ppar knockout mice had generally a lower expression of all these Ppar target genes in liver and skeletal muscle. However, in those mice, lactation did not lower the expression of genes involved in fatty acid utilization and thermogenesis in liver and skeletal muscle. Expression levels of Ppar target genes in lactating wild-type mice were similar than in lactating or nonlactating Ppar knockout mice. In conclusion, the present findings suggest that downregulation of Ppar and its coactivators in tissues with high rates of fatty acid catabolism is responsible for the reduced utilization of fatty acids in liver and skeletal muscle and the reduced thermogenesis occurring in the lactating animal, which aim to conserve energy and metabolic substrates for milk production in the mammary gland.

Thioredoxin and thioredoxin reductase influence estrogen receptor {alpha}-mediated gene expression in human breast cancer cells

Rao, A. K, Ziegler, Y. S, McLeod, I. X, Yates, J. R, Nardulli, A. M — Thu, 19 Nov 2009 09:07:54 PST

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor (ER). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ER, Trx, and TrxR interact and ER and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17β-estradiol, Trx, and TrxR alter hydrogen peroxide (H₂O₂) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H₂O₂ levels and transcription factor activity, aid ER in regulating the expression of estrogen-responsive genes in target cells.

Surface translocation and tri-iodothyronine uptake of mutant MCT8 proteins are cell type-dependent

Kinne, A., Roth, S., Biebermann, H., Kohrle, J., Gruters, A., Schweizer, U. — Thu, 19 Nov 2009 09:07:54 PST

Mutations in the gene encoding the thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), underlie severe mental retardation. We wanted to understand the functional consequences of a series of missense mutations in MCT8 in order to identify therapeutic options for affected patients. We established cell lines stably expressing 12 MCT8 variants in JEG1 and MDCK1 cells. The cell lines were characterized according to MCT8 mRNA and protein expression, tri-iodothyronine (T₃) transport activity, substrate K_M characteristics, surface expression, and responsiveness to T₃ preincubation and chemical chaperones. Functional activities of ins235V and L568P MCT8 mutants depend on the cell type in which they are expressed. These mutants and R271H exhibited considerable transport activity when present at the cell surface as verified by surface biotinylation and kinetic analysis. Most mutants, however, were inactive in T₃ transport even when present at the cell surface (e.g. S194F, A224V, F230, L512P). Preincubation of G558D with T₃ increased T₃ uptake in MDCK1 cells to a small, but significant, extent. Chemical chaperones were ineffective. The finding that the cell type determines surface expression and T₃ transport activities of missense mutants in MCT8 may be important to understand phenotypic variability among carriers of different mutations. In particular, the clinical observation that the severity of derangements of thyroid hormone levels does not correlate with mental impairments of the patients may be based on different residual activity of mutant MCT8 in different cell types.