Padmaja Tummala

Chronic Uremia Attenuates Growth Hormone–Induced Signal Transduction in Skeletal Muscle

Malnutrition and muscle wasting are common in chronic renal failure (CRF) and adversely affect morbidity and mortality. Contributing to the muscle wasting is resistance to growth hormone (GH). For testing whether impaired GH signaling is a cause of the skeletal muscle GH resistance and for elucidating its mechanisms, muscle GH signaling and action were studied in GH-deficient rats with surgically induced CRF and sham-operated pairfed control rats. GH treatment increased gastrocnemius muscle IGF-1 mRNA levels significantly in control but not in CRF rats. GH-activated Janus-associated kinase 2 (JAK2)-signal transducers and activators of transcription 5 (STAT5) signaling was impaired in CRF rats, despite normal GH receptor (GHR), JAK2, and STAT5 protein levels. Phosphorylation of the GHR, JAK2, and STAT5 in response to GH was depressed by nearly half in CRF (P < 0.05), and nuclear phospho-STAT5 levels were depressed by approximately one third (P < 0.01). GH-stimulated suppressors of cytokine signaling 2 mRNA levels were significantly higher in CRF. This may be related to inflammatory cytokine activity because C-reactive protein levels were elevated. Muscle protein-tyrosine phosphatase activity was also increased significantly by twofold. In conclusion, rats with CRF acquire skeletal muscle resistance to GH that is caused at least in part by impaired JAK2-GHR-STAT5 phosphorylation and nuclear STAT5 translocation. Furthermore, it seems that the attenuated JAK2-STAT5 phosphorylation may be caused by at least two different processes. One involves depressed phosphorylation of the signaling proteins because of increased suppressors of cytokine signaling 2 expression that may be linked to low-grade inflammation. The other may involve increased signaling protein dephosphorylation because of heightened protein-tyrosine phosphatase activity.

The Transcription Factor Neural Retina Leucine Zipper (NRL) Controls Photoreceptor-specific Expression of Myocyte Enhancer Factor Mef2c from an Alternative Promoter

Background: Mef2c gene expression is significantly diminished in the retinas of NRL (neural retina leucine zipper) knock-out mice. Results: NRL binding, RNA polymerase II association, and acetylation of histone H3, revealed a novel alternate Mef2c promoter. Conclusions: Activation of the retinal Mef2c promoter is NRL-dependent and specific to rod photoreceptor cells. Significance: Mef2c represents a novel regulatory node downstream of NRL in adult rod photoreceptor cells. Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors in the mammalian retina. In the Nrl−/− mouse retina, photoreceptor precursors fail to produce rods and generate functional cone photoreceptors that predominantly express S-opsin. Previous global expression analysis using microarrays revealed dramatically reduced expression of myocyte enhancer factor Mef2c in the adult Nrl−/− retina. We undertook this study to examine the biological relevance of Mef2c expression in retinal rod photoreceptors. Bioinformatics analysis, rapid analysis of cDNA ends (5′-RACE), and reverse transcription coupled with qPCR using splice site-specific oligonucleotides suggested that Mef2c is expressed in the mature retina from an alternative promoter. Chromatin immunoprecipitation (ChIP) studies showed the association of active RNA polymerase II and acetylated histone H3 just upstream of Mef2c exon 4, providing additional evidence for the utilization of an alternative promoter in the retina. In concordance, we observed the binding of NRL to a putative NRL-response element (NRE) at this location by ChIP-seq and electrophoretic mobility shift assays. NRL also activated the Mef2c alternative promoter in vitro and in vivo. Notably, MEF2C could support Rhodopsin promoter activity in rod photoreceptors. We conclude that Mef2c expression from an alternative promoter in the retina is regulated by NRL. Our studies also implicate MEF2C as a transcriptional regulator of homeostasis in rod photoreceptor cells.

Growth Hormone–Mediated Janus Associated Kinase–Signal Transducers and Activators of Transcription Signaling in the Growth Hormone–Resistant Potassium-Deficient Rat

Potassium deficiency (KD) is associated with severe growth failure, in part caused by growth hormone (GH) resistance. This study set out to determine whether the resistance could be caused by a defect in GH-mediated janus associated kinase-signal transducers and activators of transcription (STAT) signaling as occurs in uremia. To this end, rats were fed a K-deficient diet for 8 d and pair-fed controls received a K-replete diet. Animals from each group received GH or vehicle, and during this period, KD rats were GH resistant; GH induced body and liver weight gain and linear body growth were severely attenuated in these rats. In addition, signal transduction was studied in the liver of rats that were killed 10 or 15 min after an intravenous GH bolus or vehicle. When the rats were killed, GH receptor mRNA and protein levels were similar in the two groups. The abundance of STAT5, STAT3, and STAT1, proteins that mediate GH signaling, was significantly increased by 40 to 130% in KD. Furthermore, GH induced a far greater increase in STAT5 and STAT3 phosphorylation in this group. STAT5 phosphorylation was enhanced fourfold even when normalized for total STAT5 content. Phosphorylated STAT5 and STAT3 proteins were also increased in nuclear extracts, suggesting normal nuclear translocation of the activated signaling proteins. DNA binding of nuclear STAT5 was unaltered. Thus, in KD, there is resistance to the growth-promoting action of GH despite hyperactivation of the janus associated kinase-STAT signaling pathway. This suggests the presence of a defect distal to the nuclear binding of STAT or, alternatively, a defect in a STAT-independent GH-activated signaling pathway.

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.

Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes

Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity.

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ‐glutamylcyclotransferase activity that degrades glutathione. We found that cagA‐positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori‐infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori‐mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.