Proximal tubule PPARα attenuates renal fibrosis and inflammation caused by unilateral ureteral obstruction.

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-ß1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-ß, demonstrating PPARα signaling reduces epithelial TGF-ß production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1ß, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

Metabolic reprogramming by Dichloroacetic acid potentiates photodynamic therapy of human breast adenocarcinoma MCF-7 cells.

Aberrant glycolytic metabolism is one of the hallmarks of carcinogenesis and therefore reversal of metabolic transformation is a promising drug target in cancer treatment strategies. Dichloroacetic acid (DCA) is known to target the glycolytic pathway in cancer cells and facilitates reversal of metabolic transformation from aerobic cytosolic accumulation of pyruvic acid, "the Warburg effect", to mitochondrial oxidative phosphorylation. Recently, combination therapy particularly involving photodynamic therapy (PDT) has received considerable attention in oncology. We hypothesized that if DCA and PDT are combined, they might potentiate mitochondrial dysfunction and induce apoptosis by a reactive oxygen species (ROS) dependent pathway. We used MCF-7 cells as our in vitro model and 5-aminolevulinic acid (5-ALA) dependent PDT therapy to test our hypothesis. We found that combinatorial treatment of MCF-7 cells with PDT and DCA not only increased cell growth inhibition, but also affected mitochondrial membrane integrity perhaps via production of ROS, and enhanced apoptosis. Further, our results on ATP release during the combined treatment demonstrate that immunogenic cell death (ICD) is likely to be a potential mechanism by which PDT and DCA induce cancer cell death. Taken together, our study suggests a novel way of sensitizing MCF-7 cells for accelerated induction of apoptosis and ICD in these cells. The findings included in this study might have direct relevance in breast cancer treatment strategies.

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

Dual mTOR inhibitor MLN0128 suppresses Merkel cell carcinoma (MCC) xenograft tumor growth.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Pathologic activation of PI3K/mTOR pathway and elevated expression of c-Myc are frequently detected in MCC. Yet, there is no targeted therapy presently available for this lethal disease. Recently, MLN0128, a second-generation dual TORC1/2 inhibitor is shown to have therapeutic efficacy in preclinical studies. MLN0128 is currently in clinical trials as a potential therapy for advanced cancers. Here we characterize the therapeutic efficacy of MLN0128 in the preclinical setting of MCC and delineate downstream targets of mTORC1/2 in MCC cellular systems. MLN0128 significantly attenuates xenograft MCC tumor growth independent of Merkel cell polyomavirus. Moreover, MLN0128 markedly diminishes MCC cell proliferation and induces apoptosis. Further investigations indicate that senescence does not contribute to MLN0128-mediated repression of xenograft MCC tumor growth. Finally, we also observe robust antitumor effects of MLN0128 when administered as a dual therapy with JQ1, a bromodomain protein BRD4 inhibitor. These results suggest dual blockade of PI3K/mTOR pathway and c-Myc axis is effective in the control of MCC tumor growth. Our results demonstrate that MLN0128 is potent as monotherapy or as a member of combination therapy with JQ1 for advanced MCC.

Hyperketonemia increases tumor necrosis factor-alpha secretion in cultured U937 monocytes and Type 1 diabetic patients and is apparently mediated by oxidative stress and cAMP deficiency.

An elevated blood level of tumor necrosis factor (TNF)-alpha is a validated marker of vascular inflammation, which can result in the development of vascular disease and atherosclerosis. This study examined the hypothesis that ketosis increases the TNF-alpha secretion, both in a cell culture model using U937 monocytes and in type 1 diabetic patients in vivo. U937 cells were cultured with ketone bodies (acetoacetate [AA] and beta-hydroxybutyrate [BHB]) in the presence or absence of high levels of glucose in medium at 37 degrees C for 24 h. This study demonstrates the following points. First, hyperketonemic diabetic patients have significantly higher levels of TNF-alpha than normoketonemic diabetic patients (P < 0.01) and normal control subjects (P < 0.01). There was a significant correlation (r = 0.36, P < 0.05; n = 34) between ketosis and oxidative stress as well as between oxidative stress and TNF-alpha levels (r = 0.47, P < 0.02; n = 34) in the blood of diabetic patients. Second, ketone body AA treatment increases TNF-alpha secretion, increases oxygen radicals production, and lowers cAMP levels in U937 cells. However, BHB did not have any effect on TNF-alpha secretion or oxygen radicals production in U937 cells. Third, exogenous addition of dibutyryl cAMP, endogenous stimulation of cAMP production by forskolin, and antioxidant N-acetylcysteine (NAC) prevented stimulation of TNF-alpha secretion caused by AA alone or with high glucose. Similarly, NAC prevented the elevation of TNF-alpha secretion and lowering of cAMP levels in H(2)O(2)-treated U937 cells. Fourth, the effect of AA on TNF-alpha secretion was inhibited by specific inhibitors of protein kinase A (H89), p38-mitogen-activated protein kinase (SB203580), and nuclear transcription factor (NF)kappaB (NFkappaB-SN50). This study demonstrates that hyperketonemia increases TNF-alpha secretion in cultured U937 monocytic cells and TNF-alpha levels in the blood of type 1 diabetic patients and is apparently mediated by AA-induced cellular oxidative stress and cAMP deficiency.

Animal models of rheumatoid arthritis and their relevance to human disease.

Rodent models of rheumatoid arthritis (RA) are useful tools to study the pathogenic process of RA. Among the most widely used models of RA are the streptococcal cell wall (SCW) arthritis model and the collagen-induced arthritis (CIA). Both innate and adaptive immune mechanisms are involved in these rodent models. While no models perfectly duplicate the condition of human RA, they are easily reproducible, well defined and have proven useful for development of new therapies for arthritis, as exemplified by cytokine blockade therapies. Besides SCW and CIA models, there are numerous others including transgenic models such as K/BxN, induced models such as adjuvant-induced and pristane models, and spontaneous models in certain mouse strains, that have been used to help understand some of the underlying mechanisms. This review provides an update and analysis of RA models in mice and rats. The array of models has provided rheumatologists and immunologists a means to understand the multifactorial disease in humans, to identify new drug targets, and to test new therapies.

Elevated blood interleukin-6 levels in hyperketonemic type 1 diabetic patients and secretion by acetoacetate-treated cultured U937 monocytes.

OBJECTIVE: Diabetic patients have elevated blood levels of interleukin-6 (IL-6), which is known to increase inflammation and the development of vascular disease and atherosclerosis. This study examined the hypothesis that ketosis increases the circulating levels of IL-6 in type 1 diabetic patients as well as the secretion of IL-6 in vitro in a cell culture model using U937 monocytes. RESEARCH DESIGN AND METHODS: Fasting blood was obtained from type 1 diabetic patients and healthy siblings. To examine the effect of ketosis, U937 monocytes were cultured with ketone bodies (acetoacetate [AA], beta-hydroxybutyrate [BHB]) in the presence or absence of high glucose levels in the medium at 37 degrees C for 24 h. IL-6 was determined by the sandwich enzyme-linked immunosorbent assay method, and intracellular reactive oxygen species (ROS) generation was detected using dihydroethidium dye. RESULTS: The blood level of IL-6 was higher in hyperketonemic (HK) diabetic patients than in normoketonemic (NK) diabetic patients (P < 0.05) and normal control subjects (P < 0.05). There was a significant correlation between ketosis and IL-6 levels (r = 0.36, P < 0.04, n = 34) in the blood of diabetic patients. Cell culture studies found that exogenous addition of the ketone body AA, but not BHB, increases IL-6 secretion and ROS generation in U937 cells. N-acetylcysteine (NAC) prevented the IL-6 secretion in acetoacetate-treated U937 monocytes. CONCLUSIONS: This study demonstrates that hyperketonemia increases IL-6 levels in the blood of type 1 diabetic patients and that NAC can inhibit IL-6 secretion by U937 monocytic cells cultured in a ketotic medium.

Effect of vitamin B6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H2O2-treated U937 monocytes.

Vitamin B6 (Vit.B6) supplementation has been shown to be beneficial in reducing diabetic complications, cognitive aging, and in the prevention of coronary heart disease. It was hypothesized that Vit.B6 compounds may function as antioxidants and thus offer protection against oxidative stress under various pathophysiological and or experimental conditions. To test this hypothesis, U937 monocytes were cultured with pyridoxine (P), pyridoxal phosphate (PP) and pyridoxamine (PM) and H2O2, either alone or together for 2 h. Oxidative stress was determined by measuring superoxide radical production, lipid peroxidation, and mitochondrial transmembrane potential. Results demonstrate that Vit.B6 compounds can prevent the oxygen radical generation and lipid peroxidation caused by hydrogen peroxide in U937 monocytes, and that some of the protective effect of Vit.B6 may occur via modification of mitochondrial function.

Streptococcal cell wall induced arthritis: leukocyte activation in extra-articular lymphoid tissue.

Rheumatoid arthritis (RA) is a common systemic inflammatory disease thought to be T-helper-1 cell driven, though current controversy involves the relative role of T cells versus other leukocytes. Thus, there is a need for better understanding of the role of various leukocytes and their subsets in RA. Using the streptococcal cell wall (SCW) induced arthritis model, we examined leukocytes isolated from peripheral blood, spleen, and lymph nodes using monoclonal antibodies directed against lineage specific cell surface markers. Activation status of these cells was assessed using CD44 and CD71 as markers. T cells in general, and CD4+ T cells in particular were found to be activated in spleen and lymph nodes. B cells and monocytes in spleen demonstrated increased activation as well. The activation of cells in the myeloid and lymphoid lineages in the chronic phase of arthritis indicates ongoing involvement of innate and cognate immunity. This study quantitates specific changes in B and T lymphocytes, and myeloid cells and is consistent with findings in human RA in which specific antibodies, T cells, and myeloid cells are all implicated in the pathogenesis of RA.

FoxO3a Serves as a Biomarker of Oxidative Stress in Human Lens Epithelial Cells under Conditions of Hyperglycemia.

BACKGROUND: Forkhead box 'O' transcription factors (FoxOs) are implicated in the pathogenesis of type2 diabetes and other metabolic diseases. Abnormal activity of FoxOs was reported in the glucose and insulin metabolism. Expression of FoxO proteins was reported in ocular tissues; however their function under hyperglycemic conditions was not examined. METHODS: Human lens epithelial cell line was used to study the function of FoxO proteins. Immunofluorescence, flow cytometry and Western blotting were employed to detect the FoxO proteins under the conditions of hyperglycemia. RESULTS: In this study we examined the role of FoxO3a in hyperglycemia-induced oxidative stress in human lens epithelial cells. FoxO3a protein expression was elevated in a dose- and time-dependent fashion after high glucose treatment. Anti-oxidant defense mechanisms of the lens epithelial cells were diminished as evidenced from loss of mitochondrial membrane integrity and lowered MnSOD after 72 h treatment with high glucose. Taken together, FoxO3a acts as a sensitive indicator of oxidative stress and cell homeostasis in human lens epithelial cells during diabetic conditions. CONCLUSION: FoxO3a is an early stress response protein to glucose toxicity in diabetic conditions.

Synoviolin promotes IRE1 ubiquitination and degradation in synovial fibroblasts from mice with collagen-induced arthritis.

The E3 ubiquitin ligase synoviolin (SYVN1) functions as an anti-apoptotic factor that is responsible for the outgrowth of synovial cells during the development of rheumatoid arthritis. The molecular mechanisms underlying SYVN1 regulation of cell death are largely unknown. Here, we report that elevated SYVN1 expression correlates with decreased levels of the protein inositol-requiring enzyme 1 (IRE1)-a pro-apoptotic factor in the endoplasmic reticulum (ER)-stress-induced apoptosis pathway-in synovial fibroblasts from mice with collagen-induced arthritis (CIA). SYVN1 interacts with and catalyses IRE1 ubiquitination and consequently promotes IRE1 degradation. Suppression of SYVN1 expression in synovial fibroblasts from CIA mice restores IRE1 protein expression and reverses the resistance of ER-stress-induced apoptosis of CIA synovial fibroblasts. These results show that SYVN1 causes the overgrowth of synovial cells by degrading IRE1, and therefore antagonizes ER-stress-induced cell death.

Oxygen radical generation and endosulfan toxicity in Jurkat T-cells.

The mechanism by which endosulfan exposure causes cellular dysfunction in experimental animals and humans is not clear. In the present study, we provide experimental evidence in support of the role of oxidative stress in endosulfan toxicity. Using both cell free system and Jurkat cells as in vitro models, we demonstrate that endosulfan can generate oxygen radicals that is inhibitable by superoxide dismutase (SOD) and glutathione (GSH). In the cell culture model, oxygen radical generation in response to endosulfan was dose- (0-100 microM) and time-dependent (0-12 h). Two-color flowcytometric analysis showed that endosulfan mediated changes in delta psi(m) and generation of superoxide radicals do occur simultaneously in the affected cell population. It was hypothesized that endosulfan exerts a severe oxidative stress in Jurkat cells and this could be prevented or minimized by an antioxidant. To test this hypothesis, GSH was added exogenously and endosulfan toxicity was evaluated by alamarblue assay. In conclusion, our results demonstrate a role for reactive oxygen species (ROS) in endosulfan toxicity and that supplementation of antioxidants such as GSH may be useful in individuals who are at risk to endosulfan toxicity.

Streptococcal cell wall arthritis: kinetics of immune cell activation in inflammatory arthritis.

The streptococcal cell wall model of arthritis in Lewis rats consists of an acute, non-T-cell-dependent initiation phase, followed by a remission and then a chronic, inflammatory T-cell-dependent phase. In this report, we define pertinent changes in the cognate and noncognate immune system of the Lewis rats during various phases of the disease. We examined changes in the population size of various cell types using lineage-specific markers in three different tissues (blood, spleen, and lymph nodes) over 28 days. Our results indicate that the T cell and monocyte populations were significantly altered in PG-PS-treated rats and the activation status of these cells parallel initiation, remission, and chronic phases of joint inflammation. Activation of B cells also increases in certain tissues in the chronic phase of the disease. In summary, our results confirm the involvement of both innate and cognate immunity in the development of arthritis and demonstrate that monocytes, in addition to T cells, play a substantive role in the induction and maintenance of the inflammatory process in this rat model.

Progesterone, but not 17beta-estradiol, increases TNF-alpha secretion in U937 monocytes.

The Women Health Initiative Clinical trial results suggest that post-menopausal women receiving estrogen + progesterone are at risk for heart disease compared with estrogen alone supplemented women. We examined the hypothesis that progesterone but not 17beta-estradiol (E) increases the secretion of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. U937 human monocytes were cultured with normal or high glucose in the presence and absence of estrogen or progesterone at 37 degrees C for 24 h. Results show that estrogen inhibits IL-6 but not TNF-alpha secretion (p < 0.05) in monocytes activated by lipopolysaccharide (LPS) or high glucose. In addition, progesterone increased the TNF-alpha secretion in activated monocytes. Thus, progesterone supplementation along with estrogen may increase blood levels of pro-inflammatory cytokine TNF-alpha and thus risk of heart disease in post-menopausal women.

Splenectomy attenuates streptococcal cell wall-induced arthritis and alters leukocyte activation.

OBJECTIVE: To investigate the role of the spleen in the pathogenesis of streptococcal cell wall (SCW)-induced arthritis and determine the impact of splenectomy on monocytes and T cells involved in the arthritis. METHODS: Female Lewis rats were separated into 4 groups: 1) saline-injected, sham-operated; 2) saline-injected, splenectomized; 3) peptidoglycan-polysaccharide (PG-PS)-injected, sham-operated; and 4) PG-PS-injected, splenectomized. After a 10-day recovery period, rats received a single intraperitoneal injection of saline or PG-PS (25 microg rhamnose/gm body weight). We evaluated the effect of splenectomy on joint inflammation, histopathology, leukocyte subtypes in blood and lymph nodes, cytokines, and cell surface expression of CD44 and CD45RC in the chronic phase of the disease (day 28). RESULTS: Splenectomy dramatically decreased chronic joint inflammation and histopathologic damage as well as altered cell types in lymph nodes and peripheral blood, as analyzed by flow cytometry. Nitric oxide (NO) production, levels of interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, and a biomarker of Th1 cell predominance correlated with the level of joint inflammation. Surprisingly, in splenectomized animals, increased expression of adhesion molecules thought to track T cells to inflamed tissue were observed in lymph nodes. CONCLUSION: The result of splenectomy was attenuation of SCW-induced arthritis and changes in mediators of inflammation, including T cell subsets, proinflammatory cytokines, and NO production. Splenectomy may remove an important antigen reservoir and alter immune cell activation in the SCW-induced arthritis model.