Progression of dystrophic features and activation of mitogen-activated protein kinases and calcineurin by physical exercise,in hearts of mdx mice

We have previously demonstrated that calcineurin and p38 mitogen-activated protein kinase (MAPK) are up-regulated in the hearts of mdx mice. However, the degree of up-regulation observed was variable, which may reflect variable levels of daily physical activities among the mice. To investigate whether or not exercise affects dystrophic features and activates intracellular signaling molecules in mdx hearts, we subjected mdx and C57BL/10 mice to treadmill exercise and examined intracellular signaling molecules in cardiac muscles, at the protein level. The heart to body weight ratio was significantly increased in exercised mdx mice. Histopathology in exercised mdx hearts showed extensive infiltration of inflammatory cells, together with increases in interstitial fibrosis and adipose tissues, all of which were not observed either in exercised C57BL/10 or non-exercised mdx hearts. Phosphorylated p38 MAPK, phosphorylated extracellular signal-regulated kinase 1/2 and calcineurin, but not phosphorylated c-Jun N-terminal kinase 1, were up-regulated in exercised mdx hearts compared to exercised C57BL/10 or non-exercised mdx hearts. These data suggest that physical exercise accelerates the dystrophic process through activation of intracellular signaling molecules in dystrophin-deficient hearts.     

Developmental differences in HO-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3

The molecular mechanisms underlying H(2)O(2)-induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H(2)O(2), the antioxidant, N-acetyl-L-cysteine, blocked toxicity at both stages of development. Differentiated oligodendrocytes contained more glutathione than did progenitors and were less susceptible to decreases in glutathione concentration induced by H(2)O(2) stress. As free radicals have been considered to serve as second messengers, we examined the effect of H(2)O(2) on activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) 1/2 and p38. H(2)O(2) caused a time- and concentration-dependent increase in MAPK phosphorylation, an effect that was totally blocked by N-acetyl-L-cysteine. Further exploration of potential mechanisms involved in oligodendrocyte cell death showed that H(2)O(2) treatment caused DNA condensation and fragmentation at both stages of development, whereas caspase 3 activation and poly (ADP-ribose) polymerase cleavage were significantly increased only in oligodendrocyte progenitors. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, blocked DNA fragmentation in progenitors and produced a small but significant level of protection from H(2)O(2) toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H(2)O(2)-induced death most significantly in oligodendrocytes. The poly (ADP-ribose) polymerase inhibitor, PJ34, reduced H(2)O(2)-induced toxicity on its own but was most effective when combined with benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or PD169316. The finding that molecular mechanisms conferring resistance to reactive oxygen species toxicity are regulated during oligodendrocyte differentiation may be of importance in designing therapies for certain neurological diseases affecting white matter.     

Involvement of multiple protein kinases in cPLA2 phosphorylation, arachidonic acid release, and cell death in in vivo and in vitro models of 1-methyl-4-phenylpyridinium-induced parkinsonism – the possible key role of PKG

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP⁺-induced cytosolic phospholipase A₂ (cPLA₂) activation of dopaminergic neurons. MPP⁺ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca²⁺]_i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA₂, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF₃), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA₂ on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA₂ phosphorylation at Ser505. Inhibitors of cPLA₂ and PKG increased viability and reduced MPP⁺-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA₂ phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA₂.     

Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-kappaB and tumor necrosis factor alpha in LLC-PK1 cells

Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNFalpha), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-alpha, -delta, -epsilon, and -zeta isoforms over 24-72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 microM) was associated with inhibition of cytosol to nuclear translocation of NF-kappaB and NF-kappaB-DNA binding at 72 h. The expression of TNFalpha was significantly inhibited at 24 and 48 h in response to 1 and 10 microM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to > or =1 microM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-kappaB and TNFalpha.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

Inhibition of EGFR attenuates fibrosis and stellate cell activation in diet-induced model of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. NAFLD begins with steatosis and advances to nonalcoholic steatohepatitis (NASH) and cirrhosis. The molecular mechanisms involved in NAFLD progression are not understood. Based on recent studies showing dysregulation of epidermal growth factor receptor (EGFR) in animal models of liver injury, we sought to determine if inhibition of EGFR mitigates liver fibrosis and HSC activation in NAFLD. We utilized the high fat diet (HFD)-induced murine model of liver injury to study the role of EGFR in NAFLD. The lipid accumulation, oxidative stress, hepatic stellate cell (HSC) activation and matrix deposition were examined in the liver tissues. We also evaluated the EGFR signaling pathway, ROS activation and pro-fibrogenic phenotype in oxidized low density lipoproteins (ox-LDL) challenged cultured HSCs. We demonstrate that EGFR was phosphorylated in liver tissues of HFD murine model of NAFLD. Inhibition of EGFR prevented diet-induced lipid accumulation, oxidative stress, and HSC activation and matrix deposition. In cultured HSCs, we show that ox-LDL caused rapid activation of the EGFR signaling pathway and induce the production of reactive oxygen species. EGFR also mediated HSC activation and promoted a pro-fibrogenic phenotype. In conclusion, our data demonstrate that EGFR plays an important role in NAFLD and is an attractive target for NAFLD therapy.     

Cholesterol and bile acid-mediated regulation of autophagy in fatty liver diseases and atherosclerosis

Liver is the major organ that regulates whole body cholesterol metabolism. Disrupted hepatic cholesterol homeostasis contributes to the pathogenesis of nonalcoholic steatohepatitis, dyslipidemia, atherosclerosis, and cardiovascular diseases. Hepatic bile acid synthesis is the major catabolic mechanism for cholesterol elimination from the body. Furthermore, bile acids are signaling molecules that regulate liver metabolism and inflammation. Autophagy is a highly-conserved lysosomal degradation mechanism, which plays an essential role in maintaining cellular integrity and energy homeostasis. In this review, we discuss emerging evidence linking hepatic cholesterol and bile acid metabolism to cellular autophagy activity in hepatocytes and macrophages, and how these interactions may be implicated in the pathogenesis and treatment of fatty liver disease and atherosclerosis.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.