Effect of Maternal Exercise on Diet-induced Redox Imbalance in Hippocampus of Adult Offspring.

Physical exercise practice has been increasingly recommended in the prevention and treatment of chronic diseases, causing a positive effect from body weight/fat loss to improved cognitive function. Maternal exercise seems to induce the same positive lifelong adaptations to the offspring. We hypothesized that maternal exercise can prevent redox imbalance in adult offspring's hippocampus exposed to a high-fat diet (HFD). Female Wistar rats were divided into three groups before and during pregnancy: (1) sedentary, (2) swimming exercise, and (3) swimming exercise with overload. On 60 days of age, the male pups were divided into standard diet or HFD for one month, yielding normal and HFD subgroups for each maternal condition. Maternal interventions did not alter gestational parameters, birth outcomes, and offspring weight gain from weaning to 90 days of age. The HFD consumption increased body fat, which was not prevented by maternal exercise. Serum glucose levels were increased by HFD, an effect that was prevented by unload maternal exercise. In the hippocampus, both maternal exercise intensities could increase antioxidant defense. Hippocampal redox homeostasis was impaired by HFD, causing increased superoxide levels, which was prevented by exercise without load, while overload caused only a reduction of the effect. In summary, the practice of swimming exercise without overload during pregnancy seems to be more beneficial when evaluated in animal model, preventing HFD induced redox imbalance and increasing antioxidant defense while overload swimming exercise during pregnancy demonstrated a negative effect on offspring submitted to HFD consumption.

Transcriptional repression of IFNß1 by ATF2 confers melanoma resistance to therapy.

The resistance of melanoma to current treatment modalities represents a major obstacle for durable therapeutic response, and thus the elucidation of mechanisms of resistance is urgently needed. The crucial functions of activating transcription factor-2 (ATF2) in the development and therapeutic resistance of melanoma have been previously reported, although the precise underlying mechanisms remain unclear. Here, we report a protein kinase C-ɛ (PKCɛ)- and ATF2-mediated mechanism that facilitates resistance by transcriptionally repressing the expression of interferon-ß1 (IFNß1) and downstream type-I IFN signaling that is otherwise induced upon exposure to chemotherapy. Treatment of early-stage melanomas expressing low levels of PKCɛ with chemotherapies relieves ATF2-mediated transcriptional repression of IFNß1, resulting in impaired S-phase progression, a senescence-like phenotype and increased cell death. This response is lost in late-stage metastatic melanomas expressing high levels of PKCɛ. Notably, nuclear ATF2 and low expression of IFNß1 in melanoma tumor samples correlates with poor patient responsiveness to biochemotherapy or neoadjuvant IFN-α2a. Conversely, cytosolic ATF2 and induction of IFNß1 coincides with therapeutic responsiveness. Collectively, we identify an IFNß1-dependent, cell-autonomous mechanism that contributes to the therapeutic resistance of melanoma via the PKCɛ-ATF2 regulatory axis.

Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf(V600E)::Pten(-/-) melanoma.

Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of Braf(V600E)::Pten(-/-) or Braf(V600E)::Cdkn2a(-/-)::Pten(-/-) mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in Braf(V600E)::Pten(-/-) mice. Pdk1(-/-) melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1(-/-) melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

Increased AP-1 DNA-binding activity and nuclear REF-1 accumulation in lead-exposed primary cultures of astrocytes.

Pb was shown to perturb neuronal and glial function either directly by interacting with protein thiol groups or indirectly by mimicking Ca(2+) and increasing oxidative stress. In view of the potential action of Pb on cellular redox homeostasis we studied the regulation of activator protein-1 (AP-1) DNA binding. A 1h incubation of astrocyte primary cultures with 10 microM Pb caused a 2.5 fold increase in AP-1 DNA binding. An assessment of how Pb elicited this increase revealed the involvement of 1. transcriptional and 2. posttranslational processes. The first one was documented by an increase of c-jun mRNA content after 15 to 30 min of 10 microM Pb exposure. The second one was suggested by an enhanced nuclear accumulation of redox factor-1 after 30 to 60 min of 10 microM Pb exposure. The Pb-elicited increase of the reduction/oxidation-sensitive AP-1 signal transduction may regulate target genes operative in cell survival or cell death.

Molecular markers of oxidative stress vulnerability.

In astrocyte primary cultures of trisomy 16 mice, an animal model for Down's syndrome, protein oxidation was 50% higher than in diploid littermates. Exposure to 10 microM H2O2 or 50 microM kainic acid incremented protein oxidation in trisomic but not in diploid cultures. Studies on stress response genes showed that metallothionein (MT) level was 2-3 times higher in trisomy 16 than in diploid cultures. Kainic acid or H2O2 exposure increased the MT protein level in diploid cultures but failed to increase it in trisomy 16 mouse beyond its elevated basal level. The reduced responsiveness of MT to simulated oxidative stress may result in insufficient removal of ROS, which could partially explain the further increase of protein oxidation in trisomy 16 cultures. In contrast, Pb exposure increased MT in trisomy 16 and diploid primary cultures to a similar extent. The similar metal responsiveness of MT in both phenotypes indicated that MT in trisomic glial cultures was not yet maximally stimulated. The flawed redox sensitivity in trisomy 16 mouse suggests possible alterations in the binding activity of ROS-sensitive transcription factors on the MT promoter.

Activator protein-1 DNA binding activation by hydrogen peroxide in neuronal and astrocytic primary cultures of trisomy-16 and diploid mice.

The effect of H(2)O(2) on DNA binding activity of activator protein-1 (AP-1) was studied by electrophoretic mobility shift assay (EMSA) in cortical primary cultures of trisomy-16 mice and their diploid littermates. Exposure to 10 microM H(2)O(2) for 15 min elicited a greater and earlier occurring increase of AP-1 DNA binding in neuronal primary cultures of trisomy-16 mice than of diploid mice. When astrocyte-rich primary cultures were exposed to 10 microM H(2)O(2) a two-fold increase of AP-1 DNA binding activity was found in trisomy-16 and diploid mice. Supershift EMSA analysis revealed that c-jun was a component of AP-1 in neuronal and glial cultures of diploid and trisomic mice. A 15-min exposure to 10 microM H(2)O(2) increased c-jun mRNA in cortical neuronal cultures by six-fold, compared with a two-fold increase in cultured astrocytes. The results documented that H(2)O(2)-elicited activation of AP-1 DNA binding in trisomy-16 primary cultures is transcriptionally regulated. Since oxidative stress also activates various stress-inducible protein kinases that may phosphorylate AP-1 dimers, the increase of AP-1 DNA binding may, in part, be triggered by phosphorylation.

Effect of lead on cytoskeletal proteins expressed in E14 mesencephalic primary cultures.

Several lines of evidence indicated that Pb exposure in vivo and in vitro altered neurite morphology in central and peripheral neurons. The present report shows that neurite length in mesencephalic primary cultures, consisting of neurons and glia, was decreased by Pb exposure when serum factors, presumably essential for glial functions, were absent in the culture medium. We studied whether a serum factor might control the mechanisms involved in the uptake and accumulation of Pb and its effect on cytoskeleton proteins. The total amount of Pb taken up in cell cultures was measured by atomic absorption spectroscopy and appeared to be down-regulated by a non-albumin-like serum component. In presence of serum, Pb exposure failed to alter cytoskeletal proteins. Instead, in serum-free neurobasal medium, Pb uptake failed to reach saturation within 6 h. Western blot analysis showed that the tau, 280 kDa MAP-2b, 70 kDa MAP-2c and GAP-43 protein bands were decreased 24 h after a 3 h exposure to 3 or 6 microM Pb in absence of serum. However, if cultures were maintained in serum-containing media after a 3 h Pb exposure without serum, the immunoblots did not differ from those of controls. It can be inferred that a serum factor prevents cytoskeletal protein alterations by Pb. In serum free medium, Pb that is primarily scavenged by the metallothionein I/II isoforms present in glial cells, may bind to thiol residues of proteins involved in either oxidative stress response or transcriptional regulation of cytoskeletal proteins.

In cortical cultures of trisomy 16 mouse brain the upregulated metallothionein-I/II fails to respond to H2O2 exposure or glutamate receptor stimulation.

To assess whether a defective oxidative defense may contribute to Down's syndrome, we studied the regulation of the metallothionein(MT)-I/II isoforms in primary cultures of cerebral cortex from fetal trisomy 16 mice and their euploid littermates. Western blot analysis showed that MT-I/II was upregulated and the protein carbonyl content was higher in trisomy 16 compared with euploid cultures. Addition of N-acetyl-l-cysteine to the culture medium reduced the increment of MT-I/II in trisomy 16 cortical cells. In euploid, but not trisomic cortical cultures, kainic acid, trans-(+/-)-ACPD, or H2O2 exposure elicited a dose-dependent increase of the MT-I/II immunoblots. In trisomic cells, the MT-I/II immunoblot densities were not increased beyond their elevated basal levels. In contrast, 25 microM Pb induced MT-I/II, to a similar extent, in cortical cultures from euploid and trisomy 16 mice. This suggests that the antioxidant-but not the metal-response element of the MT-I/II promoter was altered by increased oxidative stress. Our data suggest that, in the trisomy 16 mouse, the effects of increased production of reactive oxygen species, due to the increased SOD-1, GluR5, or amyloid precursor protein gene dosage, is exacerbated by an insufficient or missing antioxidant response.

Evidence of increased oxidative stress in hippocampal primary cultures of trisomy 16 mouse. Studies on metallothionein-I/II.

In the trisomy 16 mouse the increased gene dosage of SOD-1 increases H2O2 production that results in increased oxidative stress. We report here that in hippocampal primary cultures, metallothionein (MT)-I/II immunoreactivity was present mainly in glial fibrillary acidic protein-immunolabeled cells. Western blot analysis showed a two-fold higher level of MT-I/II in trisomy 16 mice then in euploid littermates. In contrast, the immunoreactivity of glutamine synthetase, another glia-expressed protein, was similar in hippocampal cultures of trisomy 16 mouse and euploid littermates. Oxyblot analysis of hippocampal cultures showed that the carbonyl content in several protein bands was higher in trisomy 16 mice than in euploid littermates giving evidence for increased oxidative stress in trisomy 16 mouse cultures. To evaluate the responsiveness of MT-I/II to agents that increase the level of reactive oxygen species in cells we measured the effect of H2O2, kainic acid, (+/-) ACPD, and beta-amyloid peptide 1-42. Western blot analysis documented that in hippocampal cultures of euploid littermates MT-I/II was maximally increased by 50 micro M H2O2, 100 micro M kainic acid, 10 micro M (+/-)ACPD, or 1.0 mM beta-amyloid peptide 1-42, whereas in those of trisomy 16 mice no further increase above the elevated level was observed. Our data suggest that in the trisomy 16 mouse the production of reactive oxygen species may have shifted the intracellular redox environment that could have alerted the susceptibility of MT-I/II transcription. The possibility that transcription factors whose activation may be essential to initiate MT-I/II transcription get oxidized has yet to be examined.

Lead exposures increases oxidative stress in serum deprived E14 mesencephalic cultures. Role of metallothionein and glutathione.

E14 mesencephalic cultures grown 6 days in Neurobasal Medium containing 10% horse serum consist of differentiated neurons and astroglia. In these cultures, glutathione and metallothionein-I/II are enriched in astrocytes and play an important role in heavy metal scavenging and oxidative stress response. A 24 h exposure to 25 micro M Pb, in serum-containing medium, elevated the glutathione content by more than twofold and increased the metallothionein I/II-immunolabeled protein band. In contrast, exposure to 3 to 25 micro M Pb is serum-free medium increased Pb uptake by cells 2 to 4-times above the levels found in 10% serum-containing medium, reduced the glutathione level and obliterated the metallothionein-I/II protein band. The rapid decrease of metallothionein-I/II and glutathione levels in serum-free medium implies that their regulation may depend on a serum factor operative in inducing immediate early genes. Exposure to 6 micro M Pb in serum-free or in B27-supplemented medium increased the carbonyl content of several protein bands above control levels indicating that under conditions that curtail metallothionein induction Pb exposure causes increased oxidative stress.

The effect of lead exposure and serum deprivation on mesencephalic primary cultures.

The effect of Pb2+ was studied in embryonic mesencephalic primary cultures that contain neurons and glia. Pb2+ exposure in absence of serum, damaged more efficaciously the cultured cells than Pb2+ exposure in presence of serum. In serum-free medium, Pb2+ elicited mainly necrosis and apoptosis in maximally 13% of the cells in culture. The glial fibrillary acidic protein (GFAP) content was decreased by Pb2+ exposure in serum-containing medium. The abundance of GFAP was also decreased by serum deprivation that was augmented by the addition of 12.5 microM Pb2+ in serum-free medium. A 6h exposure to 6 microM Pb2+ in serum-free medium also lowered the low affinity 3H-D-aspartate uptake. A 6h exposure of mesencephalic cells to 3-25 microM Pb2+ in serum-free medium failed to alter the number of tyrosine hydroxylase- and calretinin-immunoreactive cells, whereas, 50 microM Pb2+ obliterated both cell types. A 6h exposure of cells to 3 microM Pb2+ in serum-free medium decreased 3H-dopamine uptake by 50 % and 12.5 microM Pb2+ obliterated it. Addition of albumin to serum-free medium failed to prevent the Pb2+ -elicited inhibition of [3H]-dopamine uptake suggesting that the serum-afforded delay of cell death may not be due to a removal of reactive Pb2+ by protein/chelate formation but rather to the Pb2+ -scavenging function of glial cells. Serum deprivation may exacerbate the Pb2+ -induced neurotoxicity presumably by impairing the metal scavenging function of astrocytes.