Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (W) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity. (C) 2009 Elsevier B.V. All rights reserved.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Enhanced Neural Progenitor/Stem Cells Self-Renewal via the Interaction of Stress-Inducible Protein 1 with the Prion Protein

Prion protein (PrPC), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C)-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C), with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126-1136

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrPC) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrPC trafficking and tested whether this process controls PrPC-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrPC, induced PrPC endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrPC; however, heterologous expression of PrPC reconstituted both PKA and ERK1/2 activation. In contrast, a PrPC mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrPC endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Prion Protein and Its Ligand Stress Inducible Protein 1 Regulate Astrocyte Development

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.(C) 2009 Wiley-Liss, Inc.