Inactivation of Foxo3a and subsequent downregulation of PGC-1 alpha mediate nitric oxide-induced endothelial cell migration.

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1 alpha downregulation and investigated the regulatory pathway involved. PGC-1 alpha negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1 alpha. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1 alpha downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1 alpha, and we found that a functional FoxO binding site in the PGC-1 alpha promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1 alpha is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1 alpha and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

Mutual dependence of Foxo3a and PGC-1alpha in the induction of oxidative stress genes.

Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1alpha, because it is severely curtailed in PGC-1alpha-deficient endothelial cells. Foxo3a and PGC-1alpha appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1alpha interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1alpha transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1alpha, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1alpha control of the oxidative stress response.

Mitochondrial dysfunction in human pathologies.

The integrity of mitochondrial function is fundamental to cell life. The cell demands for mitochondria and their complex integration into cell biology, extends far beyond the provision of ATP. It follows that disturbances of mitochondrial function lead to disruption of cell function, expressed as disease or even death. Mitochondria are major producers of free radical species and also possibly of nitric oxide, and are, at the same time, major targets for oxidative damage. In this review we consider recent developments in our knowledge of how the mitochondrial production of reactive oxygen species (ROS) plays a critical role in several major human pathologies. We will also consider recent advances in our understanding of the molecular mechanisms involved in mitochondrial ROS detoxification.

Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC-1alpha.

Nitric oxide (NO) has both prooxidant and antioxidant activities in the endothelium; however, the molecular mechanisms involved are still a matter of controversy. PGC-1alpha [peroxisome proliferators-activated receptor (PPAR) gamma coactivator 1-alpha] induces the expression of several members of the mitochondrial reactive oxygen species (ROS) detoxification system. Here, we show that NO regulates this system through the modulation of PGC-1alpha expression. Short-term (<12 h) treatment of endothelial cells with NO donors down-regulates PGC-1alpha expression, whereas long-term (>24 h) treatment up-regulates it. Treatment with the NOS inhibitor l-NAME has the opposite effect. Down-regulation of PGC-1alpha by NO is mediated by protein kinase G (PKG). It is blocked by the soluble guanylate cyclase (sGC) inhibitor ODQ and the PKG inhibitor KT5823, and mimicked by the cGMP analog 8-Br-cGMP. Changes in PGC-1alpha expression are in all cases paralleled by corresponding variations in the mitochondrial ROS detoxification system. Cells that transiently overexpress PGC-1alpha from the cytomeglovirus (CMV) promoter respond poorly to NO donors. Analysis of tissues from eNOS(-/-) mice showed reduced levels of PGC-1alpha and the mitochondrial ROS detoxification system. These data suggest that NO can regulate the mitochondrial ROS detoxification system both positively and negatively through PGC-1alpha.

Role of metalloproteinases MMP-9 and MT1-MMP in CXCL12-promoted myeloma cell invasion across basement membranes.

Malignant plasma cells in multiple myeloma home to the bone marrow (BM), accumulate in different niches and, in late disease, migrate from the BM into blood. These migratory events involve cell trafficking across extracellular matrix (ECM)-rich basement membranes and interstitial tissues. Metalloproteinases (MMP) degrade ECM and facilitate tumour cell invasion. The chemokine CXCL12 is expressed in the BM, and it was previously shown that it triggers myeloma cell migration and activation. In the present work we show that CXCL12 promotes myeloma cell invasion across Matrigel-reconstituted basement membranes and type I collagen gels. MMP-9 activity was required for invasion through Matrigel towards CXCL12, whereas TIMP-1, a MMP-9 inhibitor that we found to be expressed by myeloma and BM stromal cells, impaired the invasion. In addition, we show that the membrane-bound MT1-MMP metalloproteinase is expressed by myeloma cells and contributes to CXCL12-promoted myeloma cell invasion across Matrigel. Increase in MT1-MMP expression, as well as induction of its membrane polarization by CXCL12 in myeloma cells, might represent potential mechanisms contributing to this invasion. CXCL12-promoted invasion across type I collagen involved metalloproteinases different from MT1-MMP. These data indicate that CXCL12 could contribute to myeloma cell trafficking in the BM involving MMP-9 and MT1-MMP activities.

PGC-1alpha regulates the mitochondrial antioxidant defense system in vascular endothelial cells.

OBJECTIVE: Mitochondrial production of oxidants contributes to a variety of pathological conditions including the vascular complications of diabetes, neurodegenerative diseases, and cellular senescence. We postulated that a transcriptional coactivator, peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major regulator of oxidative metabolism and mitochondrial biogenesis, could be involved in the transcriptional regulation of the mitochondrial antioxidant defense system in vascular endothelial cells. METHODS AND RESULTS: We show that PGC-1alpha is present in human, bovine, and mouse endothelial cells and positively modulates the expression of the mitochondrial detoxification system. Endothelial cells that overexpress PGC-1alpha show reduced accumulation of reactive oxygen species (ROS), increased mitochondrial membrane potential, and reduced apoptotic cell death both in basal and oxidative stress conditions. Downregulation of PGC-1alpha levels by siRNA reduces the expression of mitochondrial detoxification proteins. CONCLUSIONS: These results unveil a novel regulatory pathway that links mitochondrial activity and mitochondrial oxidative stress protective systems. In addition, they suggest that PGC-1alpha could play a crucial protective role in vascular complications of diabetes, where the mitochondrial metabolism of glucose has been shown to result in oxidative stress and vascular endothelial cell dysfunction.

Streptomyces lividans contains a minimal functional signal recognition particle that is involved in protein secretion.

The bacterial version of the mammalian signal recognition particle (SRP) is well conserved and essential to all known bacteria. The genes for the Streptomyces lividans SRP components have been cloned and characterized. FtsY resembles the mammalian SRP receptor and the S. lividans SRP consists of Ffh, a homologue of the mammalian SRP54 protein, and scRNA, which is a small size RNA of 82 nt in length. Co-immunoprecipitation studies confirmed that Ffh and scRNA are probably the only components of the S. lividans SRP and that pre-agarase can co-immunoprecipitate with Ffh, suggesting that the SRP is involved in targeting secretory proteins.