mTOR and the health benefits of exercise.

Exercise is the greatest physiological stress that our bodies experience. For example, during maximal endurance exercise in elite athlete's cardiac output can increase up to 8-fold and the working muscles receive 21-times more blood each minute than at rest. Given the physiological stress associated with exercise and the adaptations that occur to handle this stress, it is not surprising that exercise training is known to prevent or effectively treat a multitude of degenerative conditions including cardiovascular disease, cancer, diabetes, depression, Alzheimer's disease, Parkinson's disease, and many others. Many of the health benefits of exercise are mediated by the mammalian/mechanistic target of rapamycin (mTOR), either in complex 1 or 2, not only within the working muscle, but also in distant tissues such as fat, liver, and brain. This review will discuss how exercise activates mTOR in diverse tissues and the ways that mTOR is important in the adaptive response that makes us bigger, stronger, and healthier as a result of exercise.

Fetuin-A (α2HS-glycoprotein) is a serum chemo-attractant that also promotes invasion of tumor cells through Matrigel.

The present study was conducted to determine whether fetuin-A, a dominant serum protein plays a role in chemo-attraction and chemo-invasion of carcinoma cells in vitro. Serum is normally used as positive chemotaxis control in Boyden chamber motility assays, prompting the need to identify the factor/s in serum that contributes the bulk of chemo-taxis and invasion. Serum has a plethora of chemotactic factors including stromal derived factor 1 also known as CXCL12. Using highly purified fetuin-A, we compared its chemo-attraction potential to culture medium containing 10% fetal bovine serum. We also investigated its ability to attract tumor cells through a bed of Matrigel (invasion assay). We demonstrated, using similar concentration range of fetuin-A found in blood, that it robustly supports both directed chemo-attraction and invasion of breast tumor cells. More importantly, we showed that at low concentrations (fetuin-A coated wells) itinteracts synergistically with CXCL12 to promote chemotaxis. The presence of plasminogen (PL) blunted the fetuin-A mediated chemotaxis. Taken together, the data suggest an in vivo chemotaxis/invasion role for fetuin-A.

Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading.

Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells. The data show that in the absence of fetuin-A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin-A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin-A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression.

Activation of CXCR4 triggers ubiquitination and down-regulation of major histocompatibility complex class I (MHC-I) on epithelioid carcinoma HeLa cells.

Many cancer cells display down-regulated major histocompatibility complex (MHC) class I antigen (MHC-I), which seems to enable them to evade immune surveillance, whereas the underlying mechanisms remain incompletely understood. Here, we demonstrate that ligand (CXCL12) stimulation of CXCR4, a major chemokine receptor expressed in many malignant cancer cells, induced MHC-I heavy chain down-regulation from the cell surface of the human epithelioid carcinoma HeLa cells, the human U251 and U87 glioblastoma cells, the human MDA-MD 231 breast cancer cells, and the human SK-N-BE (2) neuroblastoma cells. Activation of CXCR4 also induced MHC-I down-regulation in human peripheral blood mononuclear cells. The internalized MHC-I heavy chain molecules were partially co-localized with Rab7, a later endosomal marker. Activation of CXCR4 induced ubiquitination of MHC-I heavy chain, and mutation of the C-terminal two lysine residues (Lys-332, Lys-337) on one of the MHC-I alleles, HLA.B7, blocked CXCR4-evoked ubiquitination and down-regulation of HLA.B7. Moreover, purified GST-conjugated CXCR4 C terminus directly associated with the purified His-tagged beta2-microglobulin (beta2M), and MHC-I heavy chain was co-immunoprecipitated with CXCR4 in a beta2M-dependent manner. This interaction appears to be critical for CXCR4-evoked down-regulation of MHC-I heavy chain as evidenced by the data that MHC-I heavy chain down-regulation was inhibited by either truncation of the CXCR4 C terminus or knockdown of beta2M. All together, these findings shed new light on the role of CXCR4 in tumor evasion of immune surveillance via inducing MHC-I down-regulation from the cell surface.

CXCL12 induces tyrosine phosphorylation of cortactin, which plays a role in CXC chemokine receptor 4-mediated extracellular signal-regulated kinase activation and chemotaxis.

CXC chemokine receptor 4 (CXCR4) plays a role in the development of immune and central nervous systems as well as in cancer growth and metastasis. CXCR4-initiated signaling cascades leading to cell proliferation and chemotaxis are critical for these functions. The present study demonstrated that stimulation of CXCR4 by its ligand, CXCL12, induced transient translocation of cortactin from endosomal compartments to the cell periphery where it colocalized with CXCR4 followed by internalization of CXCR4 together with cortactin into endosomes. Cortactin was co-immunoprecipitated with CXCR4 in response to CXCL12 treatment in a time-dependent manner. Ligand stimulation induced phosphorylation of cortactin at tyrosine 421, and the phosphorylation was both c-Src- and dynamin-dependent. Cortactin overexpression promoted CXCR4 internalization and recycling. However, overexpression of a cortactin mutant in which tyrosine 421 was replaced with alanine (cortactin-Y421A) or knockdown of cortactin with RNA interference (RNAi) reduced CXCR4 internalization in response to CXCL12. CXCR4-mediated activation of extracellular signal-regulated kinases 1 and 2 was significantly prolonged by overexpression of wild-type cortactin but not by the cortactin-Y421A mutant and was inhibited by cortactin knockdown with RNAi. Moreover, CXCL12-induced chemotaxis was enhanced by cortactin overexpression, reduced by overexpression of the cortactin-Y421A mutant, and blocked by cortactin knockdown with RNAi. These data provide strong evidence for an important role of cortactin in CXCR4 signaling and trafficking as well in the receptor-mediated cell migration.

N-methyl-D-aspartate attenuates CXCR2-mediated neuroprotection through enhancing the receptor phosphorylation and blocking the receptor recycling.

Abnormal extracellular accumulations of beta-amyloid, a major component of the senile plaques, and of the excitatory amino acid glutamate are both believed to be associated with degeneration of nerve cells in the central nervous system of patients with Alzheimer's disease. The chemokine receptor CXCR2 has been shown to play a role in protecting neurons against beta-amyloid-induced injury in vitro, but it remains unclear whether CXCR2-mediated neuroprotection is affected by glutamate. We demonstrated that pretreatment of hippocampal neurons with a sublethal concentration of N-methyl-d-aspartate (NMDA) attenuated the macrophage inflammatory protein 2 (MIP2)-induced protection against beta-amyloid-induced neuronal death. The NMDA induced inhibition was blocked by (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), a noncompetitive NMDA receptor antagonist, indicating the involvement of NMDA receptors in this process. A sublethal dose of NMDA pretreatment induced CXCR2 phosphorylation, although to a lesser extent than the receptor phosphorylation induced by MIP2, and differential serine residues were involved in NMDA- and MIP2-induced CXCR2 phosphorylation. Moreover, NMDA treatment reduced the CXCR2-mediated Ca(2+) mobilization, suggesting that NMDA induces cross-desensitization of CXCR2. CXCR2 underwent dephosphorylation after removal of the extracellular ligand, but the dephosphorylation rate was significantly reduced in the cells pretreated with NMDA. Treatment of the neuronal cells with NMDA retarded the recycling of CXCR2. In view of the critical role of receptor phosphorylation and recycling in the functional responsiveness of the chemokine receptor, these observations indicate a novel pathway through which glutamate may interfere with the neuroprotective function of chemokines.

Macrophage inflammatory protein 2 inhibits beta-amyloid peptide (1-42)-mediated hippocampal neuronal apoptosis through activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways.

beta-Amyloid peptide accumulation in senile plaques in the brains of patients with Alzheimer's disease has been considered as a major cause of neuronal death. The present study demonstrated that the CXCR2 ligands macrophage inflammatory protein 2 (MIP-2), CXCL1, and CXCL8, protected hippocampal neurons against beta-amyloid (1-42) induced death. MIP-2-activated extracellular signal-regulated kinase (ERK)1/2 and Akt and both the mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol 3-kinase (PI3K) inhibitors 2'-amino-3'-methoxyflavone (PD98059) and wortmannin reduced the neuroprotective effect of MIP-2. MIP-2 induced weak phosphorylation of ribosomal S6 kinase (RSK) 1 but remarkable phosphorylation and nuclear translocation of RSK2. MIP-2-induced phosphorylation of RSK2 was inhibited by PD98059 but not by wortmannin. MIP-2 treatment of the neuronal cells resulted in phosphorylation of Bad at both the Ser-112 and Ser-136. The phosphorylation at Ser-112 was blocked by PD98059, whereas the phosphorylation at Ser-136 was blocked by wortmannin. The transcription factor cyclic AMP response element binding protein (CREB) was phosphorylated by MIP-2 stimulation of the neuronal cells. MIP-2-induced CREB phosphorylation was reduced by both PD98059 and wortmannin. These data demonstrate that both MEK1-ERK1/2 and PI3K-Akt signaling pathways are involved in CXCR2-mediated neuroprotection and that multiple downstream signaling events, including RSKs, Bad, and CREB, are activated in this process.