Osmotic stress regulates mammalian target of rapamycin (mTOR) complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor protein phosphorylation.

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.

Laminar shear stress enhances endothelial cell survival through a NADPH oxidase 2-dependent mechanism.

The beneficial effects of laminar shear stress (LSS) due to blood flow include inhibition of endothelial cell death, but the associated mechanism is not well understood. This issue was addressed in the present study. In a normal growth medium, the endothelial cell death rate was below 5%, but this value increased beyond 30% when the serum was depleted. However, when cells were exposed to LSS during the serum depletion period, cell viability recovered to the levels of the serum-provided cells. The pro-survival effect of LSS was not affected by l-arginine methyl ester, but it was abrogated by apocynin, indicating that NADPH oxidases (NOX) play key roles in the mechanism. The pro-survival effect of LSS was reduced by NOX2 siRNA, but not by NOX4 siRNA. LSS increased the expressions of p47(phox) and p67(phox), the subunits of NOX2 complex. These observations suggest that LSS prevents apoptotic death of endothelial cells through a NOX2-dependent mechanism.

Senescent endothelial cells are prone to TNF-α-induced cell death due to expression of FAS receptor.

The senescent endothelial cells show various phenotypes which can increase the incidence of inflammatory cardiovascular diseases, but the fundamental basis for such phenotypic changes of senescing cells remains to be elucidated. This study was undertaken to find transmembrane receptors that might be highly expressed in senescent endothelial cells and play a key role in cell death signal transduction. Comparison of mRNA expression in young and senescent human umbilical vein endothelial cells, using a cDNA microarray method, provided a list of transmembrane receptors including the FAS receptor (tumor necrosis factor receptor superfamily member 6) whose expression levels were significantly increased by cellular senescence. Additional studies focused on FAS demonstrated that a high expression of FAS receptor in senescent endothelial cells is responsible for the susceptibility to apoptotic cell death, as the siRNA-mediated suppression of FAS expression in senescent cells prevented the cell death, and overexpression of exogenous FAS in young cells increased cell death. We also verified that FAS expression level was closely associated with the activation of caspase-3 and caspase-9 involved in apoptosis. The senescence-induced transmembrane receptors including the FAS receptor may provide novel therapeutic targets to prevent cardiovascular diseases.

Analysis of serum cytokine/chemokine profiles affected by aging and exercise in mice.

Aging could be the cause of inflammation involved in the progression of many degenerative diseases while physical exercise might reduce the inflammation. This study examined the effects of aging versus exercise on serum profiles of cytokines and chemokines in mice models. Male C57BL/6N mice with different ages (2 and 20 months old) were subjected to treadmill exercise for 4 weeks. The exercise did not affect the body mass gain of the young mice but significantly reduced that of the old mice. Of 50 cytokines/chemokines analyzed using a multiplexed bead-based sandwich immunoassay, Eotaxin, Interleukin-9 and Thymus and activation-related chemokine showed significantly higher serum levels in old mice compared with young mice (p<0.05). The treadmill exercise did not alter serum cytokines/chemokines levels significantly. This study suggests that the cytokines and chemokines, whose serum levels were changed age-dependently, would provide useful markers of the systemic low-level inflammation associated with aging and age-related diseases.

Nudix-type motif 2 contributes to cancer proliferation through the regulation of Rag GTPase-mediated mammalian target of rapamycin complex 1 localization.

Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.

Combined effects of substrate topography and stiffness on endothelial cytokine and chemokine secretion.

Endothelial physiology is regulated not only by humoral factors, but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nanopatterns tested, the 1:1 and 1:2 type patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1ß, IL-3, and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling.

Deacetylated αß-tubulin acts as a positive regulator of Rheb GTPase through increasing its GTP-loading.

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αß-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αß-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αß-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αß-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αß-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αß-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.

Phospholipase D2 induces stress fiber formation through mediating nucleotide exchange for RhoA.

Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.

Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb.

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR's access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.

Phospholipase D1 regulates cell migration in a lipase activity-independent manner.

Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.