Objective Metabolic disorders increase MCP-1-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. Methods and Results Chronic exposure of monocytes to diabetic conditions induced by human low-density lipoproteins plus high D-glucose concentrations (LDL+HG) promoted Nox4 expression, increased intracellular H2O2 formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, PDGF-B and RANTES. Both, H2O2 added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 (Grx1) prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein-S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1-loaded Matrigel plugs implanted subcutaneous in these mice. Conclusions By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

Monocytic adhesion and chemotaxis are regulated by MAPK pathways, which in turn are controlled by redox-sensitive MAPK phosphatases (MKPs). We recently reported that metabolic disorders prime monocytes for enhanced recruitment into vascular lesions by increasing monocytes' responsiveness to chemoattractants. However, the molecular details of this proatherogenic mechanism were not known. Here we show that monocyte priming results in the S-glutathionylation and subsequent inactivation and degradation of MKP-1. Chronic exposure of human THP-1 monocytes to diabetic conditions resulted in the loss of MKP-1 protein levels, the hyperactivation of ERK and p38 in response to monocyte chemoattractant protein-1 (MCP-1), and increased monocyte adhesion and chemotaxis. Knockdown of MKP-1 mimicked the priming effects of metabolic stress, whereas MKP-1 overexpression blunted both MAPK activation and monocyte adhesion and migration induced by MCP-1. Metabolic stress promoted the Sglutathionylation of MKP-1, targeting MKP-1 for proteasomal degradation. Preventing MKP-1 S-glutathionylation in metabolically stressed monocytes by overexpressing glutaredoxin 1 protected MKP-1 from degradation and normalized monocyte adhesion and chemotaxis in response to MCP-1. Blood monocytes isolated from diabetic mice showed a 55% reduction in MKP-1 activity compared with nondiabetic mice. Hematopoietic MKP-1 deficiency in atherosclerosis-prone mice mimicked monocyte priming and dysfunction associated with metabolic disorders, increased monocyte chemotaxis in vivo, and accelerated atherosclerotic lesion formation. In conclusion, we identified MKP-1 as a central redox-sensitive regulator of monocyte adhesion and migration and showed that the loss of MKP-1 activity is a critical step in monocyte priming and the metabolic stress-induced conversion of blood monocytes into a proatherogenic phenotype.M etabolic disorders such as obesity and diabetes are associated with a state of chronic, low-grade inflammation (1, 2), which appears to contribute to the development of micro-and macrovascular complications such as atherosclerosis, nephropathy, and retinopathy (3-5). The cellular and molecular mechanisms involved in chronic inflammation associated with metabolic disorders are not yet fully understood, but the recruitment of blood monocytes to sites of vascular injury appears to play a central and rate-limiting role in all these complications. Metabolic disorders impact blood vessels at multiple levels, including lipid depositions, endothelial injury, and smooth muscle cell proliferation and migration, which individually or in concert initiate monocyte recruitment and promote vascular inflammation (6, 7). However, metabolic disorders also appear to affect blood monocytes directly. A number of studies reported that monocytes both in patients with metabolic disorders and in dyslipidemic or diabetic mice undergo phenotypical and functional changes that may contribute directly to the development and progression of chronic inflammatory vascular diseases (8-13)...

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Decreased growth rate, limited cell division, flat and large cell shapes, and tight binding of the cells to culture dished (1, 2) are well known characteristics of cells entering into replicative senescence (3). Primary mouse embryo fibroblasts exposed to oncogenic Ras overexpression undergo premature senescence in response to constitutive MAPK 1 kinase/MAPK mitogenic signaling (4, 5), whereas established variants lacking p53 or p19 ARF are efficiently transformed (6). Ha-Ras mutants that interact preferentially with specific Ras effector proteins are known as V 12 S 35 , V 12 C 40 , and V 12 G 37 (7, 8); V 12 S 35 binds preferentially to Raf-1 and activates MAPK without any effect on membrane ruffling (7), and the V 12 C 40 associates with phosphatidylinositol 3-kinase (Akt kinase) and promotes membrane ruffling and cell survival independent of Raf-1 (8, 9), but the V 12 G 37 binds to Ral-GDS without binding to Raf-1 or phosphatidylinositol 3-kinase (7). A distinctive feature of the cellular senescence induced by overexpression of the Ha-ras mutants as well as the replicative senescence in normal human diploid fibroblasts (HDF) is the markedly increased phosphorylation of extracellular signal-regulated protein kinase (pErk1/2) without nuclear translocation (10). However, its biochemical mechanism has not yet been clarified.MAPK activity is tightly regulated by phosphorylation and dephosphorylation. The activation of the MAPK activity requires the dual phosphorylation of the Ser/Thr and Tyr residues in the TXY kinase activation motif (11-13), and deactivation occurs through the action of either Ser/Thr protein phosphatase (14), protein-tyrosine phosphatase (PTP) (14, 15), or dual specificity phosphatases (16,17). The dual phosphatases are capable of catalyzing the removal of the phosphoryl group from Tyr(P) as well as Ser(P)/Thr(P) residues. The dual specificity phosphatases that specifically dephosphorylate and inactivate the p-Erk1/2 are called MAPK phosphatases (MKPs), and at least 9 mammalian MKPs have been identified so far (18,19). It has been reported that MKP3 (20), also termed Pyst1 (21) or VH6 (22), is predominantly localized in the cytoplasm, is highly specific for Erk1/2 inactivation, and is not inducible by either growth factor or stress (20 -23). An elegant series of studies have shown that the N-terminal domain of MKP3 can physically associate with Erk1/...

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Cellular senescence can either support or inhibit cancer progression. Here, it is shown that intratumoral infiltration of CD8+ T cells is negatively associated with the proportion of senescent tumor cells in colorectal cancer (CRC). Gene expression analysis reveals increased expression of C‐X‐C motif chemokine ligand 12 (CXCL12) and colony stimulating factor 1 (CSF1) in senescent tumor cells. Senescent tumor cells inhibit CD8+ T cell infiltration by secreting a high concentration of CXCL12, which induces a loss of CXCR4 in T cells that result in impaired directional migration. CSF1 from senescent tumor cells enhance monocyte differentiation into M2 macrophages, which inhibit CD8+ T cell activation. Neutralization of CXCL12/CSF1 increases the effect of anti‐PD1 antibody in allograft tumors. Furthermore, inhibition of CXCL12 from senescent tumor cells enhances T cell infiltration and results in reducing the number and size of tumors in azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced CRC. These findings suggest senescent tumor cells generate a cytokine barrier protecting nonsenescent tumor cells from immune attack and provide a new target for overcoming the immunotherapy resistance of CRC.

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We evaluated the nuclear actin accumulation as a new marker of cellular senescence, using human diploid fibroblast (HDF), chondrocyte primary cultures, Mv1Lu epithelial cells, and Huh7 cancer cells. Nuclear accumulation of globular actin (G-actin) and dephosphorylated cofilin was highly significant in the senescent HDF cells, accompanied with inhibition of LIM kinase (LIMK) -1 activity. When nuclear export of the actin was induced by 12-O-tetradecanoylphorbol-13-acetate, DNA synthesis of the senescent cells increased significantly, accompanied with changes of morphologic and biochemical profiles, such as increased RB protein phosphorylation and decreased expressions of p21 WAF1 , cytoplasmic pextracellular signal-regulated kinase 1/2, and caveolins 1 and 2. Significance of these findings was strengthened additionally by the fact that nuclear actin export of young HDF cells was inhibited by the treatment with leptomycin B and mutant cofilin transfection, whose LIMK-1 phosphorylation site was lost, and the old cell phenotypes were duplicated with nuclear actin accumulation, suggesting that nuclear actin accumulation was accompanied with G1 arrest during cellular senescence. The aforementioned changes were observed not only in the replicative senescence but also in the senescence induced by treatment of HDF cells, Mv1Lu, primary culture of human chondrocytes, or Huh7 cells with H-ras virus infection, hydroxyurea, deferoxamine, or H 2 O 2 . Nuclear actin accumulation was much more sensitive and an earlier event than the well-known, senescence-associated ␤-galactosidase activity.

Atmospherically stable NZVI (nanoscale zero-valent iron) particles were produced by modifying shell layers of Fe(H2) NZVI particles (RNIP-10DS) by using a controlled air contact method. Shell-modified NZVI particles were resistant to rapid aerial oxidation and were shown to have TCE degradation rate constants that were equivalent to 78% of those of pristine NZVI particles. Fe(H2) NZVI particles that were vigorously contacted with air (rapidly oxidized) showed a substantially compromised reactivity. Aging of shell-modified particles in water for one day resulted in a rate increase of 54%, implying that depassivation of the shell would play an important role in enhancing reactivity. Aging of shell-modified particles in air led to rate decreases by 14% and 46% in cases of one week and two months of aging, respectively. A series of instrumental analyses using transmission electron microscopy, X-ray diffractography, X-ray photoelectron spectroscopy, and X-ray absorption near-edge structure showed that the shells of modified NZVI particles primarily consisted of magnetite (Fe(3)O(4)). Analyses also implied that the new magnetite layer produced during shell modification was protective against shell passivation. Aging of shell-modified particles in water yielded another major mineral phase, goethite (alpha-FeOOH), whereas aging in air produced additional shell phases such as wustite (FeO), hematite (alpha-Fe(2)O(3)), and maghemite (gamma-Fe(2)O(3)).