Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes.

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.

Human metabolic response to systemic inflammation: assessment of the concordance between experimental endotoxemia and clinical cases of sepsis/SIRS.

INTRODUCTION: Two recent, independent, studies conducted novel metabolomics analyses relevant to human sepsis progression; one was a human model of endotoxin (lipopolysaccharide (LPS)) challenge (experimental endotoxemia) and the other was community acquired pneumonia and sepsis outcome diagnostic study (CAPSOD). The purpose of the present study was to assess the concordance of metabolic responses to LPS and community-acquired sepsis. METHODS: We tested the hypothesis that the patterns of metabolic response elicited by endotoxin would agree with those in clinical sepsis. Alterations in the plasma metabolome of the subjects challenged with LPS were compared with those of sepsis patients who had been stratified into two groups: sepsis patients with confirmed infection and non-infected patients who exhibited systemic inflammatory response syndrome (SIRS) criteria. Common metabolites between endotoxemia and both these groups were individually identified, together with their direction of change and functional classifications. RESULTS: Response to endotoxemia at the metabolome level elicited characteristics that agree well with those observed in sepsis patients despite the high degree of variability in the response of these patients. Moreover, some distinct features of SIRS have been identified. Upon stratification of sepsis patients based on 28-day survival, the direction of change in 21 of 23 metabolites was the same in endotoxemia and sepsis survival groups. CONCLUSIONS: The observed concordance in plasma metabolomes of LPS-treated subjects and sepsis survivors strengthens the relevance of endotoxemia to clinical research as a physiological model of community-acquired sepsis, and gives valuable insights into the metabolic changes that constitute a homeostatic response. Furthermore, recapitulation of metabolic differences between sepsis non-survivors and survivors in LPS-treated subjects can enable further research on the development and assessment of rational clinical therapies to prevent sepsis mortality. Compared with earlier studies which focused exclusively on comparing transcriptional dynamics, the distinct metabolomic responses to systemic inflammation with or without confirmed infection, suggest that the metabolome is much better at differentiating these pathophysiologies. Finally, the metabolic changes in the recovering patients shift towards the LPS-induced response pattern strengthening the notion that the metabolic, as well as transcriptional responses, characteristic to the endotoxemia model represent necessary and "healthy" responses to infectious stimuli.

Cellular metabolic regulators: novel indicators of low-grade inflammation in humans.

OBJECTIVE: The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. METHODS: Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. RESULTS: We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit α (AMPKα) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) α expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. CONCLUSIONS: These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPKα degradation, HIF-1α expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

Toll-Like Receptor 4 Induction During Renal Ischemia Correlates with Serum Creatinine in a Pig Model.

Objectives: To determine whether toll-like receptor 4 (TLR4), a mediator of organ ischemia-reperfusion injury, is overexpressed during warm ischemia in a porcine solitary kidney model, and whether its expression correlates with creatinine, a surrogate for kidney function. Materials and Methods: Eight adult Yorkshire pigs underwent initial laparoscopic nephrectomy. After 1 week, animals were randomized into two groups: group 1 underwent laparoscopic renal hilar dissection, renal ischemia by cross-clamping, and reperfusion (ischemia group); group 2 underwent laparoscopic renal hilar dissection alone (sham group). Animals were survived to day 7 postrandomization. Peripheral blood was sampled for serum creatinine (sCr) and TLR4 expression at the following time points or corresponding intervals: prenephrectomy, 1-week postnephrectomy (preischemia), after 90 minutes of ischemia, 30 minutes postreperfusion, and at sacrifice. Intragroup TLR4 expression changes were analyzed using repeated measures ANOVA. Intergroup TLR4 expression was compared using Mann-Whitney's test. Correlation between sCr and TLR4 was assessed using Spearman's test. Results: Seven animals completed the experiment (four ischemia and three sham). Relative TLR4 expression significantly increased from baseline levels during ischemia, reperfusion, and sacrifice time points only in the ischemia group, and was significantly higher for the ischemia group after 90 minutes of ischemia (p = 0.034). sCr was significantly higher for the ischemia group during the reperfusion phase (p = 0.048). Relative TLR4 expression level significantly correlated with sCr in the overall cohort (Spearman's rho = 0.69) and in the ischemia group (Spearman's rho = 0.82; p < 0.0001 for each). Conclusions: Warm ischemia in a porcine solitary kidney induces acute overexpression of TLR4 in peripheral blood leukocytes, which is detectable. Relative TLR4 expression level strongly correlated with sCr but had an observable change sooner than change in sCr. Pending further investigation, TLR4 overexpression during renal ischemia may represent a sensitive quantitative marker of unilateral renal injury sustained during nephron-sparing surgery.

Roles of SIRT1 in the acute and restorative phases following induction of inflammation.

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that in vivo endotoxin triggers a rapid and transient decline in ATP concentration in human peripheral blood leukocytes and murine peripheral blood leukocytes and liver, which is associated with a brief increase in expression of the autophagy indicator LC3-II. In both of these tissues, the ATP concentration reaches a nadir, and autophagy is induced between 2 and 4 h post-endotoxin infusion, and homeostasis is restored within 12 h. Mouse liver SIRT1 and AMP-activated protein kinase (AMPK) protein expression levels decline precipitously within 10 min and remain below detection levels for up to 12 h post-endotoxin administration. In marked contrast, the expression of HIF-1α is induced within 90 min and remains elevated for up to 12 h. The ATP recovery is delayed, and the increases in both HIF-1α expression and autophagy are prolonged in endotoxin-challenged SIRT1 liver knock-out mice. Resveratrol prevents the decline in ATP concentration and SIRT1 expression, as well as the increase in HIF-1α expression and autophagy in liver of endotoxin-challenged wild type mice but not in SIRT1 liver knock-out mice. These results provide novel insight into the state of both cellular bioenergetics and metabolic networks during the acute phase of systemic inflammation and suggest a role for SIRT1 in acute metabolic decline, as well as the restoration of metabolic homeostasis during an inflammatory challenge.

In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.

OBJECTIVES: The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. DESIGN: Clinical and laboratory investigation. SETTING: University-based research laboratory and clinical research center. SUBJECTS: Human volunteers. INTERVENTIONS: Human subjects were administered a standard dose of endotoxin (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion. MEASUREMENTS AND MAIN RESULTS: Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2 method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1epsilon, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. CONCLUSIONS: Circadian clock gene expression in peripheral blood leukocytes is dramatically altered and possibly uncoupled from the activity of the central clock during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes.

INTRODUCTION: An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration. METHODS: PBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR. RESULTS: We describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients. CONCLUSIONS: We identified a group of > 400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2.

TLR4 is implicated in diseases associated with chronic low-grade inflammation, yet homeostatic signaling mechanisms that prevent and/or are affected by chronic TLR4 activation are largely uncharacterized. We recently reported that LPS/TLR4 activates in human leukocytes signaling intermediates (SI), abbreviated TLR4-SI, which include mTORC1-specific effectors and targets, and that leukocytes of patients with T2D or after cardiopulmonary bypass (CPB) expressed similar SI. Extending these previous findings, here we show that TLR4-SI expression post-CPB was associated with low serum bilirubin and reduced preoperative expression of biliverdin reductase A (BVRA), the enzyme that converts biliverdin to bilirubin, in patient's leukocytes. Biliverdin inhibited TLR4 signaling in leukocytes and triggered phosphorylation of mTORC2-specific targets, including Akt, PKCζ, AMPKα-LKB1-TSC1/2, and their association with BVRA. Torin, PP242, and a PKCζ inhibitory peptide, but not rapamycin, prevented these biliverdin-induced responses and TLR4 inhibition. In contrast, LPS/TLR4 triggered decreases in BVRA, AMPKα and PKCζ expression, and an increase in haptoglobin, a heme binding protein, in leukocytes in vivo and in vitro, indicating that activated TLR4 may suppress biliverdin/BVRA signaling. Significantly, compared to non-diabetics, BVRA and PKCζ expression was low and haptoglobin was high in T2D patients leukocytes. Sustained TLR4 activation may deregulate homeostatic anti-inflammatory BVRA/mTORC2 signaling and thereby contribute to chronic inflammatory diseases.

The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading.

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.

Shigella and Salmonella: death as a means of survival.

Shigella and Salmonella kill host cells and trigger inflammatory responses by mechanisms that are not fully understood. The goal of this review is to reevaluate key observations reported over the past 15 years and, whenever possible, to provide a chronological perspective as to how our understanding of the pathways by which Shigella and Salmonella kill host cells has evolved.

Unilateral Partial Nephrectomy with Warm Ischemia Results in Acute Hypoxia Inducible Factor 1-Alpha (HIF-1α) and Toll-Like Receptor 4 (TLR4) Overexpression in a Porcine Model.

PURPOSE: Ischemia/reperfusion (I/R) during partial nephrectomy (PN) contributes to acute kidney injury (AKI), which is inaccurately assessed using existent clinical markers of renal function. We evaluated I/R-related changes in expression in hypoxia inducible factor 1α (HIF-1α) and toll-like receptor 4 (TLR4), within kidney tissue and peripheral blood leukocytes (PBL) in a porcine model of PN. MATERIALS AND METHODS: Three adult pigs each underwent unilateral renal hilar cross clamping for 180 min followed by a 15 min reperfusion. The contralateral kidney served as control. Biopsies of clamped kidneys were obtained at baseline (time 0), every 60 min during the hypoxic phase, and post-reperfusion. Control kidneys were biopsied once at 180 min. Peripheral blood was sampled at time 0, every 30 min during the hypoxic phase, and post-reperfusion. HIF-1α and TLR4 expression in kidney tissue and PBL were analyzed by Western blotting. I/R-related histological changes were assessed. RESULTS: Expression of HIF-1α in clamped kidneys and PBL was below detection level at baseline, rising to detectable levels after 60 min of hypoxia, and continuing to rise throughout the hypoxic and reperfusion phases. Expression of TLR-4 in clamped kidneys followed a similar trend with initial detection after 30-60 min of hypoxia. Control kidneys exhibited no change in HIF-1α or TLR-4 expression. I/R-related histologic changes were minimal, primarily mild tubular dilatation. CONCLUSIONS: In a porcine model of PN, HIF-1α and TLR4 exhibited robust, I/R-related increases in expression in kidney tissue and PBL. Further studies investigating these molecules as potential markers of AKI are warranted.

Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes.

Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression.

Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction.

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.

The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin.

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration.

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.

Virulent Shigella flexneri causes damage to mitochondria and triggers necrosis in infected human monocyte-derived macrophages.

Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% +/- 6% of the HMDM and

Neutrophil survival on biomaterials is determined by surface topography.

PURPOSE: Cardiovascular device-centered infections are a major cause of hospital morbidity, mortality, and expense. Caused by opportunistic bacteria, this phenomenon is thought to arise because of a defect in neutrophil bacterial killing. We have shown that neutrophils that adhere to polystyrene remain viable, whereas neutrophils that adhere to the vascular biomaterials expanded polytetrafluoroethylene (ePTFE) and Dacron undergo a rapid nonapoptotic death. This study was designed to test the hypothesis that surface topography is a determinant of the nonapoptotic death response of neutrophils to biomaterials. METHODS: We took advantage of the ease with which a polystyrene surface can be manipulated to examine the effect of surface topography on neutrophil viability. Neutrophils were exposed to smooth or roughened polystyrene surfaces both in vivo and in vitro. Changes in cell membrane permeability and production of reactive oxygen species by individual cells were monitored with fluorescent dyes. RESULTS: Host cells and isolated human neutrophils died rapidly after adhesion to roughened polystyrene. Neutrophils adherent to roughened surfaces produced more reactive oxygen intermediates than those adherent to smooth surfaces and were first to die. The cell death response precipitated by expanded polytetrafluoroethylene, Dacron, or the roughened surfaces was significantly reduced with treatment of the neutrophils with catalase, diphenylene iodonium, or the src kinase inhibitor PP2 before adhesion. CONCLUSIONS: Neutrophil adhesion to roughened materials triggers rapid production of reactive oxygen species and precipitates a nonapoptotic cell death. Understanding the material properties that trigger these responses is essential to development of the next generation of implantable biomaterials.

Force-dependent integrin-cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2.

As cells encounter new regions of the substrate, they develop bonds with new matrix molecules for migration, matrix remodeling and force generation. How cells orchestrate the assembly of adhesion sites is only partially understood. Here we show that fibroblasts deficient in the SH2 domain containing protein tyrosine phosphatase 2 (Shp2) have an increased number of immature focal complexes deficient in alpha-actinin. Inhibition of FAK restored alpha-actinin to focal complexes, whereas inhibition of RhoA did not. In correlation, adhesion site dynamics, measured by fluorescence recovery after photobleaching (FRAP) of GFP-paxillin and GFP-vinculin were dramatically increased in Shp2(-/-) cells and restored to normal by FAK inhibition. Shp2(-/-) cells failed to strengthen initial integrin-cytoskeleton linkages, as measured by optical tweezers and large bead assays, and were rescued by inhibition of FAK. In contrast, affinity modulation of adhesion receptors was unaffected. In addition, reinforcement correlated with alpha-actinin assembly through decreased dynamics. This shows for the first time that adhesion site dynamics are regulated during adhesion formation and that force-dependent strengthening of integrin-cytoskeleton linkages is correlated with alpha-actinin assembly and decreased adhesion site dynamics.

The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin.

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.