The influence of Pyk2 on the mechanical properties in fibroblasts

The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK^+/+, FAK^−/−, and siRNA-Pyk2 treated FAK^−/− cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.     

Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency

Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fiber cell adhesion are different in AQP0^−/−, and TgAQP1^+/+/AQP0^−/− mice that transgenically express AQP1 (TgAQP1) in fiber cells without AQP0 (AQP0^−/−). In WT, lenses were transparent with ‘Y’ sutures. Fibers contained opposite end curvature, lateral interdigitations, hexagonal shape, and were arranged as concentric growth shells. AQP0^−/− lenses were cataractous, lacked ‘Y’ sutures, ordered packing and well-defined lateral interdigitations. TgAQP1^+/+/AQP0^−/− lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fiber cells in WT whereas AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1^+/+/AQP0^−/− mice. Fiber cell AQP0 expression is required to maintain their organization, which is a requisite for lens transparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses, fiber cell disorganization was evident.     

RSK2-induced stress tolerance enhances cell survival signals mediated by inhibition of GSK3β activity

Our previous studies demonstrated that RSK2 plays a key role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor (EGF) in mouse and human skin cells. However, no direct evidence has been found regarding the relationship of RSK2 and cell survival. In this study, we found that RSK2 interacted and phosphorylated GSK3β at Ser9. Notably, GSK3β phosphorylation at Ser9 was suppressed in RSK2^−/− MEFs compared with RSK2^+/+ MEFs by stimulation of EGF and calcium ionophore A23187, a cellular calcium stressor. In proliferation, we found that RSK2 deficiency suppressed cell proliferation compared with RSK2^+/+ MEFs. In contrast, GSK3β^−/− MEFs induced the cell proliferation compared with GSK3β^+/+ MEFs. Importantly, RSK2^−/− MEFs were induced severe cellular morphology change by A23187 and enhanced G1/G0 and sub-G1 accumulation of the cell cycle phase compared with RSK2^+/+ MEFs. The sub-G1 induction in RSK2^−/− MEFs by A23187 was correlated with increase of cytochrome c release, caspase-3 cleavage and apoptotic DNA fragmentation compared with RSK2^+/+ MEFs. Notably, return back of RSK2 into RSK2^−/− MEFs restored A23187-induced morphological change, and decreased apoptosis, apoptotic DNA fragmentation and caspase-3 induction compared with RSK2^−/−/mock MEFs. Taken together, our results demonstrated that RSK2 plays an important role in stress-tolerance and cell survival, resulting in cell proliferation and cancer development.     

HtrA2/Omi influences the stability of LON protease 1 and prohibitin,proteins involved in mitochondrial homeostasis

High temperature requirement A2 (HtrA2)/Omi is a serine protease localized in mitochondria. In response to apoptotic stimuli, HtrA2 is released to the cytoplasm and cleaves many proteins, including XIAP, Apollon/BRUCE, WT1, and Ped/Pea-15, to promote apoptosis. However, the function of HtrA2 in mitochondria under normal conditions remains unclear. Here, we show that the mitochondrial proteins, LON protease 1 (LONP1) and prohibitin (PHB), are overexpressed in HtrA2^−/− mouse embryonic fibroblast (MEF) cells and HtrA2 knock-down HEK293T cells. We also confirm the effect of the HtrA2 protease on the stability of the above mitochondrial quality control proteins in motor neuron degeneration 2 (mnd2) mice, which have a greatly reduced protease activity as a result of a Ser276Cys missense mutation of the HtrA2 gene. In addition, PHB interacts with and is directly cleaved by HtrA2. Luminescence assays demonstrate that the intracellular ATP level is decreased in HtrA2^−/− cells compared to HtrA2^+/+ cells. HtrA2 deficiency causes a decrease in the mitochondrial membrane potential, and reactive oxygen species (ROS) generation is greater in HtrA2^−/− cells than in HtrA2^+/+ cells. Our results implicate that HtrA2 might be an upstream regulator of mitochondrial homeostasis.     

N-WASP plays a critical role in fibroblast adhesion and spreading

N-WASP (Neural Wiskott Aldrich Syndrome Protein) regulates actin polymerization by activating the Arp2/3 complex and promotes the formation of actin-rich structures such as filopodia. Such actin-rich structures play critical roles in cell adhesion and cell motility. Analysis of the adhesion properties of N-WASP^+/+ and N-WASP^−/− mouse embryonic fibroblasts to extracellular matrix proteins revealed that N-WASP is critical for cell adhesion to fibronectin. There was no significant difference in the localization of paxillin in the two cell lines, however the vinculin patches in WASP^+/+ cells were thicker and more prominent than those in N-WASP^−/− cells. The β1 integrins in N-WASP^+/+ cells were found in large clusters, while β1 integrins were more dispersed in N-WASP^−/− cells. The N-WASP^−/− cells migrated more rapidly than N-WASP^+/+ cells in a scratch migration assay. Thus, our data suggest that N-WASP deficiency leads to reduced adhesion to fibronectin and increased cell motility.     

Deficiency of inducible nitric oxide synthase exacerbates hepatic fibrosis in mice fed high-fat diet

The role of inducible nitric oxide synthase (iNOS) in the progression of fibrosis during nonalcoholic steatohepatitis remains to be elucidated. This study examined the role of iNOS in the progression of fibrosis during steatohepatitis by comparing iNOS knockout (iNOS^−/−) and wild-type (iNOS^+/+) mice that were fed a high-fat diet. Severe fatty metamorphosis developed in the liver of iNOS^+/+ and iNOS^−/− mice. Fibrotic changes were marked in iNOS^−/− mice. Gelatin zymography showed that pro MMP-2 and pro MMP-9 protein expressions were more highly induced in iNOS^+/+ mice than in iNOS^−/− mice. Active forms of MMP-2 and MMP-9 were clearly present only in the liver tissue of iNOS^+/+ mice. In situ zymography showed strong gelatinolytic activities in the liver tissue of iNOS^+/+ mice, but only spotty activity in iNOS^−/−mice. iNOS may attenuate the progression of liver fibrosis in steatohepatitis, in part by inducing MMP-2 and MMP-9 expression and augmenting their activity.     

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

Estrogen receptor negative (ER^−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I₂) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER^−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I₂ (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER^−ve mammary tumors could be sensitized to I₂-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I₂ treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I₂, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I₂ treatment alone. Flowcytometry of I₂ and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I₂ treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I₂ and CQ co-treated mice relative to I₂ or vehicle treated mice. These data indicate that inhibition of autophagy renders ER^−ve breast tumor cells more sensitive to I₂ induced apoptosis. Thus, I₂ together with autophagy inhibitor could have a potential tumorostatic role in ER^−ve aggressive breast tumors that may be evaluated in future studies.     

AMPK-mediated increase in myocardial long-chain fatty acid uptake critically depends on sarcolemmal CD36

CD36, also named fatty acid translocase, has been identified as a putative membrane transporter for long-chain fatty acids (LCFA). In the heart, contraction-induced 5′ AMP-activated protein kinase (AMPK) signaling regulates cellular LCFA uptake through translocation of CD36 and possibly of other LCFA transporters from intracellular storage compartments to the sarcolemma. In this study, isolated cardiomyocytes from CD36^+/+- and CD36^−/− mice were used to investigate to what extent basal and AMPK-mediated LCFA uptake are CD36-dependent. Basal LCFA uptake was not altered in CD36^−/− cardiomyocytes, most likely resulting from a (1.8-fold) compensatory upregulation of fatty acid-transport protein-1. The stimulatory effect of contraction-mimetic stimuli, oligomycin (2.5-fold) and dipyridamole (1.6-fold), on LCFA uptake into CD36^+/+ cardiomyocytes was almost completely lost in CD36^−/− cardiomyocytes, despite that AMPK signaling was fully intact. CD36 is almost entirely responsible for AMPK-mediated stimulation of LCFA uptake in cardiomyocytes, indicating a pivotal role for CD36 in mediating changes in cardiac LCFA fluxes.     

The roles of translation initiation regulation in ultraviolet light-induced apoptosis

Ultraviolet light (UV) inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α). Two eIF2α kinases, PERK and GCN2, are known to phosphorylate the Serine-51 of eIF2α in response to UV-irradiation. In this report, we present evidence that phosphorylation of eIF2α plays a role in UV-induced apoptosis. Our data show that wild-type mouse embryo fibroblasts (MEF^s/s) are less sensitive to UV-induced apoptosis than MEF^A/A cells in which the phosphorylation site, Ser51, of eIF2α is replaced with a non-phosphorylatable Ala (Ser51Ala). PARP expression in MEF^A/A cells is reduced without being cleaved after UV-irradiation. In contrast, PARP is cleaved without a significant decrease in parental PARP in MEF^S/S cells after UV-irradiation. Our data also show that MEF^GCN2−/− cells, in which GCN2 is knocked out, are more sensitive to UV-irradiation, agreeing with the observation from MEF^A/A cells. However, MEF^PERK−/− cells, in which PERK is knocked out, are less sensitive to UV-irradiation. In addition, MCF-7-PERKΔC cells, which are stably transfected with a kinase domain deleted mutant of PERK (PERKΔC), are more resistant to UV-induced apoptosis than parental MCF-7 cells. Overexpression of wild-type PERK sensitizes MCF-7 cells to UV-induced apoptosis without directly inducing cell death. These results suggest that the level of eIF2α phosphorylation impacts PARP expression upon UV-irradiation. The eIF2α kinases may mediate UV-induced apoptosis via an eIF2α dependent or independent signaling pathway.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis

Angiopoietin-like protein family 4 (Angptl 4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). We generated ApoE^−/−Angptl 4^−/− mice to study the effect of Angptl 4 deficiency on lipid metabolism and atherosclerosis. Fasting and postolive oil-loaded triglyceride (TG) levels were largely decreased in ApoE^−/−Angptl 4^−/− mice compared with and ApoE^−/−Angptl 4^+/+ mice. There was a significant (75 ± 12%) reduction in atherosclerotic lesion size in ApoE^−/−Angptl 4^−/− mice compared with ApoE^−/− Angptl 4^+/+ mice. Peritoneal macrophages, isolated from Angptl 4^−/− mice to investigate the foam cell formation, showed a significant decrease in newly synthesized cholesteryl ester (CE) accumulation induced by acetyl low-density lipoprotein (acLDL) compared with those from Angptl 4^+/+ mice. Thus, genetic knockout of Angptl 4 protects ApoE^−/− mice against development and progression of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells in vitro.     

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7^−/−) than in erythrocytes from wild type mice (anxA7^+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7^−/− and anx7^+/+-mice following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7^−/− than in anx7^+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7^−/− than in anx7^+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.     

NOCICEPTIVE RESPONSES IN INTERLEUKIN-6-DEFICIENT MICE TO PERIPHERAL INFLAMMATION AND PERIPHERAL NERVE SECTION

The cutaneous nociceptive response threshold to mechanical and thermal stimulation, the development of hyperalgesia and plasma extravasation after subcutaneous injection of carrageenan and the development of autotomy behaviour after nerve section were assessed in interleukin-6-deficient (IL-6^−/−) and age-matched wild-type (IL-6^+/+) mice. IL-6^−/−mice had significantly lower response threshold to both mechanical and thermal stimulation in comparison to IL-6^+/+controls. Both IL-6^−/−and IL-6^+/+mice developed hyperalgesia to mechanical and thermal stimulation after localized carrageenan injection, but the magnitude of the hyperalgesia was less in the IL-6^−/−than in the IL-6^+/+controls. IL-6^−/−mice also exhibited less plasma extravasation after carrageenan injection. No difference was noted between males and females in basal nociception and inflammatory hyperalgesia. However, female IL-6^−/−mice exhibited autotomy behaviour, a sign of neuropathic pain, significantly more frequently and after a shorter interval following peripheral nerve injury than male IL-6^−/−or male and female IL-6^+/+mice. It is suggested that IL-6^−/−mice exhibited numerous changes in nociceptive responses compared to controls, some of which are sex related. The mechanisms of these changes in relation to null-mutation of the IL-6 gene and the influence of genetic background are discussed.     

IKK-β/NF-κB p65 mediates p27 protein degradation in arsenite response

p27^Kip1 is a potent inhibitor of the cyclin-dependent kinases that drive G1 to S phase transition. Since deregulation of p27^Kip1 is found in many malignancies and is associated with the poor prognosis, elucidation of the molecular bases for regulation of p27^Kip1 expression is of great significance, not only in providing insight into the understanding of biological p27^Kip1, but also in the development of new cancer therapeutic tactics. We here explored the inhibitory regulation of IKKβ on p27^Kip1 expression following arsenite exposure. We found that although the basal level of p27^Kip1 expression in the IKKβ^−/− cells is much lower than that in the IKKβ^+/+ cells, the deletion of IKKβ in the MEFs led to a marked increase in p27^Kip1 protein induction due to arsenite exposure in comparison to that in the IKKβ^+/+ cells. The IKKβ regulatory effect on p27^Kip1 expression was also verified in the IKKβ^−/− and IKKβ^−/− cells with IKKβ reconstitutional expression, IKKβ^−/− (IKKβ). Further studies indicated that IKKβ-mediated p27^Kip1 downregulation occurred at protein degradation level via p65-dependent and p50-independent manner. Moreover, the results obtained from the comparison of arsenite-induced GSK3β activation among transfectants of WT, IKKβ^−/− and IKKβ^−/− (IKKβ), and the utilization of GSKβ shRNA, demonstrated that IKKβ regulation of p27 protein degradation was mediated by GSK3β following arsenite exposure.     

Impairment of survival signaling and efferocytosis in TRPC3-deficient macrophages

We have recently shown that in macrophages proper operation of the survival pathways phosphatidylinositol-3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) has an obligatory requirement for constitutive, non-regulated Ca²⁺ influx. In the present work we examined if Transient Receptor Potential Canonical 3 (TRPC3), a member of the TRPC family of Ca²⁺-permeable cation channels, contributes to the constitutive Ca²⁺ influx that supports macrophage survival. We used bone marrow-derived macrophages obtained from TRPC3^−/− mice to determine the activation status of survival signaling pathways, apoptosis and their efferocytic properties. Treatment of TRPC3^+/+ macrophages with the pro-apoptotic cytokine TNFα induced time-dependent phosphorylation of IκBα, AKT and BAD, and this was drastically reduced in TRPC3^−/− macrophages. Compared to TRPC3^+/+ cells TRPC3^−/− macrophages exhibited reduced constitutive cation influx, increased apoptosis and impaired efferocytosis. The present findings suggest that macrophage TRPC3, presumably through its constitutive function, contributes to survival signaling and efferocytic properties.     

The leukotriene B4 receptor, BLT1, is required for the induction of experimental autoimmune encephalomyelitis

Leukotriene B₄ (LTB₄) is a potent chemoattractant and activator of neutrophils, macrophages and T cells. These cells are a key component of inflammation and all express BLT1, a high affinity G-protein-coupled receptor for LTB₄. However, little is known about the neuroimmune functions of BLT1. In this study, we describe a distinct role for BLT1 in the pathology of experimental autoimmune encephalomyelitis (EAE) and T_H1/T_H17 immune responses. BLT1 mRNA was highly upregulated in the spinal cord of EAE mice, especially during the induction phase. BLT1^−/− mice had delayed onset and less severe symptoms of EAE than BLT1^+/+ mice. Additionally, inflammatory cells were recruited to the spinal cord of asymptomatic BLT1^+/+, but not BLT1^−/− mice before the onset of disease. Ex vivo studies showed that both the proliferation and the production of IFN-γ, TNF-α, IL-17 and IL-6 were impaired in BLT1^−/− cells, as compared with BLT1^+/+ cells. Thus, we suggest that BLT1 exacerbates EAE by regulating the migration of inflammatory cells and T_H1/T_H17 immune responses. Our findings provide a novel therapeutic option for the treatment of multiple sclerosis and other T_H17-mediated diseases.     

ER stress responses in the absence of apoptosome: A comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9^+/+ and casp9^−/− MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9^−/− cells as compared with casp9^+/+ MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9^−/− MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.