NHE-1 and  &lt;meta charset="utf-8" /&gt; β1 integrin dependent monocyte adhesion and migration after glucose, insulin or PPARγ stimulation

Background: LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. Aims: The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. Methods: FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. Results: LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. Conclusion: In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration.

Section: Discussionmentioning

confidence: 99%

LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a

Alowayed

Salker²,

Zeng³

et al. 2016

“…The Na + /H + exchanger is further well known to participate in the regulation of migration [39,[57][58][59]. Thus, inhibition of the Na + /H + exchanger may contribute to the inhibitory effect of thymoquinone on DC migration following stimulation with LPS.…”

Section: Discussionmentioning

confidence: 99%

Effect of Thymoquinone on Cytosolic pH and Na<sup>+</sup>/H<sup>+</sup> Exchanger Activity in Mouse Dendritic Cells

Yang

Bhandaru²,

Pasham³

et al. 2012

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na⁺/H⁺ exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 µM). Cytosolic pH (pH_i) was determined from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na⁺-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 µg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 µM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 µM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.

“…CCL21-induced migration is thus a characteristic feature of mature DCs. As migration is similarly dependent on Na + /H + exchanger activity, [51,[54][55][56], further studies were performed to elucidate the effect of azathioprine on LPS-induced DCs migration. To this end, a transwell migration assay was performed in DCs treated with LPS in the presence and absence of azathioprine.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, the Na + /H + exchanger is known to participate in the machinery of cell migration [51,[54][55][56].…”

Section: Introductionmentioning

confidence: 99%

Effect of Azathioprine on Na⁺/H⁺Exchanger Activity in Dendritic Cells

Bhandaru

Pasham

Yang

et al. 2012

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na⁺/H⁺ exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na⁺/H⁺ exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH_i) was estimated utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na⁺/H⁺ exchanger activity from the Na⁺-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 µg/ml) led to a transient increase of Na⁺/H⁺ exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 µM) did not significantly alter the Na⁺/H⁺ exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na⁺/H⁺ exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na⁺/H⁺ exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.