Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

Chen, Zhenlong; Bakhshi, Farnaz R.; Shajahan, Ayesha N.; Sharma, Tiffany; Mao, Mao; Trane, Andy; Bernatchez, Pascal; Amerongen, Geerten P. van Nieuw; Bonini, Marcelo G.; Skidgel, Randal A.; Malik, Asrar B.; Minshall, Richard D.

doi:10.1091/mbc.e11-09-0811

Cited by 113 publications

(122 citation statements)

References 53 publications

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“…Finally, to further validate the participation of eNOS in Hath6 signaling, N v -Nitro-L-arginine methyl ester hydrochloride (L-NAME), a small-molecule eNOS inhibitor (Chen et al, 2012;Go et al, 2012), was applied to the step-wise endothelial differentiation system. FACS analysis (Fig.…”

Section: Hath6 Is a Direct Transcriptional Regulator Of Enosmentioning

confidence: 99%

The role of Hath6, a novel shear stress-responsive transcription factor, in endothelial differentiation and function modulation

Fang¹,

Wasserman²,

Torres-Vázquez³

et al. 2014

Journal of Cell Science

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The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study, we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system, Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently, gain-of-function and lossof-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6, which mimics shear stress treatment, resulted in an increased CD45 2 CD31 + KDR + population, a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast, the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression, tubularstructure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis, and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover, we present a potential signaling pathway through which shear stress stimulates endothelial differentiation.

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Section: Hath6 Is a Direct Transcriptional Regulator Of Enosmentioning

confidence: 99%

The role of Hath6, a novel shear stress-responsive transcription factor, in endothelial differentiation and function modulation

Fang¹,

Wasserman²,

Torres-Vázquez³

et al. 2014

Journal of Cell Science

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“…14 We have previously shown that reduced Eph-B4 function is associated with increased basal Akt phosphorylation in endothelial cells but not in whole vein grafts, 15 suggesting a link from Eph-B4 to eNOS via the Akt pathway, at least in endothelial cells. In addition, caveolin-1 interacts with eNOS, and negatively regulates eNOS phosphorylation, 30,31 and we have previously shown that caveolin-1 is a mediator of vein graft adaptation, 8 eg the Eph-B4-eNOS pathway is quite complex, 10 and the activity of this pathway during vein graft adaptation is likely to be have additional layers of regulation in vivo. It is likely that differences between Eph-B4-eNOS pathway activity in vitro, e.g.…”

Section: Discussionmentioning

confidence: 99%

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

Wang

Collins

Bai

et al. 2015

Journal of Vascular Surgery

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Objectives-Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand Ephrin-B2, the embryonic determinant of arterial identity. eNOS is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be via regulation of downstream eNOS activity. Methods-Mouse lung endothelial cells (MLEC)were stimulated with Ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor L-NAME or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using a NO-specific chemiluminescence analyzer. Cell migration was assessed using a transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type (WT) or eNOS-knockout (KO) mice.Results-Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n=4; p<.05), with preclustered Ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n=4; p<.05). Ephrin-B2/Fc increased NO release (n=3; p<.01) as well as increased endothelial cell migration (n=6; p<. 05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n=3; p<.05). Vein grafts from eNOS-KO mice showed less dilation and less wall thickening compared with WT vein grafts (n=7; p<.05).

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“…tional modification that in turn modulates multiple processes at the cell membrane, including Src localization to focal adhesions (21), activation of TLR4-mediated signaling (30), and eNOS inhibition (6). PBEF had no effect on total expression of phosphorylated Cav-1 nor on the phosphorylation of Cav-1 that was bound to the IR (Fig.…”

Section: Ir Signaling Is Dependent Uponmentioning

confidence: 99%

Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells

Peng

Jia

Parodo

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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. Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells. Am J Physiol Endocrinol Metab 308: E324 -E333, 2015. First published December 17, 2014 doi:10.1152/ajpendo.00006.2014.-Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IR␤ chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IR␤ movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IR␤ constructs to detect interactions between PBEF, the IR␤, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IR␤-and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser 473 and Thr 308 . We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEFinduced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.pre-B cell colony-enhancing factor; nicotinamide phosphoribosyl transferase; lipid rafts; insulin; insulin receptor; caveolin-1; signaling; phosphorylation CELLULAR RESPONSES TO STIMULI from the microenvironment are dependent upon the capacity of proteins in the cell membrane to aggregate and dissociate, and so they create multiprotein platforms that initiate intracellular signaling and lead to gene expression or repression within the nucleus. How these dynamic interactions are facilitated is incompletely understood. Lipid rafts are small, self-organizing dynamic membrane domains enriched in cholesterol, sphingolipid, and other proteins that are hypothesized to contribute to protein-protein interactions within the cell membrane (64,66,66), although whether lipids per se mediate protein sorting or whether protein localization within the membrane is a function of actin associating with membrane sphingolipids remains controversial (35).Caveolae represent a particular type of lipid raft that appears as a flask-shaped invagination of the cell surface (2). The formation and maintenance of caveolae depends on the pre...

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Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

Abstract: The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

Cited by 113 publications

References 53 publications

The role of Hath6, a novel shear stress-responsive transcription factor, in endothelial differentiation and function modulation

The role of Hath6, a novel shear stress-responsive transcription factor, in endothelial differentiation and function modulation

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells

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