TIMAP is a positive regulator of pulmonary endothelial barrier function

Csortos, Csilla; Czikora, István; Bogatcheva, Natalia V.; Adyshev, Djanybek; Poirier, Christophe; Oláh, Gabor; Verin, Alexander D.

doi:10.1152/ajplung.00325.2007

Cited by 36 publications

(66 citation statements)

References 30 publications

(50 reference statements)

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“…In 2ME-challenged cells, the intracellular localization of ERM significantly differed from the localization of phospho-ERM, suggesting that only a minor fraction of ERM is phosphorylated. Our data were consistent with previous reports (17,18) showing that phospho-ERM were mostly localized to the peripheral area in endothelial cells undergoing contraction and a loss of monolayer integrity. Of interest, the colocalization of phospho-ERM with F-actin branching points was also described in endothelial cells (42).…”

Section: Discussionsupporting

confidence: 94%

“…Previously we reported that 2ME causes barrier dysfunction in HPAEC monolayers, and that this process is dependent on the disruption of MTs and the activation of Rho kinase (ROCK) and p38 cascades (16). With the realization that barrier-disruptive signaling events may not be limited to the activation of ROCK and p38, we studied the involvement of protein kinase C (PKC) in response to 2ME.Ezrin/radixin/moesin (ERM) proteins are well-known PKC substrates, emerging as potential candidates in the regulation of barrier permeability (17)(18)(19). Believed to serve as cross-linkers between the actin cytoskeleton and plasma membrane, ERM were also shown to be engaged in cell motility and adhesion.…”

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confidence: 99%

“…Ezrin/radixin/moesin (ERM) proteins are well-known PKC substrates, emerging as potential candidates in the regulation of barrier permeability (17)(18)(19). Believed to serve as cross-linkers between the actin cytoskeleton and plasma membrane, ERM were also shown to be engaged in cell motility and adhesion.…”

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confidence: 99%

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Ezrin, Radixin, and Moesin Are Phosphorylated in Response to 2-Methoxyestradiol and Modulate Endothelial Hyperpermeability

Bogatcheva

Zemskova

Gorshkov

et al. 2011

Am J Respir Cell Mol Biol

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We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylationdeficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubuledisrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.Keywords: 2-methoxyestradiol; ERM; PKC; phosphorylation; barrier dysfunction 2-methoxyestradiol (2ME) is a natural derivative of estradiol metabolism, which exhibits antiproliferative and antiangiogenic properties when used at pharmacological concentrations (1). 2ME is currently undergoing clinical trials to establish its efficacy as an anticancer drug, and to reveal its potential toxicities. One major problem encountered so far involves the low bioavailability of the orally delivered drug (2-5), which prompted the use of the nanodispersed 2ME form in trials (6, 7), and also instigated a search for 2ME analogues with better availability and a lower rate of degradation (8-11). At the same time, several studies using 2ME solo or in combination with other agents reported vascularrelated side effects such as edema, angioedema, and dyspnea (2-6). These data reveal the multifaceted nature of the responses evoked by the anticancer agent 2ME. An elucidation of the signaling pathways leading to 2ME-induced vascular barrier dysfunction will help manage the unwanted side effects of 2ME, and ensure the safe clinical use of this promising anticancer compound.Unlike receptor-interacting estrad...

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Section: Discussionsupporting

confidence: 94%

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confidence: 99%

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Ezrin, Radixin, and Moesin Are Phosphorylated in Response to 2-Methoxyestradiol and Modulate Endothelial Hyperpermeability

Bogatcheva

Zemskova

Gorshkov

et al. 2011

Am J Respir Cell Mol Biol

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“…The LAMR1 is upregulated during EC proliferation and angiogenesis (35,50) and is required for capillary formation in vitro and angiogenesis in vivo (18,31). TIMAP also regulates moesin phosphorylation in EC (11) and can dephosphorylate MLC2 in vitro (48), but its actual mechanism of action in EC is poorly understood.…”

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confidence: 99%

TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells

Obeidat

Ballermann

2014

American Journal of Physiology-Renal Physiology

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Obeidat M, Li L, Ballermann BJ. TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells. Am J Physiol Renal Physiol 307: F623-F633, 2014. First published July 9, 2014 doi:10.1152/ajprenal.00070.2014.-The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 Ϯ 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2=-deoxyuridine incorporation by 38 Ϯ 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAPdepleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/ Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN. endothelial cells; PTEN; protein phosphatase 1-regulatory subunit; PPP1R16B; apoptosis; clectric cell-substrate impedance sensing MECHANISMS THAT REGULATE ANGIOGENESIS have received massive attention since Folkman (15) first advanced the theory that tumor growth depends on new blood vessel formation. Angiogenesis involves the sprouting and elongation of new blood vessels from preexisting vessels followed by stabilization and vessel specification (28). This process is crucial for normal blood vessel formation and patterning during embryonic development, for postembryonic blood vessel repair (5), and remodeling in response to hypoxia, in the female reproductive cycle, and for the establishment of the placental vasculature. Pathological retinal and tumor vascularization also depend on angiogenesis. While endothelial cells (EC) at the tip of the angiogenic sprout determine the direction and pattern of new blood vessel growth, elongation of the new vessel stalk requires EC proliferatio...

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“…If the physiological PP1cβ activity contributes to the normal maintenance of some cellular functions such as endothelial cell barrier protection, cytoskeleton structure support [9,10,11] and DNA repair [12], its overexpression has been observed in certain cancers, like metastatic melanoma [13], malignant pancreatic tumors [14,15], and breast cancers [16,17]. Additionally, we have recently shown that hypoxia, a major regulator of angiogenesis [18], upregulates the mRNA of protein phosphatase type 1 beta (PP1cβ) in cyanotic children with tetralogy of Fallot (TOF) [19].…”

Section: Introductionmentioning

confidence: 99%

Protein Phosphatase 1 Beta is Modulated by Chronic Hypoxia and Involved in the Angiogenic Endothelial Cell Migration

Iacobazzi

Garaeva

Albertario

et al. 2015

Cell Physiol Biochem

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Background/Aim: Endothelial cell migration is required for physiological angiogenesis, but also contributes to various pathological conditions, including tumour vascularization. The mRNA expression of PP1cβ, the beta isoform of the catalytic PP1 subunit, was shown to be upregulated in chronic hypoxia. Since hypoxia is a major regulator of angiogenesis, the potential role of PP1cβ in angiogenesis was investigated. Methods: We examined PP1cβ protein level in pediatric heart following chronic hypoxia and found PP1cβ upregulation in cyanotic compared with acyanotic myocardium. By treating HUVEC cells with hypoxia mimicking agent, PP1cβ protein level increased with maximum at 8 hours. The effect of PP1cβ pharmacological inhibition, knockdown and overexpression, on endothelial cell migration and morphogenesis, was examined using in vitro wound healing scratch assay and endothelial tube formation assay. The PP1cβ knockdown effects on F-actin reorganization (phalloidin staining), focal adhesion formation (vinculin) and focal adhesion kinases (FAK) activation, were evaluated by immunocytochemical staining and immunoblotting with specific antibodies. Results: PP1cβ knockdown significantly reduces endothelial cell migration, but does not have any significant effect on endothelial tube formation. Endothelial cell migration in the knockdown group is restored to the control level upon consecutive transfection with PP1cβ cDNA. PP1cβ overexpression does not significantly affect endothelial cell migration. Furthermore, PP1cβ knockdown induces profound cytoskeletal reorganization, loss of focal adhesion sites and impairment of focal adhesion kinases (FAK) activation. Conclusions: PP1cβ is regulator of endothelial cell migration, which is critical in the angiogenic process. PP1cβ inhibition reduces endothelial cell migration through focal adhesion turnover and actin polymerization pathways.

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TIMAP is a positive regulator of pulmonary endothelial barrier function

Cited by 36 publications

References 30 publications

Ezrin, Radixin, and Moesin Are Phosphorylated in Response to 2-Methoxyestradiol and Modulate Endothelial Hyperpermeability

Ezrin, Radixin, and Moesin Are Phosphorylated in Response to 2-Methoxyestradiol and Modulate Endothelial Hyperpermeability

TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells

Protein Phosphatase 1 Beta is Modulated by Chronic Hypoxia and Involved in the Angiogenic Endothelial Cell Migration

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