Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator

Ji, Hong; Zhao, Runzhen; Komissarov, Andrey A.; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A.

doi:10.1074/jbc.m114.623496

Cited by 39 publications

(40 citation statements)

References 92 publications

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“…Practical issues are coupling long duration of action with absence of systemic effects. Therefore, our results support the continued development of safe, long-acting ENaC inhibitors1617, as well as continued investigation of endogenous ENaC inhibition2545484950. Evidence that ENaC-mediated I sc by surface epithelia can be decreased by cholinergic stimulation255152 suggests that ENaC inhibition might be a component of physiologically mediated increases in mucus clearance from the airways.…”

Section: Discussionsupporting

confidence: 77%

Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel

Joo

Jeong

Cho

et al. 2016

Sci Rep

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Mucociliary clearance (MCC) is a critical host innate defense mechanism in airways, and it is impaired in cystic fibrosis (CF) and other obstructive lung diseases. Epithelial fluid secretion and absorption modify MCC velocity (MCCV). We tested the hypotheses that inhibiting fluid absorption accelerates MCCV, whereas inhibiting fluid secretion decelerates it. In airways, ENaC is mainly responsible for fluid absorption, while anion channels, including CFTR and Ca2+-activated chloride channels mediate anion/fluid secretion. MCCV was increased by the cAMP-elevating agonists, forskolin or isoproterenol (10 μM) and by the Ca2+-elevating agonist, carbachol (0.3 μM). The CFTR-selective inhibitor, CFTRinh-172, modestly reduced MCCV-increases induced by forskolin or isoproterenol but not increases induced by carbachol. The ENaC inhibitor benzamil increased basal MCCV as well as MCCV increases produced by forskolin or carbachol. MCC velocity was most dramatically accelerated by the synergistic combination of forskolin and carbachol, which produced near-maximal clearance rates regardless of prior treatment with CFTR or ENaC inhibitors. In CF airways, where CFTR-mediated secretion (and possibly synergistic MCC) is lost, ENaC inhibition via exogenous agents may provide therapeutic benefit, as has long been proposed.

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Section: Discussionsupporting

confidence: 77%

Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel

Joo

Jeong

Cho

et al. 2016

Sci Rep

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“…All of these substrates have been implicated in cancer progression [59,60,62,63]. The requirement for uPAR in allowing uPA to cleave PDGF-DD is unclear, while the cleavage of a6 integrin and ENaC are independent of uPAR [61,62].…”

Section: Discussionmentioning

confidence: 99%

“…Under physiologically relevant conditions, uPA can directly cleave uPAR [59], PDGF-DD [60], the epithelial Na ? channel ENaC [61], and the a6 integrin of the laminin receptor [62]. All of these substrates have been implicated in cancer progression [59,60,62,63].…”

Section: Discussionmentioning

confidence: 99%

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR

Almholt

Lærum

Nielsen

et al. 2015

Clin Exp Metastasis

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Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

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“…Then, anti-V5 or anti-HA monoclonal ) antibodies were added to the samples (1:5,000 and 1:1,000 dilution, respectively). Our and other groups have demonstrated that non-ENaC bands could be detected in parental oocytes or oocytes expressing ENaC constructs without these artificial flags (Carattino et al, 2014;Ji, Zhao, Komissarov, Chang, Liu & Matthay, 2015;Passero, Mueller, Myerburg, Carattino, Hughey & Kleyman, 2012;Passero, Mueller, Rondon-Berrios, Tofovic, Hughey & Kleyman, 2008). Horseradish peroxidase-labelled secondary antibodies (Jackson Immunoresearch) were used (1:10,000).…”

Section: Biotinylation and Immunoblottingmentioning

confidence: 99%

“…However, whether exogenously administered plasmin enhances the removal of oedema fluid via ENaC and related mechanisms, is not clear. We recently identified human ENaC as a novel target of urokinase (Ji, Zhao, Komissarov, Chang, Liu & Matthay, 2015). Based on the lesser specificity of plasmin substrates in amino acid sequence, it is a challenge to identify all of cleavage sites for plasmin, in particular for in vivo lifetime exposure to plasmin.…”

Section: Introductionmentioning

confidence: 99%

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Zhao¹,

Ali²,

Nie³

et al. 2020

Preprint

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Background and Purpose: Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid.Experimental Approaches: Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits.Key Results: Plasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated abgENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (aD432-444, gD131-138, gD178-193, and gD410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human gENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both a and g subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and Implications:We demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases.

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Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator

Cited by 39 publications

References 92 publications

Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel

Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

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