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Gene expression of human keratinocyte (HaCaT) under the stress of high sodium concentration

ABSTRACT: The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a disruption of the epidermal barrier, water is lost resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as the sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. The same pathways are present in lung epithelial cells. A signal transduction pathway mediated directly through Nax and secondarily through ENaC results in production of secretory inflammatory mediators. These mediators result in epithelial proliferation and restoration of epidermal homeostasis, but can also have negative effects including excess inflammation and ultimately leads to activation of fibroblasts. Both wild type and Nax knockdown HaCaT cells were seeded in 12-well plates. 70-80% confluent cells were starved with fetal bovine serum (FBS) free Dulbecco's modiM-oM-,M-^Aed Eagle's medium (DMEM) overnight followed by a treatment with control medium (final150 mM sodium) or medium plus 10% more sodium (final 165 mM sodium). Cells were harvested at time point 0, 4, and 16 hours post treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Wei Xu

PROVIDER: E-GEOD-65366 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression of human keratinocyte (HaCaT) under the stress of high sodium concentration

Project description:The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a disruption of the epidermal barrier, water is lost resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as the sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. The same pathways are present in lung epithelial cells. A signal transduction pathway mediated directly through Nax and secondarily through ENaC results in production of secretory inflammatory mediators. These mediators result in epithelial proliferation and restoration of epidermal homeostasis, but can also have negative effects including excess inflammation and ultimately leads to activation of fibroblasts.

2015-01-29 | GSE65366 | GEO

Transcription profiling of cortical collecting duct/connecting nephron tissue from mice treated with aldosterone, canrenoate vs. controls to identify gene products regulated by aldosterone in target cells

Project description:The mineralocorticoid hormone aldosterone controls sodium reabsorption and blood pressure largely by regulating the cell-surface expression and function of the epithelial sodium channel ENaC in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of SGK1, a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. We performed a microarray study in order to investigate which other gene products are rapidly regulated by aldosterone in target cells that participate to the control of Na+ reabsorption and K+ secretion.

2007-03-07 | E-TABM-229 | biostudies-arrayexpress

Transmembrane serine protease 2 and proteolytic activation of the epithelial sodium channel in mouse kidney: Effects of TMPRSS2 knockout

Project description:The appropriate regulation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron is required for sodium homeostasis and thereby for the long-term regulation of arterial blood pressure. A unique feature of ENaC is its activation by proteolytic cleavage. However, the relevant proteases involved in proteolytic ENaC activation in the kidney remain elusive. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). A mouse cortical collecting duct cell line (mCCDcl1) was used as a model system. We performed RNA-Seq transcriptome analysis of mCCDcl1 cells, which confirmed coexpression of Tmprss2 and all three ENaC subunits (Scnn1a, Scnn1b, Scnn1g) in this cell line. Importantly, high baseline expression of TMPRSS2 was detected in these cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated transepithelial short-circuit currents, which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. From these findings we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in the kidney.

2024-10-30 | GSE274306 | GEO

Transmembrane serine protease 2 and proteolytic activation of the epithelial sodium channel in mouse kidney: Early effects of aldosterone

2024-10-30 | GSE274304 | GEO

Transmembrane serine protease 2 and proteolytic activation of the epithelial sodium channel in mouse kidney: Late effects of aldosterone

2024-10-30 | GSE274305 | GEO

Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport

Project description:Membrane-bound serine proteases are well known regulators of ENaC. More recently, they have also been identified as regulators of tight junction complexes. Here, we demonstrate that within the kidney, the serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC, whereas Furin is primarily expressed in ENaC-negative cortex cells. In mCCDcl1 kidney cells, aldosterone-induced ENaC and Scnn1a-specific overexpression is accompanied by increased Tmprss2 gene expression, whereas Scnn1a silencing does not alter Tmprss2 transcription, suggesting that ENaC depends on Tmprss2 expression. This was confirmed by CRISPR-Cas9 Tmprss2 knockout which significantly downregulates Scnn1a gene and protein expression, and thus ENaC-mediated transepithelial sodium transport. Additionally, RNA sequencing analyses unveiled a drastic downregulation in EpCAM and claudin-3 and -7 gene expression, resulting in reduced protein expression also confirmed by immunocytochemistry. In summary, our data clearly demonstrates that the serine protease Tmprss2 is important in maintaining the epithelial tight junction barrier, in addition to regulating ENaC function via Scnn1a expression.

2024-01-02 | GSE247775 | GEO

Quantitative Phosphoproteomic Identification of Novel Vasopressin-mediated Signaling Networks in Kidney Distal Convoluted Tubule Cells

Project description:The kidney distal convoluted tubule (DCT) plays an important role in body sodium regulation and thus control of blood pressure. The main sodium reabsorption pathways in the DCT are the epithelial sodium channel (ENaC) and the thiazide-sensitive NaCl cotransporter (NCC), the functions of which can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. We performed large scale stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells (mpkDCT) to map global changes in protein phosphorylation events upon acute treatment with the VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). The aim of this study is to identify unique VP signaling cascades in DCT cells that may be important for regulating ENaC or NCC activity. These pathways may be novel targets for modulation of blood pressure.

2015-08-04 | PXD001729 | Pride

Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

Project description:Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

| PRJNA544464 | ENA

ZNF750 in late keratinocyte differentiation

Project description:Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are still poorly understood. We have previously demostrated that a dominante mutation in ZNF750 leads to a clinical phenotype that reminiscent of psoriasis and seborrehic dermatitis. We defined ZNF750 as a nuclear effector that is atrongly activated in and essiential for keratinocyte terminal differentiation. ZNF750 knockdown in HaCaT keratinocytes markedly reduced the expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, ZNF750 over-expression in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differnetiation, and with its downstream targets can serve in future elucidation of therapeutics for common disease of skin barrier Gene expression analysis: To determine the differentaition signature for HaCaT keratinocytes, with ZNF750 gene silencing, total RNA was isolated in biologic triplicates from cells induced to differentiate for twelve days and hybridized to Affymerix Human Gene 1.0 ST arrays.

2012-09-24 | E-GEOD-38039 | biostudies-arrayexpress

Quantitative proteomic analysis of urinary exosomes from patients with primary aldosteronism that underwent fludrocortisone suppression test

Project description:The sodium chloride cotransporters (NCC) and the epithelial sodium channel (ENaC) have been reported to be the main effectors of aldosterone in Na+ and Cl- reabsorption in the distal nephron and dietary K+ downregulates NCC. The Cl-/HCO3- exchanger pendrin has been revealed as a key role in maintaining extracellular fluid and electrolyte homeostasis by regulating Cl- reabsorption in the aldosterone-sensitive distal nephron working in tandem with ENaC and perhaps NCC. Pendrin-mediated Cl-/HCO3- exchange is stimulated in models of metabolic alkalosis (e.g. aldosterone administration or dietary NaHCO3 intake), and plasma K+ level is thought to negatively influence pendrin. In this study we used high-throughput proteomics and bioinformatics approaches to analyze urinary exosome contents isolated from patients undergoing fludrocortisone suppression test (FST, as a means of confirming or excluding primary aldosteronism [PA]) to examine the actions of administration of aldosterone analogs, NaCl and KCl orally on transmembrane proteins existing in urinary exosomes.

2020-11-20 | PXD017083 | Pride

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