PER1 is a core component of the internal time-keeping system. In the suprachiasmatic nucleus, it serves as the primary circadian pacemaker in mammalian brains. PER1 functions with other clock components to generate a feedback loop involving the transcriptional repression of gene expression to produce a circadian rhythm with an approximately 24-hour cycle. Post-transcriptional modifications (PTMs) are a basic regulatory mechanism that both perpetuate self-sustained oscillations and interpret metabolic input into circadian physiology by affecting factors such as protein stability, interactions, localization, and activity. Here we examined whether the serine/threonine protein kinase WNK3, which is expressed in a circadian rhythm, can interact and colocalize with PER1 in the SCN. In rats, WNK3 knockdown in the SCN is associated with altered sleep patterns. Moreover, WNK3 can phosphorylate PER1 to promote its degradation and is associated with circadian oscillations when PER1 is expressed in vitro.

BACKGROUND: The lncRNA H19 is believed to act as an oncogene in various types of tumors and is considered to be a therapeutic target and diagnostic marker. However, the role of the lncRNA H19 in regulating the radiosensitivity of non-small cell lung cancer (NSCLC) cells is unknown.
METHODS: The expression profiles of lncRNAs in NSCLC were explored via transcriptome sequencing. CCK-8, EdU incorporation and clonogenic survival assays were conducted to evaluate the proliferation and radiosensitivity of NSCLC cells. Flow cytometry and Western blotting were conducted to measure the level of apoptosis. The binding relationship between the lncRNA H19 and miR-130a-3p was determined by a dual-luciferase reporter assay. A binding relationship was also identified between miR-130a-3p and With-No-Lysine Kinase 3 (WNK3).
RESULTS: Expression patterns of lncRNAs revealed that the lncRNA H19 was upregulated in radioresistant NSCLC (A549-R11) cells compared with A549 cells. Knockdown of the lncRNA H19 enhanced the sensitivity of NSCLC cell lines to X-ray and carbon ion irradiation. Mechanistically, the lncRNA H19 serves as a sponge of miR-130a-3p, which downregulates WNK3 expression. The lncRNA H19-miR-130a-3p-WNK3 axis modulates radiosensitivity by regulating apoptosis in NSCLC cell lines.
CONCLUSIONS: Knockdown of the lncRNA H19 promotes the sensitivity of NSCLC cells to X-ray and carbon ion irradiation. Hence, the lncRNA H19 might function as a potential therapeutic target that enhances the antitumor effects of radiotherapy in NSCLC.

The activation of chloride (Cl-)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental \"switch\" in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl- concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13-15) but had no effect on immature neurons (DIV6-8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA . Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the \"adult\" GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

UNASSIGNED: The primary features of malignant glioma include high rates of mortality and recurrence, uncontrollable invasiveness, strong angiogenesis, and widespread hypoxia. The hypoxic microenvironment is an important factor affecting the malignant progression of glioma. However, the molecular mechanisms underlying glioma adaption in hypoxic microenvironments are poorly understood.
UNASSIGNED: The work presented in this paper focuses on the role of WNK3 gene in glioma invasion under hypoxic conditions. Furthermore, we aim to explore its role in epithelial-to-mesenchymal transition (EMT).
UNASSIGNED: ShRNA targeting WNK3 transfection was used to knockdown the WNK3 expression in U87 cells. We used western blot analysis to detect the relative expression of proteins in U87 cells. The effect of WNK3 on cell migration was explored using a transwell assay in the U87 cell line. We also evaluated WNK3 expression levels in glioma samples by immunohistochemistry analysis.
UNASSIGNED: WNK3 expression was significantly higher in high-grade (III and IV) gliomas than in low-grade (I and II) gliomas. WNK3 expression was up-regulated in U87 cells when cultured in a hypoxic environment in addition; WNK3 knockdown inhibited the invasion of U87 glioma cells by regulating the EMT, especially under hypoxic conditions.
UNASSIGNED: These findings suggested that WNK3 plays an important role in the hypoxic microenvironment of glioma and might also be a candidate for therapeutic application in the treatment of glioma.

Intracerebral hemorrhage (ICH) is the common brain diseases in middle-aged and elderly people, with high disability and/or mortality rate, and is a serious public health concern. Both WNK3 kinase and the WNK3/SPAK/NKCC1 signaling pathway play an integral role in maintaining normal cell homeostasis. However, their role and underlying mechanisms in ICH-induced secondary brain injury (SBI) have yet to be elucidated.
We established an ICH model using male Sprague-Dawley (SD) rats by injecting autologous arterial blood into the unilateral basal ganglia. To establish ICH model in vitro, oxyhemoglobin (OxyHb; 20 μM) and neurons were cultured for 6 h at 37 °C, 5% CO2 atmosphere. To investigate the role of WNK3 and the WNK3/SPAK/NKCC1 signaling pathway in SBI, after genetic interventions, rotation and water maze test, brain edema and neuroinflammation were detected, and terminal-deoxynucleoitidyl transferase mediated dUTP nick end labeling (TUNEL), Fluoro-Jade C (FJC), and Nissl staining were performed.
Our data showed that WNK3 expression in brain tissue were upregulated after ICH induction. In addition, silencing of WNK3 reduced neuronal apoptosis, and inflammatory responses in rats that underwent ICH. Inhibition of WNK3 expression reduced the damaged blood-brain barrier (BBB), alleviated the impaired degree of cerebral edema, and improved disruptive neurobehavioral cognition caused by ICH. Moreover, overexpression of WNK3 had the opposite effects. Finally, WNK3/SPAK/NKCC1 signaling pathway may be involved in the above-mentioned processes.
In conclusion, our findings showed that WNK3 and WNK3/SPAK/NKCC1 signaling pathway play a vital biological function in ICH-induced SBI. Depletion of WNK3 attenuated brain injury after ICH both in vivo and in vitro. Thus, WNK3 and WNK3/SPAK/NKCC1 signaling pathway are potential targets for treating SBI after ICH.

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK\'s carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

WNK kinases, along with their upstream regulators (CUL3/KLHL3) and downstream targets (the SPAK/OSR1 kinases and the cation-Cl- cotransporters [CCCs]), comprise a signaling cascade essential for ion homeostasis in the kidney and nervous system. Recent work has furthered our understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling, and uncovered novel roles for this pathway in immune cell function and cell proliferation.

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.

Mutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na(+) and K(+) excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.