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Objective To investigate the effect of transforming growth factor β1 (TGF-β1) on the proliferation and migration ability of bladder cancer T24 cells and its mechanism. Methods T24 cells were treated with TGF-β1 at concentrations of 0, 1, 5, 10, 20 and 40 μg/L for 24 h. The proliferation activity of T24 cells was determined by methyl thiazolyl tetrazolium assay; their migration ability was detected by cell scratching method; the relative expression of programmed death ligand-1 (PD-L1) protein in T24 cells was detected by Western blot. T24 cells were treated with TGF-β1 at a concentration of 5 μg/L for 0, 0.5, 1, 2, 6 and 24 h. The relative expression of PD-L1 protein as well as phosphorylated serine-threonine protein kinase (p-AKT) and phosphorylated extracellular regulated kinase (p-ERK) proteins in T24 cells were detected by Western blot. T24 cells were divided into groups A to G. Group A was cultured with normal medium; group B was cultured with the medium containing TGF-β1 at a final concentration of 5 μg/L; group C was cultured with the medium containing TGF-β1 and SB431542 at a final concentration of 5 μg/L. The relative expression of PD-L1 in groups A to C was measured by Western blot. Group D was cultured with normal medium; group E was cultured with the medium containing TGF-β1 at a final concentration of 5 μg/L; group F was cultured with the medium containing TGF-β1 and AKT inhibitor MK2206 at a final concentration of 5 μg/L; group G was cultured with the medium containing TGF-β1 and ERK inhibitor U0126 at a final concentration of 5 μg/L. All T24 cells were treated for 24 h. The relative expression of p-AKT, p-ERK, and PD-L1 proteins in groups D to G was detected by Western blot. Results After T24 cells were treated with TGF-β1 at concentrations of 0, 1, 5, 10, 20 and 40 μg/L for 24 h, the proliferation ability of T24 cells was significantly enhanced compared with that of T24 cells treated with TGF-β1 at 0 μg/L (t=3.59-12.18,P0.05); the PD-L1 protein expression was significantly higher in T24 cells treated with TGF-β1 at 5 μg/L than at other concentrations (t=3.22-5.64,P

The present review gives a perspective on the Aurora kinase family members, their function in normal cells, their role in cancer progression as well as their potential as target for anticancer treatment. Mitosis has been an important target for anticancer therapy development, leading to some specific drugs mainly addressing Tubulines, as a key structure of the mitotic spindle. Vinca alkaloids, taxanes or epotilones are good examples of conventionally developed antimitotic agents. However, novel classes of antineoplastic drugs are being studied, targeting the regulatory system that controls functional aspects of mitosis, such as Aurora or Polo-like kinases or Kinespondin inhibitors. The specific role of the different Aurora kinase proteins as regulator enzymes of the mitotic process in normal cells is discussed. Some of the mechanisms that link Aurora overexpression with cancer are also considered. Thereafter, the clinical and preclinical development of the different Aurora kinase inhibitors is presented. This is nowadays a very active area of therapeutic research and at least, sixteen new compounds are being studied as potential antineoplastic drugs. Most of them are in a very early phase of clinical development. However, we summarized the most recently published findings related with these drugs: main characteristics, way of administration, dose limiting toxicities and recommended doses for further studies. Another important aspect in Aurora kinase inhibition is the study and validation of potential biomarkers to optimize the clinical development. Several studies included pharmacodynamic assessments in normal blood cells, skin or/and tumor biopsies. Several proposals included a higher mitotic index, a decreased number of mitosis with bipolar spindles or normal alignment of chromosomes and inhibition of histone H3 phosphorylation. Future strategies and challenges for trials with Aurora kinase inhibitors are also discussed. [ABSTRACT FROM AUTHOR]

The Na[sup+]:K[sup+]:2Cl[sup-] cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus Iaevis oocytes, achieved by either coexpression of the K-CI cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines(96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl[sup-]][subi]. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCC5). WNK3 is positioned upstream of SPAK and appears to be the chloride- sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption. [ABSTRACT FROM AUTHOR]

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular CI- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular CI- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrink-age-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the CI- influx and efflux branches of the SLC12A cotransporter family. [ABSTRACT FROM AUTHOR]

The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl- influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl- efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits C1 exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. [ABSTRACT FROM AUTHOR]

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCI reabsorption and decreased K-secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCI reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCI, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin. [ABSTRACT FROM AUTHOR]