60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling

Phenotype-dependent apoptosis signalling in mesothelioma cells after selenite exposure

<p>Abstract</p> <p>Background</p> <p>Selenite is a promising anticancer agent which has been shown to induce apoptosis in malignant mesothelioma cells in a phenotype-dependent manner, where cells of the chemoresistant sarcomatoid phenotype are more sensitive.</p> <p>Methods</p> <p>In this paper, we investigate the apoptosis signalling mechanisms in sarcomatoid and epithelioid mesothelioma cells after selenite treatment. Apoptosis was measured with the Annexin-PI assay. The mitochondrial membrane potential, the expression of Bax, Bcl-XL, and the activation of caspase-3 were assayed with flow cytometry and a cytokeratin 18 cleavage assay. Signalling through JNK, p38, p53, and cathepsins B, D, and E was investigated with chemical inhibitors. Furthermore, the expression, nuclear translocation and DNA-binding activity of p53 was investigated using ICC, EMSA and the monitoring of p21 expression as a downstream event. Levels of thioredoxin (Trx) were measured by ELISA.</p> <p>Results</p> <p>In both cell lines, 10 μM selenite caused apoptosis and a marked loss of mitochondrial membrane potential. Bax was up-regulated only in the sarcomatoid cell line, while the epithelioid cell line down-regulated Bcl-XL and showed greater caspase-3 activation. Nuclear translocation of p53 was seen in both cell lines, but very little p21 expression was induced. Chemical inhibition of p53 did not protect the cells from apoptosis. p53 lost its DNA binding ability after selenite treatment and was enriched in an inactive form. Levels of thioredoxin decreased after selenite treatment. Chemical inhibition of MAP kinases and cathepsins showed that p38 and cathepsin B had some mediatory effect while JNK had an anti-apoptotic role.</p> <p>Conclusion</p> <p>We delineate pathways of apoptosis signalling in response to selenite, showing differences between epithelioid and sarcomatoid mesothelioma cells. These differences may partly explain why sarcomatoid cells are more sensitive to selenite.</p

Decreased bone density and increased phosphaturia in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase 3

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.Kidney International advance online publication, 30 March 2011; doi:10.1038/ki.2011.67

Enhanced FGF23 Serum Concentrations and Phosphaturia in Gene Targeted Mice Expressing WNK-Resistant Spak

Contains fulltext : 107752.pdf (publisher's version ) (Open Access)Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na(+),K(+),2Cl(-) cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak(tg/tg)) and in mice expressing wild type SPAK (spak(wt/wt)). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak(tg/tg) mice than in spak(wt/wt) mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak(tg/tg) mice compared to spak(wt/wt) mice. Treatment of spak(wt/wt) mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca(2+) and phosphate excretion