Glucocorticoid-induced Leucine zipper 1 stimulates the epithelial sodium channel by regulating serum- and glucocorticoid-induced kinase 1 stability and subcellular localization

Serum- and glucocorticoid-induced kinase 1 (SGK1) is a multifunctional protein kinase that markedly influences various cellular processes such as proliferation, apoptosis, glucose metabolism, and sodium (Na(+)) transport via the epithelial Na(+) channel, ENaC. SGK1 is a short-lived protein, which is predominantly targeted to the endoplasmic reticulum (ER) to undergo rapid proteasome-mediated degradation through the ER-associated degradation (ERAD) system. We show here that the aldosterone-induced chaperone, GILZ1 (glucocorticoid-induced leucine zipper protein-1) selectively decreases SGK1 localization to ER as well as its interaction with ER-associated E3 ubiquitin ligases, HRD1 and CHIP. GILZ1 inhibits SGK1 ubiquitinylation and subsequent proteasome-mediated degradation, thereby prolonging its half-life and increasing its steady-state expression. Furthermore, comparison of the effect of GILZ1 with that of proteasome inhibition (by MG-132) supports the idea that these effects of GILZ1 are secondary to physical interaction of GILZ1 with SGK1 and enhanced recruitment of SGK1 to targets within an "ENaC regulatory complex," thus making less SGK1 available to the ERAD machinery. Finally, effects of GILZ1 knockdown and overexpression strongly support the idea that these effects of GILZ1 are functionally important for ENaC regulation. These data provide new insight into how the manifold activities of SGK1 are selectively deployed and strengthened through modulation of its molecular interactions, subcellular localization, and stability.     

Expression of ENaC and serum- and glucocorticoid-induced kinase 1 in the rat intestinal epithelium

Increase in epithelium sodium channel (ENaC) activity induced by aldosterone in the distal tubule of the kidney has been attributed to serum- and glucocorticoid-induced kinase 1 (sgk1). The distal colon constitutes another classical aldosterone-responsive epithelium that expresses both ENaC and sgk1 in an aldosterone-dependent manner. However, the site of expression and the temporal relationship of the aldosterone induction of these two proteins have not been investigated. Here, we examined the distribution and abundance of sgk1 in the rat intestine under basal conditions and after changes in the concentration of aldosterone and glucocorticoids. Results indicate that sgk1 is expressed in the distal colon and also in the ileum and jejunum. Abundance of sgk1 was high in control animals, and it did not change significantly after sodium depletion or after a single dose of aldosterone; however, it decreased after adrenalectomy. In contrast, the three subunits of ENaC were markedly induced in the distal colon by acute and chronic increases in aldosterone levels. Results indicate differential regulation of sgk and ENaC subunits by aldosterone in the distal colon. Distribution of sgk1 in the intestine beyond the aldosterone-responsive segments suggests that sgk1 may additionally regulate other sodium transporters in the intestinal epithelium.     

Translational regulation of the serum- and glucocorticoid-inducible kinase-1 (SGK1) in platelets

GPR21 is an orphan G-protein-coupled receptor. We found that mice deficient for the GPR21 gene were resistant to diet-induced obesity. Knockout mice were leaner than their wildtype counterpart, despite that no difference was observed in food intake. No differences were observed in the respiratory exchange rate and thermogenesis. However, knockout mice were more active than wildtype littermates, and this level of activity may be an underlying reason for the difference in energy balance. Mutant mice were more sensitive to insulin than their wildtype control and showed an improved glucose tolerance. Several inflammatory markers MCP-1, CRP and IP-10 were decreased in mutant animals, suggesting that GPR21 may also mediate its effect through anti-inflammatory mechanisms. We found that GPR21 is widely expressed in all tissues, with the highest levels found in the brain and in the spleen. Overall, these findings suggest that GPR21 may play an important role in regulating body weight and glucose metabolism.     

Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase

The subcellular location and some properties of the rat kidney 25-hydroxyvitamin D3-1 alpha-hydroxylase are described. Enzyme activity can be measured as previously discussed (Tanaka, Y., and DeLuca, H.F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 196-199) using saturating substrate (25-hydroxyvitamin D3) concentrations. The reaction is linear with time for up to 30 min at a substrate concentration of 80 microM and 9-11 mg/ml mitochondrial protein. The enzyme, located in the mitochondria, requires molecular oxygen and a source of NADPH. Succinate supplies NADPH for 1 alpha-hydroxylation through reversal of electron transport and transhydrogenation as shown by inhibition with antimycin A and dinitrophenol. Malate supplies NADPH for the reaction via the mitochondrial malic enzyme or malate dehydrogenase and transhydrogenase as indicated by the lack of inhibition by antimycin A but inhibition with dinitrophenol. Metyrapone and carbon monoxide both inhibit 1 alpha-hydroxylation indicating the involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, has no effect on 1 alpha-hydroxylation.     

Up-regulation of serum- and glucocorticoid-induced protein kinase 1 in the brain tissue of human and experimental epilepsy

Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.     

Up-regulation of serum- and glucocorticoid-induced protein kinase 1 in the brain tissue of human and experimental epilepsy

Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.     

Serum/glucocorticoid-induced protein kinase-1 facilitates androgen receptor-dependent cell survival

Androgen receptor (AR) is a critical factor in the development and progression of prostate cancer. We and others recently demonstrated that eliminating AR expression leads to apoptotic cell death in AR-positive prostate cancer cells. To understand the mechanisms of AR-dependent survival, we performed a genome-wide search for AR-regulated survival genes. We found that serum/glucocorticoid-induced protein kinase-1 (SGK-1) mRNA levels were significantly upregulated after androgen stimulation, which was confirmed to be AR dependent. Promoter analysis revealed that the AR interacted with the proximal and distal regions of the sgk1 promoter, leading to sgk-1 promoter activation after androgen stimulation. Functional assays demonstrated that SGK-1 was indispensable for the protective effect of androgens on cell death induced by serum starvation. SGK-1 overexpression not only rescued cells from AR small-interfering RNA (siRNA)-induced apoptosis, but also enhanced AR transactivation, even in the absence of androgen. Additionally, SGK-1 siRNA reduced AR transactivation, indicating a positive feedback effect of SGK-1 expression on AR-mediated gene expression and cellular survival. Taken together, our data suggest that SGK-1 is an androgen-regulated gene that plays a pivotal role in AR-dependent survival and gene expression.     

Immunofluorescence location using monoclonal antibodies of prolactin receptors in mammary epithelial cells of lactating rabbits

Immunofluorescent staining of PRL receptors on frozen sections of lactating rabbit mammary gland with a monoclonal antibody (IgG1 M110) shows that receptors are localized in the cytoplasm of epithelial cells and on short portions of plasma membrane. In vivo treatment by bromocriptine or in vitro treatment of mammary tissue slices by monensin modifies localization of receptors.     

Antiproliferative and apoptotic effect of TGF-beta 1 in bovine mammary epithelial BME-UV1 cells

Transforming growth factor beta1 (TGF-beta(1)) is regarded as an important auto/paracrine regulator of mammary gland involution, however, its apoptotic effect and inhibition of growth in bovine mammary epithelial cells (MEC) has not been documented. In the present study, laser scanning cytometry, confocal and immunoelectron microscopy techniques were used for quantitative and qualitative analyzes of apoptosis, cell cycle and expression, subcellular redistribution and interactions of apoptosis-related proteins in bovine BME-UV1 MEC exposed to TGF-beta(1). TGF-beta(1) exerted both antiproliferative and apoptotic action. The antiproliferative effect was manifested by increase of cell number in G1 phase with simultaneous decrease of cell number in S and G2/M phases. It resulted in significant increase of G1/S ratio in TGF-beta(1) treated cells, indicating partial cell cycle arrest at the G1-S transition. Apoptosis induced by TGF-beta(1) manifested by characteristic morphological changes. Among biochemical features of TGF-beta(1)-induced apoptosis in BME-UV1 cells we found: (1) an increase of cell number with lowered DNA content and condensed chromatin, (2) enhanced expression of caspase-3 and m-calpain, (3) elevated number of 89 kDa PARP degradation fragments, and (4) aggregation of Bax and its interactions with voltage dependant anion channel-1. In conclusion, antiproliferative and apoptotic action of TGF-beta(1), observed in the culture of BME-UV1 cells, suggests an essential role of this cytokine in the regulation of mammary gland involution in cow.