The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na+ channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1–/–) mice and wild-type (sgk1+/+) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1–/– mice to adequately decrease Na+ excretion despite increases in plasma aldosterone levels and proximal-tubular Na+ and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate

Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 microg/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor alpha (TNF-alpha), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4 h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-kappaB and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-kappaB and prolonged AP-1 DNA-binding activities and that the synergism of TNF-alpha and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4 h elapsed between the addition of CHI and that of TNF-alpha. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.

Mineralocorticoid regulation of the renal epithelial Na + channel (ENaC) is of paramount importance for the maintenance of extracellular fluid volume and blood pressure (1). Targeted disruption of αENaC (2, 3), βENaC (4), γENaC (5), or the mineralocorticoid receptor (6) in mice is either not compatible with survival or leads to severe salt wasting. Similarly, loss-of-function mutations of ENaC subunits (7, 8) or the mineralocorticoid receptor (9) in humans cause pseudohypoaldosteronism type 1, characterized by salt wasting and hypotension, despite elevated levels of aldosterone. Conversely, gain-of-function mutations of ENaC (Liddle syndrome) (10) or of the mineralocorticoid receptor (11) lead to severe hypertension. Low dietary Na + intake (12, 13) or exogenous application of aldosterone (14) increases the apical abundance of ENaC in the renal collecting system, the renal target for mineralocorticoid action (15). Recently, compelling evidence has been gathered pointing to an important role for the serum-and glucocorticoid-regulated kinase (Sgk1) in the regulation of ENaC by mineralocorticoids (16). The sgk1 gene was originally cloned as a glucocorticoid-sensitive (17, 18) or a cell volume-regulated (19) gene and was subsequently shown to be strongly upregulated by mineralocorticoids (20-23). In kidney, Sgk1 is highly expressed in the collecting system (14, 20, 24), where aldosteroneinduced apical translocation of ENaC is preceded by an upregulation of Sgk1 in ENaC-positive epithelial cells (14). In Xenopus oocytes expressing the α, β, and γ subunits of ENaC, coexpression of Sgk1 results in a marked upregulation of Na + channel activity (20, 21, 23-26), presumably by increasing ENaC abundance in the cell membrane (14, 23, 26). Although highly suggestive, these observations do not allow one to estimate the contribution of Sgk1 to renal Na + reabsorption and extracellular fluid homeostasis. To address the functional significance of Sgk1 expression in vivo, we generated sgk1-deficient mice. We show that Sgk1 indeed participates in, but does not fully account for, the mineralocorticoid regulation of distal-tubular Na + reabsorption. Methods Generation of sgk1-/mice. Genomic fragments of sgk1 were isolated from a λ phage genomic library (AB-1) prepared from 129/Sv(ev) embryonic stem (ES) cells.

The peptide hormone oxytocin is highly expressd In the hypothalamus within only a small number of magnocellular neurons. However, It is also expressed in a much lager number ofcells in the bovine corpus luteum at hih levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studie, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been

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