Expression and localization of serum/glucocorticoid-induced kinase in the rat ovary: relation to follicular growth and differentiation.

Expression of serum/glucocorticoid-inducible kinase (Sgk), one member of an inducible serine/threonine kinase family, is induced by FSH/cAMP in rat granulosa cells cultured in defined medium. The FSH-stimulated pattern of sgk expression is biphasic, and transcriptional activation of the sgk gene depends on an intact Sp1/Sp3 binding site within the proximal promoter. To determine whether sgk was expressed in a hormone-dependent and physiologically relevant manner in vivo, the cellular levels of sgk messenger RNA (mRNA) and protein as well as the subcellular localization of this kinase were analyzed in ovaries containing follicles and corpora lutea at specific stages of differentiation. To stimulate follicular development and luteinization, hypophysectomized (H) rats were treated with estradiol (E; HE) and FSH (FSH; HEF) followed by hCG (hCG; HEF/hCG). To analyze Sgk in functional corpora lutea, PRL was administered to HEF/hCG rats, or ovaries of pregnant rats were obtained on day 7, 15, or 22 of gestation. In situ hybridization indicated that sgk mRNA was low/undetectable in granulosa cells of H and HE rats. An acute injection (i.v.) of FSH to HE rats rapidly increased sgk mRNA at 2 and 8 h. Sgk mRNA was also elevated in granulosa cells of preovulatory follicles of HEF rats and in luteal cells of HEF/hCG and pregnant rats. Northern blots and Western blots confirmed the in situ hybridization data, indicating that the amount and cellular localization Sgk protein were related to that of sgk mRNA. When the subcellular localization of this kinase was analyzed by immunohistochemistry, Sgk protein was nuclear in granulosa cells and some thecal cells of large preovulatory follicles. In contrast, Sgk protein was cytoplasmic in luteal cells as well as some cells within the stromal compartment. Intense immunostaining was also observed in oocytes present in primordial follicles, but not in growing follicles. Collectively, these results show that FSH and LH stimulate marked increases in the cellular content of Sgk, as well as dramatic changes in the subcellular distribution of this kinase. The specific nuclear vs. cytoplasmic compartmentalization of Sgk in granulosa cells and luteal cells, respectively, indicates that Sgk controls distinct functions in proliferative vs. terminally differentiated granulosa cells.

Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.

The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.

Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway.

Serum and glucocorticoid-inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF-1 or pervanadate induced a 3- to 12-fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3-kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second-messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6-fold activation of SGK activity, whereas kinase-dead PDK1 caused no activation. GST-pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

Epithelial sodium channel regulated by aldosterone-induced protein sgk.

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.

Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways.

The serum- and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S and G2/M) and the cytoplasm (in G1) in synchrony with the cell cycle. In cells synchronously released from the G1/S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgk gene, or the nucleus, using a nuclear localization signal-containing sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serum-stimulated cells. Thus, our study implicates the nuclear-cytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.

Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity.

Recently, a family of novel, serine/threonine protein kinases has been identified. One of these transcriptionally inducible, immediate-early genes encodes serum/glucocorticoid inducible-protein kinase, sgk. By in situ hybridization, we show that sgk expression in the rat ovary is selectively localized to granulosa cells. In culture, FSH or forskolin, activators of the protein kinase A (PKA) pathway, rapidly (2 h) and transiently increased sgk mRNA levels in undifferentiated granulosa cells. Sgk mRNA exhibited a biphasic expression pattern, with maximal levels observed at 48 h of FSH/forskolin as granulosa cells differentiate to the preovulatory phenotype. Deletion analyses using sgk promoter-reporter constructs (-4.0 kb to -35 bp) identified a region between -63 and -43 bp that mediated FSH and forskolin-responsive transcription in undifferentiated and differentiated granulosa cells. This G/C-rich region 1) conferred both basal and inducible transcription to the minimal -35 sgk promoter chloramphenicol acetyltransferase reporter construct, 2) specifically bound Sp1 and Sp3 present in granulosa cell extracts, and 3) bound recombinant Sp1. Mutation of 2 bp in this region not only prevented Sp1 and Sp3 binding, but also abolished the PKA-mediated transactivation observed when using the wild type construct. Sp1 and Sp3 DNA-binding activity and protein levels did not change significantly during sgk induction. Collectively, these data indicate that Sp1/Sp3 transactivation of the sgk promoter likely involves regulated, phosphorylation-dependent interaction with other factors. Thus the novel, biphasic induction of sgk that correlates with granulosa cell progression from proliferation to differentiation appears to involve sequential, coordinated actions of FSH, PKA, and transcription factors, including Sp1 and Sp3.

FDG-PET evaluation of indeterminate pancreatic masses.

PURPOSE: The purpose of this study was to assess the ability of PET with 2-[18F]fluoro-2-deoxy-D-glucose (FDG) to differentiate benign from malignant pancreatic masses in patients with indeterminate findings on CT. METHOD: We performed FDG-PET on 12 patients with indeterminate mass lesions and 2 patients with CT findings typical for malignancy. Eight were found to have pancreatic carcinoma and six had benign lesions. The final diagnosis was histopathologically confirmed in all patients but two with a presumed diagnosis of focal pancreatitis based on stable clinical follow-up for at least 12 months. Lesion uptake of FDG was evaluated qualitatively and semiquantitatively by determination of the standardized uptake value (SUV) RESULTS: With use of a 2.5 cutoff value for SUV, all eight malignant and four of six benign lesions were correctly categorized. Qualitative evaluation gave the same results. The two false-positive lesions had elevated SUV values of 3.4 and 3.8, respectively. CONCLUSION: Our results indicate that FDG-PET has potential value for assessing patients with CT findings that are indeterminate for pancreatic carcinoma. FDG-PET may obviate invasive diagnostic procedures in many patients with benign disease.

Glucocorticoid-induced functional polarity of growth factor responsiveness regulates tight junction dynamics in transformed mammary epithelial tumor cells.

The synthetic glucocorticoid, dexamethasone, induces the "normal-like" differentiated property of tight junction formation and suppresses growth of the Con8 mammary epithelial tumor cell line, derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. Characterization of the transepithelial electrical resistance of Con8 mammary tumor cells cultured on permeable supports revealed that a novel response to dexamethasone is the generation of a polarized cell monolayer with respect to epidermal growth factor receptor responsiveness. Administration of transforming growth factor-alpha (TGF-alpha) to the basolateral, but not the apical, plasma membrane compartment disrupted the glucocorticoid-stimulated tight junction barrier. Confocal immunofluorescence microscopy revealed that dexamethasone caused the ZO-1 tight junction-associated protein to localize exclusively to the apical border of laterally adjacent membranes of the cell periphery, whereas basolateral administration of TGF-alpha caused the redistribution of ZO-1 back to disorganized aggregates along the cell periphery. In contrast, TGF-alpha was able to exert its mitogenic effects equally on both sides of the cell monolayer independent of its polarized disruption of tight junction formation. Our results represent the first evidence for a functional polarization of the epidermal growth factor receptor and strongly implicate the glucocorticoid-regulated formation of tight junctions in policing the polarized responsiveness of mammary cells to growth factors.

Transforming growth factor-alpha abrogates glucocorticoid-stimulated tight junction formation and growth suppression in rat mammary epithelial tumor cells.

The glucocorticoid and transforming growth factor-alpha (TGF-alpha) regulation of growth and cell-cell contact was investigated in the Con8 mammary epithelial tumor cell line derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. In Con8 cell monolayers cultured on permeable filter supports, the synthetic glucocorticoid, dexamethasone, coordinately suppressed [3H]thymidine incorporation, stimulated monolayer transepithelial electrical resistance (TER), and decreased the paracellular leakage of [3H]inulin or [14C]mannitol across the monolayer. These processes dose dependently correlated with glucocorticoid receptor occupancy and function. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevented the glucocorticoid growth suppression response and disrupted tight junction formation without affecting glucocorticoid responsiveness. Treatment with hydroxyurea or araC demonstrated that de novo DNA synthesis is not a requirement for the growth factor disruption of tight junctions. Immunofluorescence analysis revealed that the ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells and that TGF-alpha caused-ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, our results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha, can override both effects of glucocorticoids. These results have uncovered a novel functional "cross-talk" between glucocorticoids and TGF-alpha which potentially regulates the proliferation and differentiation of mammary epithelial cells.

Cervical esophageal web associated with a patch of heterotopic gastric mucosa.

Cervical esophageal webs are a relatively common finding on esophograms. We report a web resulting from the squamocolumnar junction produced by heterotopic gastric mucosa. The clinical significance of this lesion is discussed and the importance of differentiating it from Barrett's esophagus is stressed.

A case of rabies.

Details of the clinical and laboratory findings of the first case of rabies to be described in Australia are presented. The veterinary, public-health, travel and diagnostic problems and implications are discussed.

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro.

Glycosphingolipid biosynthesis was examined using [3H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p less than 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p less than 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse4Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.

Glycosphingolipids of an alveolar rhabdomyosarcoma and normal human muscle. A case study.

The distribution of neutral, or asialosyl-, glycosphingolipids and gangliosides in a rhabdomyosarcoma of alveolar type have been studied. Histologically, this muscle tumor is composed primarily of two cell types: one with oval or round hyperchromatic nuclei and very little cytoplasm, and one a giant cell, with multiple, peripherally placed nuclei and weakly staining eosinophillic cytoplasm. In comparing glycolipids of the rhabdomyosarcoma with normal muscle from the same leg, the striking alteration in the tumor was a virtual disappearance of ganglioside GM2. There was also a slight increase in GM3 and a decrease in GD1a. The asialosyl derivative of GM2 (GalNac-Gal-Glc-Cer) was markedly increased in the tumor. A loss of glucosylceramide was also observed. The results are discussed in terms of glycolipid metabolic changes in muscle oncogenesis and their implications.

Sequential intellectual recovery after near-drowning.

Sequential neurological and intellectual recovery after childhood near-drowning is discussed. Decisions concerning the persistence and intensity of resuscitation require a knowledge of the natural history of intellectual improvement after rescue from near-drowning. A severe case of fresh-water immersion, leading to recovery, is described. Evidence is presented to suggest that the time interval of one hour before the first spontaneous respiratory gasp forms the upper limit of the apnoeic time bracket after which survival can still be expected, and to indicate that intellectual improvement (to a measured IQ of 97) can occur even after initial decerebrate signs if vigorous therapy is prosecuted. The proportion of cases capable of sequential neurological improvement is unknown. A time base for sequential clinical and intellectual improvement after near-drowning is presented to form a yardstick with which future cases may be compared.

Melioidosis in a child in papua new guinea successful treatment with kanamycin and trimethoprim-sulphamethoxazole.

An instance of melioidosis is described in a five-year-old Papua New Guinean. The disease did not involve the lungs, but the patient presented with an abscess on the abdomen and with numerous abscesses on the feet. The disease was successfully treated with kanamycin and trimethoprim-sulphamethoxazole. This is the fourth case known to have occurred in Papua New Guinea, and the patient is the first to have survived.