The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice.

In brief: The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Abstract: Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Steroidogenic Factor 1 Regulation of the Hypothalamic-Pituitary-Ovarian Axis of Adult Female Mice.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

The myotubularin MTMR4 regulates phagosomal phosphatidylinositol 3-phosphate turnover and phagocytosis.

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum-induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.

Phosphoinositide 3-kinase and INPP4B in human breast cancer.

The PI3K/Akt signaling pathway is frequently increased in many human cancers, including breast cancer. Recent studies have identified INPP4B, which inhibits PI3K signaling, as an emerging tumor suppressor in breast cancer. This short review discusses these issues and the possibility that INPP4B is an important regulator in many cancers.

Inositol polyphosphate phosphatases in human disease.

Phosphoinositide signalling molecules interact with a plethora of effector proteins to regulate cell proliferation and survival, vesicular trafficking, metabolism, actin dynamics and many other cellular functions. The generation of specific phosphoinositide species is achieved by the activity of phosphoinositide kinases and phosphatases, which phosphorylate and dephosphorylate, respectively, the inositol headgroup of phosphoinositide molecules. The phosphoinositide phosphatases can be classified as 3-, 4- and 5-phosphatases based on their specificity for dephosphorylating phosphates from specific positions on the inositol head group. The SAC phosphatases show less specificity for the position of the phosphate on the inositol ring. The phosphoinositide phosphatases regulate PI3K/Akt signalling, insulin signalling, endocytosis, vesicle trafficking, cell migration, proliferation and apoptosis. Mouse knockout models of several of the phosphoinositide phosphatases have revealed significant physiological roles for these enzymes, including the regulation of embryonic development, fertility, neurological function, the immune system and insulin sensitivity. Importantly, several phosphoinositide phosphatases have been directly associated with a range of human diseases. Genetic mutations in the 5-phosphatase INPP5E are causative of the ciliopathy syndromes Joubert and MORM, and mutations in the 5-phosphatase OCRL result in Lowe's syndrome and Dent 2 disease. Additionally, polymorphisms in the 5-phosphatase SHIP2 confer diabetes susceptibility in specific populations, whereas reduced protein expression of SHIP1 is reported in several human leukaemias. The 4-phosphatase, INPP4B, has recently been identified as a tumour suppressor in human breast and prostate cancer. Mutations in one SAC phosphatase, SAC3/FIG4, results in the degenerative neuropathy, Charcot-Marie-Tooth disease. Indeed, an understanding of the precise functions of phosphoinositide phosphatases is not only important in the context of normal human physiology, but to reveal the mechanisms by which these enzyme families are implicated in an increasing repertoire of human diseases.

Anti-inflammatory therapy in an ovine model of fetal hypoxia induced by single umbilical artery ligation.

Perinatal morbidity and mortality are significantly higher in pregnancies complicated by chronic hypoxia and intrauterine growth restriction (IUGR). Clinically, placental insufficiency and IUGR are strongly associated with a fetoplacental inflammatory response. To explore this further, hypoxia was induced in one fetus in twin-bearing pregnant sheep (n=9) by performing single umbilical artery ligation (SUAL) at 110 days gestation. Five ewes were administered the anti-inflammatory drug sulfasalazine (SSZ) daily, beginning 24h before surgery. Fetal blood gases and inflammatory markers were examined. In both SSZ- and placebo-treated ewes, SUAL fetuses were hypoxic and growth-restricted at 1 week (P<0.05). A fetoplacental inflammatory response was observed in SUAL pregnancies, with elevated pro-inflammatory cytokines, activin A and prostaglandin E(2). SSZ did not mitigate this inflammatory response. It is concluded that SUAL induces fetal hypoxia and a fetoplacental inflammatory response and that SSZ does not improve oxygenation or reduce inflammation. Further studies to explore whether alternative anti-inflammatory treatments may improve IUGR outcomes are warranted.

Celecoxib, but not rofecoxib or naproxen, attenuates cardiac hypertrophy and fibrosis induced in vitro by angiotensin and aldosterone.

1. Cyclo-oxygenase (COX)-2 inhibitors and other non-steroidal anti-inflammatory drugs (NSAIDs) have been implicated in increased cardiovascular events. However, the direct effects of these drugs on cardiac function have not been explored extensively. Given the important role of the renin-angiotensin-aldosterone system (RAAS) in cardiac remodelling, we sought to determine the effect of COX-2 inhibitors and non-specific (NS-) NSAIDs on RAAS-induced cardiac hypertrophy and fibrosis in neonatal rat cardiac myocytes (NCM) and fibroblasts (NCF) isolated from 1-2-day-old Sprague-Dawley rat pups. 2. The NCM were pretreated for 2 h with COX-2 inhibitors (celecoxib or rofecoxib) or NS-NSAIDs (naproxen; all at 0.1-10 micromol/L) before being stimulated with 10 micromol/L aldosterone for 72 h or with 0.1 micromol/L angiotensin (Ang) II for 60 h. Hypertrophy of NCM was assessed by [3H]-leucine incorporation. 3. The NCF were pretreated with COX-2 inhibitors or naproxen as described for NCM before being stimulated with 0.1 micromol/L AngII for 48 h. Collagen synthesis was subsequently assayed by [3H]-proline incorporation. 4. Pooled cryopreserved male and female rat hepatocytes were treated with or without COX-2 inhibitors for 1 h before 1 nmol/L aldosterone ( approximately 540 pg/mL) was added to all wells. Cells were incubated for a further 60 min and culture media harvested by centrifugation. Human hepatic HepG2 cells were treated with compounds with or without serum starvation for 48 h. All cells were pretreated with COX-2 inhibitors for 2 h before the addition of aldosterone. Cell culture media were harvested after a further 3, 18, 24 or 48 h incubation. Aldosterone concentrations in the culture media were determined by enzyme immunoassay. 5. Aldosterone- and AngII-stimulated NCM hypertrophy was inhibited by celecoxib, but not by rofecoxib or naproxen. In NCF, AngII-stimulated collagen synthesis was inhibited by celecoxib and, to a lesser extent, by rofecoxib, whereas naproxen had no effect. The COX-2 inhibitors inhibited aldosterone uptake and/or metabolism by rat hepatocytes, but had no effect in human hepatic HepG2 cells. 6. These results demonstrate a potential antiremodelling effect of selective COX-2 inhibitors in the setting of RAAS stimulation in cardiac cells, whereas naproxen has no effect.