Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.     

Alteration of glutathione reductase expression in the female reproductive organs during the estrous cycle.

The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.     

Potential effect of fish oil to preserve expression of cell cycle and tight junction regulating genes in colon after di-isononyl phthalate ingestion in albino Wistar rats

Di-isononyl phthalate (DIP) is considered a high molecular weight subtype of phthalates that are commonly used (to make plastics more durable) and could easily affect the gastrointestinal tract (GIT). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the main active components of fish oil (FO), and their antiinflammatory potential was previously documented. The current study was designed to investigate the protective potential of fish oil against the impact of DIP exposure on the colon of albino Wistar rats. Sixty albino Wistar rats were divided into control group received corn oil for ten days. DIP treated group received DIP. Diisononyl phthalate + fish oil treated group received both DIP and FO three groups: the control group received corn oil for ten days, the DIP treated group received DIP, and the DIP + FO treated group received both DIP and FO.. FO was found to preserve the histological architecture, tight junction, and cell cycle of the colon. In conclusion, the current study provided an evidence that FO has a protective potential against DIP, and further examination are suggested to fully understand the molecular basis of this potential as a step for further clinical applications.Key words: Di-isononyl phthalate, gastrointestinal tract, fish oil, tight junction, apoptosis     

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.     

Metallothionein in male reproductive organs of adrenalectomized and hydrocortisone-treated Wistar rats

Adrenalectomy resulted in an increase in metallothionein (MT) levels in testes, caput and cauda epididymis and prostate of rats but not in seminal vesicles where its levels decreased significantly. Inspite of administration of hydrocortisone, MT in testes, prostate (1.2 mg), caput (0.3 mg days 2, 8; 0.6 mg and 1.2 mg) and seminal vesicles (0.3 mg day 2, 4; 0.6 mg and 1.2 mg) remained increased. Thus adrenal insufficiency/hydrocortisone has no direct influence on MT levels. However, the increased levels of MT can be related to its ability to protect the cells from free radical damage caused by atrophy of reproductive tissues in adrenalectomised rats. Exogenously administered hydrocortisone to ADX rats resulted in return to ADX state as hydrocortisone metabolizes (half-life < 12 hr) and hence MT levels remained increased. The observations could provide a clue for the physiological functioning of the male reproductive tissue in a state of adrenal deprivation and hormonal supplementation.     

Immunocytochemical localization of nitric oxide synthase-III in reproductive organs of female rats during the oestrous cycle

Summary Constitutive endothelial nitric oxide synthase (NOS III) expression during the oestrous cycle was mapped immunocytochemically on 5 μm-thick paraffin sections of rat female reproductive organs. Ovarian NOS III immunoreactivity increased with follicular maturation (strongest in dioestrus corpora lutea), suggesting that nitric oxide may regulate folliculogenesis and luteal functions. Oviductal NOS III, localized in mucosal epithelium and muscular wall, was maximal during pro-oestrus and oestrus, suggesting that nitric oxide may impart periovulatory quiescence for reception, retention and fertilization of ovulated oocytes. Uterine NOS III, localized in endometrial and glandular epithelium, and in myometrial smooth muscle cells, was abundantly expressed during pro-oestrus and oestrus. The peri-implantation period in pregnant rats corresponds to the periovulatory period and the elevated NOS, and thus nitric oxide may provide uterine relaxation to facilitate embryo implantation following fertilization. Cervical NOS III, localized in the mucus-secreting epithelium and smooth muscle cells, exhibited enzyme abundance during pro-oestrus and oestrus, probably indicating cervical preparation to facilitate sperm entry following mating. Vaginal NOS III, found in the stratified squamous epithelial lining and in smooth muscle cells, was maximal during oestrus and pro-oestrus, suggesting that nitric oxide may stimulate vaginal secretions. Differential expression of NOS III by different reproductive organs during the oestrus cycle suggests a role for nitric oxide in modulating reproduction.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.     

Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1

In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)- through the binding of PKC- to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and ³⁶Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 µg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC- to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC- binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; protein-protein interaction; slot blot assay; pulldown; PDZ domain; chloride efflux; immunoprecipitation     

Sex differences in kidney gene expression during the life cycle of F344 rats

Background

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

Methods

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

Results

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

Conclusions

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

     

The expression of the aryl hydrocarbon receptor in reproductive and neuroendocrine tissues during the estrous cycle in the pig

The sprouted wheat (SW) contains the 6-methoxy-2-benzoxazolinone (6-MBOA), a phenol compound that stimulates reproduction in certain small wild herbivorous mammals. The objective of the present study was to evaluate the effect of short-term supplemental dietary SW on libido, semen and sperm characteristics of rabbit bucks. Five-month old New Zealand White pubertal rabbits (n=18) were randomly allocated to one of two treatments: supplementation or not (control) supplemented with SW. The experimental design was completely random with nine replications, experimental unit was one buck. Semen collection for each male was conducted once a week with two ejaculations during 20 weeks. The SW was given during four consecutive days prior to each semen collection. Analysis of variance was under a mixed model: treatment, ejaculate number and season were fixed and rabbit random effects. There was no effect of treatment (P>0.05) on reaction time, gel presence, volume, pH, sperm motility, sperm number per ml and sperm number per ejaculate. The percentage of normal alive spermatozoa was 13.5% greater in SW-supplemented bucks than in the control and the percentage of abnormal alive spermatozoa was 44.1% greater in the control than in the SW-supplemented bucks. The morphology of dead spermatozoa, integrity of acrosome, number of normal alive motile sperm and semen doses per ejaculate were not influenced (P>0.05) by SW supplementation. The proportion of presence of gel and semen volume in the first ejaculate was greater than the second ejaculate (+140% and +56.4%). However, the semen quality in the latter was greater (P=0.0001) than the former in terms of an increase in motility (+29.7%). Reproductive traits were more desirable (P<0.05) in winter than autumn. Dietary wilted SW as a source of biological 6-MBOA enhanced sperm characteristics in terms of a greater percentage of normal alive and lesser percentage of abnormal alive spermatozoa but did not affect the number of normal motile live sperm and suitable semen doses in rabbit bucks in autumn and winter.     

Protective effect of Abutilon indicum against lead-induced reproductive toxicity in male Wistar rats

Despite ample literature on the toxic impact of lead on the environment and health, the exact mechanism of pathogenesis/toxicity is not clearly known. Because it is well established that lead induces oxidative stress, it is assumed that exposure to antioxidants may reduce the toxic impact of lead. In this study, we evaluated the impact of coadministration of the methanolic root extract of a plant Abutilon indicum (50, 100, 200 mg kg ⁻¹b.wt.) in mitigating the toxic impact of lead on the reproductive system of rats. In brief, Wistar rats were exposed to lead acetate in drinking water with or without coadministration of plant root extract and compared with that of control animals. After 45 days of exposure as outlined above, the animals were killed and the reproductive toxicity was assessed by sperm parameters, hormone and antioxidant enzyme assays, and testis histopathology. Significant reduction in testis weight, sperm count, testosterone levels, and antioxidant enzymes levels such as Superoxide Dismutase, Catalase, and Glutathione peroxidase was seen in lead-treated animals, confirming the toxic impact. The coadministration of A. Indicum (100 and 200 mg kg ⁻¹b.wt.) was found to bring the studied parameters close to the levels seen in untreated (control) animals. Our findings are indicative of the protective nature of A. Indicum against lead-induced reproductive toxicity in a dose-dependent manner. However, further characterization of the root extract is required to elucidate the probable mechanism of protection.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle

Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytes at all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stages of the estrous cycle.