Developmental expression and localization of IA-2 mRNA in mouse neuroendocrine tissues

Islet antigen (IA)-2 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM), and belongs to a new class within the receptor-type protein tyrosine phosphatase (PTP) family characterized by lack of PTP enzymatic activity with conventional substrates. Its expression is restricted primarily to the pancreas, pituitary, and brain with the highest level in the brain. IA-2 mRNA expressions in the brain, pituitary and pancreas of 1-, 4-, and 8-week-old mice were examined. In situ hybridization of the brain revealed that IA-2 mRNA was expressed in the cerebral cortex, hippocampus, thalamus, choroid plexus, hypothalamus, Purkinje cells, and granular layer of the cerebellum. In the pituitary, IA-2 mRNA was located in the anterior and posterior pituitary by in situ hybridization. The pattern of IA-2 mRNA expression in normal male mouse brain at 1, 4, and 8 weeks of age by the Northern blot analysis was similar to that in the pituitary by RT-PCR analysis. The expression level was higher at 4 weeks and lower at 1 week of age. In the pancreas, IA-2 mRNA expressions detected by RT-PCR were highest at 8 weeks of age. These results indicated that the amount of mRNA expression increased in accordance to development in brain, pituitary, and pancreas.     

Testosterone and progesterone metabolism in the central nervous system: Cellular localization and mechanism of control of the enzymes involved

Summary This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia.1. The activities of 5-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5-androstane-3,17-diol; 3-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5-reductase activity. A completely different localization was observed for 3-hydroxysteroid dehydrogenase; the formation of 3-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5-reductase and 3-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5-reductase, which metabolizes progesterone into 5-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-reduce progesterone. On the contrary, 3-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5-pregnane-3-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.     

Expression of enzymes involved in estrogen metabolism in human prostate.

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaP can synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17beta-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17beta-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3beta-HSD and 17beta-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells.     

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.     

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.     

Metabolism of androgens in human hyperplastic prostate: evidence for a differential localization of the enzymes involved in the metabolism

The subcellular distribution of 5 alpha-reductase, 17 beta-hydroxy steroid dehydrogenase, 3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities was studied in human hyperplastic prostate. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities are located in the nuclear envelope. 3 alpha-hydroxysteroid dehydrogenase activity was almost equally distributed between cytosol and membranes, 3 beta-hydroxysteroid dehydrogenase activity was linked to all membranes. Direct testosterone metabolism (transformation into its active metabolite 5 alpha-DHT and into androstenedione, an inactive androgen) takes place only in the nucleus whereas indirect metabolism takes place mainly in the cytoplasm. These findings add new evidence for the mechanism of action of testosterone in prostatic tissue. Testosterone diffuses into the cell, migrates toward the nucleus and is transformed at the nuclear envelope level into two metabolites, DHT and androstenedione. After transformation into its active form, the hormone enters the nucleus whereas the inactive form is released into the cytoplasm. This metabolism could be seen as a control of the amount of active hormone entering the nucleus and being able to bind the androgen receptor.     

Cellular localization of aquaporin mRNA in hybrid poplar stems

? Premise of the study: Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes. ? Methods: Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought. Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize spatial expression of three AQP genes in stem cross sections. ? Key results: The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable. High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization (control plants) and plants subjected to drought. ? Conclusions: Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.     

Immunocytological localization of two enzymes involved in berberine biosynthesis

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.     

Cellular basis for the differential response of mouse kallikrein genes to hormonal induction.

The expression of many mouse kallikrein genes in the salivary gland is sexually dimorphic and inducible in females by administration of testosterone or thyroxine. Induction is slow (3-7 days) and is accompanied by the non-uniform differentiation of the cell type expressing these genes from striated duct (SD) cells (female) to granular convoluted tubule (GCT) cells (male). One kallikrein gene, mGK-6, is expressed at an apparently constant total level in male and female and is not induced by either hormone. In situ hybridization histochemistry shows that all kallikrein genes analyzed exhibit uniform cellular distribution of expression in the SD cells of the female. The hormonally mediated differentiation of some, but not all, of these cells has different effects on kallikrein gene expression--mGK-6 is repressed while other kallikreins are induced--leading to non-uniform distribution of expression.     

Cellular location and hormonal regulation of ghrelin expression in rat testis

Ghrelin, the endogenous ligand for the growth hormone-secretagogue receptor, is a recently cloned 28-amino acid peptide, expressed primarily in the stomach and hypothalamus, with the ability to stimulate growth hormone (GH) release and food intake. However, the possibility of additional, as yet unknown biological actions of ghrelin has been suggested. As a continuation of our recent findings on the expression and functional role of ghrelin in rat testis, we report here the pattern of cellular expression of ghrelin peptide in rat testis during postnatal development and after selective Leydig cell elimination, and we assess hormonal regulation of testicular ghrelin expression, at the mRNA and/or protein levels, in different experimental models. Immunohistochemical analyses along postnatal development demonstrated selective location of ghrelin peptide within rat testis in mature fetal- and adult-type Leydig cells. In good agreement, ghrelin protein appeared undetectable in testicular interstitium after selective Leydig cell withdrawal. In terms of hormonal regulation, testicular ghrelin mRNA and protein expression decreased to negligible levels after long-term hypophysectomy, whereas replacement with human chorionic gonadotropin (CG) (as superagonist of LH) partially restored ghrelin mRNA and peptide expression. Furthermore, acute administration of human CG (25 IU) to intact rats resulted in a transient increase in testicular ghrelin mRNA levels, with peak values 4 h after injection, an effect that was not mimicked by FSH (12.5 IU/rat). In contrast, testicular expression of ghrelin mRNA remained unaltered in GH-deficient rats, under hyper- and hypothyroidism conditions, as well as in adrenalectomized animals. In conclusion, our results demonstrate that mature Leydig cells are the source of ghrelin expression in rat testis, the protein being expressed in both fetal- and adult-type Leydig cells. In addition, our data indicate that testicular expression of ghrelin is hormonally regulated and is at least partially dependent on pituitary LH.     

Syncytin-A mediates the formation of syncytiotrophoblast involved in mouse placental development.

Syncytin-A, a new mouse endogenous retroviral envelope protein expressed in placenta, can mediate cell fusion in vitro. But its physiological function was still unknown. We proposed a role for syncytin-A in syncytiotrophoblast (SynT) formation derived from the differentiation of trophoblast stem (TS) cells during placental development. To evaluate this hypothesis, we analyzed the involvement of syncytin-A in the differentiation of mouse TS cells. After withdrawing fibroblast growth factor 4 (FGF4), TS cells can fuse to form SynT cells. We found syncytin-A mRNA and protein expression are colinear with fusion index increase during TS cell differentiation. Expression of syncytin-A is localized in SynT cells through in situ immunofluorescent staining. By using specific antibody and antisense oligonucleotides, we demonstrated that inhibition of syncytin-A lead to obvious decrease of SynT cell formation. These results present evidence in support of the direct role for syncytin-A in mouse TS cell fusion and differentiation involved in placental development.     

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.     

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.     

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.     

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.