Fabio Celotti

Estrogen Receptor Is Expressed in Different Types of Glial Cells in Culture

Abstract: Estrogens derived from the aromatization of androgens are believed to be responsible for the induction of the sexual differentiation of the CNS interacting with specific estrogen receptors (ER) present in developing neurons. However, the brain cellular distribution of ER is not so well documented. The aim of this study was to investigate the qualitative and quantitative expression of ER mRNA in well characterized cultures of rat type 1 and type 2 astrocytes and of oligodendrocytes by polymerase chain reaction. A series of amplifications with a set of primers spanning along the entire ER mRNA was utilized in the different types of glial cells, in a positive control (uterus), and in a negative control (SK‐N‐BE cell line) previously shown to be devoid of ER. The data obtained show that ER mRNA is expressed in all three types of glial cell analyzed in almost equal amounts, which are 25–50 times lower than those in the uterus. The mRNA expressed in the glia is homologous with that expressed in the uterine tissue.

Transient Expression of the 5α-Reductase Type 2 Isozyme in the Rat Brain in Late Fetal and Early Postnatal Life1

The enzyme 5α-reductase plays a key role on several brain functions controlling the formation of anxiolytic/anesthetic steroids derived from progesterone and deoxycorticosterone, the conversion of testosterone to dihydrotestosterone, and the removal of excess of potentially neurotoxic steroids. Two 5α-reductase isoforms have been cloned: 5α-reductase type 1 is widely distributed in the body, and 5α-reductase type 2 is confined to androgen-dependent structures. In this study, the gene expression of the two 5α-reductase isozymes has been analyzed in fetal, postnatal, and adult rat brains by RT-PCR followed by Southern analysis. 5α-Reductase type 1 messenger RNA is always detectable in the rat brain [from gestational day 14 (GD14) to adulthood]. 5α-Reductase type 2 messenger RNA expression is undetectable on GD14, increases after GD18, peaks on postnatal day 2, then decreases gradually, becoming low in adulthood. This pattern of expression appears to be correlated with the rate of production of testosterone ...

Progesterone 5-α-reduction in neuronal and in different types of glial cell cultures: type 1 and 2 astrocytes and oligodendrocytes

Progesterone, like testosterone, can be converted in the brain into 5-alpha-reduced metabolites (5-alpha-pregnan-3,20-dione, DHP; 5-alpha-pregnan-3-alpha-ol-20-one, THP). Recently we have shown that testosterone is 5-alpha-reduced to DHT mainly in neurons, while glial cells possess this enzymatic activity only in limited amounts. On the other hand, a glial cell type (type 1 astrocytes) is almost exclusively responsible for the further metabolism of DHT into 3-alpha-diol. The aim of the present studies was that of evaluating the formation of the 5-alpha-reduced metabolites of progesterone in cultures of neurons, type 1 and 2 astrocytes and oligodendrocytes. The data here presented indicate that, similarly to what happens when testosterone is used as the substrate, the 5-alpha-reductase which metabolizes progesterone 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-alpha-reduce progesterone. On the contrary, the 3-alpha-hydroxysteroid dehydrogenase (3-alpha-HSD), the enzyme which converts DHP into THP, appears to be mainly present 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 utilizing androgens as precursors, oligodendrocytes show a considerable 3-alpha-HSD activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoforms of the enzymes involved in androgen and progesterone metabolism may explain these data.

5α-Reductase Isozymes in the Central Nervous System

Abstract The enzyme 5α-reductase (5α-R) activates several Δ4–3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5α-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5α-R type 1 and type 2. In androgen-dependent structures DHT is almost exclusively formed by 5α-R type 2; 5α-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5α-reduction, and to the two 5α-R isoforms in the CNS.

Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-β

Platelet‐enriched plasma (PRP) is used in therapy as a source of growth factors in bone fracture and wound healing; however, few data exist on its role in the different aspects of the healing process. The effect of PRP and of the two main growth factors present in this preparation (platelet‐derived growth factor [PDGF] and transforming growth factor‐β [TGF‐β]) was evaluated in vitro using the human osteoblastic cell line SaOS‐2, which was shown by reverse transcription‐polymerase chain reaction to express both PDGF‐α and ‐β receptors. Batroxobine‐activated PRP was added in different concentrations to SaOS‐2 cells to assess cell migration (by a microchemotaxis assay) and cell proliferation (by [3H]‐thymidine incorporation into the DNA). Immunoneutralization with anti‐PDGF‐β or anti‐TGF‐β antibodies allowed the assessment of the specific role of these growth factors. The overall results obtained indicate that PRP dose‐dependently stimulates both chemotaxis and cell proliferation. PDGF and TGF‐β appear to exert distinct effects on the two parameters, the former involved in stimulating cell migration and the latter in inhibiting cell proliferation. It is concluded that the different growth factors present in activated PRP can specifically contribute to the main processes of tissue regeneration.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat Part 2: Effects on reproductive parameters, on sex behavior, on memory retention and on hypothalamic expression of aromatase and 5alpha-reductases in the offspring.

The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.

Testosterone 5α-reductase activity in the rat brain is highly concentrated in white matter structures and in purified myelin sheaths of axons

Previous results obtained in this laboratory indicate that in the rat brain the 5 alpha-reductase, the enzymatic activity involved in metabolizing testosterone into 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone), is particularly concentrated in the white matter. In the present experiments, this enzymatic activity was studied in the following white matter structures, which were microdissected using the punch technique of Palkovits: anterior commissure (CA), fornix (FX), habenulo-interpeduncular tract (HP), corpus callosum (CC), stria medullaris (SM), optic chiasm (CO), fimbria of the hippocampus (FI), cerebral peduncle (PC), pontine fibers (FP), cerebellar medulla (CMD) and corticospinal tract (TCS). Moreover brain myelin was isolated and purified by sucrose density gradient ultracentrifugation. The results obtained confirm that, in the rat brain, the enzymes involved in testosterone 5 alpha-reduction are preferentially localized in the white matter. However, clearcut differences in the metabolic activity exist between the different structures examined so far. DHT formation increases rostro-caudally, so that the highest activity has been recorded in the white matter structures punched at the level of pons (FP), medulla oblungata (TCS) and cerebellum (CMD). The high metabolic activity associated with the white matter structures appears to be linked to the presence of myelin, since the specific activity of the enzyme is particularly elevated in purified preparations of myelin sheaths.

Estrogen receptor beta (ERbeta) and inhibition of prostate cancer cell proliferation : studies on the possible mechanism of action in DU145 cells

Estrogen receptor beta (ERbeta) plays a protective role against uncontrolled cell proliferation. ERbeta is lost during prostate cancer (CaP) progression suggesting its direct involvement in contrasting tumor proliferation in this disease; however, the molecular mechanism at the basis of this effect has not been clearly defined yet. Possible molecular targets of ERbeta were assessed in DU145 cells, a CaP cell line expressing only ERbeta. Cells treated from 1 to 9 days with different doses of estradiol or diarylpropionitrile (DPN, an ERbeta-selective agonist) show a time-dependent decrease in cell proliferation. The reduced proliferation rate is accompanied by the stimulation of ERbeta expression and the increase of cyclin-dependent kinase inhibitor p21. We demonstrate that the endogenous ERbeta is one of the mediator of the antiproliferative action of estrogens enhancing the synthesis of molecules such as p21 that control cell cycle, an effect amplified by the autoregulation of ERbeta expression. Our observations suggest that CaP, when expressing a functional ERbeta, might be sensitive to the antiproliferative action of estrogens; therefore, ERbeta specific agonists might be valid candidates for new pharmacological approaches to this disease.

The metabolic effects of inhibitors of 5-lipoxygenase and of cyclooxygenase 1 and 2 are an advancement in the efficacy and safety of anti-inflammatory therapy

Chronic treatment of inflammatory diseases with non-steroidal anti-inflammatory drugs is effective but not always devoid of serious side effects. In particular, the use of traditional non-steroidal aspirin-like drugs has been associated with a high incidence of gastrointestinal bleedings. The development of a new class of drugs, the selective cyclooxygenase type 2 (COX-2) inhibitors, has generated much expectation on the possibility to have safer compounds. After the initial enthusiasm of the scientific community, a re-evaluation of some large, randomized double-blind clinical studies performed with two of these compounds, has disclosed that the late serious gastrointestinal complications are not significantly reduced in comparison with non-selective inhibitors and that cardiovascular concerns might arise particularly if theses drugs are utilized in patients with underlying heart diseases. A new promising class of drugs to control inflammatory diseases is in advanced clinical development. The balanced inhibitors of 5-lipoxygenase (5-LOX) and of cyclooxygenase (both types 1 and 2) block the formation of all the enzymatically arachidonic acid-derived metabolites, both prostaglandins (like COX inhibitors) and leukotrienes (LT); these drugs have been shown to possess a very good anti-inflammatory efficacy without serious side effects. Licofelone, previously known as ML3000, is the molecule in the most advanced phase of clinical development (phase III) among this class of compounds; it is a potent, competitive, and well balanced inhibitor of 5-LOX and COX pathways. The drug has been shown to possess analgesic, anti-inflammatory, antipyretic antibronchocostrictory and antiplatelet properties at doses which are safe for the gastrointestinal tract. Moreover, the newly performed preclinical studies, here briefly reviewed, appear to indicate that the compound seems particularly suitable to protect the articular cartilage and the synovial space in degenerative joint disease and to exert a relevant antithrombotic activity. Preliminary results of clinical studies of licofelone in osteoarthritis indicate that the drug has a comparable or slightly better efficacy than that of naproxen but possesses a much better gastrointestinal safety. This latter important aspect has been also evaluated by an endoscopic study in normal volunteers randomly assigned to a 4-week treatment with licofelone, placebo or naproxen. The results indicate that no ulcers occurred in either licofelone group or the placebo group, while ulcers with unequivocal depth were present in 20% of the naproxen-treated subjects.

Differential distribution of the 5-α-reductase in the central nervous system of the rat and the mouse: Are the white matter structures of the brain target tissue for testosterone action?

In the brain of several animal species testosterone is converted into a series of 5-alpha-reduced metabolites, and especially into 17-beta-hydroxy-5-alpha-androstan-3-one (DHT), by the action of the enzyme 5-alpha-reductase. The formation of DHT has never been evaluated in the white matter structures of the brain, which are composed mainly of myelinated axons. The experiments here described were performed in order to study, in the rat and the mouse, the DHT forming activity of several white matter structures, in comparison with that of the cerebral cortex and of the hypothalamus. Two sampling techniques were used in the rat: microdissection under a stereo-microscope from frozen brain sections of fragments of corpus callosum, optic chiasm and cerebral cortex; fresh tissue macrodissection of subcortical white matter, cerebral cortex and hypothalamus. Only macrodissection was used in the mice. The data show that, independently from the sampling technique used, there are considerable quantitative differences in the distribution pattern of the 5-alpha-reductase activity within different brain structures. Both in the rat and in the mouse, the enzyme appears to be present in higher concentrations in the white matter structures, than in the cerebral cortex and in the hypothalamus. The present results clearly show that the subcortical white matter and the corpus callosum are at least three times as potent as the cerebral cortex in converting testosterone into DHT. An even higher 5-alpha-reductase activity has been found in the optic chiasm. Further work is needed in order to understand the possible physiological role of DHT formation in the white matter structures.