Paola Negri-Cesi

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 ...

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.

Presence of 5α‐Reductase isozymes and aromatase in human prostate cancer cells and in benign prostate hyperplastic tissue

Prostate trophism depends on DHT formed from T by the enzyme 5α‐R. Two 5α‐R isoforms with different biochemical characteristics have been cloned. Also estrogens might contribute to the prostate growth; however, their intraglandular formation by the enzyme aromatase is still debated. The aim of the present study was to verify whether (a) only one or both isoforms of the 5α‐Rs are expressed in the prostate cancer cell line LNCaP and in BPH, or (b) the aromatase is present in these samples.

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.

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.

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.

Expression of androgen-activating enzymes in cultured cells of developing rat brain

Abstract: Dihydrotestosterone and estradiol, two active metabolites formed locally in the brain from testosterone, modulate several functions of the developing rat CNS; these compounds derive from the 5α‐reduction or the aromatization of the A‐ring of the hormone. Also, progesterone and corticosteroids may be 5α‐reduced and subsequently 3α‐hydroxylated, becoming modulators of specific neuronal functions. Although the aromatase is a single enzyme, two types of 5α‐reductase have been cloned, showing peculiar biochemical properties and probably different functions. Therefore, the isoform(s) of the enzyme 5α‐reductase(s) present in early stage of brain development have been characterized in primary neuronal and glial cell cultures obtained from the fetal or neonatal rat brain, respectively. Aromatase expression was also studied. The results have shown that in all the brain cells examined type 1 5α‐reductase mRNA is expressed. No specific transcript of type 2 5α‐reductase is detectable in any of the cell types examined. Finally, the aromatase gene is expressed only in cultured fetal neurons and especially in those derived from the hypothalamic area of the rat embryos. It is interesting that no aromatase mRNA is detectable in mixed glia or in type 1 astrocytes and oligodendrocytes cultured separately.

5alpha-reductase isozymes and aromatase are differentially expressed and active in the androgen-independent human prostate cancer cell lines DU145 and PC3.

The presence and possible role of androgen‐metabolizing enzymes in androgen‐independent prostate carcinoma (CaP) are still unclear. The aim of the present study was: 1) to evaluate the pattern of androgen metabolism (relative production of 5α‐reduced vs. 17‐keto androgens); and 2) to analyze whether one or both the two known 5α‐reductase isoforms (5α‐R1 and 5α‐R2) and the aromatase (Aro) are expressed and active in this pathology.

Sexual differentiation of the rodent hypothalamus: hormonal and environmental influences.

Brain sexual differentiation is a complex developmental phenomenon influenced by the genetic background, sex hormone secretions and environmental inputs, including pollution. The main hormonal drive to masculinize and defeminize the rodent brain is testosterone secreted by the testis. The hormone does not influence sex brain differentiation only in its native configuration, but it mostly needs local conversion into active metabolites (estradiol and DHT) through the action of specific enzymatic systems: the aromatase and 5alpha-reductase (5alpha-R), respectively. This allows the hormone to control target cell gene expression either through the estrogen (ER) or the androgen (AR) receptors. The developmental profile of testosterone metabolizing enzymes, different in the two sexes, is therefore of the utmost importance in affecting the bioavailability of the steroids active in brain differentiation. Widely diffused pollutants, like polychlorinated biphenyls (PCBs) are able to affect the production and/or action of testosterone metabolites, exerting detrimental influences on reproduction and sex behavior. The main studies performed in our and other laboratories concerning the pattern of expression and the control of the enzymatic systems involved in brain androgen action and metabolism are shortly reviewed. Some recent data on the influence exerted by PCBs on these metabolic systems are also reported.