Effects of steroid hormones on gene expression of glial markers in the central and peripheral nervous system: variations induced by aging

The present article summarizes our data regarding: (a) the effect of sex steroids on the expression of a specific astrocytic marker in glial cell cultures (GFAP); (b) the effects of aging on two markers of the peripheral myelin (glycoprotein Po and the myelin basic protein, MBP); (c) the possible modification of the damaging effects of aging on these two markers by the in vivo administration of progesterone and its derivatives; and, finally, (d) the effect of progesterone derivatives on the gene expression of Po in cultures of rat Schwann cells. The data obtained have indicated that progesterone and its 5 alpha-reduced metabolites may play an important role in the control of gene expression of GFAP and Po, respectively, in type 1 astrocytes and Schwann cells. It has also been found that the gene expression of Po and MBP is dramatically decreased in the myelin of the sciatic nerve of aged male rats and that the aged-linked decrease of the gene expression of Po is partially reversible with steroid treatment.     

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation

Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3beta-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3beta-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.     

Purification of the insecticidal toxin in crystals of Bacillus thuringiensis

Newly transected or denervated segments of isogeneic rat tibial nerve were implanted into the rat midbrain and sampled at weekly intervals up to 6 weeks post-operation. By 3 weeks, the peripheral nervous system (PNS) grafts were well-vascularized and contained Schwann cells, axons associated with Schwann cell processes, and macrophages. From 3 to 6 weeks, many axons within both the fresh and predegenerated grafts were myelinated by Schwann cells. The nerve fiber arrangement within the implant was similar to that of regenerating peripheral nerve in situ. The central nervous system (CNS) border of the implant was clearly demarcated by a rim of astrocytes behind which was a layer of regenerating oligodendrocytes and axons. Extending from the CNS margin were radial bridges of astroglial tissue which apprarently guided regenerating axons into the implant. Between the CNS and the PNS implant, abundant collagen deposition was present. The findings suggest that regenerating CNS axons grow via astroglial bridges into transplanted PNS tissue and are capable of stimulating the implanted Schwann cells to form myelin. Even Schwann cells deprived of axonal contact for prolonged periods were still capable of PNS myelin formation.     

Development and regeneration of the nervous system: a role for neurosteroids

Several steroids, termed 'neurosteroids', are synthesized from cholesterol within both the central and peripheral nervous systems. These include pregnenolone and its sulfate ester, progesterone and its 5 alpha-reduced metabolites. Dehydroepiandrosterone, mainly in its sulfated form, also remains present in the brain long after removal of the steroidogenic endocrine glands. Its biosynthesis in brain remains an open possibility, but the pathways involved are unknown. Little information is available concerning the role of neurosteroids during the maturation of the nervous system, although they are already synthesized by glial cells and by some populations of neurons during embryonic life. Cell culture experiments suggest that neurosteroids may increase the survival and differentiation of both neurons and glial cells. In the adult nervous system, neurosteroids modulate neurotransmission by acting directly on the neuronal membrane and also produce structural changes in neurons and in astrocytes. Studies of neurosteroid levels are currently conducted to examine their possible role during aging. We have recently reported that progesterone, synthesized by Schwann cells, promotes the formation of new myelin sheaths after lesion of the mouse sciatic nerve. Thus, neurosteroids may also play an important role during regeneration of the nervous system.     

Season of birth and Alzheimer''s disease: a population-based study in Saguenay-Lac-St-Jean/Québec (IMAGE Project)

The ontogenic expression of progesterone and estrogen receptors (PR and ER) and effect of estrogen on these receptors were investigated immunohistochemically in rat uterus from the day of birth ( = 0 day) to 30 days of age. Uterine epithelial and stromal cells showed a negative PR immunoreaction at 0 day. The PR in the epithelial cell nuclei appeared by 5 days, while the stromal cells showed a negative PR reaction until 12 days. The staining of the stromal cells appeared from 12 to 15 days. In both the epithelial and stromal cells, the initiation of the PR appearance was not affected by ovariectomy performed at 0 day or 5 days prior to the appearance of PR in the epithelial and stromal cells. Estrogen injections from 0 day failed to initiate the appearance of PR in the epithelial cells, regardless of doses of estradiol-17 beta (0.1, 1 and 10 micrograms daily), but induced PR in the stromal cells. The staining of ER appeared at 5 days in the epithelial cells and at 1 day in the stromal cells, respectively. ER appeared after 2-3 daily injections of estrogen from 0 day depending upon the doses. These results suggest that steroid hormones secreted from neonatal ovary do not play any important role in ontogenic expression of PR during the postnatal uterine maturation.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

The proteolipid protein gene

Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform, DM-20, produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM-20 is also present in myelin-forming and non-myelin-forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM-20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus-Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X-linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM-20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot-Marie-Tooth type in which the peripheral myelin-22 gene is mutated.     

Teaching multiskilling in dietetics education

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This "reactive" response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a "reactive" response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms.     

2',3'-cyclic nucleotide 3'-phosphodiesterase: a novel candidate autoantigen in demyelinating diseases

Autoaggressive T-cells specific for myelin proteins like proteolipid protein (PLP) and myelin basic protein (MBP) are thought to play a major role in the pathogenesis of demyelinating diseases of the central nervous system (CNS). 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is the third most abundant myelin protein in the CNS. Due to lack of supply with enough CNPase of sufficient purity its immunologic properties have not been studied yet. We subcloned a human CNPase cDNA and expressed human recombinant CNPase (rh-CNPase) in E. coli. Purification of the protein was achieved by Ni(2+)-chelating chromatography. Furthermore we describe for the first time several rh-CNPase specific T-cell lines from a multiple sclerosis patient and a healthy control.     

Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer

The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.     

The effect of mifepristone on progesterone and estrogen receptors in human decidua and serum hormone levels

OBJECTIVE: To examine the effects of mifepristone on progesterone and estrogen receptors in human decidua and steroid hormone levels in serum for investigating the mechanisms of antigestational action of mifepristone. METHODS: Decidual progesterone receptor (PR) and estrogen receptor (ER) concentrations or binding sites were measured by both dextran coated charcoal (DCC) and histochemical methods in normal subjects and after 100 mg-mifepristone treatment. Meanwhile, the concentrations of serum beta-human chorionic gonadotropin (beta-hCG), estradiol (E2), progesterone (P) and testosterone (T) were also determined by radioimmunoassay. The reactions of these two groups were compared. RESULTS: Mifepristone therapy significantly reduced decidual cytosol PR content (P < 0.05) and increased decidual cytosol ER content (P < 0.05). Histochemical analyses indicated mifepristone treatment increased ER staining in vessel and glandular cells of decidua. The serum beta-hCG, E2 and T levels elevated significantly after mifepristone administration, while the progesterone levels were unaffected. CONCLUSION: Our data suggested that the anti-gestational effect of mifepristone may act through decreasing the decidual PR and increasing the ER concentrations, which interfered the balance between these two components, and also through increasing serum T levels.     

Regulation of cytoskeleton by myelin components: studies on shiverer oligodendrocytes carrying an Mbp transgene

Oligodendrocytes from the shiverer mutant mouse are missing most of the myelin basic protein (Mbp) gene. In axon-free cultures, they produce membrane sheets with abnormally assembled microtubule and actin-based structures. This suggests that an Mbp gene product may have an important role in regulating the organization and stability of the wild-type oligodendrocyte cytoskeleton. We now present evidence extending these observations, using cultured oligodendrocytes that carry both the shiverer mutation and the Mbp1 transgene which partially corrects their deficit. Shiverer oligodendrocytes that carry one dose of the Mbp1 transgene abnormally express MBP along major cytoskeletal vein-like structures in processes and sheets. Shiverer oligodendrocytes that carry two doses of the Mbp1 transgene contain two types of membrane sheet regions, i.e. regions filled with aberrant punctate foci of MBP, and regions with normal domains of MBP. Immunocytochemical staining data show that the distribution of cytoskeleton and associated 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is dependent upon how MBP is organized. Bundling of actin filaments occurs only around MBP domains, and the colocalization of CNPase along microtubular structures also appears to be regulated by MBP domains in sheets. Multinucleated oligodendrocytes are observed, a likely result of the inability of dividing pro-oligodendrocytes to bundle actin filaments. In addition, the ability of MBP to mediate extracellular signals that modulate cytoskeleton appears to be dependent upon MBP's organization. Transduction of the galactocerebroside signaling pathway, which results in the destabilization of microtubules but not actin filaments, occurs only in sheets containing MBP domains. The distribution of MBP, however, does not affect the myelin/oligodendrocyte-specific protein signaling pathway, which results in growth of microtubular structures and extensive destabilization of the actin cytoskeleton.     

OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures

We have established a new permanent cell line (OLN-93), derived from spontaneously transformed cells in primary rat brain glial cultures. In growth medium supplemented with 10% fetal calf serum a doubling time of 16-18 hr was determined. OLN-93 cells in their antigenic properties resemble primary oligodendrocytes in culture. As analyzed by indirect immunofluorescence, the A2B5 surface marker is absent, they express galactocerebroside and myelin-specific proteins, such as myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipidprotein (PLP), and Wolfgram protein (WP), but do not exhibit astrocytic properties, such as the expression of vimentin or the glial fibrillary acidic protein (GFAP). In their morphological features they resemble bipolar O-2A-progenitor cells and, when grown at low density or on poly-L-lysine-coated culture dishes under low serum conditions, immature oligodendrocytes with a more arborized cell morphology. The cellular processes contain microfilaments, while N-CAM/D2 immunoreactivity is localized on the cell surface of the somata and processes. Immunoblot analysis further confirmed the presence of MAG, WP and MBP immunoreactivity, and the absence of vimentin and GFAP. Only a single MBP isoform (approximately 14 kDa) was detectable in the cellular extracts. PLP mRNA expression was studied by RT-PCR. The two proteolipid-specific mRNAs, DM20 and PLP, were present in OLN-93 cell extracts. Comparisons with embryonic rat cerebral cells in culture and primary oligodendrocytes suggest that OLN-93 cells in their morphological features and their antigenic properties resemble 5- to 10-day-old (postnatal time) cultured rat brain oligodendrocytes. Thus, the new cell line described in this study should provide a useful model system to investigate the specific mechanisms regulating the proliferation and differentiation of oligodendrocytes in vitro, and the molecular interactions with other cells of the nervous system.     

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.     

Immunohistochemical analysis of distribution of estrogen receptors and progesterone receptors in the postmenopausal endometrium

OBJECTIVE: To examine the expression of sex steroid receptors (ER: estrogen receptor; PR: progesterone receptor) in the postmenopausal endometrium (PMEM) and the relationship to clinical data for studying its characters. METHODS: The immunohistochemical reactivity of the PMEM was studied using monoclonal antibodies against ER and PR, in 33 postmenopausal patients. RESULTS: The endometrium was thicker in patients who were postmenopausal for 1 to 10 years (1.48 +/- 1.31 mm) than in patients who were postmenopausal for more than 10 years (0.79 +/- 0.37 mm)(p < 0.05). Among the 33 postmenopausal endometrial samples, ER positivity was found in the glands in 26 cases (78.8%) and PR positivity was detected in 18 cases (54.5%). The average age of the patients with ER positive reactivity in the glands (61.69 +/- 7.26 years) was significantly lower than that of the patients with ER negative reactivity (66.00 +/- 3.56 years)(p < 0.05). Furthermore, the endometrial thickness of the patients with ER or PR positive reactivity in the glands (1.24 +/- 1.09 mm and 1.47 +/- 1.20 mm, respectively) was significantly greater than that of the patients with ER or PR negative reactivity (0.67 +/- 0.26 mm and 0.70 +/- 0.40 mm, respectively)(p < 0.05). CONCLUSION: ER in the glands of the PMEM was determined to decrease gradually with increased aging. The presence of ER and PR in the gland cells seemed likely to determine the thickness of the PMEM.     

Expression of oligodendrocytic mRNAs in glial tumors: changes associated with tumor grade and extent of neoplastic infiltration

We examined the expression of glial- and neuronal-specific mRNAs within human gliomas using in situ hybridization. We found that low-grade astrocytomas contained a high number of proteolipid protein (PLP) mRNA-positive cells and that the number of PLP-stained cells decreased markedly with increasing tumor grade. Interestingly, the ratio of PLP mRNA-stained cells:myelin basic protein (MBP) mRNA-stained cells in normal white matter and low-grade astrocytoma was about 2:1 but approached 1:1 with increasing tumor grade. This parameter appeared to be a good indicator of tumor infiltration in astrocytomas, so we tested this in the analysis of other gliomas. Unlike astrocytomas, oligodendrogliomas were found consistently to contain few PLP mRNA- or MBP mRNA-expressing cells. In contrast, gemistocytic astrocytomas, typically highly invasive tumors, contained high numbers of PLP-positive cells and a ratio of PLP mRNA:MBP mRNA-stained cells of about 1.5:1, similar to low-grade astrocytomas. Nonradioactive in situ hybridization also enabled the morphological identification of specific cells. For example, gemistocytic astrocytes, which were found to be strongly vimentin mRNA positive, contained little glial fibrillary acidic protein mRNA and did not stain for PLP or MBP mRNAs. Neuronal mRNAs, such as neurofilament 68, were observed in small numbers of entrapped neurons within gliomas but were uninformative with respect to predicting tumor grade. Our results suggest that oligodendrocytes survive low-grade tumor infiltration and that glial tumor cells, unlike cell lines derived from them, do not express oligodendrocyte or neuronal mRNAs. In addition, the expression of mRNAs for the two major myelin protein genes, PLP and MBP, could be used to predict the grade and extent of tumor infiltration in astrocytomas.