Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures.

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase-1 (HO-1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17ß-estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve- or twenty-four-hour epidermal growth factor (EGF) treatment significantly enhanced HO-1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO-1 expression was obtained after the last-12-hr EGF treatment in 48-hr E2-pretreated astrocyte cultures; this enhancement was particularly significant in 48-hr E2-pretreated cultures as well as in the last-12-hr insulin-treated ones pretreated for 48 hr with E2. Sixty-hour DEX-alone pretreatment as well as the last-12-hr EGF treatment in 60-hr DEX-pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last-12-hr insulin-like growth factor-I (IGF-1)-treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF-1 in 48-hr E2-pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO-1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.

Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.

Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (α-glyceryl-phosphoryl-choline [αGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 µM (+)lipoic acid or (+/-)lipoic acid and/or 10 mM αGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(+)lipoic acid, (+/-)lipoic acid, or αGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (+)lipoic acid + αGPC or (+/-)lipoic + αGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.

Cholinergic precursors modulate the expression of heme oxigenase-1, p21 during astroglial cell proliferation and differentiation in culture.

Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and α-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for α-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after α-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.

Alpha-lipoic acid modulates GFAP, vimentin, nestin, cyclin D1 and MAP-kinase expression in astroglial cell cultures.

In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 µM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.

Effect of choline-containing phospholipids on transglutaminase activity in primary astroglial cell cultures.

The aim of the present investigation was to study the effects of choline and choline-containing phospholipids CDP-choline (CDPC) and L-alpha-glyceryl-phosphorylcholine (AGPC) on transglutaminase (TG) activity and expression in primary astrocyte cultures. TG is an important Ca(2+)-dependent protein that represents a normal constituent of nervous systems during fetal stages of development, playing a role in cell signal transduction, differentiation, and apoptosis. Confocal laser scanning microscopy (CLSM) analysis showed an increase of TG activity in astrocyte cultures treated with choline, CDPC, or AGPC at 0.1 microM or 1 microM concentrations. Comparatively, AGPC induced the most conspicuous effects enhancing monodansyl-cadaverine fluorescence both in cytosol and in nuclei, supporting the evidence of the important role played by AGPC throughout differentiation processes tightly correlated to nucleus-cytosol cross- talk during astroglial cells proliferation and development. Western blot analysis showed that in 24h 1 microM AGPC and choline-treated astrocytes increased TG-2, whereas no effect was observed in 24h 1 microM CDP-choline treated astrocytes. Our data suggest a crucial role of choline precursors during different stages of astroglial cell proliferation and differentiation in cultures.

Effect of acetylcholine precursors on proliferation and differentiation of astroglial cells in primary cultures.

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5'-diphosphocholine (CDP-choline) and alpha-glyceryl-phosphorylcholine (alpha-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 microM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 microM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 microM alpha-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 microM choline or alpha-GPC, whereas in 24 h 1 microM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 microM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 microM alpha-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures.

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.