Immortalized hypothalamic luteinizing hormone‐releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor

Abstract

Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 microg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)

“…For single and double immunofluorescence labeling, cells preincubated for 60 min in blocking buffer at 4°C (0.3-0.5% Triton X-100͞5% BSA͞5% normal serum in PBS) were then incubated overnight at 4°C with primary antibodies against Mrp1 (32) and GFAP, 1:100, as detailed elsewhere (27,29). Golgi apparatus was stained with WGA-FITC (33).…”

“…Cells labeled by immunofluorescence were visualized and analyzed by using a Leica TCS NT (Ver. 1.0, Leica Lasertechnik, Heidelberg) inverted confocal laser scanning microscope equipped with an argon͞krypton laser by using ϫ10, ϫ20, ϫ40, and ϫ100 oil-immersion objectives (27)(28)(29). The pinhole was set at 1.0-1.2 for optical sections of 0.5-0.6 m. Single lower-power scans were followed by 16-22 serial optical sections of randomly chosen cells in four to five fields per coverslip by using the same settings.…”

“…Astrocytes also regulate growth, differentiation, and survival of neurons as part of bidirectional neuronal-glial interactions (27)(28)(29). Efflux of glutathione from astroglial cells, mediated by Mrp1, is vital in the defense of CNS cells from antioxidants (30).…”

Growth Factors and Steroid Mediated Regulation of Cytoskeletal Protein Expression in Serum-Deprived Primary Astrocyte Cultures

“…Nonetheless, GFs, bFGF, epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and insulin (INS) are mitogenic polypeptides participating in neuronglia cross-talk stimulating cultured nerve cell proliferation and differentiation [7][8][9]. Using a in vitro model of newborn rat astroglial cells, we have previously studied GR expression during development and we found that its mRNA levels were low at 8 days in vitro (DIV), then progressively enhanced between 12 and 20 DIV and stabilised up to 50 DIV [10].…”

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

“…INS and IGF-I are powerful mitogenic and trophic factors for both neurons and astrocytes [7][8][9]. EGF stimulates proliferation and differentiation of cultured rat primary astrocytes [10][11][12][13][14][15][16][17] and acts synergically with IGF-I [18]. Growth factors elicit their biological effects by binding to plasmalemmal tyrosine kinase receptors [19].…”