Effect of growth factors on nuclear and mitochondrial ADP-ribosylation processes during astroglial cell development and aging in culture
Introduction
Different types of astrocytes were identified in the central nervous system. They have a functional and regional heterogeneity that could reflect differences on scheduled brain development (Garcia-Abreu et al., 1996). Astrocyte differentiation is switched on by a transition program involving the vimentin-glial fibrillary acidic protein (GFAP) binome. GFAP is the main marker of mature cultured astrocytes (Bignami et al., 1972), whereas vimentin is expressed only in developmental stages (Dahl, 1981). Increased expression of GFAP and down-regulation of vimentin, indicate astrocyte maturation (Dahl et al., 1981).
Astroglial cells release peptide growth factors in surrounding environment during development and maturation. They act as stimulants of cell growth and differentiation (Morrison et al., 1982) and play an important role in glia–neuron cross-talk (Avola et al., 2000). Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and insulin (INS), are powerful mitogens and neurotrophic factors in several cell types (Morrison et al., 1986, Rotwein et al., 1988, Enfors et al., 1990, Torres-Aleman et al., 1990, Casper et al., 1991). EGF stimulates mitogenesis of astroglial cells in primary culture (Avola et al., 1988a, Avola et al., 1988b), whereas bFGF and IGF-I are crucial factors during brain development (Rotwein et al., 1988, Han et al., 1992), and influence neurite sprouting and outgrowth of neurons derived from different brain areas (Morrison et al., 1986, Walicke et al., 1986). These functions are exerted through signaling cascades triggered by their binding on cognate receptors (Carpenter and Cohen, 1990, Fantl et al., 1993). Activated receptors undergo autophosphorylation on specific tyrosine residues. They couple to downstream adaptors such as Grb-2, Shc, Gab-1, Nck and the p85 subunit of phosphatidyl-inositol (PI) 3-kinase and leading mainly to small G-protein RasGTP loading and activation. This, in turn, activates the extracellular signal-regulated kinases 1 and 2 (ERK1 and 2) cascade, the phospholipase A2-mediated arachidonic acid release, the phospholipase Cγ-mediated phosphoinositide hydrolysis, classical protein kinases C (PKC) membrane translocation and inositol-1,4,5-trisphosphate (IP3)-mediated calcium mobilization from endoplasmic reticulum stores (Berridge, 1987).
The above biochemical events activate several transcription factors, such as fos/jun activator protein-1 (AP-1), serum response factor (SRF), activation transcription factor 2 (ATF-2), early growth response 1 (Egr-1) and polyADP-ribose polymerase (PARP) (Homburg et al., 2000). These events in sequence modulate nuclear gene expression activities linked to proliferation or differentiation (Denhardt, 1996). PARP enzyme polyADP-ribosylates a great number of nuclear proteins involved not only in DNA repair and genome stabilization (e.g. p53, topoisomerases I and II, DNA lygase III), but also in cell cycle (c-fos, DNA polymerases α and β) and differentiation such as TATA box binding protein and high mobility group proteins (HMGs) 1, 2, 14 and 17 (D'Amours et al., 1999). It also plays a pivotal role in DNA transcription and replication role (De Lucia et al., 1996, Simbulan-Rosenthal et al., 1996). An association of PARP with transcriptionally active chromatin regions was found. PARP also regulates the expression of c-Myc, protein-kinase C and DNA methyl transferase genes (Bauer et al., 1996). However, at present, no direct role of PARP in astrocyte differentiation was demonstrated.
A causal relationship between ADP-ribosylation and astroglial cell proliferation including histone H1 poly ADP-ribosylation during intense proliferative early postnatal events (18 DIV), higher in cerebellum of rat embryos slices than in cerebral cortex was shown (Spina-Purrello et al., 1990, Avola et al., 1998). A significant increase of PARP in bFGF or IGF-I treated fetal rat brain slices was also observed. Moreover, IGF-I or INS treatment resulted in a dose and time-dependent increase in PARP activity in 18-day-old rat brain embryo slices (Avola et al., 1998). On the other hand, mono ADP-ribosylation is likely involved in mitochondrial metabolism, as demonstrated in testis mitochondria (Burzio et al., 1981). It was also suggested that ADP ribosyltransferase (ADPRT) activation might be linked to mitochondrial Ca2+ release induced by chemical oxidants (Richter and Frei, 1988).
The present study was designed to assess the effects of the growth factors EGF, bFGF, IGF-I and INS on nuclear PARP and mitochondrial ADPRT enzymatic activities in developing (30 DIV) or aging (90 and 190 DIV) primary rat astrocyte cultures. This is to clarify mitogenic or differentiative roles played by these neurotrophic factors during postnatal development, maturation and aging in in vitro models.
Section snippets
Astroglial cell cultures
Astroglial cell cultures were obtained from cerebral hemispheres of newborn rats according to the protocol detailed in earlier studies of our group (Avola et al., 1988a, Avola et al., 1988b). Cultures were grown in Falcon plastic Petri dishes (35, 60 or 100 mm diameter) at a plating density of 0.5–1×105 cells/cm2 and incubated at 37 °C in a humidified 5% CO2–95% air atmosphere. Culture medium was changed after 7 days and then twice a week. The morphology of this astroglial cell culture system
Results
The results of nuclear PARP activity in purified nuclei from primary rat astrocyte cultures at 30 DIV treated with IGF-I and bFGF are shown in Fig. 1. PARP activity significantly increased in IGF-I- and bFGF-treated astroglial cell cultures (by about 100% for IGF-I and 140% for bFGF) compared to control untreated cultures. The effects of INS or EGF or both together (INS+EGF) on PARP and ADPRT activities, in nuclei and mitochondria respectively purified from 30 DIV-developing astrocyte culture,
Discussion
The above data have shown that bFGF, IGF-I, EGF or INS, or the two last together (EGF+INS) greatly stimulate PARP and ADPRT activities in 30 DIV developing astroglial cell cultures. This effect can be explained by a synergistic potentiation of effects of these growth factors on cell proliferation and differentiation, through a cross talk between multiple signaling cascades triggered by them (Avola et al., 1988a, Avola et al., 1988b). These mechanisms are known to up-regulate cell cycle protein
Acknowledgements
This work was accomplished by the financial support from Italian National Research Center CNR research project no. 97.04535.CT04 and the Italian Ministry of Scientific and Technological Research (MURST) 60% 1997 Cod. 2 1040178 to R. Avola.
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