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. 1999 Nov 15;19(22):10014-25.
doi: 10.1523/JNEUROSCI.19-22-10014.1999.

Platelet-derived growth factor-mediated signal transduction underlying astrocyte proliferation: site of ethanol action

J Luo  1 , M W Miller
Affiliations

Platelet-derived growth factor-mediated signal transduction underlying astrocyte proliferation: site of ethanol action

J Luo et al. J Neurosci. .

Abstract

Platelet-derived growth factor (PDGF) is a critical regulator of cell proliferation. Because ethanol inhibits cell proliferation in vivo and in vitro, we hypothesize that ethanol-induced inhibition results from differential interference with signal transduction pathways activated by PDGF. Cultured cortical astrocytes were used to examine the effects of ethanol on PDGF-mediated signal transduction, on the expression of two PDGF monomers (A- and B-chains), and on the expression of two PDGF receptor subunits (PDGFalphar and PDGFbetar). PDGF-B chain homodimer (PDGF-BB), and to a lesser extent PDGF-A chain homodimer (PDGF-AA), stimulated the proliferation of astrocytes raised in a serum-free medium. Ethanol attenuated these actions in a concentration-dependent manner. Ethanol inhibited both PDGF-AA- and PDGF-BB-mediated phosphorylation of PDGFalphar, but it had little effect on PDGFbetar autophosphorylation. Likewise, ethanol abolished the association of PDGFalphar to Ras GTPase-activating protein (Ras-GAP), but it did not affect the binding of Ras-GAP to PDGFbetar. PDGF stimulated the activities of mitogen-activated protein kinase (MAPK) in protein kinase C (PKC) independent and dependent manners. Ethanol inhibited the PKC-independent, acute activation of MAPK; however, it stimulated the PKC-dependent, sustained activation of MAPK. The expression of neither ligand was altered by exposure to ethanol for 3 d. Moreover, such treatment specifically upregulated PDGFalphar expression in a concentration-dependent manner. It did not, however, affect the binding affinity of either receptor. Thus, the signal transduction pathways initiated by PDGF-AA and PDGF-BB were differentially affected by ethanol. This differential vulnerability resulted from the preferential effects of ethanol on PDGFalphar autophosphorylation. Hence, ethanol-induced alterations are transduced through specific receptors of mitogenic growth factors.

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Figures

Fig. 1.
Fig. 1.
PDGF-mediated astrocyte growth. Astrocytes were maintained in a serum-free medium and treated with either PDGF-AA or PDGF-BB (30 ng/ml) for 3 d. The left panel shows the mean number of cells in a culture (±SEM) before PDGF or ethanol (200–800 mg/dl) was added. The plot depicts the original (concocted) ethanol concentrations. The middle panel describes the number of cells in a culture after 3 d of treatment with PDGF and/or ethanol. The amount of ethanol-induced inhibition (expressed as a percentage) was measured against the amount of PDGF stimulation in the absence of ethanol (right panel). The formula for calculating the percent inhibition (PI) is PI = [(PDGF+)0 − (PDGF+)x]/[(PDGF+)0 − (PDGF−)x] (100%), in which (PDGF+) and (PDGF−) are the numbers of cells in cultures treated with and without PDGF, respectively, after being treated with a particular concentration of ethanol (i.e., 0 or x mg/dl). Each data point is based on six trials.
Fig. 2.
Fig. 2.
Effects of PDGF and ethanol on [3H]dT incorporation. Astrocytes were maintained in a serum-free medium. [3H]dT incorporation was measured after 3 d of treatment with PDGF (0 or 30 ng/ml) and/or ethanol (0 or 400 mg/dl). Data represent the means (±SEM) for four independent experiments. Asterisks denote statistically significant differences (p < 0.05) in comparisons with the untreated (control) cultures.
Fig. 3.
Fig. 3.
Time sequence of PDGF receptor autophosphorylation. Glia were isolated from cortices of 5-d-old rats, purified, and cultured in serum-supplemented medium for 2 weeks. Subsequently, the cells were grown in a serum-free medium for 24 hr and treated with PDGF-AA or PDGF-BB for 0, 5, 10, 20, 40, or 60 min. PDGFαr and PDGFβr were immunoprecipitated separately and assayed for tyrosine phosphorylation. The experiment was repeated three times.
Fig. 4.
Fig. 4.
Effect of ethanol on PDGF receptor autophosphorylation. Top, Astrocytes were maintained in a serum-free medium for 24 hr, then exposed to ethanol (0 or 400 mg/dl) for 1 hr. Subsequently, the cells were treated with PDGF-AA or PDGF-BB (30 ng/ml) in the presence or absence of ethanol for 10 min. Cell lysates were collected, and an equal amount of cellular protein (200 μg) was immunoprecipitated with an anti-PDGFαr or anti-PDGFβr antibody. The immunoprecipitates were electrophoretically separated and probed with an antibody (PY20) directed against phosphorylated tyrosine. Bottom, After the nitrocellulose papers were stripped, they were reprobed with either anti-PDGFαr or anti-PDGFβr antibody. These blotsshow that not only did the phosphorylation signal align with the receptor signal, but the loading of the protein on each lane was consistent. Note that protein expression of the PDGFαr was unaffected by 1 hr ethanol treatment; changes in the expression of the protein PDGFαr required a chronic exposure, e.g., 3 d (Fig. 8). Three repeats of the experiment were performed.
Fig. 5.
Fig. 5.
Association of Ras–GAP. After the isolation, purification, and growth in a medium containing 10% fetal calf serum, astrocytes were maintained in a serum-free medium for 24 hr. The cells were exposed to ethanol (0 or 400 mg/dl) for 1 hr and treated with PDGF-AA or PDGF-BB (30 ng/ml) in the presence or absence of ethanol for 10 min. Cell lysates were collected and immunoprecipitated with an anti-PDGFαr or anti-PDGFβr antibody. The immunoprecipitates were electrophoretically separated and probed with a monoclonal antibody directed against Ras–GAP. The experiment was performed three times.
Fig. 6.
Fig. 6.
Effects of PDGF, ethanol, and H-7 on time sequence of MAPK activity. MAPK activity was determined for astrocytes pretreated with ethanol (0 or 400 mg/dl) and/or H-7 (0 or 50 μm) for 1 hr in a serum-free medium (designated as time 0 min). The cells were then incubated with either PDGF-AA (30 ng/ml) or PDGF-BB (30 ng/ml) for as long as 90 min. Insets, The histograms compare MAPK activity for cultures treated with ethanol (0 or 400 mg/dl) and/or H-7 (0 or 50 μm) at time 0 min (black bars) or at time 90 min (white bars). Each data point represents the mean of four trials (±SEM). Asterisks denote statistically significant (p < 0.05) differences relative to the control cultures at time 0 min.
Fig. 7.
Fig. 7.
PKC activity. The effects of four substances (PDGF-AA, PDGF-BB, ethanol, and H-7) on PKC activity in the membranal (top) and cystolic fractions (middle) was determined. The graph at the bottomdescribes the effects of the four substances on the ratio of PKC activity in the membrane to the combined PKC activity in the membrane and cytosol. Statistically significant differences (p < 0.05) in comparisons relative to the untreated (control) cells are noted by an asterisk. Differences caused by H-7 pretreatment (i.e., comparing cells that were similarly treated with PDGF and/or ethanol) are identified by #. Ethanol-induced differences for cells that were treated with the same growth factor are labeled with +. Each experiment was performed four times.
Fig. 8.
Fig. 8.
Expression of PDGF ligands and receptors.Top, The effect of ethanol on the expression of the 18 kDa PDGF-AA and 16 kDa PDGF-BB was analyzed with Western immunoblots. Astrocytes were treated with ethanol (0–800 mg/dl) for 3 d. Thirty micrograms of cellular protein were loaded onto each lane.Bottom, Expression of the two PDGF receptor subunits was examined with immunoblots. Both receptors are 180 kDa but could be distinguished using specific antibodies that had no cross-reactivity with the other subunit.
Fig. 9.
Fig. 9.
PDGF binding characteristics. The kinetics of PDGF-AA and PDGF-BB binding was assessed by a Scatchard analysis. Astrocytes were maintained in a medium containing 10% serum and treated with a PDGF ligand (0 or 30 ng/ml) and/or ethanol (0 or 400 mg/dl) for 3 d. Each data point is the mean of four independent trials.
Fig. 10.
Fig. 10.
Schematic of signal transduction pathways of MAPK activation initiated by PDGF ligands. PDGF-AA induces only the tyrosine phosphorylation of PDGFαr, whereas PDGF-BB activates either receptor subunit. Receptor phosphorylation causes the activation of downstream effector molecules that contain an SH2 domain (e.g., Ras–GAP) and the sequential activation of Ras, Raf-1, MAPK kinase (MEK), and eventually MAPK. Alternatively, receptor phosphorylation can activate PKC, which in turn, can stimulate MAPK by either by a direct effect on Ras–Raf-1 or through an unknown intermediate molecule(s). Ethanol can affect receptor phosphorylation and PKC activity in the MAPK cascade. These positive (dashed lines) and negative (dotted lines) effects can alter the signal transduction cascade that ultimately leads to the stimulation or inhibition of MAPK.
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