Published as doi: 10.1096/fj.05-5510fje.
(The FASEB Journal. 2006;20:1263-1265.)
© 2006 FASEB
Apomorphine-induced activation of dopamine receptors modulates FGF-2 expression in astrocytic cultures and promotes survival of dopaminergic neurons
Aiqun Li*,
,
Hong Guo*,
Xiaoying Luo*,,
Jiansong Sheng*,,
Shuo Yang*,,
Yanqing Yin*,
Jianwei Zhou
and
Jiawei Zhou*,,1
* Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences and
Graduate School of the Chinese Academy of Sciences, Shanghai, China; and
Institute of Public Health, Nanjing Medical University, Nanjing, China
1Correspondence: Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Rd., Shanghai, 200031, P. R. China. E-mail: jwzhou{at}sibs.ac.cn
SPECIFIC AIMS
Apomorphine (APO), a potent D1/D2 dopamine receptor agonist, is currently in use as an antiparkinsonian drug. We investigated the molecular mechanism underlying APO-induced survival-promoting activity on nigral dopaminergic neurons in vitro. The present study was designed to investigate the effects of APO on FGF-2 expression and regulation in astrocytes, and furthermore, to identify the underlying signaling mechanism for the effects.
PRINCIPAL FINDINGS
1. Treatment of astrocyte cultures with APO enhanced biosynthesis and extracellular release of FGF-2, which contributed to elevated survival of TH-positive neurons
The conditioned medium (CM) derived from striatal astrocytic cultures increased the number of tyrosine hydroxylase (TH)-positive neurons in the recipient mesencephalic neuronal cultures in a dose-dependent manner. Western blot analysis on the concentrated CM, using a FGF-2 polyclonal antibody, which recognized four FGF-2 isoforms corresponding to higher MW forms (HMW, 24, 22.5, 22 kDa) and lower MW form (LMW, 18 kDa) FGF-2, revealed that in the CM of striatal astrocyte cultures treated with APO for 6 h, the HMW- and LMW FGF-2 were detectable at 18 h and 24 h following the treatment. Conversely, FGF-2 was not detectable in the CM of untreated astrocytic cultures at any time points examined. Incubation of APO-induced CM with FGF-2 antibody (Ab) dramatically attenuated the increase in the number of TH-positive neurons by 55.9%. These data suggest that FGF-2 may be the major contributor that mediated the APO-induced survival-promoting effect.
2. Activation of DA receptor is responsible for APO-modulated FGF-2 biosynthesis
We next asked whether DA receptor activity was required for APO-mediated induction of FGF-2. Treatment of the striatal astrocytes with a variety of concentrations of S-APO (0.02–20 µM), a derivative of R-APO lacking activity of DA receptor agonist, for 6 h did not significantly alter the concentration of FGF-2 biosynthesis. However, striatal astrocytic cultures exposed to the DA receptor agonists SKF-38393 (10 µM), or quinpirole (50 µM) for 6 h showed remarkably increased levels of FGF-2 isoforms. Moreover, preincubation with either D1 receptor antagonists SCH23390 (10 µM), SKF83566 (10 µM) or D2 receptor antagonist haloperidol (1 µM), respectively, significantly blocked APO-stimulated FGF-2 expression. Combination of D1 and D2 receptor antagonists partially abolished APO-stimulated FGF-2 expression. Our data indicated that both D1 and D2 DA receptor is involved in APO-modulated FGF-2 biosynthesis.
3. APO modulates FGF-2 biosynthesis via cAMP and PKC, not phosphoinositide 3-kinase
To determine the mechanism of the DA receptor-mediated FGF-2 induction, we blocked key components of several signal transduction pathways while costimulating the receptors with both D1- and D2-selective agonists. Exposure of the astrocytic cultures to APO resulted in 4.8-fold increase in the concentration of phosphorylation of PKA at 30 min following the treatment (Fig. 1
A, B), indicating that APO indeed stimulates the PKA activation. Addition of forskolin (5 µM), into the astrocytic cultures led to an average fourfold increase of FGF-2 compared to the untreated (Fig. 1C, D
). Conversely, pretreatment of the cultures with either hemagglutinin (HA)-100 (30 µM, a PKA/PKC inhibitor) or KT5720 (50 µM, a PKA inhibitor) completely abrogated APO-stimulated FGF-2 expression (Fig. 1C, D
). These data suggest that cAMP/PKA signaling pathway play an important role in APO-induced FGF-2 up-regulation.
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Figure 1. APO modulates FGF-2 expression in cultured astrocytes through activation of cAMP/PKA and MEK/MAPK but not PI-3K signaling pathways. A) Phosphorylation of PKA is activated by addition of APO in the cultures. Astrocytes stimulated by APO were harvested at different time points (30 min, 60 min, 2 h, and 6 h). Supernatants were assayed for PKA phosphorylation. B) Quantitation of A. *P < 0.05, compared with untreated. C) Effects of cAMP activators and inhibitors of PKA, PKC, MAPK, or PI-3K on APO-induced FGF-2 expression in striatal astrocytes. Cultures were exposed to HA-100 (30 µM, PKA/PKC inhibitor), LY294002 (20 µM, PI-3K inhibitor), PD98059 (50 µM, MAPK inhibitor), KT5720 (50 µM, PKA inhibitor), or GF109203X (2 µM, PKC inhibitor) for 2 h, followed by addition of APO for 6 h, while striatal astrocytes were treated only with forskolin (5 µM, cAMP activator) and APO. Cells were then lysed for Western blot analysis. Bottom) The levels of  -tubulin are shown as a loading control. D) Quantitation of C. *P < 0.05, compared with untreated. E) The MEK/MAPK, but not the PI-3K signaling pathway, was activated following APO stimulation. Serum-starved striatum astrocytes were stimulated with APO, and levels of phospho-MAPK, MAPK, phospho-Akt (Ser-473) and Akt were assayed by Western blot analysis. Bottom) The levels of -tubulin are shown as a loading control. F) Treatment of cultures with FGF-2 (1 ng/ml) or BDNF does not affect FGF-2 expression.
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Moreover, exposure of serum-starved striatal astrocytic cultures to APO phosphor-activated MAPK at all time points (Fig. 1E
). Treatment of the astrocytic culture with either GF109203X (2 µM, a PKC inhibitor) or PD98059 (50 µM, a MAPK inhibitor) prior to the addition of APO markedly reduced the up-regulation of FGF-2 protein (Fig. 1C, D
). These results suggest that MEK/MAPK signaling pathway is also important for APO-induced FGF-2 up-regulation. In contrast, APO did not alter the levels of phospho-Akt in astrocytic cultures for up to 6 h (Fig. 1E
), implicating that APO does not modulate Akt activity.
4. The D1 receptor activates the cAMP/PKA pathway, whereas the D2 receptor mediates the MEK/MAPK pathway following APO stimulation
As shown in Fig. 2
, DA receptors and two signaling pathways (cAMP/PKA and MEK/MAPK) were activated contributing to APO-modulated FGF-2 induction. The downstream signaling pathway to corresponding subtype of DA receptor was thus determined. The astrocytic cultures were treated with combination of D2 receptor antagonist haloperidol with APO. Notably, the phosphorylation concentration of PKA was increased, while phosphorylation levels of MAPK were not markedly altered. Likewise, combinational treatment of the astrocytes with D1 receptor antagonist, SCH23390 or SKF83566, and APO produced a significant increment of phosphorylated MAPK levels, but without marked alteration in levels of phosphorylated PKA. Indeed, treatment of astrocytes with SKF38393, a D1 receptor agonist, increased phosphorylation concentration of PKA without marked influence on concentration of phosphorylated MAPK. Moreover, exposure to quinpirole had a completely opposite effect compared with SKF38393. Furthermore, in the presence of APO, treatment of the cultures with either PD98059 or GF109203X abrogated phosphorylation concentration of MAPK having little effect on phosphorylation levels of PKA. Taken together, our data suggest that D1 receptor uses the cAMP/PKA pathway in the presence of APO, whereas the MEK/MAPK pathway is preferably activated by D2 receptor (Fig. 2
).
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Figure 2. Schematic diagram showing signal transduction pathways involved in APO-modulated biosynthesis and release of FGF-2 in striatal astrocytes. The dashed line indicates an inactivated pathway. Enhanced release of HMW- and LMW FGF-2 leads to increased survival of DA neurons.
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It was found that APO itself elevated phosphorylation levels of both PKA and MAPK. However, in the presence of forskolin, which stimulates intracellular concentration of cAMP, APO failed to increase phosphorylation of MAPK, suggesting inhibitory effect of D1 receptor-associated downstream signaling pathway on MEK/MAPK cascade. In contrast, combinational treatment with KT5720 and APO abrogated this inhibitory effect, suggesting that inhibition of MAPK phosphorylation by PKA, but not cAMP, is critical to the inhibitory effect of D1 receptor-associated pathway on D2 receptor pathway (Fig. 2)
.
CONCLUSIONS AND SIGNIFICANCE
A major finding in the present study is that APO treatment changes subcellular localization of HMW FGF-2 and enhances release of both HMW- and LMW FGF-2 into the cultured medium. The present study has shown that the most abundant forms of FGF-2 in the untreated astrocytes in cultures are the HMW isoforms. However, more HMW FGF-2 was detected in the cytoplasm and the CM of APO-treated astrocytes, suggesting that APO affects FGF-2 release in addition to its synthesis. The released HMW- and LMW FGF-2 may together contribute to promote survival of DA neurons.
There is controversy as to which intracellular signaling pathways correspond to D1 and D2 receptors in neuronal cells. A recent study has shown that D1 receptor induces phosphorylation of PKA, while D2 receptor induces phosphorylation of MAPK and cAMP-responsive element binding protein (CREB) in dopaminoceptive neurons. In contrast, D1 receptor also can phosphorylate and activate MAPK and CREB. However, very little information is available on the roles of DA receptor-mediated signaling pathways in astroglial cells. We demonstrate here in the present study that PKA was phosphorylated and activated preferentially by D1 receptor following treatment of either APO or selective D1 receptor agonist SKF38393. Similarly, MAPK is a target found to be activated by D2 receptor in response to APO stimulation in this study. We conclude that cAMP/PKA and MEK/MAPK controls the inductive effect of APO on FGF-2 in astroglia by distinctly different mechanisms from that used in neuronal cells. It is plausible that activation of cAMP/PKA signaling pathway increase FGF-2 gene expression via the activation of cis-regulatory elements located in the FGF-2 promoter. Because APO administrations increase concentration of phosphorylated CREB, APO may thus affect the transcription of the FGF-2 gene by inducing the binding activity of transcription factors to cis-elements located in the FGF-2 promoter. Importantly, we found that the activation of PI-3K pathway did not contribute to FGF-2 induction in this study.
The induction of FGF-2 in astrocytes evoked by APO may be a common mechanism elicited by catechol-derived compounds. Our data imply the dual actions of DA, a neurotransmitter with property of DA receptor agonist, i.e., mediation of neurotransmission and modulation of FGF-2 and other neurotrophic factors expression in astrocytes. Both effects are highly dependent on activation of DA receptors. Dysfunction of DA nigrostriatal pathway in PD leads to deregulation of FGF-2 in this neural pathway, which, in turn, affects the survival and function of nigral DA neurons. On the basis of these considerations, we can speculate that APO, by restoring appropriate levels of endogenous trophic factors, may prevent loss of midbrain DA neurons. The clinical benefits of APO administration can be interpreted as consequence of sustained agonism on DA receptor. It may also result from either APO-induced neuroprotection or modulation of synaptic activity. This therapeutical strategy may open up a new avenue of research for preventing or delaying the onset/progression of PD.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5510fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
