The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade.

Most forms of neuronal plasticity are associated with induction of the transcription factor Zif268 (Egr1/Krox24/NGF-IA). In a genomewide scan, we obtained evidence for potential modulation of proteasome subunit and regulatory genes by Zif268 in neurons, a finding of significance considering emerging evidence that the proteasome modulates synaptic function. Bioinformatic analysis indicated that the candidate proteasome Zif268 target genes had a rich concentration of putative Zif268 binding sites immediately upstream of the transcriptional start sites. Regulation of the mRNAs encoding the psmb9 (Lmp2) and psme2 (PA28␤) proteasome subunits, along with the proteasome-regulatory kinase serum/glucocorticoid-regulated kinase (SGK) and the proteasome-associated antigen peptide transporter subunit 1 (Tap1), was confirmed after transfection of a neuronal cell line with Zif268. Conversely, these mRNAs were upregulated in cerebral cortex tissue from Zif268 knock-out mice relative to controls, confirming that Zif268 suppresses their expression in the CNS. Transfected Zif268 reduced the activity of psmb9, SGK, and Tap1 promoter-reporter constructs. Altered psmb9, SGK, and Tap1 mRNA levels were also observed in an in vivo model of neuronal plasticity involving Zif268 induction: the effect of haloperidol administration on striatal gene expression. Consistent with these effects on proteasome gene expression, increased Zif268 expression suppressed proteasome activity, whereas Zif268 knock-out mice exhibited elevated cortical proteasome activity. Our findings reveal that Zif268 regulates the expression of proteasome and related genes in neuronal cells and provide new evidence that altered expression of proteasome activity after Zif268 induction may be a key component of long-lasting CNS plasticity.

The later phases of neuronal plasticity are invariably dependent on gene transcription. Induction of the transcription factor Zif268 (Egr-1) in neurones is closely associated with many forms of functional plasticity, yet the neuronal target genes modulated by Zif268 have not been characterized. After transfection of a neuronal cell line with Zif268 we identified genes that show altered expression using high density microarrays. Although some of the genes identified have previously been associated with forms of neuronal plasticity, the majority have not been linked with neuronal plasticity or Zif268 action. Altered expression of a representative sample of the novel target genes was confirmed in Zif268-transfected PC12 neurones, and in in vitro and in vivo models of Zif268-associated neuronal plasticity. In particular, altered expression of the protease inhibitor Cystatin C and the chemokine Cxcl10 was observed in striatal tissue after haloperidol administration. Surprisingly, the group of identified genes is enriched for components of the proteasome and the major histocompatibility complex. Our findings suggest that altered expression of these genes following Zif268 induction may be a key component of long lasting plasticity in the CNS.

We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis.

The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated-proteinkinase (p42\p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric Gprotein G i ) induced an approx. 40-50 % decrease in the activation of c-Src and p42\p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42\p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42\p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, G i , to regulate c-Src and subsequent p42\p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42\p44 MAPK activation. This is confirmed here