Mitogen activated protein kinase-dependent activation of c-Jun and c-Fos is required for neuronal differentiation but not for growth and stress response in PC12 cells

MAPK-dependent activation of AP-1 protein c-Jun is involved in PC12 cell differentiation and apoptosis. However, the role of other AP-1 proteins and their connection to MAPKs during growth, differentiation and apoptosis has remained elusive. Here we studied the activation of AP-1 proteins in response to ERK, JNK, and p38 signaling upon NGF, EGF and anisomycin exposures. All treatments caused different kinetics and strength of MAPK and AP-1 activities. NGF induced persistent ERK and AP-1 activities, whereas upon EGF and anisomycin exposures, their activities were only weakly and transiently induced. The sustained AP-1 activity was associated with concomitant c-Fos and c-Jun expression and phoshorylation, which were JNK and ERK dependent. While inhibition of the ERK, JNK, and p38 activities partially prevented AP-1 activity and suppressed differentiation, none of them was required for anisomycin-induced apoptosis. The importance of c-Fos and c-Jun as mediators of differentiation was demonstrated by the findings that the corresponding siRNAs suppressed NGF-induced neurite outgrowth. However, the capacity of c-Fos to promote differentiation required cooperation with Jun proteins. In contrast, Fra-2 expression was not required for the differentiation response. Together, the results show that sustained c-Jun and c-Fos activities mediate MAPK signaling and are essential for differentiation of PC12 cells.     

β2-adrenergic receptors inhibit the expression of collagen type II in growth plate chondrocytes by stimulating the AP-1 factor Jun-B

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through β(2)-adrenergic receptors (β(2)-AR). We have shown previously that β(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined β(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of β(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by ～50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated β(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through β(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.     

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.     

Differential expression of sodium channels and nicotinic acetylcholine receptor channels in nnr variants of the PC12 pheochromocytoma cell line

An important component of neuronal differentiation is the tightly controlled expression of a spectrum of ion channel proteins. Ion channels play a critical role in the generation and propagation of action potentials as well as in the cellular response to neurotransmitters, and thus are central in the transfer and integration of information in the nervous system. A model system amenable to analysis of ion channel expression and neuronal differentiation is the rat pheochromocytoma (PC12) cell line. Here, we have used electrophysiological and molecular biological approaches to analyze the expression of voltage-dependent sodium (Na) channels and nicotinic acetylcholine receptors (nAChR) in mutagenized variants (nnr cells) of the PC12 cell line. Our data reveal striking differences in the expression of these channels when compared to wild-type PC12 cells. Even in the absence of nerve growth factor (NGF), nnr cells express functional Na channels and Na channel mRNA at levels exceeding those in wild-type PC12 cells differentiated with NGF. In contrast, acetylcholine-induced currents were evident in only a small proportion of cells, presumably due to the altered pattern of expression of mRNAs encoding individual nAChR subunits. The altered ion channel expression in these variants provides an avenue for analyzing Na channel and nAChR channel function, as well as for identifying mechanisms governing their expression.The authors thank J. Boulter (The Salk Institute) for providing nAChR cDNA clones, C. Machida (Oregon Health Sciences University) for providing the cyclophilin cDNA, and L. Greene for providing nnr3 cells, nnr5 cells, and nnr5 cells stably transfected with trkA. This research was supported by grants awarded by the National Institutes of Health to LPH (NS28668), PDG (NS30243), and RAM (NS28767).