Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells

Abstract

In pheochromocytoma (PC12) cells nerve growth factor (NGF) and epidermal growth factor (EGF) activate similar receptor tyrosine kinase signaling pathways but evoke strikingly different biological outcomes: NGF induces differentiation and EGF acts as a mitogen. A novel approach was developed for identifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DNA recognition library to isolate specific DNA binding proteins from PC12 nuclear extracts. A protein complex from NGF-treated, but not EGF-treated, cells was identified that exhibits increased mobility and DNA binding activity in gel mobility shift assays. The binding complex was identified in supershift assays as Fra-2/JunD. The clones used as probes contain either AP-1 or cAMP response element binding (CREB) recognition elements. Time course experiments revealed further differences in NGF and EGF signaling in PC12 cells. NGF elicits a more delayed and sustained ERK phosphorylation than EGF, consistent with previous reports. Both growth factors transiently induce c-fos, but NGF evokes a greater response than EGF. NGF specifically increases Fra-1 and Fra-2 levels at 4 and 24 hr. The latter is represented in Western blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upper bands, corresponding to phosphorylated Fra-2. These findings suggest that prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD binding to AP-1 and CREB response elements common among many gene promoters could serve to trigger broadly an NGF-specific program of gene expression.

Fig. 1.

Oligonucleotide clones from a DNA recognition element library (DREL) are used as probes to detect NGF-specific changes in transcription factor binding activity. Nuclear extracts from untreated control (C) PC12 cells or cells treated for 48 hr with NGF (N) are incubated with double-stranded 65 bp oligonucleotides that contain a randomized 25 bp core. The extracts are fractionated by EMSA, and the oligonucleotides bound to the nuclear proteins are isolated. The selected oligonucleotides are amplified by PCR and then serve as input oligonucleotides for another round of the procedure. After four rounds the amplified oligonucleotide pools that bind to extracts from either or both the control and NGF-treated cells are combined and cloned into pBluescript, producing a DREL. Individual clones from the DREL are used as probes in EMSAs to identify differential gel shift patterns when incubated with nuclear extracts from control (C), EGF-treated (E), and NGF-treated (N) PC12 cells. At the bottom leftof the figure is a representative sample of the patterns observed in EMSAs in the first four rounds of the protocol. Broad smears without individual bands are observed because the oligonucleotide pool is heterogeneous. The representative sample on the bottom right shows an EMSA probed with a single clone isolated from the DREL. Individual bands are visible, and in this example NGF treatment elicits a different banding pattern (arrowhead) from that observed with either no treatment or EGF.

Fig. 2.

EGF- versus NGF-treated nuclear extracts produce differential gel shift patterns when probed with individual oligonucleotide clones isolated from the DREL. A, Core sequences are listed for four of 100 individual DREL oligonucleotides that were tested. B, Nerve growth factor-regulated DNA binding activities (NERDs) are detected as differences in individual gel shifts. The A9, C35, and C9 probes recognize complexes from PC12 nuclear extracts for which the binding activity is increased by 24–48 hr of treatment with NGF (N) as compared with EGF (E) or control (C), denoted as NERDs 1, 2, and 3 (see respective arrowheads). For the majority of probes that were tested, such as C50, nuclear extracts from control or growth factor-treated extracts exhibited the same banding pattern, indicating that proteins bound by the N25 core sequences were not regulated by EGF or NGF.

Fig. 3.

Truncated versions of the C35 and A9 probes, designated C35WT and A9WT, mutually compete for NERD1 binding.A, B, Both C35WT and A9WT demonstrate an increase in mobility and intensity of the NERD1 band (arrow) in PC12 nuclear extracts treated for 24 hr with 100 ng/ml NGF (N) as compared with 30 ng/ml EGF (E) or no drug (−). A, Binding of NERD1 to the radiolabeled C35WT probe is competed by increasing concentrations (2–100 ng) of unlabeled A9WT or C35WT.B, Binding of NERD1 to the radiolabeled A9WT probe is competed by increasing concentrations (2–100 ng) of A9WT or C35WT. This figure is a representative example from five separate experiments.

Fig. 4.

Mutational analysis demonstrates that NERD1 binds to a consensus CREB site in the A9WT probe. This experiment is a representative example of three independent EMSAs in which (A) potential binding sites in the A9WT sequence are mutated, and (B) the mutant probes are used in 100- to 200-fold excess to compete for NERD1 binding (arrowhead) from NGF-treated extracts (N) probed with labeled A9WT. B, A9WT, as well as A9m4 and A9m5 (which are mutated in the Ets binding site), blocks binding of the A9WT probe. In contrast, A91, A9m2, and A9m3 fail to compete for NERD1 binding, suggesting that the mutated CREB site is crucial to NERD1 binding. Control extracts include no treatment (−) and EGF-treatment (E).

Fig. 5.

NERD1 binding to C35WT and A9WT probes is supershifted by antibodies that specifically recognize Fra-2 and JunD in EMSAs. A, B, NERD1 binding for untreated PC12 cells (−) or cells treated for 24 hr with 30 ng/ml EGF (E) or 100 ng/ml NGF (N) is supershifted by antibodies specific for Fra-2 and JunD, but not by antibodies recognizing CREB, c-Jun, or other members of the AP-1 family. A, The C35WT probe detects a single strong band in PC12 nuclear extracts (arrow), which is supershifted partially by antibodies specifically recognizing Fra-2 or JunD.B, The NERD1 band (arrow) also is supershifted by Fra-2 and JunD antibodies when extracts are probed with A9WT. CREB- and ATF-2-specific antibodies supershift other complexes but have no effect on NERD1. This figure is representative of four independent experiments.

Fig. 6.

NFG, but not EGF, increases the expression of Fra-2 but has no effect on JunD in PC12 cells. A, An antibody that specifically recognizes Fra-2 reveals a series of bands in the 40–46 kDa range in Western blots of PC12 nuclear extracts. All lanes were loaded with 10 μg of protein. NGF (N; 100 ng/ml, 24 hr) induces a large increase in the size of upper band(s), producing a broad smear in the 43–46 kDa range. EGF (E; 30 ng/ml, 24 hr) has no effect on Fra-2 expression. B, An antibody specific for JunD p39 recognizes a doublet in the 37–40 kDa range. Neither EGF (E) nor NGF (N) affects expression of JunD in PC12 cells. This figure is representative of three independent experiments.

Fig. 7.

A time course experiment illustrates the differences in phospho-ERK and AP-1 proteins induced by EGF versus NGF in PC12 cells. Western blots of whole-cell extracts were processed with antibodies that specifically recognize phospho-ERK (A), both phosphorylated and unphosphorylated ERK (B), c-fos(C), Fra-1 (D), and Fra-2 (E). Cells were untreated (0) or were treated with EGF or NGF for 0.08–24 hr, as labeled in the figure. A, EGF elicits a very rapid transient phospho-ERK response, whereas NGF elicits a rapid but sustained response. B, Overall ERK expression is unchanged by either EGF or NGF treatment. C, Both EGF and NGF induce c-fos, with a maximal response occurring at 1 hr. For both growth factors this effect is transient, but the NGF response is more robust than that produced by EGF. D, Fra-1 (arrow; the lower band at ∼43 kDa) is induced by NGF, but not by EGF, after a 4 hr delay and the response is sustained at 24 hr. The upper bands do not correspond with the reported size for Fra-1 and are nonspecific. E, NGF, but not EGF, induces a large increase in the upper Fra-2 bands (43–46 kDa;arrow) at 4 and 24 hr. Fra-2 is detectable as two to three bands at 40–46 kDa in control, EGF-, and NGF-treated cells. All lanes were loaded with 20 μg of protein. These data are representative of three independent experiments. Inset box (bottom right), EGF increased ERK phosphorylation (p-MAPK), but not overall ERK (MAPK) expression, at 5 min over a concentration range of two log units but did not affect Fra-2 expression at 4 hr at any dose. An example of the NGF-induced Fra-2 response at 4 hr is included in the same blot for comparison. All lanes were loaded with 10 μg of protein. This illustration is representative of three independent experiments.