Regulation of brain-derived neurotrophic factor-mediated transcription of the immediate early gene Arc by intracellular calcium and calmodulin

Abstract

The induction of the immediate early gene Arc is strongly implicated in synaptic plasticity. Although the role of ERK has been demonstrated, the regulation of Arc expression is largely unknown. In this study, we investigated the major signaling pathways underlying brain-derived neurotrophic factor (BDNF)-mediated Arc transcription in cultured cortical neurons. The BDNF-stimulated Arc transcription was regulated solely by the Ras-Raf-MAPK signaling through ERK, but not by phosphoinositide 3-kinase (PI3K) and PLC-gamma activities. Although it was demonstrated that BDNF might promote calcium entry through calcium channels and NMDA receptors, chelating extracellular calcium with EGTA failed to block Arc transcription. In contrast, chelating intracellular calcium ([Ca(2+)](i)) by BAPTA-AM abolished BDNF-mediated Arc up-regulation. Surprisingly, BAPTA-AM did not block ERK activation, indicating that [Ca(2+)](i) and Ras-Raf-MAPK are not coupled, and the activation of ERK alone is not sufficient to up-regulate Arc transcription. Moreover, we found that inhibition of calmodulin (CaM) by W13 blocked both Arc transcription and ERK activation, revealing a Ca(2+)-independent function of CaM. These data suggested novel functions of [Ca(2+)](i) and CaM in BDNF signaling. Comparison of the Arc transcription profiles between Ca(2+)-stimulated and BDNF-stimulated neurons demonstrated that the regulatory mechanisms were distinctively tailored to the complex features of neuronal activity. Specifically, PI3K and CaM-dependent protein kinase (CaMK) activity were required for Ca(2+)-stimulated Arc transcription through regulating ERK signaling. Such cross-talks between PI3K, CaMK, and ERK was absent in BDNF-stimulated neurons.

Fig. 1

Sub-nanomolar BDNF activates both Arc transcription and ERK phosphorylation through the TrkB tyrosine receptor. A) Cortical neurons were treated with (in ng/ml) 5, 10, 25, 50 and 100 BDNF for 1hr. Total RNA was extracted from control and BDNF-treated neurons. The mRNA level of Arc and GAPDH was determined by semi-quantitative RT-PCR using gene specific primers. B) Neurons were treated with or without a transcription inhibitor actinomycin (ACD) (0.1 ug/ml) for 30min followed by 1hr BDNF (5ng/ml) or KCl (50mM) incubation. Representative images are shown in the left panels, and quantification in the right panels (n = 3 from separate experiments). Relative intensity of Arc was normalized to GAPDH. C, D, and E) Sub-nanomolar BDNF activates ERK phosphorylation through the TrkB tyrosine receptor. Cortical neurons were treated with (in ng/ml) 5, 10, 25, 50 and 100 BDNF for 15min. The harvested samples were separated by 10% SDS-PAGE. The phosphorylation of ERK was determined by Western blot using an antibody against p-ERK1/2. To address the role of the receptor tyrosine kinase TrkB, Trk inhibitor K252a (0.2 uM) or TrkB-specific BDNF scavenger TrkB-IgG (0.4 ug/ml) were applied 30min before BDNF (5ng/ml) incubation. Samples were collected 15min after BDNF treatment. The level of p-ERK was determined by Western blot analysis. The activation of p-ERK in neurons was also examined by immunofluorescent microscopy (E). Control and BDNF-treated cortical neurons were double labeled with antibodies against p-ERK (upper panels) and the neuronal marker NeuN (lower panels).

Fig. 2

Regulation of Arc transcription by MAPK, PLC-γ and PI3K signaling. Cortical neurons were stimulated by 5ng/ml BDNF (A, C) or KCl (50mM) (B and D). Total RNA was extracted 1h after the treatments and used for semi-quantitative RT-PCR (A to B). Samples for Western blots (C, D) were collected 15min after the treatments. All inhibitors were applied 30min before BDNF and KCl stimulation. U73122 (5uM), LY294002 (30uM), and U0126 (10uM) were used to inhibit PLC-γ, PI3K and MEK, respectively. The levels of Arc and GAPDH mRNA were measured by RT-PCR using gene specific primers. Representative images are shown in the left panels, and quantification in the right panels. The relative intensity of Arc was normalized to GAPDH. C) The BDNF-induced ERK activation was inhibited by MEK inhibitor U0126, but not by PI3K inhibitor LY294002 or PLC-γ inhibitor U73122. The activation of ERK and PI3K was determined by the level of p-ERK and p-Akt, respectively. D) PI3K activity is required for the phosphorylation of ERK induced by membrane depolarization. Quantification of p-ERK and p-Akt was calculated from 3 independent experiments after normalization to T-ERK and T-Akt. Representative Western blot images are shown in the left panels, and quantification in the right panels.

Fig. 3

BDNF-induced Arc transcription requires intracellular, but not extracellular calcium. Cortical neurons were stimulated by 5ng/ml BDNF (A) or KCl (50mM) (B) for 1hr, after which total RNA was extracted and used for semi-quantitative RT-PCR. Extracellular calcium chelator (EGTA, 2.5mM), and intracellular calcium chelator (BAPTA-AM, 33uM) were applied 30min before BDNF and KCl stimulation. The levels of Arc and GAPDH mRNA were determined by RT-PCR using gene specific primers. Representative images are shown in the left panels, and quantification in the right panels. The relative intensity of Arc was normalized to GAPDH.

Fig. 4

Activity-dependent ERK phosphorylation does not depend on intracellular calcium. Cortical neurons were stimulated by KCl (50mM) or 5ng/ml BDNF. Pre-treatments with EGTA (2.5mM) or BAPTA-AM (30uM) were applied 30min before stimulation. Samples were collected 15min after stimulation. The ERK phosphorylation was analyzed by Western blot (A and C) and immunofluorescent microscopy (B and D). For quantification, the relative intensity of p-ERK was normalized to total ERK. Representative Western blots are shown in the left panels, and quantification (n = 3 for each treatment group) is shown in the right panels.

Fig. 5

The activity-dependent Arc transcription and ERK phosphorylation depend on CaM activity. DIV6 Cortical neurons were pre-incubated with a CaM inhibitor W-13 (70uM) or CaMK inhibitor KN93 (5uM) for 30min before neuronal stimulation with BDNF (5ng/ml) or KCl (50mM). The samples for total RNA extraction were collected 1hr after stimulation. The samples for Western blot were collected 15min after the stimulations. The mRNA level of Arc and GAPDH was determined by semi-quantitative RT-PCR (A and C). ERK phosphorylation was determined by Western blots using phospho-specific antibodies (B and D). Representative RT-PCR and Western blot results are shown in the left panels, and quantification in the right panels (n = 3 for each treatment group). The signals of Arc were normalized to GAPDH. The signal of p-ERK was normalized to T-ERK.

Fig. 6

Regulation of cAMP-mediated Arc transcription and ERK phosphorylation by intracellular Ca²⁺ and CaM. Cortical neurons were pre-treated with BAPTA-AM (33uM) or W13 (70uM) for 30min before forskolin (50uM) stimulation. Samples were collected 1h after stimulation for RT-PCR, and 15min after stimulation for Western blots. A). Forskolin-induced Arc up-regulation was blocked by BAPTA-AM, but not by W13. B). Forskolin-induced ERK phosphorylation was inhibited by W13, but not by BAPTA-AM. Representative images are shown in the left panels, and quantification in the right panels.

Fig. 7

Regulation pathways of Arc transcription in BDNF- and Ca²⁺-mediated signaling. A). BDNF-mediated Arc expression is controlled through MAPK pathway and gated by intracellular calcium. Upon the activation of TrkB by BDNF, Ras-Raf-MAPK, PI3K, and PLC-γsignaling pathways are stimulated. It appeared that they are independent, no cross talk was identified among them in BDNF-stimulated neurons, and MAPK signaling is the sole regulator for Arc transcription. In addition, [Ca²⁺]_i regulates Arc transcription in parallel to MAPK. Furthermore, CaM may regulate Arc transcription through Ras-Raf-MAPK, but is independent of [Ca²⁺]_i and not through CaMKs. B) Ca²⁺-mediated Arc transcription requires MAPK, PI3K, extracellular Ca²⁺, intracellular Ca²⁺, CaM, and CaMK activity. It appeared that PI3K and CaMK regulated Arc transcription through ERK. Although a transient influx of extracellular Ca²⁺ is required for ERK activation, [Ca²⁺]_i is not required for ERK activation. Even with the activation of ERK, chelation of [Ca²⁺]_i blocks Arc up-regulation. This implicates that ERK and [Ca²⁺]_i are also parallel pathways in KCl-stimulated neurons. “?” indicates that the mechanism is unknown.