Regulación de la expresión génica de somatostatina por despolarización con potasio y el factor neurotrófico derivado de cerebro (BDNF) en cerebro de rata durante el desarrollo

Abstract

Introduction: The regulation of neuronal gene expression by electrical activity is well documented. Numerous studies have shown that membrane depolarization causes not only rapid alteration in protein phosphorylation (Nairn et al., 1985) but also activation of new programs of gene expression in neuronal type cells (Greenberg et al., 1985 and 1986; Morgan et al., 1986; Black et al., 1987; Saffen et al., 1988; Bartel et al., 1989). Genes regulated by depolarization include those that encode neuropeptides and neurotransmitter biosynthetic enzymes (Morris et al., 1988; Van Mguyen et al., 1990; Lu et al., 1991). Changes in gene expression are important mechanisms by which neurons transform short-lasting electrical events into long-lasting morphological alterations that may be responsible for plasticity related events such as memory formation (Morgan et al., 1991). Ca2+ entry into neurons through voltage ion channels serves as the trigger for electrical activity-dependent transcriptional responses (Ghosh et al., 1995). Ca2+ influx through L-type voltage-sensitive Ca2+ channels (L-VSCCs) stimulates phosphorylation of the transcription factor cAMP response element binding protein (CREB) on Ser-133 leading to an increase in cAMP response element (CRE)-dependent genes. The neuropeptide somatostatin (SS) is widely distributed throughout the nervous system. Brain SS mRNA is detectable by day seven of embryonic life (Zing et al., 1984). SS depletion during early development has profound effects on the maturation of dendritic morphology (Kungel et al., 1997). cAMP regulates SS biosynthesis in primary cultures of fetal rat hypothalamic cells and in cloned isolates of NIH-T3 fibroblast cells transfected with the rat SS gene (Montminy et al., 1986b). The rat SS gene contains the palindromic sequence, CRE, that is necessary for its transcriptional regulation by cAMP (Montminy et al., 1986a) and is recognized by the specific binding protein, CREB (Montminy et al., 1996).Previous work from our laboratory (Tolón et al., 1994 and 2000) provided evidence that elevated extracellular K+ concentrations increased the level of SS mRNA. The signalling pathways involved in K+-induced SS mRNA levels are still unknown. Brain derived neurotrophic factor (BDNF) is a neurotrophic factor predominantly expressed in the CNS, in particular in the hippocampus and cerebral cortex (Maisonpierre et al., 1990). This neurotrophic factor has a potent neuronal differentiation activity in vivo (Carnahan et al., 1995; Nawa et al., 1994) and in vitro (Carnahan et al., 1995; Loudes et al., 2000; Nawa et al.,1993; Rage et al., 1999, Villuendas et al., 2001) that increases levels of several neuropeptides such as SS. BDNF is activated by neuronal activity (Tao et al., 1998; Morgan et al., 1986). Neurotrophins have been proposed to be mediators of activity-dependent synaptic plasticity. In neurons, CREB is a major mediator of neuronal neurotrophin response (Finkbeiner et al., 1997). The aim of this work was to establish which of the signalling pathways involved in the activation of CREB by K+ mediates K+ -induced SS mRNA levels. In addition, we investigated whether K+ -induced depolarization triggers SS gene transcription and the DNA regulatory elements that mediate the transcriptional response. The signalling pathways through which BDNF induces SS gene expression have been studied. To investigate the transcriptional activity of BDNF on the SS gene and to define the DNA regulatory elements that mediate BDNF-induced SS gene transcription, PC12trkB and cultured cerebrocortical cells, transiently transfected with different constructions of SS promoter were used. Experimental procedures: Cell cultures. A) PC12trkB cells: PC12trkB cells were grown in culture medium, DMEM containing 6% FCS and 6% HS. They were seeded in 35 mm culture dishes at a density of about 7x105 cells per dish, 24 hours before transfection. The cells were kept in a humidified atmosphere of 5% CO2: 95% air at 37ºC. B) Cerebrocortical cells: Sprague-Dawley rats, obtained from our own animal facilities, were used in all the experiments. Primary cultures of embryonic cerebrocortical cells were carried out as previously described (de los Frailes et al., 1988). For the kinase studies, the dispersed cells were plated in poly-L-ornithine coated culture dishes at a density of 5-6x106 cells/35 mm. For transfection experiments, cells were plated at a density of 3x106 cells/60 mm plate. Depolarization studies: After 10-11 days in culture, media were removed and 1.5 ml of fresh serumfree DMEM containing 1µM TTX and the specific kinase inhibitors were added. 56mM K+ was added for different time periods. At the end of incubation, cells were processed and used for kinase proteins, transcriptional factor (CREB) and SS mRNA determination. BDNF studies: After 3-4 days in culture, media were removed and fetal rat cerebral cortical cells were incubated in fresh serum-free DMEM containing the specific kinase inhibitors. BDNF (50ng/ml) was added for different time periods. At the end of incubation, cells were processed and used for kinase proteins, transcriptional factor (CREB) and SS mRNA determinations. Cell extracts and Western immunoblot: The processing of the cells and kinase activity determination were done as previously described (Fernández et al., 2005; Palacios et al., 2005). Cells were collected in lysis buffer and sonicated. Total protein extracts (10-30 µg) were resolved by SDS/PAGE and transferred to a PVDF membrane. After blocking the membranes, immunodetection was performed using an antiserum specific for tyrosine- and threonine-phosphorylated forms of p44 (ERK1) and p42 (ERK2) MAPKs (1:15000 dilution), or an antibody that recognizes phosphoSer133 CREB (Ser133; 1: 500 dilution), or an antibody that recognizes phosphoSer473 Akt (Ser473; 1:1000 dilution). Membranes were systematically reprobed with phosphorylation state-independent antibodies, ERK1, ERK2, CREB and Akt. Immunocytochemistry: Immunocytochemistry was performed as previously described (Fernández et al., 2005). Cells were plated on poly-D-Lysine-coated glass coverslips in 24-well culture plates. CREB-labelling indices indicative of the activation rate of somatostatinergic cells were determined by double-labelling immunocytochemistry staining for SS and phospho-Ser133 CREB (Ser133 ). Extraction of RNA and Northern blot analysis: Total RNA extraction and Northern blot analysis were performed as previously described (Tolón et al. 1994) . Plasmids and transfections: Dr M. Vallejo kindly provided the plasmids SS65-CAT, SS65∆CRE-CAT, SS900-CAT and SS900∆CRE-CAT. The plasmids SS65-Luc, SS65∆CRE-Luc, SS900Luc and SS900 ∆CRE-Luc were constructed using DNA fragments obtained by PCR amplification of somatostatin gene sequences in the plasmid pOCAT (5.26 Kb). We used primers or amplimers to incorporate XhoI and HindIII restriction sites. The resulting fragment was digested with the appropriate restriction enzymes, purified on agarose gel, and ligated into the promoterless plasmid pGL2-Basic (5,59 Kb) that had been digested with XhoI and HindIII. pGL2-Basic contains the reporter gene Luc. A) PC12trkB cells transfections: PC12trkB cells were seeded into 35 mm culture dishes at an approximate density of 7x105 cells per dish 24 hours before transfection. DNA (SS65-CAT, SS65∆CRE-CAT, SS900-CAT and SS900∆CRE-CAT) (17-18 µg total) was transfected using the Lipofectamine reagent according to the manufacturer´s protocol. B) Cerebrocortical cell transfections: Cerebrocortical cells were seeded into 60 mm culture dishes at an approximate density of 3x106 cells/60 mm plate 24 hours before transfection. DNA (SS65-Luc, SS65∆CRE-Luc, SS900-Luc and SS900∆CRE-Luc) (12-14 µg total) was transfected using the FuGene reagent according to the manufacturer´s protocol. Following 24 hours incubation, the cells were treated with K+ (56 mM) for 24 hours or BDNF (50ng/ml) for 48 hours, after which they were washed, lysed and analysed for CAT and luciferase activity. CAT activity was normalized to galactosidase activity and luciferase activity was normalized to renilla activity, and results are expressed as a percentage of their own control group. Conclusions: The present study analyses, for the first time, the signalling pathways involved in K+- and BDNF-induced SSmRNA in primary rat cerebrocortical cell cultures. We show that K+ activates the MAPK (ERK 1/2) pathway, and that the cAMP/PKA and CaMKs pathways are involved in this activation. We also show that K+ activates the transcription factor CREB, which is necessary for K+ to induce SS expression. CREB activation occurs in two phases: early and late. The cAMP/PKA y CaMKs pathways are involved in the early phase of CREB phosphorylation and the MAPK (ERK 1/2) and CaMKs pathways in the late phase. We found that K+ -induced SS mRNA is mediated by the activation of cAMP/PKA y CaMKs pathways, thus suggesting that the early activation of CREB is involved in the induction of SS by K+. The somatostatin gene contains the CRE, that is recognized by CREB and mediates its transcriptional responses. In this study, we demonstrate for the first time, using transient transfections of primary cultures, that K+ induces the transcriptional regulation of the SS gene through the CRE sequence located in the SS promoter. In this study, we confirm that BDNF activates the MAPK (ERK 1/2) and PI3K/Akt pathways. Our results suggest that BDNF-induced MAPK (ERK 1/2) activation is cAMP-dependent and PKA-independent. The CaMKs and PI3K/Akt pathways are also involved in BDNF-induced MAPK (ERK 1/2) activation. We demonstrate that cAMP/PKA, MAPK (ERK 1/2) and CaMKs pathways are involved in the BDNF activation of the PI3K/Akt pathway. We confirm that BDNF activates CREB and demonstrate that the MAPK (ERK 1/2), cAMP/PKA and PI3K/Akt pathways are involved in its activation. Our results suggest that MAPK (ERK 1/2) is the major pathway involved in CREB activation by BDNF. This study evidences for the first time that the cAMP/PKA, CaMKs, PI3K/Akt and MAPK (ERK 1/2) pathways are involved in the induction of SS mRNA by BDNF. To determine the transcriptional regulation of the SS gene by BDNF we perfomed transient transfections of cultured cerebrocortical cells and PC12trkB cells with different constructions of the SS promoter. We present evidence for the first time, that BDNF induces the transcriptional activity of the SS gene through the CRE sequence located in the SS promoter.