Bcl-2 enhances Ca2+ signaling to support the intrinsic regenerative capacity of CNS axons
2005; Springer Nature; Volume: 24; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7600589
ISSN1460-2075
AutoresJianwei Jiao, Xizhong Huang, Rachel Ann Feit-Leithman, Rachael L. Neve, William D. Snider, Darlene A. Dartt, Dongfeng Chen,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle17 February 2005free access Bcl-2 enhances Ca2+ signaling to support the intrinsic regenerative capacity of CNS axons Jianwei Jiao Jianwei Jiao Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Program in Neuroscience, Harvard Medical School, Boston, MA, USA Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xizhong Huang Xizhong Huang Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rachel Ann Feit-Leithman Rachel Ann Feit-Leithman Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rachael Lee Neve Rachael Lee Neve Department of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, MA, USA Search for more papers by this author William Snider William Snider University of North Carolina, Neuroscience Center, Chapel Hill, NC, USA Search for more papers by this author Darlene Ann Dartt Darlene Ann Dartt Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dong Feng Chen Corresponding Author Dong Feng Chen Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Program in Neuroscience, Harvard Medical School, Boston, MA, USA Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jianwei Jiao Jianwei Jiao Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Program in Neuroscience, Harvard Medical School, Boston, MA, USA Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xizhong Huang Xizhong Huang Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rachel Ann Feit-Leithman Rachel Ann Feit-Leithman Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rachael Lee Neve Rachael Lee Neve Department of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, MA, USA Search for more papers by this author William Snider William Snider University of North Carolina, Neuroscience Center, Chapel Hill, NC, USA Search for more papers by this author Darlene Ann Dartt Darlene Ann Dartt Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dong Feng Chen Corresponding Author Dong Feng Chen Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA Program in Neuroscience, Harvard Medical School, Boston, MA, USA Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Jianwei Jiao1,4,5, Xizhong Huang1, Rachel Ann Feit-Leithman1, Rachael Lee Neve2, William Snider3, Darlene Ann Dartt1 and Dong Feng Chen 1,4,5 1Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA 2Department of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, MA, USA 3University of North Carolina, Neuroscience Center, Chapel Hill, NC, USA 4Program in Neuroscience, Harvard Medical School, Boston, MA, USA 5Department of Ophthalmology, Harvard Medical School, Boston, MA, USA *Corresponding author. Program in Neuroscience, Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA. Tel.: +1 617 912 7490; Fax: +1 617 912 0174; E-mail: [email protected] The EMBO Journal (2005)24:1068-1078https://doi.org/10.1038/sj.emboj.7600589 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info At a certain point in development, axons in the mammalian CNS undergo a profound loss of intrinsic growth capacity, which leads to poor regeneration after injury. Overexpression of Bcl-2 prevents this loss, but the molecular basis of this effect remains unclear. Here, we report that Bcl-2 supports axonal growth by enhancing intracellular Ca2+ signaling and activating cAMP response element binding protein (CREB) and extracellular-regulated kinase (Erk), which stimulate the regenerative response and neuritogenesis. Expression of Bcl-2 decreases endoplasmic reticulum (ER) Ca2+ uptake and storage, and thereby leads to a larger intracellular Ca2+ response induced by Ca2+ influx or axotomy in Bcl-2-expressing neurons than in control neurons. Bcl-xL, an antiapoptotic member of the Bcl-2 family that does not affect ER Ca2+ uptake, supports neuronal survival but cannot activate CREB and Erk or promote axon regeneration. These results suggest a novel role for ER Ca2+ in the regulation of neuronal response to injury and define a dedicated signaling event through which Bcl-2 supports CNS regeneration. Introduction In the mammalian CNS, the exuberant growth of axons during development is markedly reduced as neurons mature losing their intrinsic capacity for axon elongation (Chen et al, 1995; Goldberg et al, 2002). For decades, the intracellular events controlling this transition remained obscure. Surprisingly, recent studies have suggested a pivotal role of the antiapoptotic protein Bcl-2 in supporting the intrinsic regenerative capacity of severed CNS axons (Chen et al, 1997; Cho et al, 2005). Expression of Bcl-2 in CNS neurons correlates with axon elongation in the developing brain (Merry et al, 1994), and deletion of the Bcl-2 gene reduces the ability of embryonic neurons to extend neurites in culture (Chen et al, 1997; Hilton et al, 1997). In contrast, constitutive expression of Bcl-2 in postnatal CNS neurons reverses the loss of intrinsic growth capacity by CNS axons and leads to robust optic nerve regeneration in postnatal mice (Chen et al, 1997; Cho et al, 2005). However, the mechanism by which Bcl-2 promotes axon regeneration remains unknown. A critical question is whether Bcl-2 directly stimulates axon growth signals inside neurons to promote regeneration or merely supports cell survival, allowing surviving neurons to extend axons automatically. Bcl-xL is a member of the Bcl-2 family that is thought to be redundant with Bcl-2 in its capacity to protect cells from apoptosis (Gonzalez-Garcia et al, 1995). However, Bcl-2 expression correlates with axonal growth, whereas Bcl-xL is expressed at high levels in the mature CNS, where neurons have lost their intrinsic axonal growth capacity (Levin et al, 1997), suggesting that Bcl-xL cannot support axonal growth. Bcl-2 and Bcl-xL also differ in their subcellular localization. Bcl-2 is found in the mitochondria, endoplasmic reticulum (ER), and nuclear envelope—organelles with key roles in intracellular Ca2+ homeostasis—while Bcl-xL is targeted to the mitochondrial outer membrane (MOM) (Kaufmann et al, 2003). Thus, Bcl-xL and Bcl-2 may have distinct roles in regulating axon growth and intracellular Ca2+ dynamics. Neural injury induces an intracellular Ca2+ response that reflects Ca2+ influx across the plasma membrane or Ca2+-inducd Ca2+ release from the smooth ER. Localized, transient elevation of intracellular Ca2+ after injury has been reported to be necessary for membrane sealing, growth cone formation, and reinitiation of neuritogenesis in lower vertebrates, whose axons regenerate automatically (Ziv and Spira, 1997; Spira et al, 2001). In addition, an optimal range of intracellular Ca2+ concentration ([Ca2+]i) is required for proper axon elongation during development (Gu and Spitzer, 1995, 1997). Bcl-2 regulates ER Ca2+ content by decreasing ER Ca2+ uptake (Foyouzi-Youssefi et al, 2000; Pinton et al, 2001; Ferrari et al, 2002; Rudner et al, 2002). Bcl-xL is associated primarily with the mitochondria and has no known role in regulating ER Ca2+ content. Unlike Bcl-xL, Bcl-2 decreases the expression of calreticulin and ER Ca2+ ATPase (SERCA), two key proteins controlling ER Ca2+ influx and content (Pinton et al, 2001; Dremina et al, 2004). An emerging hypothesis suggests that Bcl-2 resides in the ER and mediates intracellular Ca2+ signaling induced by neural injury to support CNS regeneration. Elevation of [Ca2+]i activates cAMP response element binding protein (CREB) and p44/p42 mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (Erk) (Dolmetsch et al, 2001), both of which stimulate genes essential for neurite growth and plasticity. Activation of CREB by injury-induced influx of intracellular Ca2+ is critical for axon regeneration in neurons of lower vertebrates (Dash et al, 1998). In mammals, CREB expression is developmentally regulated (Lonze and Ginty, 2002). Neurons from mice harboring a null mutation in CREB display impaired axonal growth in development (Lonze et al, 2002). The MAPK/Erk pathway, which is also activated by intracellular Ca2+ and regulates neurite extension during development, can activate CREB, which in turn further supports neurite elongation (Adams and Sweatt, 2002). In this study, to elucidate the molecular basis for CNS regeneration, we compared Ca2+ dynamics and subsequent signaling events in Bcl-2- and Bcl-xL-expressing retinal ganglion cells (RGCs)—a standard model of CNS neurons—and PC12 cells. Our findings define a new role for ER Ca2+ stores and Bcl-2 in regulating the intrinsic growth potential of CNS axons and initiating the dedicated signaling and transcriptional programs required for axon regeneration. Results Distinctive roles of Bcl-2 and Bcl-xL First, we determined if overexpression of Bcl-xL prevents injury-induced RGC loss and supports axonal regrowth as effectively as Bcl-2. Mice carrying a Bcl-2 (Bcl-2tg) (Martinou et al, 1994) or a Bcl-xL (Bcl-xLtg) (Parsadanian et al, 1998) transgene under the control of neuron-specific promoters were subjected to optic nerve crush on postnatal day 3 (P3), when RGCs of Bcl-2tg mice regenerate axons automatically in vivo (Chen et al, 1997; Cho et al, 2005). At 1 day after injury, TUNEL-positive apoptotic cells were 10-fold more abundant in retinal sections of wild-type (wt) controls than in Bcl-2tg or Bcl-xLtg mice (Figure 1A and B). Within 24 h, RGC axons in the nerve fiber layer and optic nerve degenerated rapidly in wt mice but not in Bcl-2tg and Bcl-xLtg mice (Figure 1A and C). Thus, overexpression of Bcl-xL was as effective as Bcl-2 expression in preventing axotomy-induced RGC death and axon degeneration. Figure 1.Bcl-xL supports survival but not axon regeneration of postnatal RGCs. (A) Transverse retinal sections from wt, Bcl-xLtg, and Bcl-2tg mice were stained with TUNEL and GAP-43 antibody on day 1 after optic nerve injury. Asterisks indicate TUNEL-positive cells; arrows indicate the nerve fiber layer (NFL). TUNEL-positive cells are present and the NFL is absent in the retinal sections of wt mice but not Bcl-xLtg or Bcl-2tg mice. GCL, ganglion cell layer; IPL, inner plexiform layer. Scale bar, 100 μm. (B) Number of TUNEL-positive cells in retinal sections of wt, Bcl-xLtg and Bcl-2tg mice (n=4/group). (C) Longitudinal optic nerve sections from wt, Bcl-xLtg, and Bcl-2tg mice labeled with GAP-43 antibody 1–2 days after optic nerve injury. Arrows point to the crush site. Labeled axons in Bcl-xLtg mice remain anterior to the crush site, while those in Bcl-2tg mice grew robustly past the lesion site. Scale bar, 250 μm. (D) Retinal axon regrowth in retina–brain slice cocultures prepared from wt, Bcl-xLtg, and Bcl-2tg mice. Values are mean±s.d. *P 90% of those from Bcl-2tg mice (Figure 2E). Moreover, the RGCs of Bcl-2tg mice extended significantly longer neurites (Figure 2F). Thus, Bcl-2 acts intrinsically in RGCs to promote axonal regrowth, which functions independently of its support for neuronal survival. Figure 2.Bcl-2 acts intrinsically in neurons to support neuritogenesis and axon regeneration. (A–C) RGCs were isolated from wt (A), Bcl-xLtg (B), and Bcl-2tg (C) mice, incubated for 5 days, and stained with calcein. Surviving RGCs from Bcl-2tg mice extended longer axons than those from wt or Bcl-xLtg mice. (D–F) Percentage of surviving RGCs (D), percentage of surviving RGCs with axons longer than 3 body lengths (E), and average length of the longest axon from each RGC (F) (n=5 cultures/group). RGCs in Bcl-xLtg and Bcl-2tg mice had similar survival rates, but Bcl-xLtg RGCs had shorter neurites. (G) Western blot analysis of Bcl-2 and Bcl-xL expression in stably transfected PC12 cells. (H, I) Percentage of surviving cells (H) and percentage of cells bearing neurites (I) (n=5 cultures/group). Bcl-xL- and Bcl-2-expressing PC12 cells had similar survival rates, but significantly fewer Bcl-xL-expressing cells had neurites. *P<0.01 versus wt, two-tailed t-test. Download figure Download PowerPoint To determine if Bcl-2 has a general effect on neurite outgrowth, we generated PC12 cell lines stably transfected with Bcl-2 and Bcl-xL. PC12 cells differentiate and extend neurites upon stimulation with nerve growth factor (NGF) (50 ng/ml). Overexpression of Bcl-2 and Bcl-xL was confirmed by immunoblot analysis (Figure 2G). After treatment with staurosporine (Figure 2H) or serum withdrawal (not shown), the survival rate was significantly higher in cells expressing Bcl-xL or Bcl-2 than in control cells (Figure 2H). As in RGC cultures, however, expression of Bcl-xL did not enhance the ability of cells to extend neurites. At a subthreshold concentration of 1 ng/ml, NGF failed to stimulate neurite formation in control or Bcl-xL-expressing PC12 cells but induced robust neurite outgrowth in those expressing Bcl-2 (Figure 2I). Thus, Bcl-2 promotes neuritogenesis in the presence of growth-stimulating signals, although it does not itself stimulate neurite outgrowth. This effect is not a direct consequence of its antiapoptotic function, as expression of Bcl-xL supported neuronal survival without promoting the neuritogenic response. Bcl-2-mediated growth is ER-dependent To define the molecular pathways by which Bcl-2 promotes the neuritogenic response, we took advantage of the distinct structures of Bcl-2 and Bcl-xL. Bcl-2 and Bcl-xL share all four Bcl-2 homology domains (BH) but contain a distinct C-terminal, transmembrane (TM) hydrophobic helix that targets the proteins to specific subcellular locations. To determine if the subcellular localization of Bcl-2 is crucial for its neurite growth-promoting function, we generated Bcl-2 and Bcl-xL mutants or chimeric proteins. The TM domain of Bcl-2 was deleted (Bcl-2TM) or replaced with a membrane-anchoring domain containing either the mitochondrial outer member (MOM) (Bcl-2MOM) or ER (Bcl-2ER) targeting signal (Wang et al, 2001) (Figure 3A). Bcl-xL chimeric, in which its TM domain was replaced with ER targeting signal (Bcl-xLER), was also generated. These proteins were fused to the C-terminal end of enhanced green fluorescence protein (EGFP). Figure 3.The growth-promoting effect of Bcl-2 is ER-dependent. (A) Schematic of DNA structures of Bcl-2, Bcl-xL, and Bcl-2 mutants. (B) Western blot analysis of PC12 cell lines stably transfected with Bcl-2ER, Bcl-2TM, and Bcl-2MOM. (C) Subcellular location of Bcl-2ER, Bcl-2MOM, and Bcl-xL shown by confocal microscopy. EGFP-Bcl-2ER (green) colocalizes with the ER marker calnexin (blue); EGFP-Bcl-2MOM and EGFP-Bcl-xL colocalize with the mitochondrial marker (Mito) cytochrome c (red). Scale bar, 4 μm. (D) Percentage of dying cells after treatment with staurosporine (left) and percentage that extended neurites (n=4 cultures/group). ER, Bcl-2ER; TM, Bcl-2TM; MOM, Bcl-2MOM. *P<0.05 versus wt, two-tailed t-test. Download figure Download PowerPoint To compare the survival and growth effects of Bcl-2 targeted to different subcellular localizations, we generated PC12 cell lines stably transfected with constructs encoding the Bcl-2 or Bcl-xL mutants or chimeras. Protein expression was confirmed by immunoblot analysis (Figure 3B). As shown by confocal microscopy, normal Bcl-2 protein was found primarily in the ER (Kaufmann et al, 2003), while Bcl-2TM exhibited a diffused cytoplasmic cellular localization (Usuda et al, 2003) (not shown). Bcl-2ER colocalized specifically with an ER marker, and Bcl-2MOM and Bcl-xL with a mitochondrial marker (Figure 3C). Treatment with staurosporine induced massive apoptosis in control PC12 cells, but not in cells expressing Bcl-2, Bcl-xL, Bcl-xLER, or any of the Bcl-2 mutants (Figure 3D). However, cells expressing the Bcl-2 or Bcl-xL mutants differed in their ability to extend neurites. NGF (1 ng/ml) induced vigorous neurite outgrowth only in cells expressing Bcl-2 or Bcl-2ER (Figure 3D). Because a major difference between Bcl-2 and Bcl-xL is their subcellular localization, we then targeted Bcl-xL to the ER and compared its survival and growth effect with Bcl-2. Interestingly, Bcl-xLER exhibited similar growth-promoting activity as expression of Bcl-2 (Figure 3D). EGFP-Bcl-2 fusion protein displayed similar survival and neuritogenic activity as normal Bcl-2 (not shown). Thus, the ER localization is critical for the neurite growth activity of Bcl-2 or Bcl-xL. Bcl-2 regulates ER Ca2+ to promote neuritogenic response To determine if Bcl-2 promotes neuritogenesis by regulating the Ca2+ content of the ER ([Ca2+]er), we used thapsigargin (TG), an inhibitor of the ER Ca2+-ATPase, to stimulate ER Ca2+ depletion. The peak level of [Ca2+]i reached upon TG addition was used as an indirect measure of the ER Ca2+ content. Control, Bcl-2-, and Bcl-xL-expressing PC12 cells had similar resting levels of [Ca2+]i (Figure 4A and B). TG resulted in a net release of ER Ca2+ and increased [Ca2+]i (Figure 4A and C). However, the increase in [Ca2+]i was markedly lower in Bcl-2-expressing cells than in control cells or cells expressing Bcl-xL, which resides in the mitochondria (Kaufmann et al, 2003). We corroborated this result in three different clones that were stably transfected with mouse, human, and EGFP fusion Bcl-2 genes (Figure 4B and C). This finding suggests that expression of Bcl-2, but not Bcl-xL, decreases the ER Ca2+ content of neurons. Figure 4.Bcl-2-, but not Bcl-xL-, expressing neurons display reduced ER Ca2+ content. (A–C) Representative trace (A) and quantitative analysis of basal (B) and TG-induced [Ca2+]i (C), measured by Fura-2, in PC12 cells expressing a control (Cont), Bcl-2, or Bcl-xL plasmid. Measurement of TG-induced Ca2+ change was carried out in the absence of extracellular Ca2+. (D, E) TG-induced [Ca2+]i (D) and NGF-induced neurite outgrowth (E) in control (Cont), Bcl-2-, or Bcl-2+SERCA2b-expressing PC12 cells (n=4 cultures/group). (F) NGF-induced neurite outgrowth in control and Bcl-2-expressing PC12 cells treated with 0–10 mM BHQ (n=4/group). *P<0.01 versus control, two-tailed t-test. Download figure Download PowerPoint Bcl-2 has been proposed to decrease [Ca2+]er by downregulating the expression of the ER Ca2+ pump (SERCA) and thereby suppressing ER Ca2+ uptake (Dremina et al, 2004). We transfected Bcl-2-expressing cells with constructs encoding SERCA2b. While Bcl-2 expression alone reduced TG-induced [Ca2+]i (or [Ca2+]er) relative to that of control cells, expression of SERCA2b attenuated that reduction (Figure 4D) as well as neurite outgrowth from Bcl-2-expressing cells stimulated with 1 ng/ml NGF (Figure 4E). In contrast, blocking ER Ca2+ uptake mimicked the growth-promoting effect of Bcl-2. Decreasing ER Ca2+ uptake in control PC12 cells with 2,5′-di(terbutyl)-1,4,-benzohydroquinone (BHQ), an inhibitor of SERCA (Dolor et al, 1992), increased neurite outgrowth in the presence of 1 ng/ml NGF (Figure 4F), an effect similar to that achieved by overexpressing Bcl-2. These data suggest that reduction of ER Ca2+ uptake is essential and sufficient for Bcl-2-mediated neurite growth-promoting activity. Bcl-2 regulates [Ca2+]er to enhance intracellular Ca2+ signaling Neural injury often leads to an increase of extracellular Ca2+ and subsequently a surge of intracellular Ca2+ influx across the plasma membrane. This injury-induced intracellular Ca2+ signaling is critical in the regulation of neurite outgrowth and nerve regeneration in lower vertebrates (Ziv and Spira, 1997). We hypothesized that, by reducing ER Ca2+ uptake, Bcl-2 enhances the injury- or stimuli-induced intracellular Ca2+ signaling and promotes neuritogenesis. To test this hypothesis, we added Ca2+ to cells maintained in a Ca2+-free medium to mimic the injury-induced increase of extracellular Ca2+ ([Ca2+]o) and the surge of [Ca2+]i or used KCl depolarization to induce intracellular Ca2+ influx. Addition of extracellular Ca2+ (Figure 5A) or KCl (not shown) triggered a transient increase of [Ca2+]i, followed by a sustained plateau. When stimulated with extracellular Ca2+ (Figure 5A and B) or KCl (Figure 5C), PC12 cells expressing Bcl-2 had a significantly larger elevation of [Ca2+]i than control or Bcl-xL-expressing cells, which correlated inversely with their ER Ca2+ content. These results suggest that expression of Bcl-2, but not Bcl-xL, enhances the intracellular Ca2+ response of neurons to nerve stimulation or injury. Figure 5.Bcl-2 expression enhances intracellular Ca2+ signaling by reducing ER Ca2+ uptake. (A–C) Representative trace (A) and quantitative analysis of extracellular Ca2+- (B) and KCl-induced changes in [Ca2+]i (C), measured by Fura-2 (n=4/group). PC12 cells stably transfected with control (Cont), Bcl-2, or Bcl-xL plasmids were incubated in Ca2+-free medium for over an hour; where indicated, Ca2+ (1.0 mM free extracellular Ca2+ final concentration) was added. Changes in [Ca2+]i were measured from its baseline to when the elevation of [Ca2+]i reached its peak. (D) Comparison of KCl-induced [Ca2+]i in cells expressing control (Cont), Bcl-2, Bcl-xL, Bcl-2ER (ER), Bcl-2TM (TM), and Bcl-2MOM (MOM) genes (n⩾3/group). (E) Measurement of KCl-induced changes in [Ca2+]i in cells expressing a control, Bcl-2, or Bcl-2+SERCA2b plasmid. *P<0.01 versus control, two-tailed t-test. Download figure Download PowerPoint Next, we compared the changes in [Ca2+]i after KCl depolarization in PC12 cells expressing Bcl-2 mutants or chimeras. KCl-induced elevation of [Ca2+]i in PC12 cells expressing Bcl-2 or Bcl-2ER was significantly larger than in control PC12 cells (Figure 5D). KCl-induced [Ca2+]i levels in cells expressing mutated Bcl-2 targeted to the cytoplasm (Bcl-2ΔTM) or mitochondria (Bcl-2MOM) were similar to that of control cells (Figure 5D). In addition, blocking ER Ca2+ uptake in Bcl-2-expressing cells by overexpressing SERCA2b prevented the decrease of [Ca2+]er and led to a KCl-induced elevation of [Ca2+]i comparable to that in control cells (Figure 5E). These results indicate that Bcl-2 resides in the ER and enhances the intracellular Ca2+ response of neurons to injury or stimuli by reducing ER Ca2+ uptake. Bcl-2 activates CREB and Erk to stimulate neuritogenetic response An emerging hypothesis suggests that Bcl-2 enhances the intracellular Ca2+ response to nerve injury and activates Ca2+-mediated signaling proteins, such as CREB and Erk, whose prolonged activation stimulates genes essential for neurite outgrowth (Ghosh and Greenberg, 1995; Dash et al, 1998; Lonze and Ginty, 2002). Thus, expression of Bcl-2, but not Bcl-xL, might promote a neuritogenic response by potentiating CREB and Erk activation after KCl depolarization or Ca2+ influx. In the absence of stimulation, phosphorylated CREB (pCREB) or pErk was not detected in PC12 cells (Figure 6A). KCl depolarization induced transient, low-level phosphorylation of CREB and Erk in control and Bcl-xL-expressing cells, but in Bcl-2-expressing cells, it induced pronounced and sustained activation of CREB and Erk that persisted for 24 h (Figure 6A). To confirm this, we assessed the transcriptional activities of CREB and Erk by measuring the expression of CREB- and Erk-dependent luciferase reporter genes. In the absence of stimulation, reporter gene expression was low in control, Bcl-2-, and Bcl-xL-expressing cells. Stimulation with KCl significantly upregulated the expression of CREB- and Erk-dependent reporter genes in Bcl-2-expressing cells but not in control or Bcl-xL-expressing cells (Figure 6B and C). Thus, Bcl-2 expression promotes and prolongs the activation of CREB and Erk induced by KCl depolarization or Ca2+ influx. Figure 6.Bcl-2 activates CREB and Erk to promote neuritogenic response. (A) Western blot analysis of time-dependent phosphorylation of CREB and Erk in PC12 cells expressing control, Bcl-2, or Bcl-xL plasmids treated with KCl (30 mM). Antibodies recognizing the unphosphorylated forms of CREB and Erk served as controls. (B, C) CREB-dependent (B) and Erk-dependent (C) reporter gene activities in stably transfected PC12 cells in the presence or absence of KCl (30 mM) (n⩾4/group). (D–F) Neurite outgrowth (D, F) and neuronal survival (E) in control (Cont) and Bcl-2-expressing cells (Bcl-2) incubated with 1 ng/ml NGF (n=4/group). Cultures were incubated in the absence (control) or presence of either a viral vector carrying a LacZ reporter gene or dominant-negative CREB (mCREB) (D, E) or the MEK inhibitor U0126 (100 nM) (F). *P<0.01, two-tailed t-test. Download figure Download PowerPoint To determine if CREB plays a causal role in Bcl-2-mediated neuritogenic response, we blocked CREB activation by infecting cells with herpes simplex virus expressing a dominant-negative CREB (Dolor et al, 1992). Dominant-negative CREB prevented neurite outgrowth from Bcl-2-expressing cells in the presence of 1 ng/ml NGF without inducing significant cell death; infection with a control LacZ viral vector had no effect on neurite outgrowth or survival (Figure 6D and E). Similarly, suppressing the activity of Erk with the MAPK-specific inhibitor U0126 in Bcl-2-expressing cells blocked neurite outgrowth induced by 1 ng/ml NGF (Figure 6F). We conclude that Bcl-2 enhances intracellular Ca2+ signaling induced by an increase of intracellular Ca2+ influx and potentiates CREB and Erk activation to stimulate a neuritogenic response. Bcl-2 activates CREB and Erk in vivo to support RGC axon regeneration To determine if Bcl-2 supports RGC axon regeneration by a mechanism similar to that which promotes the neuritogenic response of PC12 cells, we examined KCl-induced intracellular Ca2+ responses in RGCs purified from wt, Bcl-2tg, and Bcl-xLtg mice. As in PC12 cells, overexpression of Bcl-2 or Bcl-xL in RGCs did not alter the basal [Ca2+]i (Figure 7A and B). However, only in Bcl-2tg RGCs was the KCl-induced increase in [Ca2+]i significantly greater than in wt controls (Figure 7A and C), correlating with the reduction in TG-induced [Ca2+]i (or [Ca2+]er) in Bcl-2-expressing RGCs (not shown). Thus, as in PC12 cells, overexpression of Bcl-2 in RGCs enhances the intracellular Ca2+ response after Ca2+ influx. Figure 7.Optic nerve injury in Bcl-2tg mice increases the intracellular Ca2+ response and activates CREB and Erk in RGCs. (A–C) Representative trace (A) and quantitative analysis of basal (B) and KCl-induced changes in [Ca2+]i (C), measured by Fura-2, in RGCs of wt, Bcl-2tg, and Bcl-xLtg mice (n⩾3/group). Changes in [Ca2+]i were measured from its baseline to when the elevation of [Ca2+]i reached its peak. Values are means±s.d. *P<0.01 versus wt, two-tailed t test. (D) Immunofluorescence staining for pCREB and pErk in retinal sections from adult wt and Bcl-2tg mice at day 1 after optic nerve crush. Scale bar, 50 μm. Download figure Download PowerPoint To
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