Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons - PubMed (original) (raw)

Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons

Xianrong Mao et al. J Neurochem. 2007 Mar.

Abstract

Sp-family transcription factors (Sp1, Sp3 and Sp4) contain a zinc-finger domain that binds to DNA sequences rich in G-C/T. As assayed by RT-PCR analysis of mRNA, western-blot analysis, immunofluorescence, and antibody-dependent "supershift" of DNA-binding assays, the prominent Sp-family factors in cerebral neurons were identified as Sp3 and Sp4. By contrast, glial cells were found to express Sp1 and Sp3. We previously showed that the pattern of G-C/T binding activity of Sp-family factors is rapidly and specifically altered by the calcium influx accompanying activation of glutamate receptors. Here, we demonstrate that Sp-factor activity is also lost after a cerebral ischemia/reperfusion injury in vivo. Consistent with its calcium-dependent nature, we found that glutamate's effect on Sp-family factors could be blocked by inhibitors of calpains, neutral cysteine proteases activated by calcium. Purified calpain I cleaved Sp3 and Sp4 into products that retained G-C/T-binding activity, consistent with species observed in glutamate-treated neurons. These data provide details of an impact of glutamate-receptor activation on molecular events connected to gene expression.

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Figures

Figure 1. Loss of DNA-binding activity by Sp-factors during ischemia/reperfusion

Rats were subjected to a 1-h occlusion of the middle cerebral artery, then reperfusion for 1 or 4 h. Brains were removed and immediately frozen in liquid nitrogen. Regions of the cerebrum most likely to be affected on the side ipsilateral to the injury (I) were thawed in lysis buffer for extraction of nuclei; the same procedure was performed for the corresponding region from the contralateral, unoperated side (C). EMSA was performed on extracts equilibrated for total protein, using a radiolabeled probe containing an optimal target sequence for Sp1 and related factors (top) or for RBP-Jκ (bottom). Each pair of lanes represents the contralateral and ipsilateral sample from the same brain in adjacent lanes.

Figure 2. Calpain inhibitors block the loss of Sp-factors triggered by glutamate

Protease inhibitors (ALLN and MDL28170, 3 μM; remainder, 10 μM) were applied to neocortical neurons 30 min before glutamate exposure (50 μM, 60 min). Nuclear proteins were extracted, and protein-DNA interactions were assessed by EMSA with a Sp-factor probe. Two apparent proteolytic fragments (arrowheads) arose after glutamate treatment. (The band marked “Sp3Δ” is consistent with a smaller variant of Sp3 derived from internal translation initiation site.)

Figure 3. Calpain I cleavage of neuronal Sp-factors in vitro approximates that produced in situ by glutamate exposure

Nuclear extract from untreated neurons was incubated with commercial preparations of cathepsin B, D, L or calpain I at varying concentrations of the proteases (lanes 3–12 and 22–24) or at varying pH (lanes 13–21). The reactions in lanes 3–12 were performed at pH 5.5 and calpain I cleavage (lanes 22–24) was performed at pH 7.0. After protease digestion, reactions were returned to neutral pH for DNA-binding reactions, followed by resolution on an EMSA gel. The initial, undigested extract (lane 1) was also compared to a similar extract from glutamate-treated neurons (lane 2) for comparison to the typical degradation pattern, characterized by the two prominent products (arrowheads). (None of the protease preparations showed any reaction with the probe itself in control experiments not depicted here.)

Figure 4. Calpain I expression in neural cultures

Mixed neuronglia cultures from hippocampus were fixed without treatment (A) or following exposure to 50 μM glutamate for 60 min (B). Immunofluorescence was performed with antibodies against MAP2 (red) and calpain I (green); nuclei were subsequently stained with DAPI (blue). C: Nuclear proteins were extracted from control or glutamate-treated neocortical neurons, and 35 μg protein was subjected to western-blot analysis of calpain I (large subunit, ~80 kD); NeuN was detected to assure equivalency of loading.

Figure 5. Cell-type specificity of Sp-factors in cerebral cortex

Immunofluorescence was performed on frontal sections of rat cerebral cortex with antibodies against GFAP or NeuN (red), Sp1 or Sp4 (green). Sections were also stained with DAPI to localize total cellular nuclei. Double overlay is depicted for localization of the Sp-factors to neurons (NeuN+) or astrocytes (GFAP+); triple overlays incorporate DAPI staining as well. Scale bars represent 10 μ.

Figure 6. Cell-type specificity of Sp-factors in hippocampus

Immunofluorescence was performed on frontal sections of rat brain with antibodies against GFAP or NeuN (red), Sp1 or Sp4 (green). Sections were also stained with DAPI to localize total cellular nuclei. Double overlay is depicted for localization of the Sp-factors to neurons (NeuN+) or astrocytes (GFAP+); triple overlays incorporate DAPI staining as well. Images depict CA1 of the hippocampus; scale bars represent 10 μ.

Figure 7. Cell-type specificity of Sp-factors in culture

Mixed hippocampal cultures were analyzed by immunofluorescence with antibodies against GFAP or MAP2 (red), Sp1 or Sp4 (green). Double overlay is depicted for localization of the Sp-factors to neurons (MAP2+) or astrocytes (GFAP+). Scale bar represents 30 μ.

Figure 8. Expression of Sp-factors in neurons and/or glia as detected by western-blot analysis

Whole-cell lysates of astrocytes, neocortical neurons, NTera2 neuronal cells, and BV2 microglial cells was subjected to western-blot analysis (35 μg protein per lane) with antibodies against Sp1 (A), Sp3 (B), Sp4 (C), or actin (D). Arrows mark bands consistent with the mobilities of reported variants of each respective Sp-factor. The blots were over-developed to detect low levels of the various proteins in cell types in which they were scarce. [One 55-kD band (‘**N.S.**’) was nonspecifically detected in neuronal lysates with every antibody.]

Figure 9. Expression of Sp-factor mRNAs in neurons and glia

Total RNA was extracted from cultures of astrocytes or neocortical neurons. RT-PCR was used to semiquantitatively compare the mRNA levels of Sp1, Sp3, and Sp4 (A). Equilibration was confirmed by amplification of three constitutive genes: cyclophilin, GAPDH, and β-actin. The reaction products were quantified by densitometry (B). The relative level of each mRNA in glia was set as 100% and the levels in neurons were normalized to glial values. (**p < 0.001, *p < 0.05, by Student’s t-test).

Figure 10. Expression of Sp-factors in neurons and/or glia as detected by EMSA

Nuclear proteins were extracted from various cell types (A: primary neurons; B: NTera2 neuronal cells; C: primary astrocytes; D: primary microglia; E: BV2 microglial cells; F: N9 microglial cells) and analyzed by EMSA. Antibodies against Sp1, Sp3, and Sp4 were used to supershift Sp-binding proteins. For each supershift, 1.2 μg of total antibody was added, and an equal amount of each antibody was used when combined. (arrows: full length of Sp-proteins; arrowheads: Sp3Δ; the asterisk indicates a band observed only in NTera2 cells and sensitive to Sp1 antibody).

Figure 11. Calpain I cleavage of Sp3 and Sp4 can account for the Sp-factor degradation pattern in neurons

A. Sp1, Sp3, and Sp4 were individually expressed in SL2 cells, then nuclear extracts were made. A SL2 nuclear extract containing each indicated Sp-factor was incubated with various concentrations of calpain I, and the degradation patterns were resolved by EMSA. (Extracts from untransfected SL2 showed no reaction with the probe in control assays not depicted here.) B. Nuclear extract from neocortical neurons was subjected to cell-free proteolysis with various concentrations of calpain I (lanes 2–6), and the degradation pattern was compared to that occurring in situ after treating intact cells with glutamate (lane 1). These degradation patterns were also compared to that produced by calpain I incubation with a mixture of extracts from Sp3- and Sp4-transfected SL2 cells (lanes 7–11) (arrowheads: two fragments derived from glutamate treatment). C. Neocortical neurons were lysed with or without exposure to glutamate, and 50 μg of total protein were subjected to western-blot analysis with antibody recognizing Sp3 or Sp4. Glutamate was applied at 50 μM for 60 min; ALLN and MDL (MDL28170) were applied at 10 μM, 30 min prior to glutamate application. Arrows mark bands consistent with the mobilities of reported variants of each respective Sp-factor. ‘**N.S.**’: a protein detected nonspecifically in neuronal lysates with every antibody. D. The samples analyzed in panel C were probed for RBP-Jκ as a control for loading and specificity of the effect.

References

1. Bai G, Kusiak JW. Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells. J Biol Chem. 1997;272:5936–5942. - PubMed
1. Barger SW, Basile AS. Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function. J Neurochem. 2001;76:846–854. - PubMed
1. Barger SW, Moerman AM, Mao X. Molecular mechanisms of cytokine-induced neuroprotection: NFκB and neuroplasticity. Curr Pharm Des. 2005;11:985–998. - PubMed
1. Birnbaum MJ, van Wijnen AJ, Odgren PR, Last TJ, Suske G, Stein GS, Stein JL. Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3. Biochemistry. 1995;34:16503–16508. - PubMed
1. Bobba A, Canu N, Atlante A, Petragallo V, Calissano P, Marra E. Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons. FEBS Lett. 2002;515:8–12. - PubMed

Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons - PubMed (original) (raw)