BTEB2 prevents neuronal apoptosis via promoting bad phosphorylation in rat intracerebral hemorrhage model

Abstract

Krüppel-like zinc-finger transcription factor 5 (KLF5), known as BTEB2 or IKLF, has several biological functions that involve cell proliferation, development and apoptosis. Previous studies demonstrated that BTEB2 had anti-apoptotic effect in multiple diseases such as esophageal cancer and non-small cell lung cancers (NSCLCs). However, the distribution and function of BTEB2 in CNS diseases remain unknown. In this study, we show that BTEB2 down-regulates neuronal apoptosis during pathophysiological processes of intracerebral hemorrhage (ICH). A rat ICH model was established by behavioral tests. Western blot and immunohistochemistry revealed a remarkable up-regulation of BTEB2 expression surrounding the hematoma after ICH. Double-labeled immunofluorescence showed BTEB2 was mostly co-localized with neurons, rarely with activated astrocytes and microglia. Furthermore, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with BTEB2. In addition, KLF5 knockdown in vitro specifically resulted in increasing neuronal apoptosis coupled with reduced Bad phosphorylation at both ser112 and ser136 residues. All our findings suggested that BTEB2 down-regulated neuronal apoptosis via promoting Bad phosphorylation after ICH.

Fig. 1

Assessments and scores of behavioral tests on rats after ICH. Behavioral tests were implemented in rats after ICH or sham operation. Forelimb placing test (a) and Corner turn test (b) scores at different survival times after ICH. The ICH group showed distinctly deficits compared with the sham group over the first 3 days (*p < 0.05) with no significant difference at baseline or 3 days later

Fig. 2

BTEB2 protein expression following ICH. Western blot was performed to study the protein level of BTEB2 surrounding the hematoma at different survival times (a). Quantification graphs of the intensity of staining of BTEB2 to GAPDH at each time point (b). Data are presented as mean ± SEM (n = 3, *p < 0.05)

Fig. 3

Representative microphotographs for BTEB2 immunohistochemistry surrounding the hematoma. In the sham group, BTEB2 was low (a, b). At 2 days after ICH, comparison of BTEB2 between contralateral (c, d) and ipsilateral brain caudate putamen (e, f), many cells around the hematoma showed BTEB2, the positively stained intensity (e, f). No positive signals were found in the negative control (g). The number of BTEB2 cells was largely increased comparing the ipsilateral group with the sham and contralateral groups (h). *p < 0.05. Scale bar: left column, 50 μm; right columns, 10 μm

Fig. 4

Double immunofluorescence staining for BTEB2 with different phenotype-specific markers in brain caudate putamen surrounding the hematoma. In the adult rat caudate within 3-mm distance from the hematoma at the second day after ICH, horizontal sections were labeled with BTEB2 (red a, f, k), different cell markers (green b, g, l) such as neuronal marker (NeuN), microglia marker (CD11b) and astrocyte marker (GFAP) and as well as DAPI (blue c, h, m) to show the nucleus. The yellow and white color visualized in the merged images represents the colocalization of BTEB2 with different phenotype-specific markers (d, e) and the purple indicates the colocalization of the nucleus with phenotype-specific markers (d, e, i, j, n, o). Colocalization of BTEB2 with different phenotype-specific markers in the sham group (e, j, o) are shown in the caudate. Quantitative analysis of NeuN-positive cells expressed BTEB2 (%) in the sham group and 2 days after ICH. (ipsi) indicates the perihematomal region and (sham) presents the sham group. *Significant difference at p < 0.05 compared with the sham group (p). Error bars represent SEM. Scale bars, 20 μm (a)

Fig. 5

Association of BTEB2 with neuronal expressed active caspase-3 after ICH. The expression of active caspase-3 increased and peaked at day 3 after ICH, GAPDH was used to confirm that equal amount of protein was run on gel (a).Quantification graphs (relative optical density) of the intensity of staining of active caspase-3 and GAPDH at each time points (b). Negative correlation between BTEB2 protein expression and active caspase-3 protein expression after ICH (c). The data are mean ± SEM (p < 0.05, asterisk [*] indicates statistical significance from the sham group). Sections labeled with active caspase-3 (d, g, j, m), NeuN (e, h), BTEB2 (k, n), and the co-localization of active caspase-3/NeuN and active caspase-3/BTEB2 surrounding the hematoma at day 2 after ICH (f, i, l, o). Scale bars, 20 μm (d–o)

Fig. 6

BTEB2 prevents neuronal apoptosis through Bad phosphorylation. Western blot showed siRNA knocked down BTEB2 expression in PC12 cells (a). Knocking down BTEB2 induced increasing levels of active caspase-3 and reduced the levels of P-ser112-bad and P-ser136-bad in hemin-treated PC12 cells (c). The bar chart indicates the density of active caspase-3/BTEB2/P-ser112-Bad/P-ser136-Bad versus GAPDH (b, d). Data are presented as means ± SEM (*p < 0.05, ^# p < 0.05). Immunofluorescence showed the co-localization of BTEB2/active-caspase-3 in control and non-specific siRNA or BTEB2 siRNA transfected PC12 cells after hemin stimulus; meanwhile, the phenotype changes of nuclei were also investigated via DAPI staining (e)