Inhibition of CK2 Diminishes Fibrotic Scar Formation and Improves Outcomes After Ischemic Stroke via Reducing BRD4 Phosphorylation

Abstract

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-β1 (TGF-β1), SB431542 (a TGF-β1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-β1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-β1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-β1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke.

Keywords: CK2; Cerebral ischemia; Fibroblast; Fibrotic scar formation; p-BRD4.

Fig. 1

CK2α and CK2β expression was increased in vitro in the TGF-β1-induced fibrosis model and in vivo in the MCAO/R injury model. A Timeline of fibroblast treatment. B–D RT‒PCR analysis of CK2α, CK2α′ and CK2β mRNA expression in each group of fibroblasts treated with TGF-β1 for 72 h (n = 3). E–G Representative protein expression and quantification data for CK2α and CK2β in each group of fibroblasts were obtained by Western blotting (n = 3). *P < 0.05 vs. the Con group; ^△P < 0.05 vs. the TGF-β1 group; ^#P < 0.05 vs. the TGF-β1 + DMSO group. H Timeline and group diagram of the rats. I Schematic diagram of the coronal plane of the brain. The ischemic core is represented by the yellow area. The observed regions are shown as square boxes. J Immunofluorescence staining of FN⁺/CK2α⁺ and FN⁺/CK2β⁺ cells and images of merged signals in the ischemic core after cerebral ischemia (n = 3). Red represents tissue immunostained with antibodies against FN, and green represents cells immunostained with antibodies against CK2α or CK2β. Scale bars: 50 μm. K–M Representative quantitative analysis of FN-positive areas and CK2α-positive and CK2β-positive cells. N–Q Protein expression and quantification of FN, CK2α and CK2β levels in the ischemic core after cerebral ischemia caused by MCAO/R, as detected by Western blotting (n = 3). *P < 0.05 vs. the sham group; ^△P < 0.05 vs. the I/R 7 d group

Fig. 2

Effects of TBB on the proliferation, migration and activation of fibroblasts induced by TGF-β1 in vitro. A Timeline of fibroblast treatment. B, C Fibroblast proliferation in each group induced by TGF-β1 for 72 h was detected by EdU analysis. Green and blue represent EdU-positive cells and nuclei, respectively (n = 3). Scale bars: 50 μm. D, E The migration of fibroblasts in each group was detected by the scratch wound assay (n = 3). Scale bars: 200 μm. F–H The activation of fibroblasts in each group was detected by immunofluorescence staining with antibodies against α-SMA and FN. Green indicates the α-SMA, and red indicates the FN. (n = 3). Scale bars: 50 µm. I–K Representative protein expression and quantification analysis of α-SMA and FN protein levels in fibroblasts induced by TGF-β1 for 72 h, as detected by Western blotting (n = 3). *P < 0.05 vs. the Con group; ^△P < 0.05 vs. the TGF-β1 group; ^#P < 0.05 vs. the TGF-β1 + DMSO group

Fig. 3

TBB reduces fibrosis after MCAO/R injury. A Timeline and group diagram of the rats. B Schematic diagram of the coronal plane of the brain. The ischemic core is represented by the yellow area. The regions of interest are shown as square boxes. C, D Expression and quantitative analysis of collagen fibers with Sirius red staining at 7 days after MCAO/R (n = 3). Red represents collagen fibers. Scale bars: 1 µm. E–H Protein expression levels and quantitative analysis of FN and α-SMA at 7 days after cerebral ischemic injury determined by immunofluorescence staining (n = 3). Red represents FN-positive areas. Green represents α-SMA-positive cells. Blue represents nuclei. Scale bars: 40 μm. I–K Protein expression levels and quantitative analysis of α-SMA and FN in the ischemic core at 7 days after MCAO/R injury determined by Western blotting (n = 3). *P < 0.05 vs. the sham group; ^△P < 0.05 vs. the Ctrl group; ^#P < 0.05 vs. the vehicle group

Fig. 4

TBB ameliorates histopathological damage, protects Nissl bodies, decreases infarct volume and ameliorates neurological deficits after MCAO/R injury. A Timeline and group diagram of the rats. B Schematic diagram of the coronal plane of the brain. The ischemic core is represented by the yellow area. The observed regions are shown as square boxes. C, D Representative histological structure assessed by HE staining and Nissl body (bluish violet) expression assessed by Nissl staining of the ischemic core at 7 days after MCAO/R injury (n = 3). Scale bars: 2 µm. E Quantitative analysis of Nissl bodies expressed as the area ratio of the Nissl bodies to the image. F, G Representative images and quantitative analysis of the infarct volume in brain slices after MCAO/R determined by TTC staining (n = 3). White indicates infarction, while red indicates normal tissue. Analysis of neurological function according to the mNSS (H), Bederson score (I), and Longa score (J) at 1, 7 and 14 days after MCAO/R injury (n = 12). *P < 0.05 vs. the sham group; ^△P < 0.05 vs. the Ctrl group; ^#P < 0.05 vs. the vehicle group

Fig. 5

Effects of TBB on BRD4 phosphorylation in vitro and in vivo. A Timeline of fibroblast treatment. B–D Representative protein expression and quantitative analysis of p-BRD4 and BRD4 protein levels in fibroblasts incubated with TGF-β1 and/or TBB for 72 h, determined by Western blotting (n = 3). *P < 0.05 vs. the Con group; ^△P < 0.05 vs. the TGF-β1 group; ^#P < 0.05 vs. the TGF-β1 + DMSO group. E Timeline and group diagram of the rats. F–H Representative protein expression and quantitative analysis of p-BRD4 and BRD4 protein levels in the ischemic core at 7 days after cerebral ischemia injury, as assessed by Western blotting (n = 3). *P < 0.05 vs. the sham group; ^△P < 0.05 vs. the Ctrl group; ^#P < 0.05 vs. the vehicle group