CK2 activity is crucial for proper glucagon expression.

AIMS/HYPOTHESIS: Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells. METHODS: We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition. RESULTS: We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans. CONCLUSIONS/INTERPRETATION: These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

C-Myc/H19/miR-29b axis downregulates nerve/glial (NG)2 expression in glioblastoma multiforme.

Nerve/glial antigen (NG)2 is highly expressed in glioblastoma multiforme (GBM). However, the underlying mechanisms of its upregulated expression are largely unknown. In silico analyses reveal that the tumor-suppressive miR-29b targets NG2. We used GBM-based data from The Cancer Genome Atals databases to analyze the expression pattern of miR-29b and different target genes, including NG2. Moreover, we investigated the regulatory function of miR-29b on NG2 expression and NG2-related signaling pathways. We further studied upstream mechanisms affecting miR-29b-dependent NG2 expression. We found that miR-29b downregulates NG2 expression directly and indirectly via the transcription factor Sp1. Furthermore, we identified the NG2 coreceptor platelet-derived growth factor receptor (PDGFR)α as an additional miR-29b target. As shown by a panel of functional cell assays, a reduced miR-29b-dependent NG2 expression suppresses tumor cell proliferation and migration. Signaling pathway analyses revealed that this is associated with a decreased ERK1/2 activity. In addition, we found that the long noncoding RNA H19 and c-Myc act as upstream repressors of miR-29b in GBM cells, resulting in an increased NG2 expression. These findings indicate that the c-Myc/H19/miR-29b axis crucially regulates NG2 expression in GBM and, thus, represents a target for the development of future GBM therapies.

Hypoxia-induced downregulation of microRNA-186-5p in endothelial cells promotes non-small cell lung cancer angiogenesis by upregulating protein kinase C alpha.

The tumor microenvironment stimulates the angiogenic activity of endothelial cells (ECs) to facilitate tumor vascularization, growth, and metastasis. The involvement of microRNA-186-5p (miR-186) in regulating the aberrant activity of tumor-associated ECs has so far not been clarified. In the present study, we demonstrated that miR-186 is significantly downregulated in ECs microdissected from human non-small cell lung cancer (NSCLC) tissues compared with matched non-malignant lung tissues. In vitro analyses of primary human dermal microvascular ECs (HDMECs) exposed to different stimuli indicated that this miR-186 downregulation is triggered by hypoxia via activation of hypoxia-inducible factor 1 alpha (HIF1α). Transfection of HDMECs with miR-186 mimic (miR-186m) significantly inhibited their proliferation, migration, tube formation, and spheroid sprouting. In contrast, miR-186 inhibitor (miR-186i) exerted pro-angiogenic effects. In vivo, endothelial miR-186 overexpression inhibited the vascularization of Matrigel plugs and the initial growth of tumors composed of NSCLC cells (NCI-H460) and HDMECs. Mechanistic analyses revealed that the gene encoding for protein kinase C alpha (PKCα) is a bona fide target of miR-186. Activation of this kinase significantly reversed the miR-186m-repressed angiogenic activity of HDMECs. These findings indicate that downregulation of miR-186 in ECs mediates hypoxia-stimulated NSCLC angiogenesis by upregulating PKCα.

Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model.

Chronic lymphedema after cancer treatment is common and there is still no cure for this disease. We herein investigated the lymphangiogenic capacity of adipose tissue-derived microvascular fragments (MVF), which contain stem cells and lymphatic vessel fragments. Secondary lymphedema was induced in the hindlimbs of C57BL/6J mice. Green fluorescence protein (GFP)+ MVF were isolated from transgenic C57BL/6Tg (CAG-EGFP)1Osb/J mice, suspended in collagen hydrogel, and injected in the lymphadenectomy defect of wild-type animals. This crossover model allowed the detection of MVF-derived blood and lymphatic vessels after transplantation. The MVF group was compared with animals receiving collagen hydrogel only or a sham intervention. Lymphangiogenic effects were analyzed using volumetry, magnetic resonance (MR) lymphography, histology, and immunohistochemistry. MVF injection resulted in reduced hindlimb volumes when compared to non-treated controls. MR lymphography revealed lymphatic regeneration with reduced dermal backflow after MVF treatment. Finally, MVF transplantation promoted popliteal angiogenesis and lymphangiogenesis associated with a significantly increased microvessel and lymphatic vessel density. These findings indicate that MVF transplantation represents a promising approach to induce therapeutic lymphangiogenesis.

Inhibition of CK2 Reduces NG2 Expression in Juvenile Angiofibroma.

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve-glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2'-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA.

Targeting Pancreatic Islet NLRP3 Improves Islet Graft Revascularization.

Hypoxia-induced islet cell death, caused by an insufficient revascularization of the grafts, is a major obstacle for successful pancreatic islet transplantation. Recently, it has been reported that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is expressed in pancreatic islets and that its loss protects against hypoxia-induced cell death. Therefore, we hypothesized that the inhibition of NLRP3 in islets improves the survival and endocrine function of the grafts. The transplantation of Nlrp3-/- islets or wild-type (WT) islets exposed to the NLRP3 inhibitor CY-09 into mouse dorsal skinfold chambers resulted in an improved revascularization compared with controls. An increased insulin release after NLRP3 inhibition caused the enhanced angiogenic response. Moreover, the inhibition of NLRP3 in hypoxic ß-cells triggered insulin gene expression by inducing the shuttling of MafA and pancreatic and duodenal homeobox-1 into the nucleus. This was mediated by a reduced interaction of NLRP3 with the thioredoxin-interacting protein (TXNIP). Transplantation of Nlrp3-/- islets or WT islets exposed to CY-09 under the kidney capsule of diabetic mice markedly improved the restoration of normoglycemia. These findings indicate that the inhibition of NLRP3 in isolated islets represents a promising therapeutic strategy to improve engraftment and function of the islets.

Protein Kinase CK2 Contributes to Glucose Homeostasis by Targeting Fructose-1,6-Bisphosphatase 1.

Glucose homeostasis is of critical importance for the survival of organisms. It is under hormonal control and often coordinated by the action of kinases and phosphatases. We have previously shown that CK2 regulates insulin production and secretion in pancreatic ß-cells. In order to shed more light on the CK2-regulated network of glucose homeostasis, in the present study, a qRT-PCR array was carried out with 84 diabetes-associated genes. After inhibition of CK2, fructose-1,6-bisphosphatase 1 (FBP1) showed a significant lower gene expression. Moreover, FBP1 activity was down-regulated. Being a central enzyme of gluconeogenesis, the secretion of glucose was decreased as well. Thus, FBP1 is a new factor in the CK2-regulated network implicated in carbohydrate metabolism control.

CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression.

Nerve/glial antigen (NG)2 expression crucially determines the aggressiveness of glioblastoma multiforme (GBM). Recent evidence suggests that protein kinase CK2 regulates NG2 expression. Therefore, we investigated in the present study whether CK2 inhibition suppresses proliferation and migration of NG2-positive GBM cells. For this purpose, CK2 activity was suppressed in the NG2-positive cell lines A1207 and U87 by the pharmacological inhibitor CX-4945 and CRISPR/Cas9-mediated knockout of CK2α. As shown by quantitative real-time PCR, luciferase-reporter assays, flow cytometry and western blot, this significantly reduced NG2 gene and protein expression when compared to vehicle-treated and wild type controls. In addition, CK2 inhibition markedly reduced NG2-dependent A1207 and U87 cell proliferation and migration. The Cancer Genome Atlas (TCGA)-based data further revealed not only a high expression of both NG2 and CK2 in GBM but also a positive correlation between the mRNA expression of the two proteins. Finally, we verified a decreased NG2 expression after CX-4945 treatment in patient-derived GBM cells. These findings indicate that the inhibition of CK2 represents a promising approach to suppress the aggressive molecular signature of NG2-positive GBM cells. Therefore, CX-4945 may be a suitable drug for the future treatment of NG2-positive GBM.

Protein Kinase CK2 Regulates Nerve/Glial Antigen (NG)2-Mediated Angiogenic Activity of Human Pericytes.

Protein kinase CK2 is a crucial regulator of endothelial cell proliferation, migration and sprouting during angiogenesis. However, it is still unknown whether this kinase additionally affects the angiogenic activity of other vessel-associated cells. In this study, we investigated the effect of CK2 inhibition on primary human pericytes. We found that CK2 inhibition reduces the expression of nerve/glial antigen (NG)2, a crucial factor which is involved in angiogenic processes. Reporter gene assays revealed a 114 bp transcriptional active region of the human NG2 promoter, whose activity was decreased after CK2 inhibition. Functional analyses demonstrated that the pharmacological inhibition of CK2 by CX-4945 suppresses pericyte proliferation, migration, spheroid sprouting and the stabilization of endothelial tubes. Moreover, aortic rings of NG2-/- mice showed a significantly reduced vascular sprouting when compared to rings of NG2+/+ mice, indicating that NG2 is an important regulator of the angiogenic activity of pericytes. In vivo, implanted Matrigel plugs containing CX-4945-treated pericytes exhibited a lower microvessel density when compared to controls. These findings demonstrate that CK2 regulates the angiogenic activity of pericytes through NG2 gene expression. Hence, the inhibition of CK2 represents a promising anti-angiogenic strategy, because it does not only target endothelial cells, but also vessel-associated pericytes.