Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Aberrant BMP2 Signaling in Patients Diagnosed with Osteoporosis.

The most common bone disease in humans is osteoporosis (OP). Current therapeutics targeting OP have several negative side effects. Bone morphogenetic protein 2 (BMP2) is a potent growth factor that is known to activate both osteoblasts and osteoclasts. It completes these actions through both SMAD-dependent and SMAD-independent signaling. A novel interaction between the BMP type Ia receptor (BMPRIa) and casein kinase II (CK2) was discovered, and several CK2 phosphorylation sites were identified. A corresponding blocking peptide (named CK2.3) was designed to further elucidate the phosphorylation site's function. Previously, CK2.3 demonstrated an increased osteoblast activity and decreased osteoclast activity in a variety of animal models, cell lines, and isolated human osteoblasts. It is hypothesized that CK2.3 completes these actions through the BMP signaling pathway. Furthermore, it was recently discovered that BMP2 did not elicit an osteogenic response in osteoblasts from patients diagnosed with OP, while CK2.3 did. In this study, we explore where in the BMP pathway the signaling disparity or defect lies in those diagnosed with OP. We found that osteoblasts isolated from patients diagnosed with OP did not activate SMAD or ERK signaling after BMP2 stimulation. When OP osteoblasts were stimulated with BMP2, both BMPRIa and CK2 expression significantly decreased. This indicates a major disparity within the BMP signaling pathway in patients diagnosed with osteoporosis.

Iron-induced oxidative stress contributes to α-synuclein phosphorylation and up-regulation via polo-like kinase 2 and casein kinase 2.

α-Synuclein plays a central role in synucleinopathies pathogenesis such as Parkinson's disease (PD). Phosphorylation is the most common and important protein modification linked to α-synuclein pathologies. There is mounting evidence suggested iron and α-synuclein are closely related in PD. We previously reported iron up-regulated α-synuclein mRNA levels and induced α-synuclein aggregation. In the present study, we aimed to investigate whether and how phosphorylation was involved in iron-induced α-synuclein regulations. The results showed that iron could induce pS129 α-synuclein (phosphorylation at Ser129) and α-synuclein upregulation in the substantia nigra of iron-overloaded rats and iron-treated SH-SY5Y cells, accompanied by the elevated levels of polo-like kinase 2 (PLK2) and casein kinase 2 (CK2). Over-expression of CK2 or PLK2 induced pS129 α-synuclein up-regulation and inhibitors of CK2 or PLK2 could suppress iron-induced α-synuclein phosphorylation. Antioxidant NAC could fully block iron-induced upregulation of CK2, PLK2 and pS129 α-synuclein levels, indicating oxidative stress plays a critical role in iron-induced α-synuclein phosphorylation. However, iron-induced α-synuclein up-regulation could only be partially blocked by CK2/PLK2 inhibitor or NAC. These findings demonstrate that iron-induced oxidative stress is largely responsible for α-synuclein phosphorylation and upregulation via CK2 and PLK2, and α-synuclein upregulation is not fully phosphorylation-dependent.

Casein Kinase 2 Inhibitor, CX-4945, as a Potential Targeted Anticancer Agent in Gastric Cancer.

BACKGROUND/AIM: Casein kinase 2 (CK2) is involved in multiple cellular processes. Furthermore, its overexpression in several human cancers has been associated with tumor progression. In this study, we evaluated the efficacy of the CK2 inhibitor, CX-4945, in gastric cancer cell lines and explored the potential predictive biomarkers for CX-4945 sensitivity. MATERIALS AND METHODS: The sensitivity to CX-4945 was screened in 49 gastric cancer cell lines by the MTT assay. The mRNA and protein expression of CK2 subunits (α and α') were determined using qRT-PCR and western blot. Furthermore, the activity of CK2α was measured by ELISA. Gene expression and mutations were analyzed via whole-exome and RNA sequencing. RESULTS: The sensitivity to CX-4945 was determined by the inhibition rate (%) at the effective dose (10 µM) which ranged from -1% to 89% in 49 gastric cancer cell lines. CK2α', but not CK2α, mRNA expression was correlated with CX-4945 sensitivity. CONCLUSION: In this study, CX-4945 showed modest antitumor efficacy in gastric cancer cell lines. CK2 might represent a potential therapeutic target for gastric cancer.

Protein kinase CK2 regulates redox homeostasis through NF-κB and Bcl-xL in cardiomyoblasts.

Oxygen consumption is particularly elevated in cardiac cells as they are equipped with a large number of mitochondria and high levels of respiratory chain components. Consequently, production of reactive oxygen species (ROS) is tightly controlled as an imbalance in redox reactions can lead to irreversible cellular damage. siRNA-mediated down-regulation of protein kinase CK2 has been implicated in the accumulation of ROS in cells. The present study was undertaken in order to investigate the role of CK2 in redox homeostasis in cardiomyoblasts. We found that inhibition or silencing of CK2 causes elevated levels of ROS, notably superoxide radical, and this is accompanied by suppression of NF-κB transcriptional activity and mitochondrial dysfunction. We show that CK2 regulates the expression of manganese superoxide dismutase, the enzyme catalyzing the dismutation of superoxide, in cancer cells but not in cardiomyoblasts. Furthermore, we report evidence that impaired expression of CK2 results in destabilization of the Bcl-2 mammalian homolog Bcl-xL, which is known to stabilize the mitochondrial membrane potential, through a mechanism involving disruption of the chaperone function of heat shock protein 90. Analysis of differential mRNA expression related to oxidative stress revealed that CK2 silencing caused a statistically significant deregulation of four genes associated with the oxidative damage, i.e., Fmo2, Ptgs1, Dhcr24, and Ptgs2. Overall, the results reported here are consistent with the notion that CK2 plays a role in conferring protection against oxidative stress by positively regulating pro-survival signaling molecules and the protein folding machinery in cardiomyoblasts.

Examination of CK2α and NF-κB p65 expression in human benign prostatic hyperplasia and prostate cancer tissues.

Protein kinase CK2 plays a critical role in cell growth, proliferation, and suppression of cell death. CK2 is overexpressed, especially in the nuclear compartment, in the majority of cancers, including prostate cancer (PCa). CK2-mediated activation of transcription factor nuclear factor kappa B (NF-κB) p65 is a key step in cellular proliferation, resulting in translocation of NF-κB p65 from the cytoplasm to the nucleus. As CK2 expression and activity are also elevated in benign prostatic hyperplasia (BPH), we sought to increase the knowledge of CK2 function in benign and malignant prostate by examination of the relationships between nuclear CK2 and nuclear NF-κB p65 protein expression. The expression level and localization of CK2α and NF-κB p65 proteins in PCa and BPH tissue specimens was determined. Nuclear CK2α and NF-κB p65 protein levels are significantly higher in PCa compared with BPH, and these proteins are positively correlated with each other in both diseases. Nuclear NF-κB p65 levels correlated with Ki-67 or with cytoplasmic NF-κB p65 expression in BPH, but not in PCa. The findings provide information that combined analysis of CK2α and NF-κB p65 expression in prostate specimens relates to the disease status. Increased nuclear NF-κB p65 expression levels in PCa specifically related to nuclear CK2α levels, indicating a possible CK2-dependent relationship in malignancy. In contrast, nuclear NF-κB p65 protein levels related to both Ki-67 and cytoplasmic NF-κB p65 levels exclusively in BPH, suggesting a potential separate impact for NF-κB p65 function in proliferation for benign disease as opposed to malignant disease.

Molecular characterization of clear cell renal cell carcinoma identifies CSNK2A1, SPP1 and DEFB1 as promising novel prognostic markers.

The prognosis associated with clear cell renal carcinoma (ccRCC) can vary widely and novel molecular prognostic markers are needed to assess prognosis at an earlier stage. Several gene products have been investigated for this purpose, but none of them have been implemented in clinical practice. Here we hypothesized that we, using TaqMan® Array, could identify superior prognostic messenger RNA (mRNA)s in long-term follow-up. Messenger RNA level of 19 candidate genes was investigated in 97 patients with ccRCC. Three genes impacted significantly on prognosis in both univariate and multivariate analysis. In univariate analysis, CSNK2A1 was a strong indicator of a poor overall survival (OS) (HR = 5.01, p < 0.001), disease specific survival (DSS) (HR = 6.21, p = 0.007) and progression free survival (PFS) (HR = 5.93, p = 0.005). High expression of SPP1 was associated to poor PFS (HR = 4.41, p = 0.04). DEFB1 was associated with a better PFS (HR = 0.24, p = 0.006). In multivariate analysis, CSNK2A1 was associated to a worse OS (HR = 3.56, p = 0.008) and PFS (HR = 3.84, p = 0.005), whereas SPP1 was an independent predictor of a worse PFS (HR = 3.46, p = 0.007) and DEFB1 of a better PFS (HR = 0.37, p = 0.027). These results show that with TaqMan®) Array we could identify three superior gene products related to prognosis. Further studies are needed to elucidate the pathways and roles of these genes in renal cacer development.

Differential proteome association study of freeze-thaw damage in ram sperm.

In this study proteomics analysis was performed to investigate damage caused to ram sperm by the freeze-thaw process. Sperm motility, viability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) content were measured to evaluate sperm quality. Compared with fresh groups, motility, viability and ATP content were all lower in freeze-thawed sperm (P < 0.001), and ROS content was higher (P < 0.001). Moreover, 25 differential protein spots were detected in two-dimensional gels using PDQuest 8.0 software and the corresponding proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) coupled with searching of the NCBI protein sequence database. Among these proteins, hexokinase1 (HXK1), the enzyme that catalyzes the first step of glycolysis in the sperm glycolytic pathway, is known to be associated with sperm motility. Casein kinase II subunit alpha (CSNK2A2), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. We used immunoblotting and immunofluorescence to analyze the expression and localization of these two proteins. HXK1 and CSNK2A2 expression levels in fresh sperm were significantly higher than that in freeze-thawed sperm (P < 0.001). HXK1 and CSNK2A2 were detected in the main part of the sperm flagellum, and the immunofluorescence signal from these proteins was weakened in the freeze-thawed group. Decreased expression of HXK1 and CSNK2A2 may be associated with decreased sperm motility and viability following freeze-thawing.

Protein kinase CK2α catalytic subunit is overexpressed and serves as an unfavorable prognostic marker in primary hepatocellular carcinoma.

Protein kinase CK2 alpha (CK2α), one isoform of the catalytic subunit of serine/threonine kinase CK2, has been indicated to participate in tumorigenesis of various malignancies. We conducted this study to investigate the biological significances of CK2α expression in hepatocellular carcinoma (HCC) development. Real-time quantitative polymerase and western blotting analyses revealed that CK2α expression was significantly increased at mRNA and protein levels in HCC tissues. Immunohistochemical analyses indicated that amplified expression of CK2α was highly correlated with poor prognosis. And functional analyses (cell proliferation and colony formation assays, cell migration and invasion assays, cell cycle and apoptosis assays) found that CK2α promoted cell proliferation, colony formation, migration and invasion, as well as inhibited apoptosis in hepatoma cell lines in vitro. CK2α-silenced resulted in significant apoptosis in cells that was demonstrated been associated with downregulation of expression of Bcl-2, p-AKT (ser473) and upregulation of expression of total P53, p-P53, Bax, caspase3 and cleaved-caspase3 in HCC cells. In addition, experiments with a mouse model revealed that the stimulative effect of CK2α on tumorigenesis in nude mice. Our results suggest that CK2α might play an oncogenic role in HCC, and therefore it could serve as a biomarker for prognostic and therapeutic applications in HCC.

Suppression of casein kinase 2 sensitizes tumor cells to antitumor TRAIL therapy by regulating the phosphorylation and localization of p65 in prostate cancer.

In the United States, prostate cancer (PCa) is the most commonly diagnosed cancer in males. For PCa at the late hormone-refractory stage, substantial improvement in treatment strategies is critically needed. TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, but both intrinsic and acquired resistance to TRAIL poses a huge problem in establishing clinically effective TRAIL therapies. In the present study, we examined the role played by casein kinase 2 (CK2) in the TRAIL­induced nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) pathway in a PCa cell line. Downregulation of CK2 combined with a sub-dose of TRAIL suppressed p65 phosphorylation at serine 536. The combination treatment of TRAIL and the CK2 inhibitor decreased p65 nuclear translocation. Under the treatment of a sub-dose of TRAIL, downregulation of CK2, using both genetic and pharmacological approaches, decreased the transcriptional activity of NF-κB and the expression of NF-κB downstream anti-apoptosis genes. Therefore, we provided novel molecular mechanistic insight reporting that CK2 regulates the sensitivity of PCa cells to the antitumor effect of TRAIL. This is important for understanding how the TRAIL pathway is disrupted in PCa and may help to develop an effective combinatorial therapy for PCa.

Casein kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm.

Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during developmental epithelial-mesenchymal transition (EMT) and has been associated with EMT in malignant epithelia. Our laboratory has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2 (CK2) phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and phosphorylates this site in vitro. Knockdown or inhibition of the CK2α/α' kinase subunit in epithelial cells causes de novo accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial maintenance. In malignant breast cancer cells, the CK2ß regulatory subunit is downregulated and FOXC2 is found in the nucleus, correlating with an increase in α-smooth muscle actin (SMA) expression. Restoration of CK2ß expression in these cells results in cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell migration and invasion. In contrast, knockdown of CK2ß in normal breast epithelial cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on these findings, we propose that Foxc2 is functionally maintained in the cytoplasm of normal epithelial cells by CK2α/α'-mediated phosphorylation at serine 124, which is dependent on proper targeting of the holoenzyme via the CK2ß regulatory subunit.

Inhibition of P-TEFb by DRB suppresses SIRT1/CK2α pathway and enhances radiosensitivity of human cancer cells.

BACKGROUND: Positive transcription elongation factor-b (P-TEFb) is a complex containing CDK9 and a cyclin (T1, T2 or K). The effect of inhibition of P-TEFb by 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB) on cell radiosensitivity and the underlying mechanisms were investigated. MATERIALS AND METHODS: Six human cancer cell lines were subjected to (3)H-uridine incorporation, cell viability and clonogenic cell survival assays; cell-cycle redistribution and apoptosis assay; western blots and nuclear 53BP1 foci analysis after exposing the cells to DRB with/without γ-radiation. RESULTS: DRB suppressed colony formation and enhanced radiosensitivity of all cell lines. DRB caused a further increase in radiation-induced apoptosis and cell-cycle redistribution depending on p53 status. DRB prolonged the presence of radiation-induced nuclear p53 binding protein-1 (53BP1) foci and suppressed the expression of sirtuin-1 (SIRT1) and casein kinase 2-alpha (CK2α), suggesting an inhibition of DNA repair processes. CONCLUSION: Our findings indicate that DRB has the potential to increase the efficacy of radiotherapy and warrants further investigation using in vivo tumor models.

Overexpressions of CK2ß and XIAP are associated with poor prognosis of patients with cholangiocarcinoma.

To investigate the expressions of casein kinase II ß (CK2ß) and X-Linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma (CCA) and evaluated their correlations with major clinicopathologic features and patients' survival. Fifty CCA specimens and 20 normal liver tissues were included in the study. Immunohistochemical staining was used to determine the expression levels of CK2ß, XIAP in normal and CCA tissues. The relationships of CK2ß and XIAP expressions with clinicopathologic parameters and clinical outcome were evaluated. High immunostaining of CK2ß and XIAP were observed in 66% (33/50) and 68% (34/50) of CCA tissues, which were significantly higher than that of normal liver tissues 0% (0/20) and 25% (5/20). The high expression of CK2ß was significantly associated with TNM stage (P = 0.036), histological grade (P = 0.020) and high serum CEA level (P = 0.010), while high expression of XIAP was only associated with TNM stage (P = 0.014) and high serum CEA level (P = 0.001). By univariant analysis, patients with high expression of CK2ß and XIAP demonstrate significantly poorer overall survival (P = 0.003 vs P = 0.018). Cox regression model showed that positive expression of CK2ßis an independent factor of prognosis (P = 0.004). The expressions of CK2ß and XIAP in CCA tissues showed strong correlations with the tumor progression, CK2ß may be applied as a potential prognostic marker for CCA.

Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2.

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

MiR-186, miR-216b, miR-337-3p, and miR-760 cooperatively induce cellular senescence by targeting α subunit of protein kinase CKII in human colorectal cancer cells.

We previously demonstrated that downregulation of protein kinase CKII induces cellular senescence in human colon cancer HCT116 cells. To investigate the role of microRNAs (miRNAs) in CKII downregulation during senescence, we employed computational algorithms. Four miRNAs (miR-186, miR-216b, miR-337-3p, and miR-760) were predicted to be miRNAs against CKIIα mRNA. Mimics of all four miRNAs jointly downregulated CKIIα expression in HCT116 cells. Reporter analysis and RT-PCR have suggested that these four miRNAs may stimulate degradation of CKIIα mRNA by targeting its 3' untranslated regions (UTRs). The four miRNA mimics increased senescent-associated ß-galactosidase (SA-ß-gal) staining, p53 and p21(Cip1/WAF1) expression, and reactive oxygen species (ROS) production. In contrast, concomitant knockdown of the four miRNAs by antisense inhibitors increased the CKIIα protein level and suppressed CKII inhibition-mediated senescence. Finally, CKIIα overexpression antagonized senescence induced by the four miRNA mimics. Therefore, the present results show that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively promote cellular senescence through the p53-p21(Cip1/WAF1) pathway by CKII downregulation-mediated ROS production in HCT116 cells.

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2ß and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

Using bacteria to determine protein kinase specificity and predict target substrates.

The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.

Sox4 stimulates ß-catenin activity through induction of CK2.

ß-catenin is a key component of the Wnt signaling pathway and the abnormal accumulation of ß-catenin is characteristic of various types of cancer. Here we demonstrate that overexpression of Sox4 enhances ß-catenin/TCF activity by increasing the stability of ß-catenin. Sox4 increased the protein level of ß-catenin and its target gene cyclin D1 in a dose-dependent manner. An siRNA experiment for Sox4 also demonstrated that Sox4 increases the protein levels of ß-catenin and thus activates the Wnt signaling pathway. We found that induction of ß-catenin/TCF activity by Sox4 is caused by stabilization of the ß-catenin protein, but not by induction of ß-catenin transcription. We further demonstrate that the increased level of ß-catenin is caused by induction of CK2. In light of recent evidence that Sox4 expression is activated in the colon and in other tumors with ß-catenin dysregulation, our findings suggest that Sox4 acts as an agonist of Wnt signaling in cancer cells.

Glioblastoma-specific protein interaction network identifies PP1A and CSK21 as connecting molecules between cell cycle-associated genes.

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis.