Casein Kinase 2 Interacting Protein-1 Expression Research Articles

CKIP-1 inhibits M2 macrophage polarization to suppress the progression of gastric cancer by inactivating JAK/STAT3 signaling.

Gastric cancer (GC) is a frequently occurring malignancy with poor prognosis. Casein kinase 2 interacting protein-1 (CKIP-1) is a PH domain-containing protein implicated in regulating tumorigenesis and macrophage homeostasis. This study aimed to elucidate the role and potential mechanism of CKIP-1 in the progression of GC. CKIP-1 expression in GC tumor and para-carcinoma tissues was detected using RT-qPCR. Then, human monocyte cell line THP-1 was treated with PMA, interleukin (IL)-4 and IL-13 to induce M2-polarized macrophages. CD206, arginase-1 (Arg-1) and transforming growth factorβ1 (TGFβ1) expression in M2-polarized macrophages with or without CKIP-1 overexpression was evaluated. Moreover, GC cell lines (MKN45 and HGC27 cells) were co-cultured with CKIP-1-overexpressed M2-polarized macrophages, and the viability, migration and invasion of GC cells were measured. Additionally, immunoblotting assessed the expression of JAK/STAT3 signaling-related proteins and STAT3 agonist Colivelin was used to treat GC cells to perform the rescue experiments to analyze the changes of malignant phenotypes of GC cells. Results showed that CKIP-1 was downregulated in GC tissues and M2-polarized macrophages. CKIP-1 overexpression inhibited M2 macrophage polarization and decreased TGFβ1 secretion. Besides, elevated CKIP-1 expression in M2-polarized macrophages inhibited the viability, migration and invasion of GC cells. Furthermore, CKIP-1 overexpression inactivated JAK2/STAT3 signaling in GC cells by inhibiting TGFβ1 level. Specifically, Colivelin treatment abrogated the influences of CKIP-1 upregulation on the malignant phenotypes of GC cells. Collectively, CKIP-1 inhibits M2 macrophage polarization to suppress the progression of GC by inactivating JAK/STAT3 signaling pathway.

CKIP-1 silencing suppresses OSCC via mitochondrial homeostasis-associated TFAM/cGAS-STING signalling axis.

Limited effective targets have challenged the treatment of oral squamous cell carcinoma (OSCC). Casein kinase 2 interacting protein 1 (CKIP-1) is a scaffold protein involved in various diseases. However, the role of CKIP-1 in OSCC remains unclear. The aim of this study was to explore the regulatory role of CKIP-1 in OSCC, as well as the involved mechanism. First, higher expression of CKIP-1 in OSCC tissues and cell lines were found. Series of gain- and loss-of-function experiments demonstrated suppressed malignant behaviours and enhanced apoptosis of OSCC cells when CKIP-1 was silenced. Also, inhibited tumour growth in CKIP-1-silenced group were proved. Further, mitochondrial transcription factor A (TFAM) downregulation, increased ROS production, decreased mitochondrial membrane potential and cGAS-STING activation in CKIP-1-silenced group were observed. The involvement of mitochondrial homeostasis-related TFAM/cGAS-STING axis in CKIP-1-silenced OSCC cells was finally demonstrated by tetramethylpyrazine (TMP) that inhibits TFAM degradation. Taken together, our study demonstrated that CKIP-1 silencing could significantly antagonize OSCC via TFAM/cGAS-STING axis, which may provide a candidate target for OSCC treatment.

CKIP-1-Loaded Cartilage-Affinitive Nanoliposomes Reverse Osteoarthritis by Restoring Chondrocyte Homeostasis.

Osteoarthritis (OA) is a chronic joint disease characterized by cartilage imbalance and disruption of cartilage extracellular matrix secretion. Identifying key genes that regulate cartilage differentiation and developing effective therapeutic strategies to restore their expression is crucial. In a previous study, we observed a significant correlation between the expression of the gene encoding casein kinase-2 interacting protein-1 (CKIP-1) in the cartilage of OA patients and OA severity scores, suggesting its potential involvement in OA development. To test this hypothesis, we synthesized a chondrocyte affinity plasmid, liposomes CKIP-1, to enhance CKIP-1 expression in chondrocytes. Our results demonstrated that injection of CAP-Lipos-CKIP-1 plasmid significantly improved OA joint destruction and restored joint motor function by enhancing cartilage extracellular matrix (ECM) secretion. Histological and cytological analyses confirmed that CKIP-1 maintains altered the phosphorylation of the signal transduction molecule SMAD2/3 of the transforming growth factor-β (TGF-β) pathway by promoting the phosphorylation of the 8T, 416S sit. Taken together, this work highlights a novel approach for the precise modulation of chondrocyte phenotype from an inflammatory to a noninflammatory state for the treatment of OA and may be broadly applicable to patients suffering from other arthritic diseases.

CKIP-1 mediates P. gingivalis-suppressed osteogenic/cementogenic differentiation of periodontal ligament cells partially via p38 signaling pathway

ABSTRACT Objectives Casein kinase 2 interacting protein-1 (CKIP-1) is a versatile player involved in various biological processes. However, whether CKIP-1 mediates the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) under Porphyromonas gingivalis (Pg) stimulation remains unknown. Material and Methods The effect of Pg on PDLC differentiation was first verified. CKIP-1 expression in Pg-infected PDLCs or in PDL of apical periodontitis (AP) mice was detected. The changes of CKIP-1 during PDLC differentiation was also determined. PDLC differentiation capacity in CKIP-1 knockout (KO) mice and CKIP-1-silenced PDLCs with or without Pg stimulation were further studied. Inhibitor was finally applied to verify the involvement of p38 signaling pathway in PDLC differentiation. Results The suppression effect of Pg on PDLC differentiation was demonstrated. CKIP-1 increased in the PDL of AP mice and Pg-induced PDLCs, and decreased gradually during PDLC differentiation. Increased OSX and RUNX2 expression in PDL were observed in CKIP-1 KO mice. Also, CKIP-1 silencing facilitated and rescued Pg-inhibited PDLC differentiation. Inhibitor for p38 signaling pathway blocked CKIP-1 silencing-facilitated PDLC differentiation. Conclusions CKIP-1 mediated the osteogenic/cementogenic differentiation of PDLCs partially through p38 signaling pathway, which may provide evidence for the regeneration of periodontal hard tissues damaged by Pg.

CKIP-1 Promotes P. gingivalis-Induced Inflammation of Periodontal Soft Tissues by Inhibiting Autophagy.

As a chronic inflammatory disease, periodontitis involves many biological processes including autophagy. At the same time, casein kinase 2 interacting protein-1 (CKIP-1) was reported to play a role in regulation of inflammation. But whether CKIP-1 and autophagy interact in periodontitis remains unclear. In this paper, our research team verified the levels of CKIP-1 expression and autophagy increase in the periodontal tissues of a ligature-induced periodontitis mouse model. And this result was also confirmed in Porphyromonas gingivalis (Pg)-induced human gingival fibroblasts (HGF) and human periodontal ligament cells (PDLC). We also showed the autophagy level in periodontal tissues is higher in Ckip-1 knockout (KO) mice than wild type (WT). At the same time, CKIP-1 knockdown lentivirus was used in PDLC and HGF, and it was found that silencing CKIP-1 significantly activated autophagy. Unfortunately, the regulatory role of autophagy in periodontitis is still unclear. Then, the autophagy agonist Rapamycin and inhibitor 3-MA were used in a periodontitis mouse model to investigate periodontal tissue destruction. We found the inflammation in periodontal tissue was reduced when autophagy activated. All these conclusions have been verified both in vivo and in vitro experiments. Finally, our research proved that silencing CKIP-1 reduces the expression of inflammatory cytokines in Pg-induced PDLC and HGF by regulating autophagy. Overall, a new role for CKIP-1 in regulating periodontal tissue inflammation was demonstrated in our study, and it is possible to treat periodontitis by targeting the CKIP-1 gene.

The ubiquitination of CKIP-1 mediated by Src aggravates diabetic renal fibrosis (original article)

Renal chronic inflammation is an important hallmark of diabetic renal fibrosis. Casein kinase 2 interacting protein 1 (CKIP-1) performs a nephroprotective role in the pathogenesis of diabetic nephropathy (DN), which is dramatically decreased in diabetic kidneys. However, whether CKIP-1 regulates inflammation to ameliorate renal fibrosis remains unclear and it is interesting to clarify the degradation mechanism of CKIP-1. Here, we identified CKIP-1 expression was down-regulated in diabetic kidneys and knockout (KO) of CKIP-1 increased c-Jun expression and extra cellular matrix (ECM) in kidneys of normal mice, and knockout (KO) of CKIP-1 further exacerbated renal inflammatory fibrosis in diabetic mice. Moreover, the activated Src kinase interacted with CKIP-1 at Lys252 and increased K48 linked polyubiquitination and proteasome degradation of CKIP-1 in HG induced GMCs and diabetic kidneys. Mechanistically, Src facilitating the binding of c-Cbl with CKIP-1 by promoting the phosphorylation of c-Cbl, thereby increasing Cbl-mediated ubiquitination of CKIP-1 to down-regulate CKIP-1 protein expression. Thus, our study highlighted the anti-inflammation role of CKIP-1 and clarified the mechanism of CKIP-1 degradation in DN.

Casein Kinase-2 Interacting Protein-1 Regulates Physiological Cardiac Hypertrophy via Inhibition of Histone Deacetylase 4 Phosphorylation.

Different kinds of mechanical stimuli acting on the heart lead to different myocardial phenotypes. Physiological stress, such as exercise, leads to adaptive cardiac hypertrophy, which is characterized by a normal cardiac structure and improved cardiac function. Pathological stress, such as sustained cardiac pressure overload, causes maladaptive cardiac remodeling and, eventually, heart failure. Casein kinase-2 interacting protein-1 (CKIP-1) is an important regulator of pathological cardiac remodeling. However, the role of CKIP-1 in physiological cardiac hypertrophy is unknown. We subjected wild-type (WT) mice to a swimming exercise program for 21 days, which caused an increase in myocardial CKIP-1 protein and mRNA expression. We then subjected CKIP-1 knockout (KO) mice and myocardial-specific CKIP-1-overexpressing mice to the 21-day swimming exercise program. Histological and echocardiography analyses revealed that CKIP-1 KO mice underwent pathological cardiac remodeling after swimming, whereas the CKIP-1-overexpressing mice had a similar cardiac phenotype to the WT controls. Histone deacetylase 4 (HDAC4) is a key molecule in the signaling cascade associated with pathological hypertrophy; the phosphorylation levels of HDAC4 were markedly higher in CKIP-1 KO mouse hearts after the swimming exercise program. The phosphorylation levels of HDAC4 did not change after swimming in the hearts of CKIP-1-overexpressing or WT mice. Our results indicate that swimming, a mechanical stress that leads to physiological hypertrophy, triggers pathological cardiac remodeling in CKIP-1 KO mice. CKIP-1 is necessary for physiological cardiac hypertrophy in vivo, and for modulating the phosphorylation level of HDAC4 after physiological stress. Genetically engineering CKIP-1 expression affected heart health in response to exercise.

Let-7i-5p functions as a putative osteogenic differentiation promoter by targeting CKIP-1.

The online version of this article (10.1007/s10616-020-00444-1) contains supplementary material, which is available to authorized users.

Inflammation-mediated age-dependent effects of casein kinase 2-interacting protein-1 on osteogenesis in mesenchymal stem cells.

BackgroundThe casein kinase 2-interacting protein-1 (CKIP-1) is important in the development of osteoblasts and cardiomyocytes. However, the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells (MSCs) remain unclear. This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.MethodsBone marrow MSCs of CKIP-1 wild type (WT) and knockout (KO) mice were cultivated in vitro. Cell phenotype was analyzed by flow cytometry, colony formation was detected to study the proliferative ability. Osteogenic and adipogenic induction were performed. The osteogenic ability was explored by alizarin red staining, alkaline phosphatase (ALP) staining and ALP activity detection. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to determine the mRNA expression levels of osteoblast marker genes. The adipogenic ability was detected by oil red O staining. Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume (BV/TV), bone surface area fraction/trabecular BV, trabecular number (Tb.N), and trabecular spacing (Tb.sp). Interleukin (IL)-1β was injected on WT mice of 2 months old and 18 months old, respectively. Difference in CKIP-1 expression was detected by RT-PCR and western blot. The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.ResultsALP assays, alizarin red staining, and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis. Micro-computed tomography detection showed that among 18-month-old mice, CKIP-1 KO mice presented significantly higher bone mass compared with WT mice (P = 0.02). No significant difference was observed in 2-month-old mice. In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice (4.3-fold increase at the mRNA level, P = 0.04). Finally, the expression levels of CKIP-1 in bone marrow (3.2-fold increase at the mRNA level, P = 0.03) and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1β.ConclusionsCKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects. Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.

Casein kinase 2 interacting protein 1 positively regulates caudal-related homeobox 1 in intestinal-type gastric cancer.

BackgroundGastric cancer (GC) is one of the most common malignancies, and intestinal-type GC is the main histopathologic type of GC in China. We previously reported that casein kinase 2 interacting protein 1 (CKIP-1) acts as a candidate tumor suppressor in intestinal-type GC. CKIP-1 participates in the regulation of multiple signaling pathways, including the Wnt/β-catenin pathway, of which caudal-related homeobox 1 (CDX1) may be a downstream target gene. The purpose of this study was to investigate the relationship between CKIP-1 and CDX1 in intestinal-type GC.MethodsSixty-seven gastroscopy biopsy specimens and surgically resected gastric specimens were divided into four groups: gastric mucosa group, intestinal metaplasia (IM) group, dysplasia group, and intestinal-type GC group. The expression levels of CKIP-1 and CDX1 were detected in these groups and GC cell lines, and the correlations between these expression levels were analyzed. SGC7901 and BGC823 cells were divided into CKIP-1 shRNA groups and CKIP-1 over-expression groups, and CDX1 expression was detected. β-Catenin expression was detected in intestinal-type GC tissue samples and CKIP-1 shRNA and CKIP-1 over-expression SGC7901 cells, and its correlation with CKIP-1 expression in intestinal-type GC tissue was analyzed. The Wnt/β-catenin pathway inhibitor DKK-1 and activator LiCl were incubated with SGC7901 cells, BGC823 cells, and CKIP-1 shRNA and CKIP-1 over-expression SGC7901 and BGC823 cells, following which CDX1 and Ki-67 expression were detected.ResultsThe expression levels of CKIP-1 and CDX1 were lower in patients with intestinal-type GC than in patients with IM and dysplasia (both P < 0.05). CKIP-1 and CDX1 expression levels were positively correlated in IM, dysplasia, and intestinal-type GC tissue and cell lines (r = 0.771, P < 0.01; r = 0.597, P < 0.01; r = 0.654, P < 0.01; r = 0.811, P < 0.01, respectively). CDX1 expression was decreased in the CKIP-1 shRNA groups and increased in the CKIP-1 over-expression groups of SGC7901 and BGC823 cells compared to that in the corresponding control groups (both P < 0.05). CKIP-1 expression was negatively correlated with β-catenin expression in intestinal-type GC patients (r = −0.458, P < 0.01). Compared to the control group, β-catenin expression was increased in the CKIP-1 shRNA SGC7901 cell group and decreased in the CKIP-1 over-expression SGC7901 cell group (P < 0.05). CDX1 expression was increased in SGC7901 and BGC823 cells treated with DKK-1, DKK-1 increased CDX1 expression and decreased Ki-67 expression in the CKIP-1 shRNA group; the opposite result was observed in SGC7901 and BGC823 cells treated with LiCl, and LiCl decreased CDX1 expression and increased Ki-67 expression in the CKIP-1 over-expression group (both P < 0.05).ConclusionsThrough the Wnt/β-catenin signaling pathway, CKIP-1 may positively regulate CDX1 in intestinal-type GC.

Prostatic epithelial cells and their high expressions of CKIP-1 affect the TGF-\u03b21 expression levels which might reduce the scar formation in remodeling stage at prostatic urethral wounds after wound repair

ObjectiveThere are less scar formations in some wounds after wound repair. Our earlier study had shown that the amount of collagen fibers in canine prostatic urethra wound were less than in bladder neck wound after 2-μm laser resection of the prostate (TmLRP) and partial bladder neck mucosa at 4 weeks. The purpose of this study was to observe the amount of scar tissue and characterize the probable causes of “less scar healing” in prostatic urethra wound.MethodsA total of 12 healthy adult male crossbred canines underwent resection of prostate and partial bladder neck mucosa using 2-μm laser. The prostatic urethra and bladder neck wound specimens were harvested at 3, 4, 8 and 12 weeks after operation, respectively. The histopathologic characteristics were observed by hematoxylin and eosin(HE)staining, and the expression of transforming growth factor-β1 (TGF-β1) and casein kinase-2 interacting protein-1 (CKIP-1) were examined by immunohistochemistry in prostatic urethra and bladder neck wound, respectively. Overexpressed CKIP-1 human prostate epithelial cells (BPH-1 cells) were established and the expression of TGF-β1 was detected by Western blotting. Furthermore, a non-contact co-culture system of BPH-1 cells and human fibroblast (HFF-1) cells was used to observe the effects of BPH-1 cell and their high CKIP-1 levels on the expression of TGF-β1 in HFF-1 in vitro.ResultsThe histology showed that there were a large number of prostatic epithelium and a small amount of scar tissue in prostatic urethra wound, while no epithelial cells and more scar tissue in bladder neck wound at 4, 8 and 12 weeks after repair. There were a higher expression level of TGF-β1 in prostate epithelial cells and fibroblasts and a lower expression level of CKIP-1 in prostate epithelial cells at 3 weeks after surgery in prostatic urethral wound. Compared to week 3, the TGF-β1 expression decreased both in prostate epithelial cells and fibroblasts at 4, 8 and 12 weeks in prostatic urethral wound (p < 0.05 or p < 0.01). The CKIP-1 expression increased in prostate epithelial cells at 4, 8 and 12 weeks compared to 3 weeks in prostatic urethra wound (p < 0.01). A higher TGF-β1 expression level of fibroblasts was observed in bladder neck wound at 3 weeks. And there was no significant change in the expression of TGF-β1 of fibroblasts in 3, 4, 8 and 12 weeks after operation in bladder neck wound. Both the prostate urethra and bladder neck wound fibroblasts showed weak expression of CKIP-1 and there was no significant change in 3, 4, 8 and 12 weeks. The vitro experiments showed that the TGF-β1 expression in BPH-1 cells with CKIP-1 overexpression decreased 25% compared with control group (p < 0.05). Furthermore, the expression of TGF-β1 in HFF-1 cells of co-cultured group decreased by 20% compared with Control group (p < 0.05); the expression of TGF-β1 in HFF-1 cells of overexpression co-culture group were reduced by 15% compared with co-cultured group (p < 0.01).ConclusionsA large number of prostate epithelial cells in prostatic urethra wound may be one of the causes of less formation of scar tissue after repair. The prostate epithelial cells might reduce expression level of TGF-β1 by raising CKIP-1 expression and inhibit expression of TGF-β1 in peripheral fibroblasts at remodeling stage to reduce the excessive proliferation of fibrous cells and the excessive scar formation.

Overexpression of CKIP-1 alleviates hypoxia-induced cardiomyocyte injury by up-regulating Nrf2 antioxidant signaling via Keap1 inhibition

Acute myocardial infarction causes irreversible myocardial damage and is a leading cause of death and disability worldwide. Casein kinase 2 interacting protein-1 (CKIP-1) has been suggested to confer cytoprotection against various pathologic injuries. However, it remains unclear whether CKIP-1 regulates myocardial infarction-induced cardiomyocyte injury. This study aimed to explore the potential role of CKIP-1 in regulating hypoxia-induced cardiomyocyte injury and reveal the underlying mechanism. The results demonstrated that hypoxia-exposed cardiomyocytes showed lower CKIP-1 expression. CKIP-1 restoration by transfecting a CKIP-1 expression vector significantly improved viability and reduced apoptosis in hypoxia-treated cardiomyocytes. Moreover, CKIP-1 overexpression suppressed hypoxia-induced oxidative stress in cardiomyocytes. Mechanism research revealed that CKIP-1 overexpression reduced the expression of kelch-like ECH-associated protein 1 (Keap1) and increased the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), actions which resulted in an increase in the transcription of Nrf2 target genes. However, Keap1 overexpression partially reversed CKIP-1-mediated Nrf2 promotion and cardioprotection. Notably, the blockade of Nrf2 signaling also significantly abolished CKIP-1-mediated cardioprotection. Overall, our findings demonstrate that CKIP-1 alleviates hypoxia-induced cardiomyocyte injury through the up-regulation of Nrf2 antioxidant signaling via the down-regulation of Keap1, suggesting a potential role for CKIP-1 in myocardial infarction.

CKIP-1 regulates the immunomodulatory function of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are self-renewing multipotent cells with immunoregulatory function, which makes them attractive candidates for regenerative medicine. However, the detailed mechanisms of their immunomodulatory capacity are not fully characterized. Here, we found that casein kinase 2 interacting protein-1 (CKIP-1) expression was induced in the murine MSC cell line C3H/10T1/2 by LPS. Knockdown of CKIP-1 did not cause significant differences on the cell cycle or immunophenotype of MSCs. However, MSCs with CKIP-1 knockdown showed enhanced immunosuppressive capacity. Real-time PCR and western blot analyses revealed that compared with the control group, MSCs with CKIP-1-knockdown exhibited higher IL-10 production and p38 MAPK phosphorylation following LPS treatment. Interestingly, the expression of CKIP-1 was decreased in MSCs following high glucose treatment. Furthermore, MSCs became more immunosuppressive after high glucose treatment, as shown by higher IL-10 production and enhanced inhibition of T cell proliferation. Collectively, our data reveal a novel role for CKIP-1 in regulating MSC-mediated immunomodulation, and indicate that MSCs become more immunosuppressive under high glucose conditions. These new insights may help in the development of future applications of MSCs.

CKIP-1 alleviates oxygen-glucose deprivation/reoxygenation-induced apoptosis and oxidative stress in cultured hippocampal neurons by downregulating Keap1 and activating Nrf2/ARE signaling

Accumulating evidence has shown that casein kinase 2 interacting protein-1 (CKIP-1) is a pivotal regulator of apoptosis and oxidative stress. However, whether CKIP-1 is involved in regulating neuronal injury during the progression of cerebral ischemia/reperfusion injury remains unknown. In the present study, we aimed to investigate the potential role and underlying mechanism of CKIP-1 in regulating neuronal apoptosis and oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment in vitro. Herein, we found that OGD/R treatment resulted in a significant increase in CKIP-1 expression in cultured hippocampal neurons. The silencing of CKIP-1 exacerbated OGD/R-induced neuronal apoptosis and production of reactive oxygen species. By contrast, CKIP-1 overexpression reduced the apoptosis and reactive oxygen species production induced by the OGD/R treatment. Mechanistically, CKIP-1 inhibited the expression of Kelch-like ECH-associated protein 1 (Keap1) and promoted the expression of nuclear factor E2-related factor 2 (Nrf2). In addition, CKIP-1 increased the activation of antioxidant response element and the expression of downstream antioxidant genes. However, Keap1 overexpression or Nrf2 knockdown partially reversed the neuroprotective effect of CKIP-1 overexpression. Taken together, our results demonstrate that CKIP-1 overexpression alleviates OGD/R-induced neuronal injury by enhancing the Nrf2-mediated anti-oxidative stress signaling pathway, revealing a neuroprotective role of CKIP-1. Our study suggests CKIP-1 as a potential therapeutic target for neuroprotection.

CKIP-1 suppresses odontoblastic differentiation of dental pulp stem cells via BMP2 pathway and can interact with NRP1

ABSTRACTAim: Casein kinase 2 interacting protein-1 (CKIP-1) is a recently discovered intracellular regulator of bone formation, muscle cell differentiation, and tumor cell proliferation. Our study aims to identify the inhibition of BMP2-Smad1/5 signaling by CKIP-1 in odontoblastic differentiation of human dental pulp stem cells (DPSCs). Materials and Methods: DPSCs infected CKIP-1 siRNA or transfected CKIP-1 full-length plasmid were cultured in odontoblastic differentiation medium or added noggin (200 ng/mL) for 21 days. We examined the effects of CKIP-1 on odontoblastic differentiation, mineralized nodules formation, and interaction by western blot, real-time polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP) staining, alizarin red S staining, and immunoprecipitation. Results: Firstly, we have demonstrated that CKIP-1 expression markedly decreased time-dependently along with cell odontoblastic differentiation. Indeed, the silence of CKIP-1 upregulated odontoblastic differentiation via BMP2-Smad1/5 signaling, while CKIP-1 over-expression had a negative effect on odontoblastic differentiation of DPSCs. Furthermore, CKIP-1 could interact with Neuropilin-1 (NRP1). Conclusions: This work provides data that advocates a novel perception on odontoblastic differentiation of DPSCs. Therefore, inhibiting the expression of CKIP-1 may be of great significance to the development of dental caries.

Effect of CKIP-1 siRNA lentivirus transfection on proliferation of glioma U87-MG cells

Objective To investigate the effects of Casein kinase 2-interacting protein 1 (CKIP-1) gene silencing on the proliferation of glioma cells U87-MG. Methods The recombinant lentiviral vectors targeting CKIP-1 gene or negative control were constructed and then used to infect glioma U87-MG cell line.The effects of knock-down on the mRNA or protein expression of CKIP-1 were evaluated by real-time qPCR and western blotting.Cell cycle was detected by the flow cytometry assay, and cell proliferation changes were evaluated by cell counting, MTT, and BrdU assay, respectively.Lastly, the colony formation was used to investigate the effect of CKIP-1 knock-down on the clone formation. Results Compared with the group of Ctrl, CKIP-1 siRNA was observed to significantly inhibit CKIP-1 expression at the mRNA levels (Ctrl (1.01±0.13) vs CKIP-1 siRNA (0.23±0.02), P<0.01) and protein levels in the U87-MG cells.Also, CKIP-1 suppression mediated by RNAi decreased the ratio of G0/G1 phase (Ctrl (69.64±0.79) vs CKIP-1 siRNA(62.64±0.66), P<0.01), increased that of G2/M phase (Ctrl (8.36±0.52) vs CKIP-1 siRNA(13.87±2.90), P<0.05), and significantly inhibited the cell proliferation and clone formation (Colony number: Ctrl (25±2) vs CKIP-1 siRNA(2±1), P<0.05) of transfected U87-MG cells. Conclusion Knocking down the expression of CKIP-1 significantly inhibit cell proliferation in human U87-MG glioma cells, indicating that CKIP-1 is involved in the development of gliomas and could promote the cell proliferation. Key words: Casein kinase 2-interacting protein 1; Glioma; Gene silencing; Cell proliferation

Myocardial CKIP-1 Overexpression Protects from Simulated Microgravity-Induced Cardiac Remodeling.

Human cardiovascular system has adapted to Earth's gravity of 1G. The microgravity during space flight can induce cardiac remodeling and decline of cardiac function. At present, the mechanism of cardiac remodeling induced by microgravity remains to be disclosed. Casein kinase-2 interacting protein-1 (CKIP-1) is an important inhibitor of pressure-overload induced cardiac remodeling by decreasing the phosphorylation level of HDAC4. However, the role of CKIP-1 in the cardiac remodeling induced by microgravity is unknown. The purpose of this study was to determine whether CKIP-1 was also involved in the regulation of cardiac remodeling induced by microgravity. We first detected the expression of CKIP-1 in the heart from mice and monkey after simulated microgravity using Q-PCR and western blotting. Then, myocardial specific CKIP-1 transgenic (TG) and wild type mice were hindlimb-suspended (HU) to simulate microgravity effect. We estimated the cardiac remodeling in morphology and function by histological analysis and echocardiography. Finally, we detected the phosphorylation of AMPK, ERK1/2, and HDAC4 in the heart from wild type and CKIP-1 transgenic mice after HU. The results revealed the reduced expression of CKIP-1 in the heart both from mice and monkey after simulated microgravity. Myocardial CKIP-1 overexpression protected from simulated microgravity-induced decline of cardiac function and loss of left ventricular mass. Histological analysis demonstrated CKIP-1 TG inhibited the decreases in the size of individual cardiomyocytes of mice after hindlimb unloading. CKIP-1 TG can inhibit the activation of HDAC4 and ERK1/2 and the inactivation of AMPK in heart of mice induced by simulated microgravity. These results demonstrated CKIP-1 was a suppressor of cardiac remodeling induced by simulated microgravity.

Casein Kinase 2 Interacting Protein-1 regulates M1 and M2 inflammatory macrophage polarization

The importance of macrophage plasticity, albeit being discovered recently, has been highlighted in a broad spectrum of biological processes operative in physiological and pathological environments. Macrophage polarized activation and inactivation has profound effects on immune and inflammatory responses with several major pathways being elucidated in the past few years. However, transcriptional regulation mechanisms governing macrophage polarization is still preliminary. In this study, we identify the Casein Kinase 2 Interacting Protein 1 (CKIP-1) as a molecular toggle manipulating macrophage speciation. CKIP-1 expression was strongly induced by pro-inflammatory M1 stimuli (LPS and IFN-γ) and robustly suppressed by M2 stimuli (IL-4 and IL-13) in human and murine macrophage. Gain and loss of function studies suggest that CKIP-1 is a prerequisite for optimal LPS-induced pro-inflammatory gene activation, which exhibits its roles in a NF-κB dependent manner. Furthermore, CKIP-1 inhibits anti-inflammatory gene expression by negatively regulating JAK1-STAT6 activation in macrophages. Taken together, these data integrated CKIP-1 expression and function as a novel transcriptional regulator of macrophage polarization and identified a double feedback loop consisting of CKIP-1 and the key regulators of the M1 and M2 macrophage effectors in polarization pathway. Moreover, the inhibitory roles of CKIP-1 in LPS-mediated sepsis and TPA-mediated cutaneous provide a new target for treatments of acute inflammation.

Role of the CKIP1 gene in proliferation and apoptosis of the human lung cancer cell line H1299.

Casein kinase 2 interacting protein 1 (CKIP1) is a specific interacting protein of the casein kinase 2 (CK2) α subunit, and, by binding CK2 and other proteins, functions as an adaptor to regulate a series of cellular functions. Previous studies suggested that CKIP1 might play an important role in regulating oncogenic activities. However, few studies examining the function of CKIP1 in cancer cells have been performed. The present study aimed to investigate the role of CKIP1 in lung cancer. CKIP1 mRNA expression was detected in 5 human lung cancer cell lines (H-125, H1299, LTEP-A-2, SPC-A-1, and NCL-H446) by semi-quantitative RT-PCR, and in 10 noncancerous lung tissues and 30 non-small lung cancer tissues by real-time quantitative PCR. A lentivirus-mediated small interfering RNA (siRNA) was used to knock down CKIP1 expression in the H1299 cell line. To elucidate the impact of CKIP1 downregulation on H1299 cells, cell proliferation, DNA synthesis, and cell cycle distribution and apoptosis were measured by high content screening assay, BrdU incorporation, and flow cytometric analyses, respectively. CKIP1 mRNA was highly expressed both in H1299 cells and lung cancer tissues. We found that downregulation of CKIP1 resulted in suppression of proliferation and colony-forming ability of H1299 cells, and led to S phase cell cycle arrest and G2 phase promotion, as well as a significant enhancement of H1299 cell apoptosis. Our study indicated that high expression levels of CKIP1 were associated with the development of lung cancer, and that CKIP1 knockdown may block tumor cell growth mainly by promoting cell apoptosis.

Integrated analysis of genomics and proteomics reveals that CKIP-1 is a novel macrophage migration regulator

Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to play an important role in cell morphology, differentiation and apoptosis. However, the role of CKIP-1 in other cellular processes is still unknown. Here we investigated transcriptome profiles of WT and CKIP-1-deficient mouse embryonic fibroblasts (MEFs), and found that innate immunity and cell migration related pathways were significantly correlated with CKIP-1 expression. As macrophage is a key cell type in innate immunity, we then used murine macrophage RAW264.7 cells to discover CKIP-1 interacting proteins by immunoprecipitation/mass spectrometry (IP/MS). Analysis of these proteins revealed migration related pathways were enriched. Further experiments indicated that knockdown of CKIP-1 in RAW264.7 cells resulted in impaired cell migration. Our study suggests that CKIP-1 is a novel regulator of macrophage migration.