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-ß (TGF-ß) 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.

The construction of a novel xenograft bovine bone scaffold, (DSS)6-liposome/CKIP-1 siRNA/calcine bone and its osteogenesis evaluation on skull defect in rats.

BACKGROUND: Xenograft bone scaffolds have advantages such as mechanical strength, sufficient source and safety. Combined with siRNA properly targeting CKIP-1, a negative regulator of osteogenesis, may contribute to the repair result of calcine bone alone. METHODS: Herein, we constructed a novel xenograft bovine bone scaffold namely (DSS)6-liposome/CKIP-1 siRNA/calcine bone, the characteristics of which were investigated by confirming the effect of (DSS)6-liposome, observing the appearance and testing mechanical strength of calcine bone, and observing the combined result of CKIP-1 siRNA by FAM immunofluorescence. In addition, cytotoxicity by CCK-8 and LDH activity of L929 â€‹cells and MC3T3-E1 osteoblasts cultured with the scaffold were tested in vitro, primary osteoblasts proliferation, the mRNA expressions of CKIP-1, ALP, COL1-α and OCN, the protein expressions of CKIP-1, BMP-2, COL-1 and Runx2 and calcium nodules were also determined by CCK-8, RT-qPCR, western-blot and Alizarin Red staining in vitro. Then, we successively established the skull defect model for evaluating the repair result of the novel scaffold by HE staining of 2, 4, 8 and 12 weeks, immumohistochemical stainings of 2, 4, 8 and 12 weeks such as ALP, COL-1α and OCN, Mirco-CT scanning of 4 and 12 weeks and the relative parameters and so on in vivo. RESULTS: It indicated that (DSS)6-liposome/CKIP-1 siRNA/calcine bone could successfully knock down the CKIP-1 mRNA and protein expressions, promote osteoblasts proliferation with the little cytotoxicity in vitro, increase the protein expressions of BMP-2, COL-1 and Runx2 in vitro, increase mRNA expressions of ALP, COL-1α and OCN in vitro and in vivo, and have a better bone defect repair effect with few side effects in rats after 12 weeks. CONCLUSION: Our research indicates (DSS)6-liposome/CKIP-1 siRNA/calcine bone could repair skull defects well in rats, and it may lay the foundation of applicating the novel xenograft bone scaffold in the clinical. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings provide evidence that (DSS)6- liposome/CKIP-1 siRNA/calcine bone could be used as a novel xenograft bone scaffold for osteogenesis with the good safety.

The effects of casein kinase 2 interacting protein-1 on the growth and development of craniomaxillofacial soft and hard tissues in mice / 口腔疾病防治

Objective@# To investigate the effect of casein kinase 2 interacting protein-1 (CKIP-1) on craniofacial soft tissues and hard tissues, to provide the basis for the study and treatment of craniomaxillofacial related diseases.@*Methods@#6-month- old male CKIP-1 knockout (KO) mice were selected as the experimental group, and wild-type (WT) mice were selected as the control group. The craniomaxillofacial hard tissues (parietal bone, nasal bone, incisors and molars) were analyzed through micro- CT, and the morphological changes of maxillofacial soft tissues (nasal cartilage, lip mucosa and tongue) were analyzed through HE staining and toluidine blue staining.@* Results@#CKIP-1 negatively regulated bone mass of cancellous bone of cranial and maxillofacial bones and dentin mineralization. Compared with the WT mice, the thickness of the parietal baffle layer increased by 93% in KO mice, while cortical bone showed no significant difference between the two groups. The nasal cancellous bone thickness increased by 160% in KO-mice, while cortical bone showed no significant difference between the two groups; the enamel thickness was normal, but the pulp cavity became smaller and the dentin thickness increased by 48%. Compared with the WT mice, the HE staining and toluidine blue staining analyses of the soft tissues revealed that the thickness of the alar cartilage plate of KO mice increased by 57%, and local ossification was found within the cartilage plate. The thickness of the keratinized layer of the labial mucosa increased by 170% in KO mice and the muscle fiber diameter of the lingual muscle increased by 45%. @*Conclusion@#CKIP-1 genes have different effects on the growth and development of various soft and hard tissues in the maxillofacial region of mice.

CKIP-1 augments autophagy in steatotic hepatocytes by inhibiting Akt/mTOR signal pathway.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by aberrant accumulation of intrahepatic triglycerides and lipid droplets (LDs) in the liver cells, is becoming increasingly prevalent at an alarming rate worldwide. LDs can be consumed by either hydrolysis or autophagy, which is shown to be of importance in the regulation of hepatic lipid metabolism. We have shown that deficiency of pleckstrin homology domain-containing casein kinase 2 interacting protein-1 (CKIP-1), a scaffold protein that interacts with various proteins in multiple signal pathways, in mice aggravates high-fat diet induced fatty liver. However, its underlying mechanisms remain largely unknown. In this study, we found that the mRNA and protein levels of CKIP-1 decreased dramatically in steatotic HepG2 cells induced by oleic acid (OA) treatment. Coincidently, hepatic autophagy was also dynamically regulated in steatotic HepG2 cells. In addition, overexpression of CKIP-1 activated autophagy by suppression of Akt/mTOR signaling, which in turn reduced lipid accumulation. Moreover, these phenomena were reversed in CKIP-1-shRNA transfected steatotic hepatocytes. To further evaluate the potential role of CKIP-1 in autophagy, we determined the level of autophagy related proteins in CKIP-1 knockout mice. These results supported our findings in vitro. In summary, we found CKIP-1 to be a positive regulator of hepatic autophagy and a promising therapeutic target for treatment of NAFLD.

Effects of casein kinase 2 interacting protein-1 on the osteogenic differentiation ability of human periodontal ligament stem cells / 口腔疾病防治

Objective @#To investigate the effects of casein kinase 2 interacting protein-1 (CKIP-1) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs).@*Methods @#The hPDLSCs were obtained by primary culture with periodontal ligament tissues that were collected from normal humans. Then, a lentiviral vector containing a CKIP-1-specific siRNA sequence was constructed, and the transcriptional level of CKIP-1 in hPDLSCs was downregulated after vector infection. The P4 cells were divided into four groups: the control group, negative control group (infected with a control vector), CKIP-siRNA group (infected by a CKIP-1 siRNA lentivirus) and CKIP-1 group (infected by a CKIP-1 overexpression virus). All of the cells were cultured under osteogenic induction for 21 days. Then, alizarin red staining and quantitative determination were performed to detect the osteogenic differentiation ability of the hPDLSCs. In addition, qPCR was used to detect the transcriptional level of osteogenesis-related regulatory factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL), and the osteogenesis-related regulatory factors of the bone morphogenetic protein (BMP) signaling pathway.@*Results@#There were no differences in the indexes between the negative control group and the control group (P > 0.05). Compared with the negative control group, the CKIP-siRNA group demonstrated more mineralized nodules (P < 0.05), significantly increased calcium salt deposition (P < 0.05), and increased mRNA levels of osteogenesis-related regulatory factors, such as Runx2 , ALP, OCN, and RANKL, and the osteogenesis-related regulatory factors of BMP signaling pathway (P < 0.05). @*Conclusion@#Downregulation of CKIP-1 could promote the osteogenic differentiation of hPDLSCs, which is related to the transcription level of osteogenic-related regulatory factors.

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

AIM: 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.

miR­98­5p promotes osteoblast differentiation in MC3T3­E1 cells by targeting CKIP­1.

Casein kinase 2-interacting protein 1 (CKIP-1) is a negative regulator for bone formation. Previously, using bioinformatics analysis, CKIP­1 has been predicted to serve the role of target gene of miR­98­5p. In the present study, the potential role of miR­98­5p in regulating osteoblast differentiation through CKIP­1 was investigated. Following pre­treatment with microRNA (miR)­98­5p agomir or miR­98­5p antagomir, MC3T3­E1 cells were cultured in an osteoinductive medium. Subsequently, the expression of miR­98­5p, CKIP­1 and levels of osteoblast differentiation markers, including alkaline phosphatase, matrix mineralization, osteocaicin, collagen type I, runt­related transcription factor 2 and osteopontin were assayed. Using a dual­luciferase reporter assay, it was demonstrated that CKIP­1 was the target gene of miR­98­5p. miR­98­5p was upregulated as a result of treatment with miR­98­5p agomir and promoted osteoblast differentiation. Conversely, miR­98­5p antagomir inhibited miR­98­5p expression and osteoblast differentiation. miR­98­5p targeted CKIP­1 by binding to its 3'­untranslated region. Furthermore, miR­98­5p overexpression decreased the protein levels of CKIP­1 and inhibition of miR­98­5p increased the protein levels of CKIP­1. The results of the present study indicated that CKIP­1 was a target gene of miR­98­5p and that miR­98­5p regulated osteoblast differentiation in MC3T3­E1 cells by targeting CKIP­1.

Effect of CKIP-1 siRNA lentivirus transfection on proliferation of glioma U87-MG cells / 中华行为医学与脑科学杂志

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.

Deficiency of CKIP-1 aggravates high-fat diet-induced fatty liver in mice.

Casein kinase 2 interacting protein-1(CKIP-1) is widely expressed in a variety of tissues and cells, and plays an important role in various critical cellular and physiological processes including cell growth, apoptosis, differentiation, cytoskeleton and bone formation. Here, we found: (1) CKIP-1 deficient mice exhibited increased body weight, liver weight, number and size of lipid droplets, and TG content comparing with WT mice after being exposed to high fat diet (HFD); (2) the levels of serum insulin, liver glycogen, phosphorylated C-Jun-N-terminal kinase-1 (pJNK1) and phosphorylated insulin receptor substrate -1(pIRS1) in CKIP-1-/- mice were higher than those of WT mice; (3) CKIP-1 interacted with JNK1 in vitro. Our results indicate that CKIP-1 deficiency in mice aggravates HFD-induced fatty liver by upregulating JNK1 phosphorylation and further upregulating IRS-1 phosphorylation and RI.

Function and regulation of serine/threonine phosphatases in the healthy and diseased heart.

Protein phosphorylation is a major control mechanism of a wide range of physiological processes and plays an important role in cardiac pathophysiology. Serine/threonine protein phosphatases control the dephosphorylation of a variety of cardiac proteins, thereby fine-tuning cardiac electrophysiology and function. Specificity of protein phosphatases type-1 and type-2A is achieved by multiprotein complexes that target the catalytic subunits to specific subcellular domains. Here, we describe the composition, regulation and target substrates of serine/threonine phosphatases in the heart. In addition, we provide an overview of pharmacological tools and genetic models to study the role of cardiac phosphatases. Finally, we review the role of protein phosphatases in the diseased heart, particularly in ventricular arrhythmias and atrial fibrillation and discuss their role as potential therapeutic targets.

Crystallization and preliminary X-ray crystallographic analysis of the human CKIP-1 pleckstrin homology domain.

The casein kinase 2 interacting protein-1 (CKIP-1) is involved in many cellular functions, including apoptosis, signalling pathways, cell growth, cytoskeleton and bone formation. Its N-terminal pleckstrin homology (PH) domain is thought to play an important role in membrane localization and controls shuttling of CKIP-1 between the plasma membrane and nucleus. In this study, the human CKIP-1 PH domain was purified but problems were encountered with nucleic acid contamination. An S84D/S86D/S88D triple mutant designed to abolish nucleic acid binding was purified and successfully crystallized. Single crystals diffracted to 1.7 Šresolution and belonged to space group P43212 with unit-cell parameters a=53.0, b=53.0, c=113.8 Å, α=ß=γ=90.0°.

A preliminary study on recombinant expression and function in vitro of proteasome activator REGγ / 军事医学科学院院刊

Objective To study the expression of the fused proteasome activator REGγ(11S regulator complex gamma subunit) using gene recombination technology and to further study the interaction between REGγ and casein kinase-2 interacting protein-1(CKIP-1)in vitro.Methods Firstly, the full length cDNA fragment of REGγ was amplified through PCR using the plasmid pCMV-Myc-REGγ as template and subcloned into the prokaryotic expression vector pGEX-4T-2 before being transformed into E.coli BL21 cells. The protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG) .Secondly, the protein expression was monitored by SDS-PAGE and Western blotting after ultrasonication. Finally, the GST Pull-down assay was performed to investigate the interaction between REGγ and CKIP-1 in vitro.Results The prokaryotic expression construct pGEX-4T-2-REGγ was generated successfully and confirmed by DNA sequencing. Expression analysis showed that the GST-REGγ protein was easily expressed and isolated mainly in the lysate supernatant after sonication and centrifugation. The GST Pull-down assay revealed the strong mutual interaction between REGγ and CKIP-1 in vitro.Conclusion The proteasome activator REGγ could interact with the negative regulator of osteoblastogenesis CKIP-1 in vitro and the current study has shed light on further investigations of their physiological relevance.