MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling.

Background: Mixed lineage kinase 3 (MLK3) is a member of a serine/threonine MAP3K family, and it has been demonstrated to play critical roles in various biological activities and disease progression. Previous studies showed that impaired skeletal mineralization and spontaneous tooth fracture in the MLK3-deficient mice, suggesting MLK3 actively participated in the bone formation. However, the detailed function and underlying mechanisms remain obscure. Methods: The MLK3 knockout (KO) mouse was applied in the present study, and multi-omics were performed to compare the metabolites and gene expression between wild type (WT) and KO mice. The bone fracture model was successfully established, and the healing process was evaluated by X-ray, micro-CT examination, histomorphometry and immunohistochemistry (IHC) staining. On the other hand, the effects of MLK3 on osteogenic differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining and qRT-PCR examination. Finally, the downstream signaling pathways were screened out by RNA-sequencing (RNA-seq) and then validated by Western blotting. Results: In the present study, imbalanced bone metabolism was observed in these MLK3 KO mice, suggesting MLK3 may participate in bone development. Moreover, MLK3 -/- mice displayed abnormal bone tissues, impaired bone quality, and delayed fracture healing. Further investigation showed that the inhibition of MLK3 attenuated osteoblast differentiation in vitro. According to the RNA-seq data, MAPK signaling was screened out to be a downstream pathway, and its subfamily members extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal protein kinase (JNK) were subjected to Western blotting examination. The results revealed that although no differences in their expression were observed between MSCs derived from WT and KO mice, their phosphorylated protein levels were all suppressed in MLK3 -/- MSCs. Conclusion: In conclusion, our results demonstrated that loss of MLK3 suppressed osteoblast differentiation and delayed bone formation via influencing metabolism and disturbing MAPK signaling. The translational potential of this article: The findings based on the current study demonstrated that MLK3 promoted osteogenesis, stimulated new bone formation and facilitated fracture healing, suggesting that MLK3 may serve as a potential therapeutic target for bone regeneration. MLK3 activator therefore may be developed as a therapeutic strategy for bone fracture.

Preoperative quantitative diffusion tensor imaging on the spinal nerve root is a predictor for the surgical outcome of lumbar disc herniation: a prospective study of 117 patients.

BACKGROUND: The diffusion tensor imaging (DTI) parameters (fractional anisotropy [FA] and apparent diffusion coefficient [ADC]) are commonly used to provide quantitative information on tissues. This study aimed to evaluate the predictive value of preoperative DTI of the spinal nerve roots in the surgical outcome of lumbar disc herniation (LDH). METHODS: A total of 117 LDH patients were included. According to the postoperative improvement rate, the patients were dichotomized into the unfavorable group (N.=35) and favorable group (N.=82). RESULTS: The favorable group had a younger age (P=0.005) and a shorter disease course (P<0.001) than the unfavorable group. The favorable group had higher affected side FA and ADC and lower healthy/affected FA and ADC ratio than the unfavorable group (all P<0.05). Logistic regression analysis showed that younger age, shorter disease course, higher affected side FA and ADC, lower healthy/affected FA and ADC ratio, and lower healthy side ADC were the independent factors associated with positive surgical outcome. ROC analysis showed that the affected side FA had an excellent predictive performance for the surgical outcome (AUC=0.900). The Healthy/affected FA ratio had a good predictive performance (AUC=0.846). The overall predictive accuracy ranged from 0.91 to 0.92. However, ADC had poor predictive performance (AUC ranged from 0.626 to 0.663). CONCLUSIONS: These results suggested the preoperative affected side FA value had an excellent predictive performance for the surgical outcome of LDH patients. The LDH patients with a higher preoperative affected side FA value were more likely to have a positive surgical outcome.

Effects of Pretreatment of the Dorsal Meningovertebral Ligaments on the Incidence of Intraoperative Cerebrospinal Fluid Leakage: A Comparative Study.

Cerebrospinal fluid leakage can lead to postoperative refractory headaches and meningitis. Dural injury is the main cause of postoperative cerebrospinal fluid leakage. Previously, we performed a comprehensive anatomic study on the dorsal meningovertebral ligaments in the lumbosacral regions and concluded that these ligaments are an anatomic factor leading to dural laceration. However, no clinical study has examined the relationship between dorsal meningovertebral ligaments and the incidence of intraoperative cerebrospinal fluid leakage. The goal of this study was to investigate the effect of prophylactic intraoperative pretreatment of the meningovertebral ligaments on the incidence of cerebrospinal fluid leakage during surgery. [Orthopedics. 2023;46(1):e66-e71.].

Comparison of region-of-interest delineation methods for diffusion tensor imaging in patients with cervical spondylotic radiculopathy.

BACKGROUND: Diffusion tensor imaging is a promising technique for determining the responsible lesion of cervical radiculopathy, but the selection and delineation of the region of interest (ROI) affect the results. This study explored the impact of different ROI sketching methods on the repeatability and consistency of DTI measurement values in patients with cervical spondylotic radiculopathy (CSR). METHODS: This retrospective study included CSR patients who underwent DTI imaging. The images were analyzed independently by two radiologists. Four delineation methods were used: freehand method, maximum roundness, quadrilateral method, and multi-point averaging method. They re-examined the images 6 weeks later. The intra-class correlation coefficient (ICC) was used to investigate the consistency between the two measurements and the reproducibility between two radiologists. RESULTS: Forty-two CSR patients were included in this study. The distribution of the compressed nerve roots was five C4, eight C5, sixteen C6, eleven C7, and two C8. No differences were found among the four methods in fractional anisotropy (FA) or apparent diffusion coefficient (ADC), irrespective of radiologists (all P>0.05). Similar results were observed between the first and second measurements (all P>0.05), but some significant differences were observed for radiologist 2 for the four-small rounds method (P=0.033). The freehand and single largest circle methods were the two methods with the highest ICC between the two measurements and the two radiologists (all ICC >0.90). CONCLUSION: The freehand and single largest circle methods were the most consistent methods for delineating DTI ROI in patients with CSR.

Biomechanical effects of an oblique lumbar interbody fusion combined with posterior augmentation: a finite element analysis.

BACKGROUND: Oblique lateral interbody fusion (OLIF) is widely used to treat lumbar degenerative disc disease. This study aimed to evaluate the biomechanical stability of OLIF, OLIF including posterior pedicle screw and rod (PSR), and OLIF including cortical screw and rod (CSR) instrumentation through finite element analysis. METHODS: A complete L2-L5 finite element model of the lumbar spine was constructed. Surgical models of OLIF, such as stand-alone, OLIF combined with PSR, and OLIF combined with CSR were created in the L3-L4 surgical segments. Range of motion (ROM), end plate stress, and internal fixation peak stress were compared between different models under the same loading conditions. RESULTS: Compared to the intact model, ROM was reduced in the OLIF model under all loading conditions. The surgical models in order of increasing ROM were PSR, CSR, and stand-alone; however, the difference in ROM between BPS and CSR was less than 0.4° and was not significant under any loading conditions. The stand-alone model had the highest stress on the superior L4 vertebral body endplate under all loading conditions, whereas the end plate stress was relatively low in the BPS and CSR models. The CSR model had the highest internal fixation stress, concentrated primarily at the end of the screw. CONCLUSIONS: OLIF alone significantly reduces ROM but does not provide sufficient stability. Addition of posterior PSR or CSR internal fixation instrumentation to OLIF surgery can significantly improve biomechanical stability of the segment undergoing surgery.

Gene correlation network analysis to identify regulatory factors in sciatic nerve injury.

BACKGROUND: Sciatic nerve injury (SNI), which frequently occurs under the traumatic hip and hip fracture dislocation, induces serious complications such as motor and sensory loss, muscle atrophy, or even disabling. The present work aimed to determine the regulating factors and gene network related to the SNI pathology. METHODS: Sciatic nerve injury dataset GSE18803 with 24 samples was divided into adult group and neonate group. Weighted gene co-expression network analysis (WGCNA) was carried out to identify modules associated with SNI in the two groups. Moreover, differentially expressed genes (DEGs) were determined from every group, separately. Subsequently, co-expression network and protein-protein interaction (PPI) network were overlapped to identify hub genes, while functional enrichment and Reactome analysis were used for a comprehensive analysis of potential pathways. GSE30165 was used as the test set for investigating the hub gene involvement within SNI. Gene set enrichment analysis (GSEA) was performed separately using difference between samples and gene expression level as phenotype label to further prove SNI-related signaling pathways. In addition, immune infiltration analysis was accomplished by CIBERSORT. Finally, Drug-Gene Interaction database (DGIdb) was employed for predicting the possible therapeutic agents. RESULTS: 14 SNI status modules and 97 DEGs were identified in adult group, while 15 modules and 21 DEGs in neonate group. A total of 12 hub genes was overlapping from co-expression and PPI network. After the results from both test and training sets were overlapped, we verified that the ten real hub genes showed remarkably up-regulation within SNI. According to functional enrichment of hub genes, the above genes participated in the immune effector process, inflammatory responses, the antigen processing and presentation, and the phagocytosis. GSEA also supported that gene sets with the highest significance were mostly related to the cytokine-cytokine receptor interaction. Analysis of hub genes possible related signaling pathways using gene expression level as phenotype label revealed an enrichment involved in Lysosome, Chemokine signaling pathway, and Neurotrophin signaling pathway. Immune infiltration analysis showed that Macrophages M2 and Regulatory T cells may participate in the development of SNI. At last, 25 drugs were screened from DGIdb to improve SNI treatment. CONCLUSIONS: The gene expression network is determined in the present work based on the related regulating factors within SNI, which sheds more light on SNI pathology and offers the possible biomarkers and therapeutic targets in subsequent research.

Troxerutin Stimulates Osteoblast Differentiation of Mesenchymal Stem Cell and Facilitates Bone Fracture Healing.

Troxerutin (TRX), a semi-synthetic derivative of the natural bioflavonoid rutin, is a bioactive flavonoid widely abundant in various fruits and vegetables. Known as vitamin P4, TRX has been demonstrated to have several activities including anti-inflammation, anti-oxidants, vasoprotection, and immune support in various studies. Although rutin, the precursor of troxerutin, was reported to have a protective role against bone loss, the function of TRX in skeletal system remains unknown. In the present study, we found that TRX promoted osteogenic differentiation of human mesenchymal stem cells (MSCs) in a concentration-dependent manner by stimulating the alkaline phosphatase (ALP) activity, calcium nodule formation and osteogenic marker genes expression in vitro. The further investigation demonstrated that TRX stimulated the expression of the critical transcription factor ß-catenin and several downstream target genes of Wnt signaling, thus activated Wnt/ß-catenin signaling. Using a femur fracture rats model, TRX was found to stimulate new bone formation and accelerate the fracture healing in vivo. Collectively, our data demonstrated that TRX could promote osteogenesis in vitro and facilitate the fracture healing in vivo, indicating that TRX may be a promising therapeutic candidate for bone fracture repair.

The Wnt pathway regulator expression levels and their relationship to bone metabolism in thoracolumbar osteoporotic vertebral compression fracture patients.

OBJECTIVE: To investigate of the Wnt pathway serum regulator expression levels and their relationship with bone metabolism (BM) in thoracolumbar osteoporotic vertebral compression fracture (OVCF) patients. METHODS: In this study, 40 healthy controls (group A), 33 osteoporotic patients (group B), and 47 thoracolumbar OVCF patients (group C) were recruited as the study cohort during the same period. The Wnt pathway serum regulator levels, bone density, BM-related inflammatory cytokines, bone formation markers, and bone resorption markers were compared among the three groups, and the correlation between the Wnt pathway serum regulators and BM was analyzed. RESULTS: The ß-catenin levels, the BALP, the densities at the femoral neck and lumbar spine, and the PINP, IL-10, OPG and BGP in groups B and C were lower than they were in group A, and the above indices in group C were lower than they were in group B (P < 0.05). Groups B and C showed higher CDKK-1, RANKL, TRACP-5b, ß-CTX, IL-2, IL-6, MMP-2, MMP-9, Leptin, and TNF-α levels than group A, and the above indicators in group C were higher than they were in group B (P < 0.05). A Pearson's correlation analysis showed that the MMP-2, MMP-9, RANKL, ß-CTX, and TRACP-5b levels were negatively correlated with ß-catenin (r < 0, P < 0.05) and were positively correlated with DKK-1 (r > 0, P < 0.05). The BGP, PINP, OPG, and BALP levels were positively correlated with ß-catenin (r > 0, P < 0.05) and were negatively correlated with DKK-1 (r < 0, P < 0.05). CONCLUSION: Patients with thoracolumbar OVCF have abnormal Wnt pathway serum regulator expression levels, low bone density, and abnormal BM, and the patients' Wnt/ß-catenin and DKK-1 levels are closely related to BM, so they may be potential targets for the prevention and treatment of metabolic bone diseases.

Application of Magnetic Resonance Diffusion Tensor Imaging in the Clinical Diagnosis of Disc Herniation after Lumbar Spine Injury.

Magnetic resonance diffusion-weighted imaging (DTI) provides a unique perspective on the pathophysiological and microstructural changes during spinal cord injury, with high spatial specificity; meanwhile, NM reflects the conduction and integrity of neuroelectrical signals in spinal cord fiber tracts, with time-specific and dynamic evaluation effects. The fractional anisotropy (FA) value, SEP amplitude, and neurological function score or improvement rate are correlated. The combination of DTI and NM can more reliably quantify the spinal cord function, evaluate the effectiveness of treatment, and determine the patient's prognosis, which can provide reference for clinical decision making and future research for SCI patients. That is, the lower the preoperative FA value and the lower the SEP amplitude, the worse the preoperative and postoperative neurological function, the lower the improvement rate, and the worse the prognosis of patients. Therefore, we believe that spinal cord function can be graded according to JOA scores to find the corresponding FA and SEP amplitude ranges and that, by measuring FA and SEP amplitude in the future, we can reverse the assessment of spinal cord function, expected postoperative improvement, and long-term prognosis. At the same time, FA values can also help determine the nature of the lesion to some extent.

COL3A1, COL6A3, and SERPINH1 Are Related to Glucocorticoid-Induced Osteoporosis Occurrence According to Integrated Bioinformatics Analysis.

BACKGROUND Glucocorticoid-induced osteoporosis (GIOP) represents the most frequently seen type of secondary osteoporosis, a systemic skeleton disorder. Numerous factors are associated with GIOP occurrence, but there are no specific diagnostic and therapeutic biomarkers for GIOP so far. MATERIAL AND METHODS In this work, gene modules related to GIOP were screened through weighted gene coexpression network analysis. Moreover, protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were carried out for hub genes. In addition, microarray GSE30159 dataset was used as a training set to analyze gene expression within bone biopsy samples from patients with endogenous Cushing's syndrome with GIOP and from normal controls. GSE129228 was used as the test set for investigating the hub gene involvement within GIOP. RESULTS According to our results, the turquoise module showed clinical significance, and 10 genes (COL3A1, POSTN, COL6A3, COL14A1, SERPINH1, ASPN, OGN, THY1, NID2, and TNMD) were discovered to be the "real" hub genes within coexpression as well as PPI networks. GSEA showed that the interaction of extracellular matrix receptors together with the focal adhesion pathway had significant enrichment within samples with high COL3A1 and COL6A3 expression. After the results from both test and training sets were overlapped, SERPINH1 was also significantly altered between GIOP and normal control samples. CONCLUSIONS COL3A1, COL6A3, and SERPINH1 were identified to be the candidate biomarkers for GIOP.

LncRNA HOTTIP enhances human osteogenic BMSCs differentiation via interaction with WDR5 and activation of Wnt/ß-catenin signalling pathway.

To uncover the underlying molecular mechanism of long non-coding RNA in the osteogenic differentiation process of bone marrow mesenchymal stem cells (BMSCs), HOXA transcript at the distal tip (HOTTIP) was selected by using a lncRNA microarray assay. Results showed that HOTTIP was significantly upregulated during osteogenic differentiation of human BMSCs. Downregulation of HOTTIP by shRNA inhibited the osteogenic differentiation of BMSCs. Overexpression of HOTTIP by lentiviral vector promoted human BMSCs osteogenic differentiation by increasing the transcription of ß-catenin. RIP assay and RNA pulldown assay confirmed the interaction between HOTTIP and WDR5, a transcription factor binding to the promoter of ß-catenin. The interaction promoted the translocation of WDR5 into the nucleus and increased the transcription of ß-catenin. Implanted HOTTIP-overexpressing BMSCs increased ectopic bone formation in nude mice. HOTTIP is a conservative long noncoding RNA that is essential for osteogenic differentiation of BMSC. HOTTIP enhances osteogenic differentiation via interaction with WDR5 and up-regulation of ß-catenin gene expression, therefore activating Wnt/ß-catenin signalling pathway.

MiR-27a promotes the autophagy and apoptosis of IL-1ß treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling.

Osteoarthritis (OA) is a common degenerative joint disorder, which involves articular cartilage degeneration as well as joint inflammatory reactions. The recent studies have identified microRNA (miRNA) as one of the epigenetic mechanisms for the regulation of gene expression. Here we aim to reveal the role of miRNA in the regulation of gene expression in articular chondrocytes and its significance in the OA pathogenesis. In the present study, miRNA profiling was performed using OA cartilage and normal healthy cartilage tissues. As compared to their levels in normal cells and tissues, miR-27a expression was found to be upregulated in OA cartilage and IL-1ß-treated articular chondrocytes. TUNEL staining, as well as flow cytometry with Annexin V-FITC/PI double labeling indicated that miR-27a inhibition reduced the apoptosis of IL-1ß-treated articular chondrocytes. Bioinformatics prediction and the dual-luciferase reporter assay indicated that miR-27a targeted the 3'-UTR of the PI3K gene to silence it. The PI3K mRNA level in OA cartilage and IL-1ß-treated articular chondrocytes was also downregulated, comparing with normal cells and tissues. Transfection of chondrocytes transfected with the miR-27a inhibitor upregulated the PI3K expression. This study demonstrated miR-27a is a regulator of the PI3K-Akt-mTOR axis in human chondrocytes and could participate in OA pathogenesis.

STAG2 loss-of-function mutation induces PD-L1 expression in U2OS cells.

BACKGROUND: A tumor suppressor protein, stromal antigen 2 (STAG2), has recurrent mutations or loss of expression in many tumors including in bladder cancer, osteosarcoma (OS), and leukemia. However, the mechanism of STAG2 mutations promoting tumorigenesis is still unclear. METHODS: The distribution of STAG2 mutations in cancer was determined through the COSMIC database; we also generated a STAG2 truncating mutation in OS cell line U2OS cells to mimic a common mutation in OS. CCK-8 assay was employed to evaluate the effect of STAG2 on proliferation and chemo-resistance in OS cells. Cell apoptosis and cell cycle assays were used to assess the effect of STAG2 on apoptosis and the cycle of OS cells. A high throughput RNA sequencing (RNA-Seq) strategy using the Illumina Hiseq 2500 platform was applied to characterize the transcriptome profile from STAG2 knockout and STAG2 WT OS cell lines. RESULTS: We found that STAG2 deficient-cells exhibited reduced cell proliferation and growth; however, they enhanced cell metastasis and invasion, and increased tolerance to chemotherapeutic drugs. We also found that PD-L1, a molecule involved in tumor immune evasion, was up-regulated in the SATG2-lost cells. Expression profile analysis by RNA-seq revealed that there were changes in the expression of many immune-related genes. CONCLUSIONS: Our findings indicated that STAG2 contributes to cell survival and chemo-resistance to cisplatin of OS, suggesting that deletion of STAG2 may promote tumorigenesis by enhancing the immune evasion capacity of cancer cells.

ZIC2 promotes viability and invasion of human osteosarcoma cells by suppressing SHIP2 expression and activating PI3K/AKT pathways.

Osteosarcoma is a malignant tumor of the skeletal system. The zinc finger transcription factor ZIC2 has been reported to be highly expressed in human cancers. The present study evaluated the effects of ZIC2 and the possible underlying mechanisms in the human osteosarcoma cells. The expression levels of ZIC2 in human fetal osteoblastic cell line (hFOB1.19), osteosarcoma cell lines (U-2OS, SaoS2, and MG63), normal bone tissue, and osteosarcoma tumor were analyzed by Western blot, and real-time quantitative RT-PCR (qRT-PCR). Osteosarcoma cells with either overexpressed ZIC2 or suppressed ZIC2 were analyzed to determine cell viability, colony formation, and cell invasion. The expressions of SHIP2 and PI3K/AKT signal pathway-related proteins were analyzed by Western blot and qRT-PCR. We first showed that ZIC2 is highly expressed in osteosarcoma cells and tissues. Then we demonstrated that overexpression of ZIC2 promoted viability, migration, and invasion of osteosarcoma cells, whereas suppression of ZIC2 showed opposite effects. Furthermore, SHIP2 expression was negatively regulated by ZIC2. Importantly, ZIC2 overexpression activated the PI3K/AKT signal pathway; however, overexpressed SHIP2 inhibited these effects. Lastly, we showed that activation of the PI3K/AKT signal pathway is essential for the effects of ZIC2 on osteosarcoma cells, as the effects of ZIC2 on the osteosarcoma cells were reversed by a PI3K/AKT inhibitor. Overall, ZIC2 is highly expressed in osteosarcoma cells and tissues, and its overexpression promotes viability, invasion of osteosarcoma cells via SHIP2 suppression, and PI3K/AKT activation. Thus, ZIC2 can be considered as a novel drug target for osteosarcoma management.

MicroRNA­206 contributes to the progression of steroid­induced avascular necrosis of the femoral head by inducing osteoblast apoptosis by suppressing programmed cell death 4.

The expression of microRNA­206 (miR­206) is aberrantly induced in steroid­induced avascular necrosis of femoral head (SANFH). Therefore, investigating the function of miR­206 in SANFH and uncovering the functional mechanism associated with the condition will promote the understanding and treatment of the disease. The purpose of the present study was to investigate the pro­osteoclasteogenic effect of miR­206 that occurs through regulation of programmed cell death 4 (PDCD4). The expression of miR­206 and PDCD4 was analyzed in the clinical SANFH specimens. The level of miR­206 and PDCD4 was regulated in human osteoblast lineage hFOB1.19 and the effect of different treatments on cell viability, proliferation, apoptosis and differentiation potential of osteoblasts were analyzed with a Cell Counting kit­8, 5­ethynyl­2'­deoxyuridine staining, flow cytometry and Hoechst staining. The expression of miR­206 was upregulated while PDCD4 was downregulated in the SANFH specimens. Induced expression of miR­206 decreased cell viability and proliferation, while apoptosis was induced. At the molecular level, overexpression of miR­206 inhibited the expression of PDCD4, alkaline phosphatase (ALP) and B­cell lymphoma 2 (Bcl­2), and increased the expression of apoptosis regulator Bcl2­X­associated protein (Bax). Inhibiting the expression of miR­206 increased cell viability and proliferation but had no effect on cell apoptosis, as detected by flow cytometry and Hoechst staining. However, at the molecular level, inhibiting the expression of miR­206 induced expression of PDCD4, ALP and Bcl­2, while it decreased the expression of Bax. Additionally, knockdown of PDCD4 blocked the effect of miR­206 inhibition on hFOB1.19 cells, representing a PDCD4­dependent manner of miR­206 in inducing apoptosis of osteoblasts. Therefore, miR­206 promoted the onset of SANFH by inducing apoptosis and suppressed the proliferation of osteoblasts, which was dependent on the inhibition of PDCD4.

TGF-ß1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway.

Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-ß1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-ß1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-ß1-treated LF cells. Cell growth assay revealed that TGF-ß1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-ß1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-ß1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway.

Resistin Promotes Intervertebral Disc Degeneration by Upregulation of ADAMTS-5 Through p38 MAPK Signaling Pathway.

STUDY DESIGN: Rat nucleus pulposus (NP) cells were activated with resistin with or without p38 mitogen-activated protein kinase (MAPK) pathway inhibition. The expression of a disintegrin and metalloprotease with thrombospondin motif-5 (ADAMTS-5), which plays an important role in intervertebral disc degeneration (IDD), was determined. OBJECTIVE: The aim of this study was to demonstrate whether resistin can influence the ADAMTS-5 expression and to further investigate the underlying mechanisms. SUMMARY OF BACKGROUND DATA: Obesity has been demonstrated to promote IDD, whereas the exact mechanism remains poorly understood. Resistin, as an important adipokine, is increased with obesity and has been shown to play pro-inflammatory and catabolic role in cartilage metabolism. However, the effect of resistin on the catabolic enzymes within NP cells remains unknown. METHODS: We exposed NP cells to resistin, and the transcriptional activity, gene expression, and protein levels of ADAMTS-5 were measured by luciferase reporter assay, qRT-polymerase chain reaction, immunofluorescence, and western blot, respectively. The activation of p38 MAPK pathways was detected using western blot analysis. RESULTS: Resistin had no effect on cell viability. Resistin increased ADAMTS-5 expression in rat NP cells time and dose dependently. The p38 MAPK signaling pathway was activated after exposure to resistin. Treatment with p38 inhibitor decreased the upregulation of ADAMTS-5 by resistin. CONCLUSION: The current study, for the first time, investigated the role of resistin in ADAMTS-5 regulation in IDD. These findings provide novel evidence supporting the causative role of obesity in IDD, which is important to develop novel preventative or therapeutic treatment in disc degenerative disorders. LEVEL OF EVIDENCE: N/A.

Polo-like kinase 2 acting as a promoter in human tumor cells with an abundance of TAp73.

BACKGROUND: TAp73, a member of the p53 tumor suppressor family, is frequently overexpressed in malignant tumors in humans. TAp73 abundance and phosphorylation modification result in variations in transcriptional activity. In a previous study, we found that the antitumor function of TAp73 was reactivated by dephosphorylation in head and neck squamous cell carcinomas. Polo-like kinase 2 (PLK2) displayed a close relationship with the p53 family in affecting the fate of cells. Herein, we investigate the hypothesis that PLK2 phosphorylates TAp73 and inhibits TAp73 function. MATERIALS AND METHODS: Head and neck squamous cell carcinoma cell lines and osteosarcoma cell lines were used as natural models of the different expression levels of TAp73. Phosphorylation predictor software Scansite 3.0 and the predictor GPS-polo 1.0 were used to analyze the phosphorylation sites. Coimmunoprecipitation, phosphor-tag Western blot, metabolic labeling, and indirect immunofluorescence assays were used to determine the interactions between PLK2 and TAp73. TAp73 activity was assessed by Western blot and reverse transcription polymerase chain reaction, which we used to detect P21 and PUMA, both downstream genes of TAp73. The physiological effects of PLK2 cross talk with TAp73 on cell cycle progress and apoptosis were observed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. RESULTS: PLK2 binds to and phosphorylates TAp73. PLK2 phosphorylates TAp73 at residue Ser48 and prohibits TAp73 translocation to the nucleus. Additionally, PLK2 inhibition combined with a DNA-damaging drug upregulated p21 and PUMA mRNA expression to a greater extent than DNA-damaging drug treatment alone. Inhibiting PLK2 in TAp73-enriched cells strengthened the effects of the DNA-damaging drug on both G1 phase arrest and apoptosis. Pretreatment with TAp73-siRNA weakened these effects. CONCLUSION: These findings reveal a novel PLK2 function (catalyzed phosphorylation of TAp73) which suppresses TAp73 functions. PLK2 promotes the survival of human tumor cells, a novel insight into the workings of malignant tumors characterized by TAp73 overexpression, and one that could speed the development of therapies.

Effects of sequentially released BMP-2 and BMP-7 from PELA microcapsule-based scaffolds on the bone regeneration.

Osteoinductive biomaterials are helpful for the therapy of large bone defects and provide an alternative to autogenous bone and allografts. Recently, multiple growth factors are delivered to mimic the natural process of bone healing in the bone tissue engineering. Herein, we investigated the effects of sequential released bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-7 (BMP-7) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on the bone regeneration. Through improving the double emulsion/solvent evaporation technique, BMP-7 was encapsulated in PELA microcapsules, to the surface of which BMP-2 was attached. Then, the scaffold (BMP-2/PELA/BMP-7) was fused by these microcapsules with dichloromethane vapor method. In vitro, it sequentially delivered bioactive BMP-2 and BMP-7 and partially imitated the profile of BMPs expression during the fracture healing. To determine the bioactivity of released BMP-2 and BMP-7, alkaline phosphatase (AKP) activity was analyzed in MC3T3-E1 cells. When compared with simple BMP-2 plus BMP-7group and pure PELA group, the AKP activity in BMP-2/PELA/BMP-7 group significantly increased. MTT assay indicated the BMP-loaded PELA scaffold had no adverse effects on cell activity. In addition, the effects of BMP-loaded scaffolds were also investigated in a rat femoral defect model by micro-computed tomographic (mCT) and histological examination. At 4 and 8 weeks post-implantation, BMP-2/PELA/BMP-7 significantly promoted osteogenesis as compared to other groups. The scaffold underwent gradual degradation and replacement by new bones at 8 weeks. Our findings suggest that the sequential release of BMP-2 and BMP-7from PELA microcapsule-based scaffolds is promising for the therapy of bone defects.