Osteobiologies for Spinal Fusion from Biological Mechanisms to Clinical Applications: A Narrative Review.

Degenerative lumbar spinal disease (DLSD), including spondylolisthesis and spinal stenosis, is increasing due to the aging population. Along with the disease severity, lumbar interbody fusion (LIF) is a mainstay of surgical treatment through decompression, the restoration of intervertebral heights, and the stabilization of motion segments. Currently, pseudoarthrosis after LIF is an important and unsolved issue, which is closely related to osteobiologies. Of the many signaling pathways, the bone morphogenetic protein (BMP) signaling pathway contributes to osteoblast differentiation, which is generally regulated by SMAD proteins as common in the TGF-ß superfamily. BMP-2 and -4 are also inter-connected with Wnt/ß-catenin, Notch, and FGF signaling pathways. With the potent potential for osteoinduction in BMP-2 and -4, the combination of allogenous bone and recombinant human BMPs (rhBMPs) is currently an ideal fusion material, which has equalized or improved fusion rates compared to traditional materials. However, safety issues in the dosage of BMP remain, so overcoming current limitations will provide significant advancement in spine surgery. In the future, translational research and the application of clinical study will be important to overcome the current limitations of spinal surgery.

RNF2 regulates Wnt/ß-catenin signaling via TCF7L1 destabilization.

The Wnt signaling pathway is a crucial regulator of various biological processes, such as development and cancer. The downstream transcription factors in this pathway play a vital role in determining the threshold for signaling induction and the length of the response, which vary depending on the biological context. Among the four transcription factors involved in canonical Wnt/ß-catenin signaling, TCF7L1 is known to possess an inhibitory function; however, the underlying regulatory mechanism remains unclear. In this study, we identified the E3 ligase, RNF2, as a novel positive regulator of the Wnt pathway. Here, we demonstrate that RNF2 promotes the degradation of TCF7L1 through its ubiquitination upon activation of Wnt signaling. Loss-of-function studies have shown that RNF2 consistently destabilizes nuclear TCF7L1 and is required for proper Wnt target gene transcription in response to Wnt activation. Furthermore, our results revealed that RNF2 controls the threshold, persistence, and termination of Wnt signaling by regulating TCF7L1. Overall, our study sheds light on the previously unknown degradation mechanism of TCF7L1 by a specific E3 ligase, RNF2, and provides new insights into the variability in cellular responses to Wnt activation.

Transcriptomic Profiling Analysis of Castration-Resistant Prostate Cancer Cell Lines Treated with Chronic Intermittent Hypoxia.

Castration-resistant prostate cancer (CRPC) is still a major concern in men's health, with 375,000 cancer deaths annually. Hypoxia, which is a marked characteristic of advanced solid tumors, has been suggested to induce prostate cancer towards CRPC, metastasis and treatment resistance. To evaluate the effect of hypoxia on prostate cancer, two and five cycles of hypoxia and reoxygenation were administered using 22Rv1 cell lines and denominated as 22Rv1-CI and 22Rv1-PCI, respectively. Cancer cell migration was promoted in 22Rv1-CI compared to controls, and the expression of COL13A1 was significantly up-regulated in 22Rv1-CI according to differentially expressed gene analysis of RNA sequencing among groups. Cancer cell migration was impeded in a wound healing assay after transfecting si-COL13A1. Moreover, the expression of COL13A1 was also higher in the cell line originating from bone metastatic prostate cancer compared to other cell lines. Using the open database GEO, we also confirmed that the expression of COL13A1 was higher in bone metastatic prostate cancer tissue than in localized prostate cancer tissue in patients. Therefore, COL13A1 may be closely related to the bony metastasis of prostate cancer, and our findings may provide valuable information on the pathophysiology of the metastatic niche induced by hypoxia in patients with CRPC.

Genome-wide RNA interference screening reveals a COPI-MAP2K3 pathway required for YAP regulation.

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.

Expression of bladder α1-adrenoceptor subtype after relief of partial bladder outlet obstruction in a rat model.

Purpose: Many patients with benign prostatic hyperplasia require treatment for persistent storage symptoms, even when the obstruction is successfully relieved by surgery. Previous studies identified a characteristic increase in α1D-adrenoceptor levels in the bladder in a bladder outlet obstruction (BOO) model. Here, we investigated the expression of α1-adrenoceptor subtypes in the bladder after relief of partial BOO (pBOO) in a rat model. Materials and Methods: A total of 60 female Sprague-Dawley rats were randomly divided into three groups (sham-operated, pBOO, and pBOO relief groups), and the expression of α1-adrenoceptor subtypes in the urothelium and detrusor muscle tissues was examined by western blot. Results: The expression of the α1D-adrenoceptor was significantly higher in the urothelium and detrusor muscle tissue of the pBOO and pBOO relief groups than in the corresponding tissue of the sham-operated group. Additionally, the α1A-adrenoceptor was predominant in the sham-operated group but significantly decreased in the urothelium in the pBOO group. No significant differences were found in α1A-adrenoceptor levels in detrusor muscle or whole bladder. Conclusions: Our results showed that α1D-adrenoceptor levels were consistently increased with pBOO, even after relief, suggesting that the α1D-adrenoceptor might be a cause of persistent storage symptoms after relief of pBOO.

Synergistic anticancer effect of acteoside and temozolomide-based glioblastoma chemotherapy.

Temozolomide (TMZ) is an alkylating agent commonly used as a first­line treatment for high­grade glioblastoma. However, TMZ has short half­life and frequently induces tumor resistance, which can limit its therapeutic efficiency. In the present study, it was hypothesized that combined treatment with TMZ and acteoside has synergistic effects in glioblastoma therapy. Using cell viability and wound­healing assays, it was determined that this treatment regimen reduced cell viability and migration to a greater extent than either TMZ or acteoside alone. Following previous reports that TMZ affected autophagy in glioma cells, the present study examined the effects of TMZ + acteoside combination treatment on apoptosis and autophagy. The TMZ + acteoside combination treatment increased the cleavage of caspase­3 and levels of B­cell lymphoma 2 (Bcl­2)­associated X protein and phosphorylated p53, and decreased the level of Bcl­2. The combination treatment increased microtubule­associated protein 1 light chain 3 and apoptosis­related gene expression. It was also determined that TMZ + acteoside induced apoptosis and autophagy through the mitogen­activated protein kinase signaling pathway. These findings suggest that acteoside has beneficial effects on TMZ­based glioblastoma therapy.

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202.

BACKGROUND: Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis. METHODS: Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed. RESULTS: OMV secretion was increased > twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which ß-lactamase OXA-23, various proteases, outer membrane proteins, ß-barrel assembly machine proteins, peptidyl-prolyl cis-trans isomerases and inherent prophage head subunit proteins were significantly upregulated. CONCLUSION: In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

Allopurinol Protects against Ischemia/Reperfusion-Induced Injury in Rat Urinary Bladders.

Bladder ischemia-reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS) and markedly elevates the risk of lower urinary tract symptoms (LUTS). Allopurinol is an inhibitor of xanthine oxidase (XO) and thus can serve as an antioxidant that reduces oxidative stress. Here, a rat model was used to assess the ability of allopurinol treatment to ameliorate the deleterious effects of urinary bladder I/R injury. I/R injury reduced the in vitro contractile responses of longitudinal bladder strips, elevated XO activity in the plasma and bladder tissue, increased the bladder levels of tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase, reduced the bladder levels of extracellular regulated kinase (ERK), and decreased and increased the bladder levels of Bcl-2 and Bax, respectively. I/R injury also elevated lipid peroxidation in the bladder. Allopurinol treatment in the I/R injury was generated significantly ameliorating all I/R-induced changes. Moreover, an in situ fluorohistological approach also showed that allopurinol reduces the generation of intracellular superoxides enlarged by I/R injury. Together, the beneficial effects of allopurinol reducing ROS production may be mediated by normalizing the activity of the ERK, JNK, and Bax/Bcl-2 pathways and by controlling TNF-α expression.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Protective effect of N-acetylcysteine against ischemia/reperfusion injury in rat urinary bladders.

Ischemia/reperfusion (I/R) injury represents an important cause of bladder contractile dysfunction. One of the major causes leading to this dysfunction is thought to be reactive oxygen species formation. In this study, we investigated the potential benefit of N-acetylcysteine (NAC), a potent antioxidant that neutralizes free radicals, in a rat model of urinary bladder injury. NAC treatment rescues the reduction of contractile response to I/R injury in a dose-dependent manner. In addition, all levels of reactive oxygen species, lipid peroxidation, and NADPH-stimulated superoxide production in the I/R operation+NAC (I/R+NAC) group also decreased compared with a marked increase in the I/R operation+saline (I/R+S) group. Moreover, an in situ fluorohistological approach also showed that NAC reduces the generation of intracellular superoxides enlarged by I/R injury. Together, our findings suggest that NAC has a protective effect against the I/R-induced bladder contractile dysfunction via radical scavenging property.

Differential expression of MicroRNAs in patients with glioblastoma after concomitant chemoradiotherapy.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, and notorious for resistance to chemoradiotherapy. MicroRNAs (miRNAs) are significantly involved in the initiation and progression of numerous cancers; however, the role of miRNAs in recurrence of tumors remains unknown. Here we tried to identify novel miRNAs that are differentially expressed in recurrent GBM. Tissue samples were obtained from patients with primary and recurrent GBM treated with chemoradiotherapy, and the expression changes of miRNAs were measured by microarray. A total of 318 miRNAs were expressed in the GBM patients. The expression of 43 miRNAs were significantly altered at least 2-fold in primary and recurrent GBMs. Bioinformatic analysis revealed that the differentially expressed miRNAs and their putative target genes were mainly involved in cell death, cellular development, and cellular growth and proliferation, which are the key regulators for stem cells. Pathway analysis supported that the miRNAs may regulate signaling associated with induction and maintenance of cancer and stem cell, such as p53, ErbB1, Notch, Wnt, and TGF-ß signaling pathways. These data suggest that, in recurrent GBM, growth factor and anti-apoptotic signalings for cancer cell growth and proliferation are regulated by miRNAs. Our findings will aid future research in understanding the pathophysiology of recurrent GBM and identifying diagnostic markers and/or therapeutic targets for recurrence of GBM.

Role of PKA as a negative regulator of PCP signaling pathway during Xenopus gastrulation movements.

Convergent extension (CE) movements in gastrulation are essential for the establishment of the body axis during early vertebrate development. Although the precise molecular mechanisms of CE movements are not clearly understood, noncanonical Wnt pathway is known to be important for the control of CE movements. Here, we present evidence that PKA is implicated in noncanonical Wnt pathway. Overexpression and specific depletion of PKA inhibit CE movements. PKA depletion also disrupts cell morphology, protrusive activity, and cortical actin formation in dorsal mesodermal cells. Moreover, PKA activity is negatively regulated by major components of planar cell polarity (PCP) pathway. In line with this, overexpression of PKA can rescue the inhibition of CE movements caused by overexpression of these molecules. We also demonstrate that this regulation of PKA activity is dependent upon Galphai signaling. As a negative component of PCP signaling, PKA inhibits not only the activation of RhoA and JNK but also the Dsh-Daam1-RhoA complex formation which is essential for the regulation of RhoA activity. Together, our study suggests a molecular pathway from Wnt/Dsh/PKA signaling to Rho activation in PCP signaling.