Inhibition of USP14 enhances anti-tumor effect in vemurafenib-resistant melanoma by regulation of Skp2.

BACKGROUND: The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown. METHODS: In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status. RESULTS: In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.

TLR9 Exerts an Oncogenic Role in Promoting Osteosarcoma Progression Depending on the Regulation of NF-κB Signaling Pathway.

Osteosarcoma (OS) is the most common primary malignant bone tumor and is mainly diagnosed in children. Toll-like receptor 9 (TLR9) is expressed in various tumor cells and was correlated with cancer progression. However, the underlying mechanism of TLR9 on the OS progression remains unclear. Our previous study demonstrated that the expression of TLR9 was positively correlated with the development stage of OS. Herein, we further evaluated the actual roles and the molecular mechanism of TLR9 on regulating OS cell proliferation and metastasis. Our data showed that TLR9 was upregulated in OS cells compared to normal osteoblastic cells, and knockdown of TLR9 inhibited OS cell proliferation and induced cell cycle arrest by the decreased expression of cyclin D1, CDK2, and p-Rb, while TLR9 overexpression exerted the inverse effects. Furthermore, TLR9 overexpression could enhance the migration and invasion activities of the OS cells by the upregulation of matrix metalloproteinases 2 (MMP2) and MMP9, and the opposite result was observed in TLR9-silenced cells. Moreover, the nuclear factor kappa B (NF-κB) signaling pathway was activated by TLR9, and TLR9-induced malignant phenotype of OS cells was abrogated by the NF-κB antagonist BAY11-7082. Our study indicated that TLR9 might play a critical role in facilitating OS progression by activating the NF-κB signaling pathway, which may provide a valuable therapeutic target for OS.

Etiology of Spontaneous Extensor Tendon Injury in Northern-China Rice Farmers.

BACKGROUND: Spontaneous extensor tendon injury is caused by repeated finger flexion and wrist movement. This rare disease has a relatively high incidence in northern China, mostly among rice farmers. This study aimed to elucidate the etiology of spontaneous extensor tendon injury in rice farmers. METHODS: Morphologic changes in the extensor tendons and wrist extensor retinaculum in wrist dorsiflexion were studied via ultrasound examination of the hands of 30 healthy adult men, and 34 patients with a non-rupture extensor tendon injury and 11 patients with spontaneous ruptures of the extensor tendons were also enrolled in the study. The daily workload and potential causes of injuries were assessed. RESULTS: Ultrasound images of healthy male hands showed that during wrist dorsiflexion, the extensor retinaculum of the wrist forms differently shaped trochleas as the dorsiflexion angle changes. Histopathologic examination of the wrist extensor retinaculum revealed that the inner face was abraded evenly, the synovial membrane on the surface of the wrist extensor retinaculum disappeared, the internal coarse fibers were exposed and there was fibrous debris, suggesting that dry friction occurred before the rupture. CONCLUSIONS: From clinical observation it could be concluded that the severity and progress of swelling and pain are related to the force applied during rice transplantation as abrasions were found at the front of the wrist extensor retinaculum.

Synovial fibroblast-targeting liposomes encapsulating an NF-κB-blocking peptide ameliorates zymosan-induced synovial inflammation.

Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF-κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF-targeting liposomal system that encapsulates the NF-κB-blocking peptide (NBD peptide) HAP-lipo/NBD. HAP-lipo/NBDs demonstrated efficient SF-specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP-lipo/NBD on NF-κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP-lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF-targeting NF-κB-blocking strategy is a potential approach for the development of alternative, targeted anti-RA therapies.

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice.

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion.

Assessment of the degradation rates and effectiveness of different coated Mg-Zn-Ca alloy scaffolds for in vivo repair of critical-size bone defects.

Surgical repair of bone defects remains challenging, and the search for alternative procedures is ongoing. Devices made of Mg for bone repair have received much attention owing to their good biocompatibility and mechanical properties. We developed a new type of scaffold made of a Mg-Zn-Ca alloy with a shape that mimics cortical bone and can be filled with morselized bone. We evaluated its durability and efficacy in a rabbit ulna-defect model. Three types of scaffold-surface coating were evaluated: group A, no coating; group B, a 10-µm microarc oxidation coating; group C, a hydrothermal duplex composite coating; and group D, an empty-defect control. X-ray and micro-computed tomography(micro-CT) images were acquired over 12 weeks to assess ulnar repair. A mechanical stress test indicated that bone repair within each group improved significantly over time (P < 0.01). The degradation behavior of the different scaffolds was assessed by micro-CT and quantified according to the amount of hydrogen gas generated; these measurements indicated that the group C scaffold better resisted corrosion than did the other scaffold types (P < 0.05). Calcein fluorescence and histology revealed that greater mineral densities and better bone responses were achieved for groups B and C than for group A, with group C providing the best response. In conclusion, our Mg-Zn-Ca-alloy scaffold effectively aided bone repair. The group C scaffold exhibited the best corrosion resistance and osteogenesis properties, making it a candidate scaffold for repair of bone defects.

Development and performance analysis of Si-CaP/fine particulate bone powder combined grafts for bone regeneration.

BACKGROUND: Although autogenous bone grafts as well as several bone graft substitute material have been used for some time, there is high demand for more efficient and less costly bone-substitute materials. Silicon-substituted calcium phosphates (Si-CaP) and fine particulate bone powder (FPBP) preparations have been previously shown to individually possess many of the required features of a bone graft substitute scaffold. However, when applied individually, these two materials fall short of an ideal substitute material. We investigated a new concept of combining Si-CaP with FPBP for improved performance in bone-repair. METHODS: We assessed Si-CaP/FPBP combined grafts in vitro, by measuring changes in pH, weight loss, water absorption and compressive strength over time. RESULTS: Si-CaP/FPBP combined grafts was found to produce conditions of alkaline pH levels compared to FPBP, and scaffold surface morphology conducive to bone cell adhesion, proliferation, differentiation, tissue growth and transport of nutrients, while maintaining elasticity and mechanical strength and degradation at a rate closer to osteogenesis. CONCLUSION: Si-CaP/FPBP combined grafts was found to be superior to any of the two components individually.

Titanium surfaces loaded with puerarin and exosomes derived from adipose stem cells promote the proliferation and differentiation of pre-osteoblasts.

The study is to evaluate the effects of collagen/hyaluronic acid coating with or without puerarin and exosomes (Exos) derived from adipose stem cells (ADSCs-Exos) on pre-osteoblast proliferation and differentiation on the surface of titanium materials. Titanium materials with different coatings were prepared by layer-by-layer technique, evaluating the surface characterization. Cell functions were assessed by cell biology experiments. Related genes and proteins were assessed by RT-qPCR and Western blot. Puerarin or ADSCs-Exos coating had better effects on promoting the adhesion, proliferation and differentiation of pre-osteoblasts, and the strongest effect was found after their co-coatings, manifesting as the up-regulations of alkaline phosphatase (ALP) activity, collagen type I alpha 1 (Col1a1), runt-related transcription factor 2 (Runx2), osterix and activating transcription factor-2 (ATF-2). Levels of phosphorylated-P38 (p-P38) and p-ATF-2 were up-regulated in pre-osteoblasts grown on puerarin and ADSCs-Exos-loaded titanium surfaces. Titanium surfaces loaded with puerarin and ADSCs-Exos promotes the proliferation and differentiation of pre-osteoblasts.

MitoCur-1 induces ferroptosis to reverse vemurafenib resistance in melanoma through inhibition of USP14.

Melanoma is an aggressive malignant tumor with a poor prognosis. Vemurafenib (PLX4032, vem) is applied to specifically treat BRAF V600E-mutated melanoma patients. However, prolonged usage of vem makes patients resistant to the drug and finally leads to clinical failure. We previously tested the combination regimen of tubulin inhibitor VERU-111 with vem, as well as USP14 selective inhibitor b-AP15 in combination with vem, both of which have showed profound therapeutic effects in overcoming vem resistance in vitro and in vivo. Most importantly, we discovered that vem-resistant melanoma cell lines highly expressed E3 ligase SKP2 and DUB enzyme USP14, and we have demonstrated that USP14 directly interacts and stabilizes SKP2, which contributes to vem resistance. These works give us a clue that USP14 might be a promising target to overcome vem resistance in melanoma. MitoCur-1 is a curcumin derivative, which was originally designed to specifically target tumor mitochondria inducing redox imbalance, thereby promoting tumor cell death. In this study, we have demonstrated that it can work as a novel USP14 inhibitor, and thus bears great potential in providing an anti-tumor effect and sensitizing vem-resistant cells by inducing ferroptosis in melanoma. Application of MitoCur-1 dramatically induces USP14 inhibition and inactivation of GPX4 enzyme, meanwhile, increases the depletion of GSH and decreases SLC7A11 expression level. As a result, ferrous iron-dependent lipid ROS accumulated in the cell, inducing ferroptosis, thus sensitizes the vem-resistant melanoma cell. Interestingly, overexpression of USP14 antagonized all the ferroptosis cascade events induced by MitoCur-1, therefore, we conclude that MitoCur-1 induces ferroptosis through inhibition of USP14. We believe that by inhibition of USP14, vem resistance can be reversed and will finally benefit melanoma patients in future.

Efficacy and safety of a "radical" surgical strategy in the treatment of parasagittal sinus meningioma.

Background: No standardized criteria for surgical resection of parasagittal sinus meningiomas (PSM) have been established, and different surgical strategies have been proposed. The aim of the present study was to investigate the efficacy and safety of a "radical" surgical strategy in the treatment of PSM. Methods: The clinical histories, radiological findings, pathologic features, and surgical records of 53 patients with PSM admitted by the same surgical team using the "radical" surgical strategy were retrospectively analyzed between 2018 and 2023. Results: Among the 53 PSM cases, 16 (30.2%) had a patent sinus proper, 28 (52.8%) had partial obstruction of the sinus proper, and 9 (17.0%) had complete obstruction of the sinus proper before the operation. During operation, Simpson grade I resection was performed in 34 (64.2%) cases and Simpson grade II in 19 (35.8%) cases. Postoperative pathologic examination suggested tumors of WHO grade I in 47 (88.7%) cases, WHO grade II in 4 (7.5%) cases, and WHO grade III in 2 (3.8%) cases. Postoperative complications primarily included a small amount of delayed intracerebral hemorrhage in 3 (5.7%) cases, exacerbation of cerebral edema in 3 (5.7%) cases, exacerbation of motor and sensory deficits in 4 (7.5%) cases, and intracranial infection in 2 (3.8%) cases. There were no cases of death or new-onset neurological dysfunction. Dizziness and headache symptoms improved to varying degrees, and a seizure-free status was achieved postoperatively. Excluding one case lost to follow-up, the average follow-up period was 33 months, and there were no cases of recurrence. Conclusion: A "radical" strategy for the surgical management of PSM is effective, safe, and simple to perform, provided that the sagittal sinus is properly managed and its associated veins are protected.

Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds.

OBJECTIVE: To investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats. METHODS: The subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin-eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN). RESULTS: Upon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups. CONCLUSIONS: The synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

Clinical and Radiographic Features of the Atlantoaxial Dislocation Associated With Kashin-Beck Disease.

OBJECTIVES: Keshin-Beck disease (KBD) is a particular type of osteoarthritis that affects many joints. However, the deformity of atlantoaxial joint has been rarely reported in KBD, and therefore its clinical and radiograph features have not been identified. METHODS: We reviewed data in 14 patients who were diagnosed with atlantoaxial dislocation (AAD) in KBD at our institution. The demographic data, clinical history, imaging data, operative data, and Japanese Orthopaedic Association score were collected for evaluation. RESULTS: The mean age at presentation was 50 ± 1.7 years old. The most common features of AAD in KBD were the osteoarthritis, characterized by hypertrophic dens and anterior arch of the atlas. The average inner anteroposterior diameter (IAPD) of C1 was 28 ± 3.5 mm and the average spinal canal diameter was 14 ± 3.3 mm, which were respectively lower than the control level. Five patients had severe C1 stenosis (IAPD < 26mm). Separated odontoid process, like os odontoideum, was seen 9 patients. The tip of dens fused to C1 was observed in 4 patients; 12 patients had high-riding vertebral artery; and 5 patients had severe C1 stenosis, and they underwent C1 laminectomy with C1-C2 interarticular fusion or occipital-cervical fusion. All the patients displayed neurologic improvement after surgery. CONCLUSIONS: The atlantoaxial level could be affected by KBD, which may lead to typical abnormalities and cause AAD. A C1 laminectomy with an C1-C2 interarticular fusion or occipital-cervical fusion is recommended for the patient with severe stenosis.

Nuclear Receptor PXR Confers Irradiation Resistance by Promoting DNA Damage Response Through Stabilization of ATF3.

Low response rate to radiotherapy remains a problem for liver and colorectal cancer patients due to inappropriate DNA damage response in tumors. Here, we report that pregnane X receptor (PXR) contributes to irradiation (IR) resistance by promoting activating transcription factor 3 (ATF3)-mediated ataxia-telangiectasia-mutated protein (ATM) activation. PXR stabilized ATF3 protein by blocking its ubiquitination. PXR-ATF3 interaction is required for regulating ATF3, as one mutant of lysine (K) 42R of ATF3 lost binding with PXR and abolished PXR-reduced ubiquitination of ATF3. On the other hand, threonine (T) 432A of PXR lost binding with ATF3 and further compromised ATM activation. Moreover, the PXR-ATF3 interaction increases ATF3 stabilization through disrupting ATF3-murine double minute 2 (MDM2) interaction and negatively regulating MDM2 protein expression. PXR enhanced MDM2 auto-ubiquitination and shortened its half-life, therefore compromising the MDM2-mediated degradation of ATF3 protein. Structurally, both ATF3 and PXR bind to the RING domain of MDM2, and on the other hand, MDM2 binds with PXR on the DNA-binding domain (DBD), which contains zinc finger sequence. Zinc finger sequence is well known for nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) playing E3 ligase activity to degrade nuclear factor κB (NFκB)/p65. However, whether zinc-RING sequence grants E3 ligase activity to PXR remains elusive. Taken together, these results provide a novel mechanism that PXR contributes to IR resistance by promoting ATF3-mediated ATM activation through stabilization of ATF3. Our result suggests that targeting PXR may sensitize liver and colon cancer cells to IR therapy.

Bioinformatic Data Mining for Candidate Drugs Affecting Risk of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) in Cancer Patients.

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) leads to significant morbidity. Other coadministered drugs may modulate the risk for BRONJ. The present study aimed to leverage bioinformatic data mining to identify drugs that potentially modulate the risk of BRONJ in cancer. Methods: A GEO gene expression dataset of peripheral blood mononuclear cells related to BRONJ in multiple myeloma patients was downloaded, and differentially expressed genes (DEGs) in patients with BRONJ versus those without BRONJ were identified. A protein-protein interaction network of the DEGs was constructed using experimentally validated interactions in the STRING database. Overrepresented Gene Ontology (GO) molecular function terms and KEGG pathways in the network were analysed. Network topology was determined, and 'hub genes' with degree ≥2 in the network were identified. Known drug targets of the hub genes were mined from the 'drug gene interaction database' (DGIdb) and labelled as candidate drugs affecting the risk of BRONJ. Results: 751 annotated DEGs (log FC ≥ 1.5, p < 0.05) were obtained from the microarray gene expression dataset GSE7116. A PPI network with 633 nodes and 168 edges was constructed. Data mining for drugs interacting with 49 gene nodes was performed. 37 drug interactions were found for 9 of the hub genes including TBP, TAF1, PPP2CA, PRPF31, CASP8, UQCRB, ACTR2, CFLAR, and FAS. Interactions were found for several established and novel anticancer chemotherapeutic, kinase inhibitor, caspase inhibitor, antiangiogenic, and immunomodulatory agents. Aspirin, metformin, atrovastatin, thrombin, androgen and antiandrogen drugs, progesterone, Vitamin D, and Ginsengoside 20(S)-Protopanaxadiol were also documented. Conclusions: A bioinformatic data mining strategy identified several anticancer, immunomodulator, and other candidate drugs that may affect the risk of BRONJ in cancer patients.

Mild Hypothermia Protects Brain Injury After Intracerebral Hemorrhage in Mice Via Enhancing the Nrdp1/MyD88 Signaling Pathway.

BACKGROUND: Mild hypothermia has been identified to reduce brain injury following intracerebral hemorrhage (ICH) by protecting neuron cells through several pathways. However, the role of hypothermia in brain function following ICH and the related mechanisms have not been well identified. Ubiquitination-mediated inflammation plays important roles in the pathogenesis of immune diseases. The experiment analyzed anti-inflammatory effects of mild hypothermia following ICH. METHODS: The model of ICH was induced by injecting autologous blood. Neuregulin receptor degradation protein-1 (Nrdp1) and downstream molecule were analyzed. In addition, brain inflammatory response, brain edema, and neurological functions of ICH mice were also assessed. RESULTS: We found that mild hypothermia attenuated proinflammatory factors production after ICH. Mild hypothermia significantly inhibited BBB injury, water content, and neurological damage following ICH in vivo. Moreover, mild hypothermia also increased Nrdp1/MyD88 levels and thus affect neuronal apoptosis and inflammation. CONCLUSIONS: Taken together, these results suggest that mild hypothermia can attenuate the neuroinflammatory response and neuronal apoptosis after ICH through the regulation of the Nrdp1 levels.

Combination of Paclitaxel and PXR Antagonist SPA70 Reverses Paclitaxel-Resistant Non-Small Cell Lung Cancer.

Paclitaxel (PTX) is one of the most efficient drugs for late-stage non-small cell lung cancer (NSCLC) patients. However, most patients gradually develop resistance to PTX with long-term treatments. The identification of new strategies to reverse PTX resistance in NSCLC is crucially important for the treatment. PTX is an agonist for the pregnane X receptor (PXR) which regulates PTX metabolism. Antagonizing PXR, therefore, may render the NSCLC more sensitive to the PTX treatment. In this study, we investigated the PXR antagonist SPA70 and its role in PTX treatment of NSCLC. In vitro, SPA70 and PTX synergistically inhibited cell growth, migration and invasion in both paclitaxel-sensitive and paclitaxel-resistant A549 and H460 lung cancer cells. Mechanistically, we found PTX and SPA70 cotreatment disassociated PXR from ABCB1 (MDR1, P-gp) promoter, thus inhibiting P-gp expression. Furthermore, the combination regimen synergistically enhanced the interaction between PXR and Tip60, which abrogated Tip60-mediated α-tubulin acetylation, leading to mitosis defect, S-phase arrest and necroptosis/apoptosis. Combination of PXT and SPA70 dramatically inhibited tumor growth in a paclitaxel-resistant A549/TR xenograft tumor model. Taken together, we showed that SPA70 reduced the paclitaxel resistance of NSCLC. The combination regimen of PTX and SPA70 could be potential novel candidates for the treatment of taxane-resistant lung cancer.

Proline-Serine-Threonine Phosphatase-Interacting Protein 2 Alleviates Diabetes Mellitus-Osteoarthritis in Rats through Attenuating Synovial Inflammation and Cartilage Injury.

OBJECTIVE: To explore the possible way of proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) influencing diabetes mellitus-osteoarthritis (DM-OA) progression. METHODS: In vivo, eight-week-old male Sprague Dawley rats were induced with DM-OA by intraperitoneal injection of streptozotocin with high-fat diet feeding and intra-articular injection of monoiodoacetate. PSTPIP2 overexpression was achieved by intra-articular injection of lentivirus vectors. PSTPIP2 expression was verified by real-time polymerase chain reaction and Western blotting. Histological changes were examined by hematoxylin/eosin and safranin-O/fast-green staining. In vitro, rat synovial fibroblasts were induced DM-OA by stimulation of high glucose (HG) and interleukin (IL)-1ß. PSTPIP2 overexpression was achieved by lentivirus infection. U0126 was added as an ERK inhibitor. Levels of tumor necrosis factor (TNF)-α, IL-6, and IL-1ß were detected using enzyme-linked immunosorbent assay. Expression of matrix metalloproteinase (MMP)-3, MMP-13, aggrecanase-2 (ADAMTS-5), intercellular cell adhesion molecule (ICAM)-1, extracellular regulated protein kinase (ERK) and phospho-ERK (p-ERK) was detected by Western blotting. RESULTS: In DM-OA rats, PSTPIP2 relative messenger RNA (mRNA) level was significantly decreased compared to control rats. The protein expression was also decreased obviously. Inflammation score in synovium was dramatically increased, accompanying with increased TNF-α, IL-6, and IL-1ß levels. Osteoarthritis research society international (OARSI) score in cartilage was markedly increased, along with increased MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK expression. In PSTPIP2-overexpressed DM-OA rats, PSTPIP2 mRNA level and protein expression was increased compared to DM-OA rats received negative-control lentivirus vectors. The inflammation score, as well as TNF-α, IL-6, and IL-1ß levels were dramatically decreased. Also, the OARSI score and protein expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were decreased. In HG+IL-1ß-treated rat synovial fibroblasts, PSTPIP2 protein expression was decreased compared to normal glucose (NG)-treated cells. Levels of TNF-α, IL-6, and IL-1ß, as well as expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were increased. After cells were infected with PSTPIP2-overexpressed lentivirus, levels of TNF-α, IL-6, and IL-1ß, and expression of MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK were obviously decreased compared to cells infected with NC lentivirus. In addition, ERK inhibitor U0126 treatment also decreased the TNF-α, IL-6, and IL-1ßlevels and MMP-3, MMP-13, ADAMTS-5, ICAM-1, ERK and p-ERK expression in HG + IL-1ß treated rat synovial fibroblasts. CONCLUSION: Overexpression of PSTPIP2 alleviates synovial inflammation and cartilage injury during DM-OA progression via inhibiting ERK phosphorylation.

Dendritic cells transduced with TIPE-2 recombinant adenovirus induces T cells suppression.

INTRODUCTION: TIPE-2 has been identified as a negative regulator of both innate and adaptive immunity and is involved in several inflammatory diseases. However, the role of immune suppression of dendritic cells (DCs) transduced with TIPE-2 has not been well studied. METHODS: In this study, DCs were transduced with TIPE-2 recombinant adenovirus, and then were cocultured with allogeneic CD4+ or CD8 + T cells. The proliferation, cytokine production and activation marker levels of CD4+ or CD8 + T cell were detected. RESULTS: The data demonstrated that T cell proliferation, cytokine production and activation marker levels were attenuated after treated with TIPE-2 transduced DCs. CONCLUSIONS: These results suggested that TIPE-2 transduced DCs are capable of inducing allogeneic CD4+ or CD8 + T cell immune suppression, which provide a promising way for the therapeutical strategies of transplantation or autoimmune diseases.

The miR-455-5p/ERα36 axis regulates mammalian neuronal viability and axonal regeneration.

Estrogen signal transduction plays an important role in regulating neuronal cell growth and axonal regeneration. Estrogen receptor alpha 36 (ERα36) is a truncated isoform of the estrogen receptor that inhibits aspects of estrogen transduction. Here, we investigated the role of ERα36 in neuronal viability as well as axonal growth and regeneration using SH-SY5Y neuroblastoma cells and murine sensory neurons. We identified an ERα36-targeting microRNA, miR-455-5p, that negatively regulates ERα36 levels. miR-455-5p inhibition increased cell viability, PCNA, cyclin D1, and Bcl-2 levels and decreased apoptosis and Bax levels in SH-SY5Y neuroblastoma cells. Moreover, miR-455-5p inhibition upregulated the phosphorylation of the intracellular signaling mediators MEK, ERK, Akt, and mTOR in SH-SY5Y neuroblastoma cells. miR-455-5p inhibition promoted axonal growth and regeneration and downregulated activation of the glycogen synthase kinase-3ß (GSK3ß)/Tau protein pathway in murine sensory neurons. ERα36 silencing was sufficient to reverse the phenotypic characteristics produced by miR-455-5p inhibition, suggesting that ERα36 is mechanistically linked to miR-455-5p. We provide additional mechanistic evidence showing that GSK3ß is the mediator of miR-455-5p/ERα36-induced axonal growth. Together, these findings elucidate a novel miR-455-5p/ERα36 axis that regulates mammalian neuronal viability and axonal regeneration.

Heme Induces BECN1/ATG5-Mediated Autophagic Cell Death via ER Stress in Neurons.

Intracerebral hemorrhage (ICH) is a serious medical problem, and effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme, which is mainly released from hemoglobin, plays a vital role in neurotoxicity. However, the specific mechanism involved in heme-mediated neurotoxicity has not been well studied. In this study, we investigated the neurotoxicity of heme in neurons. Neurons were treated with heme, and cell death, autophagy, and endoplasmic reticulum (ER) stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also assessed. We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacological inhibitors (3-methyladenine) or RNA interference of essential autophagy genes (BECN1 and ATG5) decreased heme-induced cell death in neurons. Moreover, the ER stress activator thapsigargin increased cell autophagy and the cell death ratio following heme treatment. Autophagy promoted heme-induced cell apoptosis and cell death through the BECN1/ATG5 pathway. Our findings suggest that heme potentiates neuronal autophagy via ER stress, which in turn induces cell death via the BECN1/ATG5 pathway. Targeting ER stress-mediated autophagy might be a promising therapeutic strategy for ICH.