Xinhui Zhu

Overexpression of glucose-regulated protein 94 after spinal cord injury in rats.

Glucose-regulated protein (GRP) 94 is a member of the stress protein family, which is localized in the endoplasmic reticulum (ER). Spinal cord injury (SCI) induced ER stress that results in apoptosis. However, the role of GRP94 in injury of the central nervous system remains unknown. In this study, we performed SCI in adult rats and investigated acutely the protein expression and cellular localization of GRP94 in the spinal cord. Western blot analysis revealed that GRP94 was low in normal spinal cord. It rose at 6h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that GRP94 immunoactivity was expressed at low levels in gray matter and white matter in normal condition and increased after SCI. Double immunofluorescence staining showed that GRP94 was co-expressed with NeuN (neuronal marker), and GFAP (astroglial marker). In addition, caspase-12, caspase-3 and phospho-c-Jun NH2-kinase (p-JNK) levels increased at 6h, peaked at 1day, and then gradually reduced to normal levels for 2 weeks after SCI by western blot analysis. Co-localization of GRP94/caspase-12 and GRP94/p-JNK was detected in neurons and glial cells. Taken together, these data suggest GRP94 involvement in the injury response of the adult spinal cord of the rats.

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.

Expression of SGTA correlates with neuronal apoptosis and reactive gliosis after spinal cord injury

Small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) is a novel TPR-containing protein involved in various biological processes. However, the expression and roles of SGTA in the central nervous system remain unknown. We have produced an acute spinal cord injury (SCI) model in adult rats and found that SGTA protein levels first significantly increase, reach a peak at day 3 and then gradually return to normal level at day 14 after SCI. These changes are striking in neurons, astrocytes and microglia. Additionally, colocalization of SGTA/active caspase-3 has been detected in neurons and colocalization of SGTA/proliferating cell nuclear antigen has been detected in astrocytes and microglial. In vitro, SGTA depletion by short interfering RNA inhibits astrocyte proliferation and decreases cyclinA and cyclinD1 protein levels. SGTA knockdown also reduces neuronal apoptosis. We speculate that SGTA is involved in biochemical and physiological responses after SCI.

Matrix Metalloproteinase-1 (MMP-1) Expression in Rat Spinal Cord Injury Model

Abstract Matrix metalloproteinase-1 (MMP-1), a member of the matrix metalloproteinases family, plays an integral role in extracellular matrix degradation and has been reportedly involved in the regulation of the brain or spinal cord traumatic neurovascular remodeling. Although the critical involvement of MMP-1 in the metastasis of tumors has been extensively documented, the role of MMP-1 in the pathology of neurological diseases remains largely elusive. In the present study, we established an adult rat spinal cord injury (SCI) model and investigated a potential role of MMP-1 in the pathological process of SCI. Using Western blot analysis, we identified notable expression change of MMP-1 after SCI. Immunohistochemistry showed that MMP-1 was distributed widely in rat spinal cord. Double immunofluorescence staining revealed that MMP-1 immunoreactivity was predominantly increased in neurons and astrocytes following SCI. Moreover, after injury, colocalization of MMP-1/active caspase-3 in neurons (NeuN-positive), and colocalization of MMP-1/PCNA in astrocytes (GFAP-positive) were clearly observed. We also examined the protein expression of PCNA, active caspase-3, Bcl-2, and Bax and found that the expression of the proteins was closely correlated with that of MMP-1. Taken together, our findings indicate that MMP-1 might play an important role in the regulation of neuronal apoptosis and astrocyte proliferation after SCI.

C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in osteosarcoma cells

Chemotherapy has significantly improved the prognosis of high-grade osteosarcoma (OS), but over 30% of OS patients can still not be cured. Pemetrexed, the newly-developed anti-folate chemotherapy drug, exerted lower efficacy against OS cells. Here, we aimed to increase pemetrexed efficiency, and found that the cell-permeable short-chain ceramide (C6) significantly enhanced pemetrexed-induced viability reduction and death in cultured OS cell lines (U2OS and MG-63). Pemetrexed induced moderate apoptosis in OS cells, which was dramatically augmented by C6 ceramide. The apoptosis inhibitor z-VAD-fmk largely inhibited C6 ceramide plus pemetrexed-induced cytotoxicity and apoptosis in OS cells. By using pharmacological and siRNA-knockdown strategies, we showed that Akt-mammalian TOR (mTOR) over-activation was an important pemetrexed resistance factor in OS cells, and C6 ceramide-mediated pemetrexed sensitization effect was mediated, at least in part, by Akt-mTOR inhibition. Finally, we found that Akt-S6 Kinase 1 (S6K1, an indicator of mTOR activation) was over-activated in human OS tissues. On the other hand, the osteoblastic MC3T3-E1 cells, which expressed lower Akt-S6K1 phosphorylation, were resistant to pemetrexed and/or C6 ceramide. Together, we conclude that C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in OS cells probably through in-activation of Akt-mTOR signaling.

microRNA-455 targets cullin 3 to activate Nrf2 signaling and protect human osteoblasts from hydrogen peroxide

Over-production of hydrogen peroxide (H2O2) will lead to human osteoblast dysfunction and apoptosis, causing progression of osteoporosis and osteonecrosis. NF-E2-related factor 2 (Nrf2) is a well-characterized anti-oxidant signaling. Cullin 3 (Cul3) ubiquitin E3 ligase dictates Nrf2 degradation. We demonstrate that microRNA-455 (“miR-455”) is a putative Cul3-targeting microRNA. Forced-expression of miR-455 in both hFOB1. 19 osteoblast cell line and primary human osteoblasts induced Cul3 degradation and Nrf2 protein stabilization, which led to subsequent transcription of ARE (anti-oxidant response element)-dependent genes (NQO1, HO1 and GCLC). Cul3 silencing, by expressing miR-455 or targeted-shRNA, protected human osteoblasts from H2O2. Reversely, miR-455 anti-sense caused Cul3 accumulation and Nrf2 degradation, which exacerbated H2O2 damages in hFOB1. 19 cells. Moreover, forced over-expression of Cul3 in hFOB1. 19 cells silenced Nrf2 and sensitized H2O2. Together, we propose that miR-455 activated Nrf2 signaling and protected human osteoblasts from oxidative stress possibly via targeting Cul3.

The β4GalT1 affects the fibroblast-like synoviocytes invasion in rheumatoid arthritis by modifying N-linked glycosylation of CXCR3

OBJECTIVE The level of β-1,4-galactosyltransferase 1 (β4GalT1) is up-regulated in collagen-induced arthritis (CIA) mice. It is reported that CXC chemokine receptor 3 (CXCR3) can enhance the invasiveness of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). This study aims to investigate the specific mechanism of β4GalT1 and relationship between β4GalT1 and CXCR3 in RA. METHODS The model of CIA mice was established to explore the role of β4GalT1. The N-glycosylation of CXCR3 was detected by mass spectrometry and western-blot. The interaction between β4GalT1 and CXCR3 was tested by immunoprecipitation. The truncted MMP-1 was detected by ELISA. Flow cytometry analysis was applied to measure ligand-receptor interaction between CXCR3 and CXCL10. RESULTS β4GalT1 can promote the inflammatory process of arthritis. CXCR3 was N-glycosylated and its glycosylation regulated by β4GalT1. β4GalT1 can enhance the invasiveness of FLS by modifying CXCR3. N-glycosylation of CXCR3 influences the ligand-receptor interaction between CXCR3 and CXCL10. CONCLUSIONS β4GalT1 can regulate N-glycans of CXCR3 in RA. N-glycans of CXCR3 affects CXCL10/CXCR3 ligand-binding which enhancing FLS invasion.

microRNA-25 targets PKCζ and protects osteoblastic cells from dexamethasone via activating AMPK signaling

AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζs 3’ untranslated regions (UTRs). Forced-expression of miR-25 downregulated PKCζ and activated AMPK in human osteoblastic cells (OB-6 and hFOB1.19 lines), which thereafter protected cells from Dex. Reversely, expression of antagomiR-25, the miR-25 inhibitor, upregulated PKCζ and inhibited AMPK activation, exacerbating Dex damages. Notably, PKCζ shRNA knockdown similarly activated AMPK and protected osteoblastic cells from Dex. AMPK activation was required for miR-25-induced osteoblastic cell protection. AMPKα shRNA or dominant negative mutation almost completely blocked miR-25-induced cytoprotection against Dex. Further studies showed that miR-25 expression increased NADPH activity and suppressed Dex-induced oxidative stress in osteoblastic cells. Such effects by miR-25 were abolished with AMPKα knockdown or mutation. Significantly, miR-25-5p level was increased in patients’ necrotic femoral head tissues, which was correlated with PKCζ downregulation and AMPK hyper-activation. These results suggest that miR-25-5p targets PKCζ and protects osteoblastic cells from Dex possibly via activating AMPK signaling.

PDK1 promotes the inflammatory progress of fibroblast-like synoviocytes by phosphorylating RSK2

This study investigated the role of PDK1 in inflammatory response which is initiated by TNF-α and analyzed the association between PDK1 and RSK2. TNF-α were added into MH7A cells to induce inflammation condition. Through overexpressing or suppressing PDK1 in MH7A cells, the role of PDK1 in cell invasiveness and inflammatory factors was determined. Levels of MMPs protein and inflammatory cytokines were assessed with PDK1 siRNA and TNF-α treatment. Inhibition of RSK2 was used to investigate the function of RSK2 on PDK1-induced inflammation. The phosphorylation of RSK2 was detected when PDK1 was inhibited. Luciferase reporter assay was performed to detect the transcriptional activity of NF-κB. We found highly expressed PDK1 could promote cell invasion and secretion of IL-1β and IL-6 in MH7A cells. Inhibition of RSK2 reduced the PDK1-induced cell invasion and cytokines secretion in MH7A cells. In response to TNF-α, PDK1 could phosphorylate RSK2 and activated RSK2, then promoting the activation of NF-κB. This may be a possible therapeutic option of rheumatoid arthritis.

Elevated Expression of β1,4-Galactosyltransferase-I in Cartilage and Synovial Tissue of Patients with Osteoarthritis

Osteoarthritis (OA) is considered a complex illness, characterized by cartilage degeneration, secondary synovial membrane inflammation, and subchondral bone sclerosis. Previous studies have shown β1,4-galactosylransferase-I (β1,4-GalT-I) to be a key inflammatory mediator that participates in the initiation and maintenance of inflammatory reaction in diseases. In the present study, we investigated the expression and possible biological function of β1,4-GalT-I in the cartilage and synovial tissue of OA patients. Cartilage and synovial tissue samples from OA patients and healthy controls were stained for β1,4-GalT-I. Reverse transcription-polymerase chain reaction was used to observe the expression of β1,4-GalT-I, and western blot was carried out for E-selectin. The interaction between β1,4-GalT-I and E-selectin was analyzed by double labeling immunohistochemistry and immunoprecipitation. The expression of β1,4-GalT-I increased in the cartilage and synovial tissue of OA patients compared with healthy controls. E-selectin was overexpressed and was correlated with β1,4-GalT-I in OA cartilage and synovial tissue. These data suggest that β1,4-GalT-I may play an important role in the inflammatory processes in cartilage and synovial tissue of patients with OA.