pLG72 induces superoxide radicals via interaction and aggregation with SOD1.

G72 has been characterised as a susceptibility gene that can have wide-ranging effects in a number of neurodegenerative diseases, including schizophrenia and major depression. Indeed, its product, pLG72, is a potential serum biomarker for schizophrenia. Previous transcriptomic and biochemical studies have indicated that pLG72 may induce the production of mitochondrial reactive oxygen species (ROS), resulting in cell damage. Here, we investigated the mechanism of pLG72 by transfecting a human U87 glioblastoma cell line with a G72 construct. By employing ROS-specific scavengers, we discovered that superoxide radicals were specifically induced in the pLG72-expressing cells. We also found that pLG72 interacted and co-localised with superoxide dismutase 1 (SOD1), resulting in aggregation of SOD1 with a concomitant 23% or 74% reduction of total SOD activity, depending on the amount of G72 transfection plasmid. Finally, we found that transfection of U87 cells with the G72 construct caused a 29% decrease in cell proliferation. The observed loss of SOD1 function in pLG72-expressing cells may explain the elevated ROS levels and inhibition of U87 cell proliferation and has implications for understanding the onset of neurodegenerative diseases in humans.

Pressure Combined with Ischemia/Reperfusion Injury Induces Deep Tissue Injury via Endoplasmic Reticulum Stress in a Rat Pressure Ulcer Model.

Pressure ulcer is a complex and significant health problem in long-term bedridden patients, and there is currently no effective treatment or efficient prevention method. Furthermore, the molecular mechanisms and pathogenesis contributing to the deep injury of pressure ulcers are unclear. The aim of the study was to explore the role of endoplasmic reticulum (ER) stress and Akt/GSK3ß signaling in pressure ulcers. A model of pressure-induced deep tissue injury in adult Sprague-Dawley rats was established. Rats were treated with 2-h compression and subsequent 0.5-h release for various cycles. After recovery, the tissue in the compressed regions was collected for further analysis. The compressed muscle tissues showed clear cellular degenerative features. First, the expression levels of ER stress proteins GRP78, CHOP, and caspase-12 were generally increased compared to those in the control. Phosphorylated Akt and phosphorylated GSK3ß were upregulated in the beginning of muscle compression, and immediately significantly decreased at the initiation of ischemia-reperfusion injury in compressed muscles tissue. These data show that ER stress may be involved in the underlying mechanisms of cell degeneration after pressure ulcers and that the Akt/GSK3ß signal pathway may play an important role in deep tissue injury induced by pressure and ischemia/reperfusion.

[Effects of endoplasmic reticulum stress related proteins and their mediated apoptosis in the formation of deep tissue injury of pressure ulcer in rats].

OBJECTIVE: To explore the effects of endoplasmic reticulum stress (ERS) related proteins and their mediated apoptosis in the formation of deep tissue injury of pressure ulcer in rats. METHODS: Forty male Sprague-Dawley rats were divided into normal control group and groups A, B, C, D according to the random number table, with 8 rats in each group. Rats in group A were loaded with 22.47 kPa pressure with a special pressure apparatus for 2.0 h in the region over gracilis, and then unloaded for 0.5 h. Rats in group B were treated with the same manoeuvre as that in group A for 3 times in one day. Rats in groups C and D were treated with the same manoeuvre as that in group B for 2 and 3 days. Rats in normal control group were free from pressure loading. Rats in groups A, B, C, and D were sacrificed after pressure loading, and then the central part of pressure loaded muscular tissues were harvested for observation of histomorphological change with HE staining; apoptotic nucleoli per millimeter pressure loaded muscular tissue were counted with Hoechst 33258 staining; the levels of binding protein (BIP), protein disulfide isomerase (PDI), C/EBP homologous protein (CHOP), and caspase-12 were assessed with Western blotting (denoted as gray level ratio of target protein to GAPDH). The same parts of gracilis of rats in normal control group were harvested for determination of all the indexes as above. Data were processed with one-way analysis of variance, LSD-t test was applied for paired comparison. RESULTS: (1) Histomorphological observation. Some pathological changes, including inflammatory cell infiltration, myofibers lysis, and vacuolar degeneration, etc. were observed in pressure loaded muscular tissue of rats in groups A, B, C, and D, but not in the same parts of gracilis muscle of rats in normal control group. Compared with those in normal control group [(2.7 ± 1.4) per millimeter muscular tissue], the number of apoptotic nuclei was significantly increased in pressure loaded muscular tissue of rats in groups A, B, C, and D [(14.5 ± 4.4), (11.0 ± 2.9) , (13.8 ± 5.1), (21.3 ± 6.0) per millimeter pressure loaded muscular tissue, with t values from 4.223 to 6.000, P values all below 0.01). (2) Western blotting. The protein expressions of BIP and PDI in rats of normal control group and groups A, B, C, D were respectively 0.64 ± 0.12, 1.20 ± 0.34, 1.59 ± 0.24, 1.17 ± 0.28, 1.44 ± 0.33; 0.48 ± 0.15, 0.61 ± 0.19, 1.23 ± 0.38, 0.37 ± 0.19, 0.29 ± 0.15, and they showed significant statistical difference (with F values respectively 5.32, 7.95, P < 0.05 or P < 0.01). The protein expressions of CHOP and caspase-12 in rats of normal control group and groups A, B, C, D were respectively 0.58 ± 0.18, 1.48 ± 0.27, 1.03 ± 0.21, 0.95 ± 0.30, 1.69 ± 0.34; 0.55 ± 0.12, 1.08 ± 0.31, 0.69 ± 0.24, 1.79 ± 0.20, 2.06 ± 0.47, with significant statistical difference (with F values respectively 8.17, 15.48, P values all below 0.01). CONCLUSIONS: ERS related proteins and their apoptotic pathway may play an important role in the formation of deep tissue injury of pressure ulcer in rats.

[Expressions of tumor necrosis factor-alpha and NF-kappa B in skeletal muscles and its effect on apoptosis in deep tissue injury of rats].

OBJECTIVE: To evaluate the changes of tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) expression in muscle of pressure ulcer rats and explore the relationship with apoptosis. METHODS: Fifty-four male SD rats were randomly divided into nine groups (n = 6), the experiment groups were pressed 9 circles (3 circles/day, 3 days), then observed on the 1st, 3rd, hematoxylin and eosin staining under the microscope; the expression of TNF-alpha was detected by Western blot; the expressions of NF-kappaB and caspase-3 were determined by immunohistochemistry, and evaluated the relationship of TNF-alpha with NF-kappaB and caspase-3; the number of apoptotic cells in compressed muscle tissue was detected by Hoechst 33258 staining under the fluorescence microscope. RESULTS: Compared with the control group, histology examination showed that the tissue structure in experiment groups was in disorder, inter-space was wider, cell edema and the number of inflammatory cells were increased, the tissue was arranged in order and inflammatory cell recruitment was gradually attenuated. The expressions of TNF-alpha, NF-kappaB and caspase-3 were higher in the experiment groups than those in the control group (P < 0.05), reached their peak on the first day, gradually decreased on the 3nd day, but still had a significantly higher level than that in the control group (P < 0.01) on the 7th day; The number of apoptotic cells of experiment groups had a downward trend after the first rise under the fluorescence microscope; the expressions of TNF-alpha and NF-kappaB caspase-3 were found to have positive correlationship (P < 0.05), the expressions of NF-kappaB and caspase-3 were found to have positive correlationship (P < 0.01). CONCLUSION: Apoptosis is closely correlated with inflammation in deep tissue injury of pressure ulcer, NF-kappaB plays a role not only in the formation of inflammation, but also triggering apoptosis, which may induce the pathological change and clinical progress of pressure ulcer.

[Expressions of vascular endothelial growth factor and basic fibroblast growth factor in the late stage of pressure ulcer].

OBJECTIVE: To study the distribution and expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the III-IV stage of pressure ulcer wound, and to explore their correlation with ulceration. METHODS: Forty-one patients hospitalized in the two Affiliated Hospital of Wenzhou Medical College from June 2010 to March 2012 were recruited, including twenty-one patients with 23 pressure ulcer of stage III-IV, 14 acute injury patients, and 6 donors of normal skin. Samples harvested from the 41 patients through surgery were divided into four groups, including pressure ulcer centre group (n = 23), pressure ulcer margin group (n = 23), acute wound group (n = 14), and normal skin group (n = 6). The histological changes in wounds were observed after HE staining. The distribution of collagen fiber in wound was observed with Masson staining. Expressions of VEGF and bFGF in wounds were detected with immunohistochemical staining. Data were processed with independent samples t test and paired samples t test. RESULTS: (1) In the two pressure ulcer groups, large number of inflammatory cells were found in aggregation; the expression of collagen fiber was decreased or disappeared; the positive expressions of VEGF and bFGF were mainly located in fibroblasts and endothelial cells. The expression levels of VEGF and bFGF were respectively 100 ± 39, 132 ± 46 in pressure ulcer centre group, and 228 ± 48, 299 ± 80 in pressure ulcer margin group. The differences between the two pressure ulcer groups were statistically significant (with t values respectively 13.497 and 13.020, P values below 0.01). (2) In acute wound group, a large number of fibroblasts but a small amount of collagen fibers were observed; the positive expressions of VEGF and bFGF were mainly located in fibroblasts, with respective expression levels of 292 ± 59 and 443 ± 194, which were significantly higher than those of the two pressure ulcer groups (with t values from 2.370 to 11.570, P < 0.05 or P < 0.01). (3) In normal skin group, structure of tissue was appropriate, and abundant collagen fibers were observed; the expression levels of VEGF and bFGF were respectively 45 ± 18 and 54 ± 22, which were significantly lower than those of the other three groups (with t values from 3.983 to 14.087, P values all below 0.01). CONCLUSIONS: In contrast with those of the acute wounds, the expression levels of VEGF and bFGF are significantly decreased in the pressure ulcer wound at stage III-IV. It may be closely correlated with the decrease or cessation of the synthesis of collagen fiber.

Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation.

The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.