In this communication, we examined the role of the MAP kinase pathway in the G2/M phase of the cell cycle. Activation of the Plk1 and MAP kinase pathways was initially evaluated in FT210 cells, which arrest at G2 phase at the restrictive temperature (391C), due to a mutation in the cdc2 gene. Previous studies had shown that these cells enter mitosis at the nonpermissive temperature upon incubation with okadaic acid, a protein phosphatase 1 and 2A inhibitor. We show that treatment of FT210 cells at 391C with okadaic acid activated Plk1, as shown by hyperphosphorylation and elevated protein kinase activity, and also induced activation of the MAP kinase pathway. The specific Mek inhibitor PD98059 antagonized the okadaic acid-induced activation of both Plk1 and MAP kinases. This suggests that activation of the MAP kinase pathway may contribute to the okadaic acid-induced activation of Plk1 in FT210 cells at 391C. We also found that PD98059 strongly attenuated progression of HeLa cells through mitosis, and active Mek colocalizes with Plk1 at mitotic structures. To study the potential function of the MAP kinase pathway during mitosis, RNAi was used to specifically deplete five members of this pathway (Raf1, Mek1/2, Erk1/2). Each of these five protein kinases is required for cell proliferation and survival, and depletion of any of these proteins eventually leads to apoptosis. Treatment with Mek inhibitors also inhibited cell proliferation and caused apoptosis. A dramatic increase of Plk1 activities and a moderate increase of Cdc2 activities in Raf1-depleted cells indicate that Raf1-depleted cells arrest in the late G2 or M phase. Mek1 and Erk1 depletion also caused cell cycle arrest at G2, suggesting that these enzymes are required for the G2/M transition, whereas the loss of Mek2 or Erk2 caused arrest at G1.
Persistent inflammatory response in the diabetic wound impairs the healing process, resulting in significant morbidity and mortality. Mounting evidence indicate that the activation of Nod-like receptor protein (NLRP) 3 inflammasome in macrophages (MΦ) contributes to the sustained inflammatory response and impaired wound healing associated with diabetes. However, the main trigger of NLRP3 inflammasome in the wounds is not known. Neutrophils, as sentinels of the innate immune system and key stimulators of MΦ, are immune cells that play the main role in the early phase of healing. Neutrophils release extracellular traps (NETs) as defense against pathogens. On the other hand, NETs induce tissue damage. NETs have been detected in the diabetic wound and implicated in the impaired healing process, but the mechanism of NETs suspend wound healing and its role in fostering inflammatory dysregulation are elusive. Here, we report that NLRP3 and NETs production are elevated in human and rat diabetic wounds. NETs overproduced in the diabetic wounds triggered NLRP3 inflammasome activation and IL-1β release in MΦ. Furthermore, NETs up-regulated NLRP3 and pro-IL-1β levels via the TLR-4/TLR-9/NF-κB signaling pathway. They also elicited the generation of reactive oxygen species, which facilitated the association between NLRP3 and thioredoxin-interacting protein, and activated the NLRP3 inflammasome. In addition, NET digestion by DNase I alleviated the activation of NLRP3 inflammasome, regulated the immune cell infiltration, and accelerated wound healing in diabetic rat model. These findings illustrate a new mechanism by which NETs contribute to the activation of NLRP3 inflammasome and sustained inflammatory response in the diabetic wound.
Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclindependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30 ± 40 population doublings cells became growtharrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point de®ned as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescenceassociated b-galactosidase (SA-b-gal
Objective. Studies were performed to determine if p53 mutations identified in rheumatoid arthritis (RA) synovial tissue are dominant negative.Methods. Site-directed mutagenesis was used to produce 2 RA-derived mutants: asparagine3serine at codon 239 (N239S) and arginine3stop at codon 213 (R213*). HS68 dermal fibroblasts were transfected with either empty vector, wild-type p53 cDNA (wt), or the N239S or R213* mutant p53 cDNA clones. Interleukin-6 (IL-6) and bax gene expression were determined by Northern blot analysis. Bax transcription was determined using a bax promoter/reporter gene construct (bax-luc).Results. Transfection of HS68 cells with wt increased bax mRNA levels. This process was blocked by cotransfection with either mutant. The mutant p53 genes also increased IL-6 gene expression. Low levels of bax promoter activity were detected in HS68 cells cotransfected with bax-luc and empty vector, N239S, or R213*, indicating that the RA mutants lacked transcriptional activity. Transfection with wt and bax-luc led to a 10-fold increase in luciferase expression. When the wt gene was cotransfected with either of the mutants, there was a dose-dependent inhibition of bax promoter activity.Conclusion. These data indicate that at least 2 of the p53 mutants identified in RA joint samples are dominant negative and suppress endogenous wild-type p53 function.
Objective. The p53 tumor suppressor gene is overexpressed in synovial tissue (ST) from patients with longstanding rheumatoid arthritis (RA), and may contain somatic mutations. The aim of this study was to determine p53 expression in ST from RA patients in different stages of the disease, compared with disease controls. Methods. ST biopsy specimens were obtained from the knee joints of 31 RA patients in varying disease phases, 8 patients with reactive arthritis (ReA), 10 patients with inflammatory osteoarthritis (OA), and 6 control patients (4 with meniscus pathology, 2 with vascular insufficiency). ST was also obtained from the clinically uninvolved knee joints of 9 RA patients. Expression of p53 was determined by immunohistology with DO1 monoclonal antibody (mAb) in all patients and by Western blot analysis with DO7 mAb in a subgroup of the patients. Results. The p53 protein was detected by immu-nohistology in 10 of the 13 patients with early RA (duration <6 months) and in 12 of the 14 patients with longstanding RA (duration >5 years). The p53 protein was also demonstrated in clinically uninvolved knee joints. Western blots revealed immunoreactive p53 in ST extracts from all RA patients. Expression of p53 was about twice as high in ST from patients with longstand-ing RA as in early RA samples, but the difference did not reach statistical significance. Small amounts of p53 were also detected in ST from ReA and OA patients, although the expression in RA synovium was significantly higher. Immunohistologic analysis of normal ST gave negative results for p53. Conclusion. This study shows that p53 overex-pression is specific for RA, compared with OA and ReA. This phenomenon is probably secondary to increased production of wild-type p53 protein in response to DNA damage and secondary to somatic mutations caused by the genotoxic local environment in inflamed ST. Of interest, p53 overexpression can also be found in the earliest stages of RA and in clinically uninvolved joints.
Oxaliplatin is included in a number of effective combination regimens used as first and subsequent lines of therapy for metastatic colorectal cancer. Accumulating evidence indicates that autophagy plays a significant role in response to cancer therapy. However, the role of autophagy in oxaliplatin-induced cell death remains to be clarified. In this study, we showed that oxaliplatin induced cell death and autophagy in Caco-2 colorectal cancer cells. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or Beclin1) enhanced the cell death and reactive oxygen species (ROS) production induced by oxaliplatin in Caco-2 cells. Blocking oxaliplatin-induced ROS production by using ROS scavengers (NAC or Tiron) decreased autophagy. Furthermore, numerous dilated endoplasmic reticula (ER) were present in oxaliplatin-treated Caco-2 cells, and blocking ER stress by RNA interference against candidate of metastasis-1 (P8) and C/EBP-homologous protein (CHOP) decreased autophagy and ROS production. Taken together, these data indicate that oxaliplatin activates autophagy as a cytoprotective response via ER stress and ROS in human colorectal cancer cells.
Acute lung injury (ALI) is the leading cause of death in sepsis patients. Exosomes participate in the occurrence and development of ALI by regulating endothelial cell inflammatory response, oxidative stress and apoptosis, causing serious pulmonary vascular leakage and interstitial edema. The current study investigated the effect of exosomal miRNAs on endothelial cells during sepsis. We found a significant increase in miR-1-3p expression in cecal ligation and puncture (CLP) rats exosomes sequencing and sepsis patients’ exosomes, and lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) in vitro. However, the specific biological function of miR-1-3p in ALI remains unknown. Therefore, mimics or inhibitors of miR-1-3p were transfected to modulate its expression in HUVECs. Cell proliferation, apoptosis, contraction, permeability, and membrane injury were examined via cell counting kit-8 (CCK-8), flow cytometry, phalloidin staining, Transwell assay, lactate dehydrogenase (LDH) activity, and Western blotting. The miR-1-3p target gene was predicted with miRNA-related databases and validated by luciferase reporter. Target gene expression was blocked by siRNA to explore the underlying mechanisms. The results illustrated increased miR-1-3p and decreased stress-associated endoplasmic reticulum protein 1 (SERP1) expression both in vivo and in vitro. SERP1 was a direct target gene of miR-1-3p. Up-regulated miR-1-3p inhibits cell proliferation, promotes apoptosis and cytoskeleton contraction, increases monolayer endothelial cell permeability and membrane injury by targeting SERP1, which leads to dysfunction of endothelial cells and weakens vascular barrier function involved in the development of ALI. MiR-1-3p and SERP1 may be promising therapeutic candidates for sepsis-induced lung injury.
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