Mesenchymal stem cells (MSCs) have been shown to be involved in bone injury repair. Programmed cell death 4 (PDCD4) is not only a tumor suppressor gene but also plays roles in the regulation of MSC function. The aim of the study was to uncover PDCD4 potential regulatory roles and mechanisms in the osteogenic differentiation and bone defect repair of MSCs. shRNA technique was used to knock down PDCD4 expression in umbilical cord-derived mesenchymal stem cells (shPDCD4-UCMSCs). Their phenotype was characterized by flow cytometry and the differentiation potential was verified. We found that PDCD4 knockdown did not affect the surface molecule expression of UCMSCs, but significantly enhanced their osteogenic differentiation and osteogenesis-related molecule expression. Mechanistically, glycogen synthase kinase-3β (GSK-3β) phosphorylation and β-catenin expression were significantly increased in shPDCD4-UCMSCs during the osteogenic differentiation process. The β-catenin inhibitor PNU-74654 reversed shPDCD4-increased osteogenesis and osteogenesis-related molecule expression. The results of animal experiments showed that shPDCD4-UCMSCs markedly improved the defect healing in rabbits. Our findings suggest that PDCD4 acts as a negative regulator of MSC osteogenic differentiation through GSK-3β/β-catenin pathway. Targeting PDCD4 may be a way to improve MSC-mediated therapeutic effects on bone injury.

Programmed cell death 4 (Pdcd4) was found to be related to apoptosis upon first discovery. It was later found to play the role of tumor suppressor gene in a variety of tumors by inhibiting transcription and translation. Recently, it has been proposed that it may play an important role in some inflammatory diseases and in the immune response. In our previous study, deficiency of Pdcd4 was found to attenuate the formation of atherosclerotic plaques. This might be because deficiency of Pdcd4 may increase IL-10 expression and lipoautophagy by macrophages and attenuate the formation of foam cells. However, the effect of Pdcd4 on the subsets of T cells in hyperlipidemic mice still remained unclear. In the present study, results showed that Pdcd4 deficiency decreased the percentage of CD8+ T cells and increased that of regulatory T cells (Tregs) under hyperlipidemic conditions both in vitro and in vivo, which may be due to the reduced expression of co-stimulatory molecules CD28 and CD137, and the enhancive expression of co-inhibitory molecules CTLA-4. These results indicated that endogenous Pdcd4 promotes immune response mediated by T cells through regulation of the co-stimulatory molecules expression, which may contribute to the development of advanced atherosclerotic plaques. The current work provides new data to understand the role of Pdcd4 in different T cell subsets under hyperlipidemic microenvironment.

The molecule of programmed cell death 4 (PDCD4) is named based on its upregulation during apoptosis. It is now known that PDCD4 is highly conserved during evolution and widely expressed in the immune cells and non-immune cells of humans and other animals. PDCD4, as an inhibitor of gene transcription and translation, is very important in maintaining the normal function of cells.1, 2 PDCD4 protein contains an N-terminal domain and two MA-3 domains in the central and C-terminal regions. The MA-3 domains can competitively bind to the translation initiation factor eIF4A with eIF4G to inhibit eIF4A activity and to impact the translation of specific mRNAs containing structured 5′-untranslated regions (5′UTRs).2 In this eIF4A-dependent manner, PDCD4 is able to inhibit the translational initiation of multiple genes, including tumor suppressor gene p53, apoptosis-related gene pro-caspase 3, autophagy-related gene Atg5 and metabolism-related gene LXR-α.3, 4, 5, 6 With its N-terminal domain, PDCD4 interacts with specific RNA secondary structures, such as c-myb and A-myb (proto-oncogene) mRNAs, and thereby suppresses translation elongation.7 The N-terminal domain is also responsible for PDCD4 functional interference by binding to certain proteins, such as poly (A)-binding protein (PABP), Daxx (a scaffold protein with roles in diverse processes, including transcriptional regulation and DNA-damage signaling), transcription factor Twist1 and the p65 subunit of NF-κB.8, 9, 10, 11 In addition, PDCD4 can also inhibit the activation of the ERK/P38 MAPK pathway and thereby suppress the expression of cytokines, such as Interleukin-10 (IL-10) (Figure 1a).12

Programmed cell death 4 (Pdcd4) is a newly defined inhibitor of transcription and translation and a tumor suppressor. Recent studies have suggested that Pdcd4 may also be involved in some inflammatory diseases. However, its role in atherosclerosis, a chronic inflammation of the arterial wall, remains to be investigated. Here, we found that Pdcd4 deficiency in mice increased the expression of IL-10 in macrophages and decreased the expression of IL-17 in T cells in the presence of an atherosclerosis-associated stimulator in vitro and in high fat-induced atherosclerotic plaques. Importantly, knocking out Pdcd4 led to a decrease in atherosclerotic lesions in Apoe −/− mice fed a high fat diet. This effect could be partly reversed by blocking IL-10 with a neutralizing antibody but not by the application of exogenous IL-17. Further mechanistic studies revealed that Pdcd4 negatively regulated the expression of IL-10 in an ERK1/2- and p38-dependent manner. These results demonstrate that Pdcd4 deficiency attenuates atherosclerosis in hyperlipidemic mice in part through the upregulation of the anti-inflammatory cytokine IL-10. This indicates that endogenous Pdcd4 promotes atherosclerosis and therefore represents a potential therapeutic target for patients with atherosclerosis.

BACKGROUND:Diabetes mellitus (DM) is an incurable metabolic disease constituting a major threat to human health. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) hold great promise in the treatment of DM. The development of an efficient IPC induction system is a crucial step for the clinical application of IPCs for DM. Laminin 411 is a key component of the basement membrane and is involved in the regulation of cell differentiation; however, little is known about a role of laminin 411 in the regulation of IPC differentiation from human MSCs.METHODS:MSCs were isolated from human umbilical cord (UC-MSCs) and expanded in an in vitro culture system. UC-MSCs were then cultured in the IPC induction and differentiation medium in the presence of laminin 411. Flow cytometry, Quantitative realtime PCR, immunofluorescence staining, ELISA, Western blotting and other techniques were applied to determine IPC generation, insulin expression and related mechanisms. To evaluate potential therapeutic efficacy of IPCs induced from UC-MSCs, a type-1 diabetes (T1DM) rat model was generated using streptozotocin. Blood glucose, insulin levels, and survival of rats were monitored periodically following intravenous injection of the tested cells.RESULTS:Laminin 411 markedly induced the expression of the genes Foxa2 and Sox17, markers for pancreatic precursor cells, efficiently induced IPC differentiation from MSCs, and up-regulated insulin expression at both mRNA and protein levels. Furthermore, the expression of the genes known to govern insulin expression including Pdx1 and Ngn3 was markedly induced by laminin 411, which suggests that Pdx1 and Ngn3 signaling pathways are involved in laminin 411 induced-insulin expression machinery. More importantly, administration of laminin 411-induced IPCs rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of T1DM rats.CONCLUSIONS:Our results demonstrate that laminin 411 acts as a potent differentiation inducer of IPCs from UC-MSCs via the Pdx1 and Ngn3 signaling pathways. Moreover, transfusion of laminin 411 induced-IPCs more efficiently improves symptoms and survival of T1DM rats. These novel finding highlights a potential clinical application of laminin 411 induced-IPCs in the treatment of T1DM, which calls for further studies.

Programmed cell death-4 (PDCD4), a selective protein translation inhibitor, has shown proinflammatory effect in some inflammatory diseases, but its roles in obesity remain unestablished. This study aims to investigate the effects of PDCD4 on obesity, inflammation, and insulin resistance. Surprisingly, high-fat diet (HFD)-fed PDCD4-deficient (PDCD4−/−) mice exhibited an absolutely lean phenotype together with improved insulin sensitivity. Compared with wild-type obese mice, HFD-fed PDCD4−/− mice showed higher energy expenditure, lower epididymal fat weight, and reduced macrophage infiltration inflammatory cytokine secretion in white adipose tissue (WAT). Alleviated hepatic steatosis along with decreased plasma levels of triglyceride and cholesterol was also observed in these mice. Importantly, PDCD4 appeared to disturb lipid metabolism via inhibiting the expression of liver X receptor (LXR)-α, a master modulator of lipid homeostasis, which was elevated in HFD-fed PDCD4−/− mice accompanied by upregulation of its target genes and relieved endoplasmic reticulum stress in WAT. These data demonstrate that PDCD4 deficiency protects mice against diet-induced obesity, WAT inflammation, and insulin resistance through restoring the expression of LXR-α, thereby proposing PDCD4 as a potential target for treating obesity-associated diseases.

Programmed cell death 4 (PDCD4) acts as a tumor suppressor gene, which suppresses tumor growth, infiltration and metastasis. Our previous studies demonstrated that PDCD4 had an important role in the development of ovarian cancer and glioma. Recent studies show that PDCD4 is also involved in various inflammatory diseases. However, its exact effect on inflammation remains unclear. In our current study, we explored the role of PDCD4 in acute liver injury induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN) using wild-type (WT) mice and PDCD4-deficient mice. Our results showed that liver-to-body weight ratios, as well as serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were significantly increased in PDCD4-deficient mice than WT mice. Histological examination, immunohistochemical and TUNEL analysis revealed PDCD4-deficient mice had more necrotic and apoptotic hepatocytes, inflammatory cells infiltration and liver internal hemorrhage than WT mice. In addition, some inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) in the serum and liver tissues were also significantly increased in PDCD4-deficient mice. More importantly, we found that the aggravation of liver tissue injury in PDCD4-deficient mice was due to excessive mitogen-activated protein kinase and NF-κB activation, which induced the release of more inflammatory factors, and consequently resulted in higher levels of hepatocyte necrosis and apoptosis. These results indicate that PDCD4 has a protective role in LPS/D-GalN-induced acute liver injury. This finding may present new opportunities for PDCD4 to be explored as a therapeutic target in acute liver injury.

RATIONALE:Apoptosis and fatty acid-binding protein-4 (FABP4) induced-endoplasmic reticulum (ER) stress in macrophage is an important pathological process in several vascular occlusive diseases, including atherosclerosis, both of which are accelerated by lipids or inflammatory cytokines.OBJECTIVE:To determine whether interleukin 17A (IL-17A) accelerates atherosclerosis through activating FABP4-mediated ER stress in macrophage.METHODS AND RESULTS:We show here that IL-17A induced ER stress in both murine and human-derived macrophages in vitro, and in the atherosclerotic lesions of ApoE-/- mice. Treating ApoE-/- mice with a chemical chaperone alleviated IL-17A-mediated ER stress and macrophage apoptosis, which was accompanied by recovered atherogenesis. Mechanistically, IL-17A up-regulated the expression of FABP4 (aP2), a cytosolic lipid chaperone that is able to promote lipid-induced macrophage ER stress, through NF-κB and ERK/p38 mitogen-activated protein kinase (MAPK) pathways in macrophages. The inhibition of aP2 expression with a specific chemical inhibitor significantly blocked IL-17A-accelerated ER stress and apoptosis in plaque, and partially rescued IL17A-induced atherogenesis.CONCLUSIONS:The data collectively establish a previously unrecognized link between IL-17A and ER stress through cytosolic lipid chaperone aP2 in macrophages and provide a new insight for understanding the role of IL-17A in atherosclerosis.

Objective To investigate the influence of PDCD4 gene to the differentiation of T lymphocyte subsets.Methods Detect the expression differences of CD8+T,CD4+T and its subsets such as Th1、Th2,Th17 and CD25+Foxp3+Treg in the splenocytes and lymph nodes of pdcd4-/-mice and C57BL/6 mice by flow cytometry.And analyse the ratio change of the lymphocyte subsets.Results Compared with that of the C57BL/6 mice,the number of CD8+T cells decreased in the pdcd4-/-mice(P<0.05).CD4+T and Th1 cells also showed a descending tendency but had no statistic significance(P>0.05).The proportion of CD4+CD25+Foxp3+Treg significantly increased in the pdcd4-/-mice(P<0.05).Conclusion PDCD4 can influence the differentiation of partial T lymphocyte subsets,the low expression of CD8+T cells as well as high proportion of CD4+CD25+Foxp3+Treg in the pdcd4-/-mice suggest that PDCD4 gene may play a role in the immune regulation.However,PDCD4 gene has no significant influence on the differentiation of CD4+T and Th1、Th2 and Th17 cells.

Objective To investigate the changes of the gene expressive profile of the human ovarian cancer cell line (SKOV3) after transfection with PDCD4. Methods The human ovarian cancer cell line SKOV3 was transfected with plasmids (pDsRed2-N1 and pDsRed2-N1-PDCD4) by using lipofectamine transfection reagent. SKOV3 cells stably expressing PDCD4 were established. Changes of the gene expression were analyzed by using gene chip technology. The related gene expression was detected by RT-PCR to confirm the results of the gene chip. Results PDCD4 transfection induced widespread changes of the gene expressive profile of the human ovarian cancer cell line SKOV3. The chip data suggested that there were 467 differentially expressed genes,in which 255 genes were up-regulated and 212 genes were down-regulated. Five of the differentially expressed genes were detected by RT-PCR,which was consistent with the chip data. Conclusion Gene chip technology was successfully used to screen the genes differentially expressed in the human ovarian cancer cell line SKOV3 transfected with PDCD4. This study provides laboratory evidence for further studying the tumor suppression mechanism of PDCD4.

Considerable evidence supports that the CD4+ T cell-mediated immune response contributes to the development of atherosclerotic plaque. However, the effects of Th17 cells on atherosclerosis are not thoroughly understood. In this study, we evaluated the production and function of Th17 and Th1 cells in atherosclerotic-susceptible ApoE−/− mice. We observed that the proportion of Th17 cells, as well as Th1, increased in atherosclerotic ApoE−/− mice compared with nonatherosclerotic wild-type littermates. In ApoE−/− mice with atherosclerosis, the expression of IL-17 and retinoic acid-related orphan receptor γt was substantially higher in the arterial wall with plaque than in the arterial wall without plaque. Increased Th17 cells were associated with the magnitude of atherosclerotic plaque in ApoE−/− mice. Importantly, treatment of ApoE−/− mice with neutralizing anti–IL-17 Ab dramatically inhibited the development of atherosclerotic plaque, whereas rIL-17 application significantly promoted the formation of atherosclerotic plaque. These data demonstrate that Th17 cells play a critical role in atherosclerotic plaque formation in mice, which may have implications in patients with atherosclerosis.

Objective To construct the human programmed cell death 4 promotor luciferase reporter gene vector and detect its activity in cells.Methods The PDCD4 promotor from human genomic DNA was amplified by PCR,and was inserted into the luciferase report gene pGL4-basic vector.The amplified DNA sequence was confirmed by sequencing.To detect the transcriptional activity of human PDCD4-P1 in the plasmid,transient transfection was performed in different cell lines,and pRL-TK was used to determine the transfection efficiency.Results The sequencing results indicated that the amplified sequence was correct.The results of transient transfection showed that the recombinant plasmid could be highly expressed in the OVCAR3 cell line which could highly express endogenous PDCD4,but lowly in the SKOV3 cell line in which lowly expressed endogenous PDCD4 could be detected.Conclusion The human PDCD4 promotor luciferase reporter gene vector was successfully constructed,which lays an experimental foundation for further study.