Dongmei Zhang

The suppressor of cytokine signaling SOCS1 promotes apoptosis of intestinal epithelial cells via p53 signaling in Crohn's disease.

The suppressor of cytokine signaling SOCS1 is a member of the cytokine signaling pathway inhibitor family, which is induced by the IFN-γ induced JAK signaling pathway. The expression of SOCS1 has been found to increase in Crohns disease (CD) patients, but the role of SOCS1 in intestinal epithelium is unclear. This study was designed to investigate whether SOCS1 has a role in the death of intestinal epithelial cells and intestinal injury. The results showed that the expression of SOCS1 increased in CD patients, and the expression of SOCS1, p-p53 and PUMA increased in the mouse TNBS induced colitis model. Using IFN-γ treated HT-29 cells as an apoptotic model of intestinal epithelial cells in vitro, we confirmed that SOCS1 promoted apoptosis of intestinal epithelial cells by activating p53. In HT-29 cells which were treated with IFN-γ, the interaction between p53 and SOCS1 and phosphorylation of p53 were significantly higher than untreated cells. When knocking SOCS1 down by using SOCS1 siRNA, phosphorylation of p53 and apoptosis of intestinal epithelial cells which was induced by IFN-γ were significantly inhibited. In summary, our findings suggest that SOCS1 may promote apoptosis of intestinal epithelial cells at least partly through mediating p53 signaling.

Epithelial-specific ETS-1 (ESE1/ELF3) regulates apoptosis of intestinal epithelial cells in ulcerative colitis via accelerating NF-κB activation.

Epithelial-specific ETS-1 (ESE1), also named as ELF3, ERT and ESX, belonging to the ETS family of transcription factors, exerts multiple activities in inflammation, epithelial differentiation and cancer development. Previous data demonstrated that ESE1 synergizes with NF-κB to induce inflammation and drive tumor progress, and the nuclear translocation of ESE1 promotes colon cells apoptosis. However, the expression and biological functions of ESE1 in ulcerative colitis (UC) remain unclear. In this study, we reported for the first time that ESE1/ELF3 was over-expressed in intestinal epithelial cells (IECs) of patients with UC. In DSS-induced colitis mouse models, we observed the up-regulation of ESE1/ELF3 accompanied with the elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and NF-κB activation indicators [phosphorylated NF-κB p65 subunit (p-p65) and p-IκB] in colitis IECs. Increased co-localization of ESE1/ELF3 with active caspase-3 (and p-p65) in IECs of the DSS-induced colitis group further indicated the possible involvement of ESE1/ELF3 in NF-κB-mediated IEC apoptosis in UC. Employing the TNF-α-treated HT-29 cells as an IEC apoptosis model, we confirmed the positive correlation of ESE1/ELF3 with NF-κB activation and caspase-dependent IEC apoptosis in vitro. Immunoprecipitation and immunofluorescence assay revealed the physical interaction and increased nuclear translocation of ESE1/ELF3 and the NF-κB p65 subunit in TNF-α-treated HT-29 cells. Knocking ESE1/ELF3 down by siRNA significantly alleviated TNF-α-induced NF-κB activation and cellular apoptosis in HT-29 cells. Taken together, our data suggested that ESE1/ELF3 may promote the UC progression via accelerating NF-κB activation and thus facilitating IEC apoptosis.

Vacuolar protein sorting 4B regulates apoptosis of intestinal epithelial cells via p38 MAPK in Crohn's disease.

Vacuolar protein sorting 4B (VPS4B), a member of ATPase family proteins, reportedly possesses multiple biological functions, such as regulating the development of breast cancer and non-small-cell lung cancer, participating in Parkinsons disease, and modulating neuronal apoptosis after cerebral ischemia. However, its expression and potential functions in Crohns disease (CD) has not been understood. In this study, we reported for the first time that VPS4B was over-expressed in intestinal epithelial cell (IECs) of patients with CD. In TNBS-induced mouse colitis models, we observed the up-regulation of VPS4B was accompanied with the elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and phosphorylated p38 in colitis IECs. Co-localization of VPS4B and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of VPS4B in IEC apoptosis. Employing the TNF-α-treated HT29 cells as an in vitro IEC apoptosis model, we confirmed the positive correlation of VPS4B with caspase-dependent cellular apoptosis. Knocking VPS4B down by siRNA significantly alleviated TNF-α-induced p38 phosphorylation and cellular apoptosis in HT29 cells. Taken together, our findings suggested that VPS4B may facilitate the IEC apoptosis in CD via p38 MAPK signaling pathway.

KPNA2 interacts with P65 to modulate catabolic events in osteoarthritis.

OBJECTIVEnKaryopherin alpha 2 (KPNA2) is a member of the importin α family, which acts as an adaptor to deliver P65 to the nucleus by recognizing the classic nuclear localization signal (NLS) of the cargo protein, and which has been reported as being involved in the pathogenesis of many diseases. This study was undertaken to determine the expression and possible functions of KPNA2 in osteoarthritis (OA).nnnMETHODSnKPNA2 expression in cartilage tissues of OA patients and normal controls was detected by RT-PCR and immunohistochemistry. SW1353 cells were stimulated with IL-1β to establish the chondrocyte injury model in vitro. The expression of KPNA2 and catabolic genes in IL-1β-treated SW1353 cells were determined by Western blot. The interaction between KPNA2 and P65 was analyzed by co-immunoprecipitation, the subcellular distribution and transportation of P65 were detected by the subcellular fractionation followed by immunoblot analysis and immunofluorescence. Furthermore, we used RNA interference to analyze the role of KPNA2 in IL-1β-induced P65 nuclear importation and MMP13, ADAMTS-5 expression in SW1353 cells.nnnRESULTSnCartilage expression of KPNA2 was higher in patients with OA compared with normal controls and mainly locating in chondrocytes. In IL-1β-treated SW1353 cells, up-regulation of KPNA2 was accompanied by the elevated expression of the catabolic marker protein levels, including MMP13 and ADAMTS-5, and increased NF-κB P65 nuclear importation. Knock-down of KPNA2 resulted in decreased catabolic marker protein levels in IL-1β-treated SW1353 cells. KPNA2 interacted with p65, and loss of KPNA2 caused decreased nuclear translocation of the active p50/p65 NF-κB complex.nnnCONCLUSIONSnThese findings suggested that KPNA2 may promote NF-κB activation via facilitating P65 nuclear transportation, and thus subsequently accelerate the catabolic events of osteoarthritis.

Sam68 modulates apoptosis of intestinal epithelial cells via mediating NF-κB activation in ulcerative colitis.

Sam68 (Src-associated substrate during mitosis of 68 KDa), also known as KHDRBS1 (KH domain containing, RNA binding, signal transduction associated 1), belongs to the prototypic member of the signal transduction activator of RNA (STAR) family of RNA-binding proteins. Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, signal transduction. Previous researches demonstrated that Sam68 regulated nuclear transcription factor kappa B (NF-κB) to induce inflammation. However, the expression and biological functions of Sam68 in ulcerative colitis (UC) are not clear. In this study, we reported for the first time that Sam68 was up-regulated in intestinal epithelial cells (IECs) of patients with UC. In DSS-induced mouse colitis model, we observed the overexpression of Sam68 accompanied with increased levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and NF-κB activation indicators (p-p65 and p-IκB) in colitis IECs. Co-localization of Sam68 with active caspase-3 (and p-p65) in IECs of the DSS-induced colitis group further indicated the possible involvement of NF-κB-mediated IEC apoptosis. Applying TNF-α-treated HT-29 cells as an in vitro IEC inflammation model, we confirmed the positive correlation amomg Sam68, NF-κB activation and caspase-dependent apoptosis. Immunofluorescence and immunoprecipitation assay identified nuclear translocation and physical interaction of Sam68 and NF-κB subunits in TNF-α-treated HT-29 cells. Besides, depletion of Sam68 by RNA interference greatly alleviated NF-κB activation and apoptosis in TNF-α-treated HT-29 cells. Taken together, our results indicated that Sam68 modulates apoptosis of intestinal epithelial cells via mediating NF-κB activation in UC.

KPNA2 Contributes to the Inflammatory Processes in Synovial Tissue of Patients with Rheumatoid Arthritis and SW982 Cells

Karyopherin-α2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. We investigated the function and possible mechanisms of KPNA2 involved in rheumatoid arthritis (RA). Western blotting and immunohistochemistry showed the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Double immunofluorescent staining indicated that KPNA2 co-localized with T cells, macrophage-like synoviocytes, fibroblast-like synoviocytes, and neutrophils in synovial tissue of RA patients. Moreover, the expression of KPNA2 in SW982 cells was increased in a time-dependent manner in response to TNFα stimulation. Both Western blotting and immunofluorescent staining assay revealed the co-localization of KPNA2 and P65 and their translocation from cytoplasma in TNFα-treated SW982 cells. Furthermore, knocking down the expression of KPNA2 by siRNA inhibited TNFα-induced expression of IL-6, MMP-1, and MMP-13 and, more importantly, decreased the P65 phosphorylation in SW982 cells. We therefore suggested that KPNA2 may play a key role in the inflammation process of RA via NF-κB P65 signal transduction pathway.

PRDM5 promotes the apoptosis of epithelial cells induced by IFN-γ during Crohn’s disease

Elevated apoptosis of intestinal epithelial cells (IECs) greatly impairs the epithelial barrier integrity and contributes to the pathogenesis of Crohns Disease (CD). Overproduction of pro-inflammatory cytokine Interferon-γ (IFN-γ) induces the excessive apoptosis of IECs and is involved in CD development. PRDM5 (PR domain containing 5 PFM2) a member of PRDM family, reportedly acts as a transcriptional regulator involved in tissue specific differentiation and tumor development. In this study, we investigated PRDM5 expression and its potential functions in both human CD (Crohns disease) and TNBS (2,4,6-trinitrobenzenesulfonic acid sol)-induced mice experimental colitis. As shown by western blot and immunohistochemistry, significant up-regulation of PRDM5 was found in the inflamed intestinal tissues of CD patients and TNBS-treated mice, and the molecule was mainly located in IECs. To explore the biological functions of PRDM5 in IEC apoptosis, we established the interferon-γ (IFN-γ) induced cellular apoptosis model on human IEC line HT29 in vitro. IFN-γ significantly increased the expression of PRDM5 in a both time-dependent and concentration-dependent manner in HT29 cells, which was accompanied with an up-regulated expression of apoptotic markers (active caspase-3 and cleaved PARP(poly (ADP-ribpse) polymerase)). Inhibiting PRDM5 expression by siRNA attenuated the IFN-γ-triggered accumulation of active caspase-3 and cleaved PARP in IECs. Moreover, flow cytometry assay and CCK-8 analysis revealed that PRDM5 knockdown significantly alleviated the IFN-γ-induced cellular apoptosis in HT29 cells. Taken together, these data suggest that highly expressed PRDM5 may promote the IFN-γ-induced IEC apoptosis in the progression of CD.

ErbB3 binding protein 1 (EBP1) participates in the regulation of intestinal inflammation via mediating Akt signaling pathway.

ErbB3 binding protein-1 (EBP1) belongs to a family of DNA/RNA binding proteins implicated in cell growth, differentiation and apoptosis. Previous data demonstrated that EBP1 regulates phosphorylation of Akt to drive tumor progress. However, the expression and biological functions of EBP1 in ulcerative colitis (UC) remain unclear. In this study, we reported for the first time that EBP1 was down-regulated in intestinal epithelial cell (IECs) of patients with UC. In DSS-induced colitis, we observed the down-regulation of EBP1 accompanied with the elevated levels of proinflammatory cytokines (IL-1β, IL-6 and IL-8) and Akt activation indicators (phosphorylated Akt) in colitis IECs, indicating the possible involvement of EBP1 in regulation of intestinal inflammation via mediating Akt in UC. Employing the TNF-α-treated HT-29 cells as an IEC inflammatory model, we confirmed the negative correlation of EBP1 with Akt activation and Akt-dependent inflammation progress in vitro. EBP1 knocking down and over-expression significantly regulated TNF-α-induced Akt activation and proinflammatory cytokines expression in HT-29 cells. Taken together, our data suggested that EBP1 participates in the regulation of intestinal inflammation via mediating Akt signaling pathway.

FKBP11 protects intestinal epithelial cells against inflammation‑induced apoptosis via the JNK‑caspase pathway in Crohn's disease

Endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) has an important role in the pathogenesis of Crohns disease (CD). FK506 binding protein 11 (FKBP11), a member of the peptidyl-prolyl cis-trans isomerase family, is involved in the unfolded protein response (UPR) and is closely associated with inflammation. Previous bioinformatics analysis revealed a potential association between FKBP11 and human CD. Thus, the present study aimed to investigate the potential significance of FKBP11 in IEC homeostasis and CD. In the present study, increased expression of FKBP11 was detected in the intestinal inflammatory tissues of patients with CD. Furthermore, the results of the present study revealed that overexpression of FKBP11 was accompanied by increased expression levels of the ER stress marker 78 kDa glucose-regulated protein in the colon tissues of a 2, 4, 6-trinitrobenzenesulphonic acid-induced mouse colitis model. Using interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α)-stimulated IECs as an ER stress and apoptosis cell model, the associated of FKBP11 with ER stress and apoptosis levels was confirmed in IECs. Overexpression of FKBP11 was revealed to significantly attenuate the elevated expression of pro-apoptotic proteins (Bcl2 associated X apoptosis regulator, caspase-12 and active caspase-3), suppress the phosphorylation of c-Jun N-terminal kinase (JNK), and decrease apoptosis of IFN-γ/TNF-α stimulated IECs. Knockdown of FKBP11 by transfection with small interfering RNA further validated the aforementioned results. In conclusion, these results suggest that the UPR protein FKBP11 may protect IECs against IFN-γ/TNF-α induced apoptosis by inhibiting the ER stress-associated JNK/caspase apoptotic pathway in CD.

Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease

OBJECTIVEnTo investigate the role of chromosome region maintenance-1 (CRM1) in Crohns disease (CD) and its potential pathological mechanisms.nnnMETHODSnThe expression and distribution of CRM1 in mucosal biopsies from patients with active CD and normal controls were detected by immunohistochemistry (IHC). We established a murine model of acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Western blot was performed to investigate the expression levels of CRM1, apoptotic markers (active caspase-3 and cleaved PARP), p27kip1 and p-p27ser10. IHC was performed to evaluate the distribution of CRM1, and double immunofluorescence (IF) was performed to evaluate the co-localization of CRM1 and active capase-3. Cells of the human intestinal epithelial cell line HT-29 were incubated with tumor necrosis factor-α (TNF-α) to establish an apoptotic in vitro model. Western blot was performed to determine the expression levels of CRM1, active caspase-3, cleaved PARP and p-p27ser10. Cytoplasmic and nuclear extracts were assessed to examine the translocation of CRM1. The interaction between CRM1 and p27kip1 was assessed by co-immunoprecipitation (co-IP) assays. Furthermore, we used small interfering RNA (siRNA) to knock down the protein expression of CRM1 in HT-29 cells and then measured the expression of active caspase-3, cleaved PARP and p-p27ser10. Flow cytometry was used to determine the effect of CRM1 on intestinal epithelial cell (IEC) apoptosis.nnnRESULTSnWe observed up-regulation of CRM1 accompanied by elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and p-p27ser10 in IECs of patients with active CD and in TNBS-induced colitis model cells. However, the expression of p27kip1 was negatively correlated with the expression patterns of CRM1, p-p27ser10 and apoptotic biochemical markers. Co-localization of CRM1 and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of CRM1 in IEC apoptosis. By employing TNF-α-treated HT-29 cells as an in vitro IEC apoptosis model, we found that the expression levels of CRM1 and p-p27ser10 were in accordance with active caspase-3 and cleaved PARP. In addition, immunoprecipitation confirmed the physical interaction between CRM1 and p27kip1. siRNA knockdown of CRM1 significantly inhibited the phosphorylation of p27kip1 and the expression of active caspase-3 and cleaved PARP. In addition, flow cytometry analysis also showed that silencing CRM1 by siRNA inhibited TNF-α-induced cellular apoptosis in HT-29 cells.nnnCONCLUSIONSnUp-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.