Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB5 toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-κB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-κB was observed with a peak at 6–12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-κB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-κB activation, suggesting that activation of Akt is an event upstream of NF-κB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-κB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1α, XBP1, or PERK did not attenuate activation of NF-κB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-κB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-κB through the ATF6 branch of the UPR.

We investigated the potential involvement of connexin hemichannels in cadmium ions (Cd 2+ )-elicited cell injury. Transfection of LLC-PK1 cells with a wild-type connexin43 (Cx43) sensitized them to Cd 2+ -elicited cell injury. The cell susceptibility to Cd 2+ was increased by depletion of glutathione (GSH) with DL-buthionine-[S,R]-sulfoximine, and decreased by N-acetyl-cysteine or glutathione reduced ethyl ester. Fibroblasts derived from Cx43 wild-type (Cx43+/+) and knockout (Cx43-/-) fetal littermates displayed different susceptibility to Cd 2+

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Postlaminectomy epidural adhesion is implicated as a main cause of ''failed back surgery syndrome'' and associated with increased risk of complications during revision surgery. Various materials acting as mechanical barriers to reduce fibroblasts infiltration into epidural space have met with limited success. In present research, amniotic membrane (AM) was studied to investigate its effects on reducing epidural scar adhesion after laminectomy in a canine model. Laminectomy sites were created at L-1, L-3, L-5, and L-7 levels in 24 adult mongrel dogs. Freeze dried AM (FAM), cross-linked AM (CAM), and autologous free fat (AFF) were implanted, respectively, at a randomly assigned site in each dog with the remaining untreated site serving as internal control. The animals were sacrificed at 1, 6, and 12 weeks postoperatively. Then, gross pathologic observation including scar amount and adhesion tenacity, qualitative histology evaluation, and quantitative histology analysis were compared. Gross observation demonstrated that scar amount and adhesion tenacity of CAM group were significantly lower in comparison with those of FAM and non-treatment groups. A white, slightly vascularized CAM layer covered the dura mater without tenacious scar adhesion. The histology analysis also indicated reduced fibroblasts infiltration and consequent epidural fibrosis, which were similar to the results of AFF group. In conclusion, the CAM is effective in reducing epidural fibrosis and scar adhesion after laminectomy in canine model. It is a promising biomaterial for future clinical applications.

Recent studies show that the unfolded protein response (UPR) within the endoplasmic reticulum is correlated with breast cancer drug resistance. In particular, human X-box binding protein-1(XBP1), a transcription factor which participates in UPR stress signaling, is reported to correlate with poor clinical responsiveness to tamoxifen. In this study, we develop a tamoxifen-resistant MCF-7 cell line by treating the cell line with low concentration of tamoxifen, and we find that XBP1 is indeed up-regulated at both the mRNA and protein levels compared to normal MCF-7 cells. STF-083010, a novel inhibitor which specifically blocks the XBP1 splicing, reestablishes tamoxifen sensitivity to resistant MCF-7 cells. Moreover, co-treatment with STF-083010 and tamoxifen can significantly delay breast cancer progression in a xenograft mammary tumor model. We next investigate the expression of XBP1s in over 170 breast cancer patients' samples and the results demonstrate that XBP1s expression level is highly correlated with overall survival in the ER+ subgroup, but not in the ER− subgroup, suggesting a potential therapeutic application of XBP1 inhibitors in ER+breast cancer treatment.

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Immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) inhibit cytokine production by activated lymphocytes through interfering with calcineurin. However, little is known about their effects on the function of nonlymphoid cells. We found that, in renal tubular cells, induction of MCP-1 by inflammatory cytokines was blunted by CsA and FK506. This suppression was correlated with induction of unfolded protein response (UPR) evidenced by endogenous and exogenous indicators. The induction of UPR by these agents was reversible and observed generally in other nonimmune cells. Furthermore, administration with CsA in reporter mice caused rapid, systemic induction of UPR in vivo. In TNF-α-treated cells, suppression of MCP-1 by CsA or FK506 was associated with blunted responses of NF-κB, the crucial regulator of MCP-1. The suppression of NF-κB was reproduced by other inducers of UPR including AB5 subtilase cytotoxin, tunicamycin, thapsigargin, and A23187. CsA and FK506, as well as other UPR inducers, caused up-regulation of C/EBP family members, especially C/EBPβ and CHOP (C/EBP homologous protein), and overexpression of either C/EBPβ or CHOP significantly attenuated TNF-α-triggered NF-κB activation. Furthermore, down-regulation of C/EBPβ by small interfering RNA substantially reversed the suppressive effect of CsA on TNF-α-induced MCP-1 expression. These results suggested that CsA and FK506 confer insensitiveness to TNF-α on resident cells through UPR-dependent induction of the C/EBP family members.

Metformin exhibits diverse protective effects against diabetic complications, such as bone loss. Here, we investigated the effect of metformin on vascular calcification, another type 2 diabetes complication. In female rat aortic smooth muscle cells (RASMCs), we observed that metformin significantly alleviated β-glycerophosphate-induced Ca deposition and alkaline phosphatase activity, corresponding with reduced expression of some specific genes in osteoblast-like cells, including Runx2 and bone morphogenetic protein-2, and positive effects on α-actin expression, a specific marker of smooth muscle cells. Mechanistic analysis showed that phosphorylation levels of both AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) were increased with NO overproduction. After inhibition of either AMPK or eNOS with the pharmacologic inhibitors, compound C or Nω-Nitro-L-arginine methyl ester, NO production was lowered and metformin-meditated vascular protection against β-glycerophosphate-induced Ca deposition was removed. Our results support that metformin prevents vascular calcification via AMPK-eNOS-NO pathway.