The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress ...signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.
Orthotopic liver transplantation (OLT) continues to be the only remedy for end‐stage liver disease. In an attempt to decrease the ever‐widening gap between organ donor and recipient numbers, and ...ultimately make more livers amenable to transplantation, we characterized the healthy human liver's response to ischemia and reperfusion‐induced injury during transplantation. This was carried out by transcriptional profiling using cDNA microarray to identify genes whose expression was modulated at the 1‐h postreperfusion time point. We observed that the map kinase phosphatase‐1/dual‐specificity phosphatase‐1 (MKP‐1/DUSP1) mRNA was strongly and significantly upregulated. Validation of this observation was carried out using reverse transcriptase‐polymerase chain reaction (RT–PCR), immunoblotting and immunohistochemistry. In addition, we characterized the signaling pathways regulating MKP‐1 expression using the human hepatoma cell line HepG2. Finally, by combining MKP‐1 silencing with reperfusion‐associated stresses, we reveal the preferential role of this protein in attenuating the activity of the JNK and p38MAPK pathways, and the resulting apoptosis, making MKP‐1 a potential target for therapeutic intervention.
Microarray results in human livers and studies in the human hepatoma cell line HepG2 indicate a potential role of MKP‐1 in the human liver response to injury.
Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the ...identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.
In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell ...cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.
The cellular events following liver ischemia/reperfusion (I/R) during transplantation are largely unknown. The spectrum of I/R damage to the liver can be clinically revealed by the development of ...primary graft dysfunction or nonfunction. Because viral-induced liver necrosis has been associated with the development of calcifications in an animal model, we investigated the spectrum of I/R changes identified at an ultrastructural level among livers after liver transplant (LT).
Random liver biopsies from five recipients with different degrees of liver dysfunction (LD) were processed for light (LM) and electron (EM) microscopic examination. The degree of calcification was estimated as mild–moderate or severe. The degree of cell vacuolization, used as a surrogate marker of cell necrosis, was reported as mild–moderate or severe.
Two patients with severe LD had obvious calcifications by LM and EM examinations. Both showed significant vacuole formation, suggesting a severe degree of cell necrosis, and both succumbed to the sequelae of their LD. One patient showed evidence of mild calcifications at EM (but not LM) examination, with mild vacuole formation. The remaining two patients displayed no microcalcifications. Both presented only mild vacuole formation. Both patients recovered from LD and are currently alive.
In this preliminary report, we conclude that the clinically observed degree of LD after orthotopic liver transplant (OLT)correlates well with ultrastructural modifications. These include calcification and vacuole formation. We believe that both findings can be used as surrogate markers of a clinically significant hepatic I/R injury.
•Detailed instability of the dam revealed by high-resolution X-band Interferometry.•InSAR-derived deformation complements traditional surveying.•Time-series analysis reveals linear deformation in ...time in areas of major instability.•Detailed deformation assessment favours pixel-wise InSAR processing.
Geodetic monitoring of infrastructures is one of the key tasks in surveying and engineering geology. Systematic monitoring and assessment of the exterior deformation of embankment dams for safety analysis are often difficult when using classical surveying techniques due to time-consuming surveying procedures and high labour costs. Modern remote sensing techniques play an important role in efficiently assessing deformation: changes in the geometry, position and orientation of dams. In this study, we present the feasibility of effective post-construction deformation monitoring of the Masjed-Soleyman dam in Iran using high-resolution (∼1m) synthetic aperture radar (SAR) imagery in SpotLight (SL) mode from the X-band TerraSAR-X (TSX) satellite. This dam has been monitored over the last 15years using classical surveying techniques, which provide horizontal and vertical deformation measurements of the structure. We show that high-resolution X-band SAR data provide a much more detailed identification of dam deformation in the crest and downstream that is not possible to infer from classical surveying techniques with few sparse geodetic monuments. High-resolution TSX data reveal that the dam is currently subject to two different deformation regimes: one is related to the crest and its adjacent area downstream, with a maximum rate of deformation of approximately 13cm/yr in the radar line-of-sight (LOS). The other is related to the lower part of the downstream, with a maximum LOS velocity of 7cm/yr. The effect of this centimetre displacement rate has been shown through several damage features on the dam body, including minor to large dislocation cracks on the crest and a significant deformation zone on the downstream slope.
NFκB (Nuclear Factor-
-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate ...regulatory elements called "super enhancers". BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions.
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