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Toll-like receptor 4 (TLR4) plays a central role in host responses to bacterial infection, but the precise mechanism(s) by which its downstream signaling components coordinate the bone marrow response to sepsis is poorly understood. Using mice deficient in TLR4 downstream adapters MYD88 or TRIF, we demonstrate that both cell-autonomous and non-cell-autonomous MYD88 activation are major causes of myelosuppression during sepsis, while having a modest impact on hematopoietic stem cell (HSC) functions. In contrast, cell-intrinsic TRIF activation severely compromises HSC self-renewal without directly affecting myeloid cells. Lipopolysaccharide-induced activation of MYD88 or TRIF contributes to cell-cycle activation of HSC and induces rapid and permanent changes in transcriptional programs, as indicated by persistent downregulation of Spi1 and CebpA expression after transplantation. Thus, distinct mechanisms downstream of TLR4 signaling mediate myelosuppression and HSC exhaustion during sepsis through unique effects of MyD88 and TRIF.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Nuclear LASP-1 (LIM and SH3 protein-1) has a direct correlation with overall survival of breast cancer patients. In this study, immunohistochemical analysis of a human breast TMA showed that LASP-1 is absent in normal human breast epithelium but the expression increases with malignancy and is highly nuclear in aggressive breast cancer. We investigated whether the chemokines and growth factors present in the tumor microenvironment could trigger nuclear translocation of LASP-1.Treatment of human breast cancer cells with CXCL12, EGF and HRG, and HMEC-CXCR2 cells with CXCL8 facilitated nuclear shuttling of LASP-1. Data from the biochemical analysis of the nuclear and cytosolic fractions further confirmed the nuclear translocation of LASP-1 upon chemokine and growth factor treatment. CXCL12-dependent nuclear import of LASP-1 could be blocked by CXCR4 antagonist, AMD-3100. Knock down of LASP-1 resulted in alterations in gene expression leading to an increased level of cell-junction and extracellular matrix proteins and an altered cytokine secretory profile. Three-dimensional cultures of human breast cancer cells on Matrigel revealed an altered colony growth, morphology and arborization pattern in LASP-1 knockdown cells. Functional analysis of the LASP-1 knockdown cells revealed increased adhesion to collagen IV and decreased invasion through the Matrigel. Proteomic analysis of immunoprecipitates of LASP-1 and subsequent validation approaches revealed that LASP-1 associated with the epigenetic machinery especially UHRF1, DNMT1, G9a and the transcription factor Snail1. Interestingly, LASP-1 associated with UHRF1, G9a, Snail1 and di- and tri-methylated histoneH3 in a CXCL12-dependent manner based on immunoprecipitation and proximity ligation assays. LASP-1 also directly bound to Snail1 which may stabilize Snail1. Thus, nuclear LASP-1 appears to functionally serve as a hub for the epigenetic machinery.
Sumoylation is a post-translational modification that is oftentimes deregulated in diseases such as cancer. Transcription factors are frequent targets of sumoylation and modification by SUMO can affect subcellular localization, transcriptional activity, and stability of the target protein. C/EBPbeta1 is one such transcription factor that is modified by SUMO-2/3. Non-sumoylated C/EBPbeta1, p52-C/EBPbeta1, is expressed in normal mammary epithelial cells but not breast cancer cell lines and plays a role in oncogene-induced senescence, a tumor suppressive mechanism. Although p52-C/EBPbeta1 is not observed via immunoblot in breast cancer cell lines, higher molecular weight bands are observed when breast cancer cell lines are subjected to immunoblot analysis with a C/EBPbeta1-specific antibody. We show that exogenously expressed C/EBPbeta1 is sumoylated in breast cancer cells, and that the higher molecular weight bands we observe in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades are activated in breast cancer cells. We demonstrate that phosphorylation of C/EBPbeta1Thr235 by Erk-2 enhances sumoylation of C/EBPbeta1 in vitro. In addition, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and mutation of Thr235 to alanine leads to a decrease in sumoylation of C/EBPbeta1. Finally, using a C/EBPbeta1-SUMO fusion protein we show that constitutive sumoylation of C/EBPbeta1 completely blocks its capability to induce senescence in WI38 fibroblasts expressing hTERT. Thus, sumolylation of C/EBPbeta1 in breast cancer cells may be a mechanism to circumvent oncogene-induced senescence.
Blood vessel epicardial substance (Bves) is a transmembrane adhesion protein that regulates tight junction (TJ) formation in a variety of epithelia. The role of TJs within epithelium extends beyond the mechanical properties. They have been shown to play a direct role in regulation of RhoA and ZONAB/DbpA, a y-box transcription factor. We hypothesize that Bves can modulate RhoA activation and ZONAB/DbpA activity through its regulatory effect on TJ formation. Immortalized human corneal epithelial (HCE) cells were stably transfected with Flag-tagged full length chicken Bves (w-Bves) or C-terminus truncated Bves (t-Bves). We found that stably transfected w-Bves and t-Bves were interacting with endogenous human Bves. However, interaction with t-Bves appeared to disrupt cell membrane localization of endogenous Bves and interaction with ZO-1. w-Bves cells exhibited increased TJ function reflected by increased trans-epithelial electrical resistance, while t-Bves cells lost TJ protein immunolocalization at cell-cell contacts and exhibited decreased trans-epithelial electrical resistance. In parental HCE and w-Bves cells ZONAB/DbpA and GEF-H1 were seen at cell borders in the same pattern as ZO-1. However, expression of t-Bves led to decreased membrane localization of both ZONAB/DbpA and GEF-H1. t-Bves cells had increased RhoA activity, as indicated by a significant 30% increase in FRET activity compared to parental HCE cells. ZONAB/DbpA transcriptional activity, assessed using a luciferase reporter probe, was increased in t-Bves cells. These studies demonstrate that Bves expression and localization can regulate RhoA and ZONAB/DbpA activity.
Bioluminescence resonance energy transfer (BRET) has become a widely used technique to monitor protein-protein interactions. It involves resonance energy transfer between a bioluminescent donor and a fluorescent acceptor. Because the donor emits photons intrinsically, fluorescence excitation is unnecessary. Therefore, BRET avoids some of the problems inherent in fluorescence resonance energy transfer (FRET) approaches, such as photobleaching, autofluorescence, and undesirable stimulation of photobiological processes. In the past, BRET signals have generally been too dim to be effectively imaged. Newly available cameras that are more sensitive coupled to image splitter now enable BRET imaging in plant and mammalian cells and tissues. In addition, new substrates and enhanced luciferases enable brighter signals that allow even subcellular BRET imaging. Here, we report methods for BRET imaging of (1) localization of COP1 dimerization in plant cells and tissues and (2) subcellular distributions of interactions of the CCAAT/Enhancer Binding Protein α (C/EBPα) in single mammalian cells. We also discuss methods for the correction of BRET images for tissues that absorb light of different spectra. This progress should catalyze further applications of BRET for imaging and high-throughput assays.
PURPOSE - To investigate the role of tight junction (TJ)-associated signaling pathways in the proliferation of uveal melanoma.
METHODS - Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1-associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways.
RESULTS - Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity.
CONCLUSIONS - TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma.
Overexpression of Ras(V12) in MCF10A cells, an immortalized mammary epithelial cell line, leads to transformation of these cells. We demonstrate that this is accompanied by degradation of C/EBPbeta1. C/EBPbeta is a transcription factor in which three protein isoforms exist because of alternative translation at three in-frame methionines. When C/EBPbeta1 is expressed in MCF10A-Ras(V12) cells, immunoblot analysis reveals that C/EBPbeta1 is degraded in these cells. Treatment of MCF10A-Ras(V12)-C/EBPbeta1 cells with the cdk inhibitor roscovitine leads to stabilization of C/EBPbeta1. It has been previously shown that cdk2 phosphorylates C/EBPbeta on Thr235. We demonstrate that mutation of Thr235 to alanine in C/EBPbeta1 is sufficient to restore the stability of C/EBPbeta1 expression in MCF10A-Ras(V12) cells. Overexpression of Ras(V12) in primary cells induces senescence rather than transformation, thus suppressing tumorigenesis. C/EBPbeta is required for Ras(V12)-induced senescence in primary mouse embryonic fibroblasts. Upregulation of interleukin-6 (IL6) by C/EBPbeta has been shown to be necessary for oncogene-induced senescence, but the specific isoform of C/EBPbeta has not been investigated. We show that the C/EBPbeta1 isoform upregulates IL6 when introduced into normal fibroblasts. In addition, we show that C/EBPbeta1 induces senescence. Taken together, degradation of C/EBPbeta1 by Ras activation may represent a mechanism to bypass OIS.
FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.
Studies in our laboratory have shown that a fraction of apolipoprotein (apo) E internalized by hepatocytes escapes degradation and is resecreted. Although the intracellular routing is not fully understood, our studies suggest that a portion of apoE recycles through the Golgi apparatus. Given the role of the Golgi apparatus in lipoprotein secretion and the fact that apoE modulates the hepatic secretion of very low-density lipoprotein, we hypothesized that recycling apoE has an effect on hepatic very low-density lipoprotein assembly and/or secretion. To test this hypothesis, apoE-/- mice were transplanted with bone marrow from wild-type mice. In this model, extrahepatic (macrophage-derived) apoE is internalized by the hepatocytes in vivo and is resecreted when the hepatocytes are placed in culture. Unexpectedly, our studies demonstrate that recycling apoE has little effect on hepatic lipid content or hepatocyte triglyceride secretion. In addition, recycling apoE has little effect on the expression of enzymes and proteins involved in lipid synthesis as well as plasma lipoprotein apoproteins. We conclude that the physiological relevance of apoE recycling may not be related to cell-specific functions, such as lipoprotein assembly in the liver. Rather, recycling may provide a mechanism for modulating general cellular effects such as intracellular cholesterol transport or cholesterol efflux.
We previously demonstrated that cholesterol deprivation increases endothelial cyclooxygenase-2 (COX-2)-dependent prostacyclin [prostaglandin I2 (PGI2)] production in vitro. Cholesterol directly regulates gene transcription through the sterol response element binding protein (SREBP). In this work, we demonstrate that SREBP directly regulates COX-2 expression. Cholesterol reduces human COX-2 promoter-luciferase reporter construct activity in transiently transfected endothelial cells. Conversely, cotransfection with a constitutively active mutant SREBP increases COX-2 promoter activity. SREBP-1a and -2 specifically bind a putative sterol response element (SRE) sequence in the COX-2 promoter. This sequence competes for SREBP binding to a low density lipoprotein receptor consensus sequence in an electromobility-shift assay. These data indicate that endothelial COX-2 is regulated by cholesterol via the SREBP pathway. The present study identifies COX-2 as the first vascular gene without a clear role in lipid metabolism transactivated by SREBP, and suggests that enhanced production of PGI2 through this pathway may be an additional benefit of cholesterol-lowering therapies.