Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia

PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.

A-kinase anchoring protein Lbc coordinates a p38 activating signaling complex controlling compensatory cardiac hypertrophy

In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous prohypertrophic signaling pathways in cultured cardiomyocytes. However, it remains to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3, and p38α that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. These results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Regulation of peripheral classical and non-classical monocytes on infliximab treatment in patients with rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVE To investigate the regulatory effect of tumour necrosis factor (TNF) blockade with infliximab on the distribution of peripheral blood monocyte subpopulations in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS Purified CD11b+CD14+ monocytes from 5 patients with RA and 5 AS were analysed ex vivo before and after infliximab treatment by flow cytometry for CD16, CD163, CD11b, C-C chemokine receptor type 2 (CCR2) and CXC chemokine receptor 4 (CXCR4) at baseline and at days 2, 14, 84 and 168 after the first infliximab administration. Serum levels of the stromal cell-derived factor (SDF)-1 and monocyte chemotactic peptide (MCP)-1 at different time points were measured in either patient group before and on infliximab treatment. RESULTS Anti-TNF treatment with infliximab led to a significant increase of circulating CD11b+ non-classical and a concomitantly decrease of CD11b+ classical monocytes, to a decline in SDF-1 levels and reduced expression of CCR2 and CXCR4 on non-classical monocyte subpopulation. CONCLUSIONS Our study shows, that TNFα blockade by infliximab resulted in a dichotomy of the regulation of classical and non-classical monocytes that might have substantial impact on inhibition of osteoclastogenesis and of subsequent juxta-articular bone destruction and systemic bone loss in RA and AS.

Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

The effect of high-energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo

The aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm(2)) was applied to femoral heads of 18 adult Sprague-Dawley rats. Two sham-treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n = 6 per treatment group) and scored on safranin-O-stained sections. Expression of tenascin-C and chitinase 3-like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real-time polymerase chain reaction (PCR) was used to examine collagen (II)alpha(1) (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin-C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough-surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High-energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA.

Processing of procollagen III by meprins: new players in extracellular matrix assembly?

Meprins ? and ?, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins ? and ? release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin ? is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin ? increases and meprin ? begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Transforming growth factor-beta inhibits the expression of clock genes

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.

Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth

We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA).

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.