In a previous study, we demonstrated that sodium salicylate (NaSal) selectively inhibits tumor necrosis factor (TNF)-induced activation of the p42 and p44 mitogenactivated protein kinases (MAPKs) (known as extracellular signal-regulated kinases). Here we show that in normal human FS-4 fibroblasts NaSal inhibits TNF-induced activation of another member of the MAPK family, the c-Jun N-terminal kinase͞stress-activated protein kinase. c-Jun N-terminal kinase activation induced by interleukin 1 or epidermal growth factor was less strongly inhibited by NaSal. Unexpectedly, treatment of FS-4 cells with NaSal alone produced a strong activation of p38 MAPK and cell death by apoptosis. NaSalinduced apoptosis was blocked by the selective p38 MAPK inhibitor SB-203580, indicating that p38 MAPK serves as a mediator of NaSal-induced apoptosis in human fibroblasts. Activation of p38 MAPK and the resulting induction of apoptosis may be important in the demonstrated antineoplastic actions of nonsteroidal anti-inf lammatory drugs.

Lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive disease caused by mutations in CFTR. In CF, chronic infection and dysregulated neutrophilic inflammation lead to progressive airway destruction. The severity of CF lung disease has significant heritability, independent of CFTR genotype1. To identify genetic modifiers, we performed a genome-wide single nucleotide polymorphism (SNP) scan in one cohort of CF patients, replicating top candidates in an independent cohort. This approach identified IFRD1 as a modifier of CF lung disease severity. IFRD1 is a histone deacetylase (HDAC)-dependent transcriptional co-regulator expressed during terminal neutrophil differentiation. Neutrophils, but not macrophages, from Ifrd1-deficient mice exhibited blunted effector function, associated with decreased NF-κB p65 transactivation. In vivo, IFRD1 deficiency caused delayed bacterial clearance from the airway, but also less inflammation and disease—a phenotype primarily dependent on hematopoietic cell expression, or lack of expression, of IFRD1. In humans, IFRD1 polymorphisms were significantly associated with variation in neutrophil effector function. These data suggest that IFRD1 modulates the pathogenesis of CF lung disease through regulation of neutrophil effector function.

Abstract-Functional proteome analysis is not restricted to the sequence information but includes the broad spectrum of structural modifications and quantitative changes of proteins to which they are subjected in different tissues and cell organelles and during the development of an organism. Cell biology has provided the means required for the analysis of the composition and properties of purified cellular elements. Subcellular fractionation is an approach universal across all cell types and tissues, including cardiac and vascular system. Subcellular fractionation and proteomics form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach resulting in reduced sample complexity and is most efficiently combined with high-resolution 2D gel/mass spectrometry analysis as well as with gel-independent techniques. In this study we introduce state of the art subcellular fractionation techniques and discuss their suitability, advantages, and limitations for proteomics research.

Mesenchymal stem cells (MSCs) are of particular interest because of their potential in regenerative medicine. Stem cell-based therapies cast a new hope for neurodegenerative disease treatment as a regeneration strategy, including treatment for Alzheimer's disease (AD). A multitude of cytokines and factors secreted from MSCs are known to give such multifunctional properties, but associated mechanisms of these factors have yet to be entirely understood. To better understand the in vitro effect of MSCs on a neurodegenerative disorder, we treated primary cortical and hippocampal neural cells with amyloid β (Aβ) as an in vitro cell line model for AD. For this purpose, bone marrow-derived MSCs (BMSCs) were cocultured with Aβ-treated neural cells, collected at day 3, and subjected to absolute telomere length measurement and telomerase activity assay. Next, the gene and protein expression levels of mTOR, p-mTOR, AMPK, p-AMPK, GSK-3β, p-GSK-3β, Wnt3, and β-catenin were investigated. Also, after 3 days of coculture treatment, the supernatant was collected from both groups for cytokine measurement. It was found that telomere length as a biomarker in neurodegenerative disorder as well as telomerase activity had significantly increased in the experimental group, and the presence of IL-6, IL-10, and TGF-β was obviously significant in the cocultured media. Also, BMSCs significantly changed the gene and protein expression of mTOR, AMPK, GSK-3β, and Wnt3/βcatenin signaling pathways components. It was concluded that the mentioned effects of MSCs on neural cells as an in vitro cell line model for AD as a therapeutic agent can be related to the signaling network.

A major obstacle to obtaining more detailed insights into the diversity of phenotypic and molecular changes occurring in colon cancer cells is the lack of low-passage colon cancer cell lines, which would still closely reflect the phenotype of the colon cancer cells in vivo. Here, we characterize eight novel, low passage number human colon carcinoma cell lines, originating from colorectal cancers extensively characterized in the clinics. All cell lines closely resemble the original tumors with respect to phenotype, markers and detectable genetic changes. Cell morphology and marker expression is highly variable, ranging from fully polarized cells correctly expressing all basolateral epithelial markers, to cells with mesenchymal characteristics and a complete loss of polarity due to delocalization or loss of junction complex proteins. The alterations in phenotype and epithelial marker expression correspond to changes already detectable in the primary tumor in vivo. Seven of the cell lines show chromosomal instability, while one cell line is characterized by microsatellite instability. p53 associated with K-ras mutations were detected in three cell lines. Hitherto non-described E-cadherin mutations were found at both alleles in one cell line whereas in another cell line the E-cadherin protein was down-regulated. A stabilizing beta-catenin mutation (S45F) appears in the same cell line that carried the mutated E-cadherin gene. Six cell lines carried APC mutations, which in five of the lines led to an activated beta-catenin/Tcf/LEF signaling pathway. In accordance with beta-catenin/Tcf/LEF activation, the cell lines show increased migration and invasiveness. Our results show that the characterized, low-passage cell lines mirror the diversity of the individual tumors from which they were derived. Through molecular analyses of these cell lines we demonstrate that tumorgenicity events are much more diverse in human colon cancer than expected, despite the common origin of the tumors from a small patient group with similar tumor grading and clinical prognosis.

The mammalian SIN3 complex consists of histone deacetylases (HDAC1, HDAC2), several known proteins (SAP30, N-CoR) and as yet unidentified proteins. Here we show that the mouse tetradecanoyl phorbol acetate induced sequence 7 (TIS7) protein is a novel transcriptional co-repressor that can associate with the SIN3 complex. We have identified tis7 as a gene that is up-regulated upon loss of polarity in a mouse mammary gland epithelial cell line expressing an estrogen-inducible c-JunER fusion protein. In unpolarized cells, TIS7 protein levels increase and TIS7 translocates into the nucleus. Overexpression of tis7 causes loss of polarity and represses a set of genes, as revealed by cDNA microarray analysis. We have shown that TIS7 protein interacts with several proteins of the SIN3 complex (mSin3B, HDAC1, N-CoR and SAP30) by yeast two-hybrid screening and co-immunoprecipitations. TIS7 co-immunoprecipitated HDAC complex is enzymatically active and represses a GAL4-dependent reporter transcription. The transcriptional repression of endogenous genes by tis7 overexpression is HDAC dependent. Thus, we propose TIS7 as a transcriptional co-repressor affecting the expression of specific genes in a HDAC activity-dependent manner during cell fate decisions, e.g. scattering.

The tetradecanoyl phorbol acetate-induced sequence 7 gene (tis7) is regulated during cell fate processes and functions as a transcriptional coregulator. Here, we describe the generation and analysis of mice lacking the tis7 gene. Surprisingly, TIS7 knockout mice show no gross histological abnormalities and are fertile. Disruption of the tis7 gene by homologous recombination delayed muscle regeneration and altered the isometric contractile properties of skeletal muscles after muscle crush damage in TIS7 ؊/؊ mice. Cultured primary myogenic satellite cells (MSCs) from TIS7 ؊/؊ mice displayed marked reductions in differentiation potential and fusion index in a strictly cell-autonomous fashion. Loss of TIS7 caused the down-regulation of musclespecific genes, such as those for MyoD, myogenin, and laminin-␣2. Fusion potential in TIS7 ؊/؊ MSCs could be rescued by TIS7 expression or laminin supplementation. Therefore, TIS7 is not essential for mouse development but plays a novel regulatory role during adult muscle regeneration.Cell proliferation and differentiation are governed by different stimuli, including soluble growth factors, the extracellular matrix (1, 12), and direct cell-cell interactions (8). While each of these signals uniquely regulates mitogenic responses and gene activity, the proliferation, differentiation, or apoptosis of a cell is an integrated response to its adhesive and growth factor environments (18,19).The mouse tis7 (PC4) gene was identified as an immediateearly gene specifically induced by tetradecanoyl phorbol acetate, epidermal growth factor, and fibroblast growth factor in Swiss 3T3 mouse cells and cultured rat astrocytes (2, 20). Vietor et al. showed previously that TIS7 is up-regulated after c-Jun activation in epithelial cells, translocates into the nucleus, and acts as a transcriptional coregulator (22). TIS7 can interact with mSin3B and histone deacetylase (HDAC) 1 as well as other members of the HDAC complex and is capable of specific transcriptional repression in an HDAC-dependent manner (22).TIS7 was shown to be expressed in C2C12 myoblasts and in differentiated myotubes, with a transient decrease after the onset of differentiation (7). In vitro studies with C2C12 myoblasts and antisense tis7 DNA transfection or microinjection of anti-TIS7 polyclonal antibodies caused a delay in myoblast differentiation (7). To define the essential functions of TIS7 in vivo, we have generated mice lacking a functional tis7 gene by homologous recombination. TIS7 Ϫ/Ϫ mice are viable and fertile but develop an interesting muscle regeneration phenotype upon muscle crush damage (MCD). In addition, we identified several myoblast-specific genes involved in muscle regeneration as being regulated by TIS7. This phenotype can be recapitulated in vitro in TIS7Ϫ/Ϫ primary myogenic satellite cell (MSC) cultures and is characterized by an almost complete absence of fusion-competent MSCs. In addition, we identified several myoblast-specific genes involved in satellite cell function as being regulated by TIS7. These d...

Highlights d Dedicated genes govern paligenosis, a conserved cellular regeneration program d DDIT4 first blocks mTORC1, inducing massive autophagy to downscale the cell d p53 activation continues mTORC1 suppression to maintain cell quiescence d IFRD1 suppresses p53 to reinduce mTORC1 and license progression into the cell cycle