Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARa/c, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-kB activation were noted. TGFb1 and proinflammatory cytokines like IL1, IL6 and TNFa were upregulated and stress-related signalingpathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.
(Dohrman et al., 2005) and TRAF2 (Kataoka and Tschopp, 2004) together with the postulated interaction of cFLIP p22 with IKKγ (Chaudhary et al., 1999; Golks et al., 2006) support the hypothesis that different cFLIP isoforms might be able to differentially regulate death receptor-induced non-apoptotic signalingpathways such as NF-κB or MAPK. These may result in activation of distinct sets of target genes upon death ligand stimulation that potentially facilitate tumor progression or metastasis, as reported in a pancreatic tumor model (Trauzold et al., 2006). In order to carefully evaluate tumor therapy conditions it is necessary to not only enhance apoptosis induction but in addition prevent activation of pro- invasive and pro-inflammatory signalingpathways. Nevertheless it is suggested that interference with cFLIP expression in combination with TRAIL agonist stimulation may represent an attractive therapy at least for melanoma treatment (Chawla-Sarkar et al., 2004; Geserick et al., 2008; Hamai et al., 2006; Ivanov and Hei, 2006). On the other hand, the contradictive data concerning cFLIP and CD95-induced NF-κB activation arrogates further investigation of cFLIP proteins in death receptor-mediated signalingpathways, before there modulation can be used in therapeutic approaches. So far as it known, cFLIP is supposed to decrease (Kreuz et al., 2004; Wachter et al., 2004), not influence (Legembre et al., 2004), or increase (Golks et al., 2006) CD95-induced NF-κB activity. A direct link between CD95 stimulation and NF-κB activation got recently new evidence (Neumann et al., 2010). Authors of original paper showed that the p43 cleavage fragment of cFLIP L interacts also with the IKK
The family of Rho-GTPases consists of 25 members divided roughly into 6 subfamilies based on their primary sequence and known functions: Rho-like, Rac-like, Cdc42-like, Rnd, RhoBTB and Miro, except RhoD, Rif and TTF/RhoH, which do not obviously fall into any of these subfamilies (Wennerberg and Der 2004). Like all GTPases, Rho-GTPases cycle between active (GTP-bound) and inactive (GDP-bound) conformations regulated by guanine-nucleotide exchange factors (GEFs), and GTPase activating proteins (GAPs). In addition, Rho-GTPases are regulated further by guanine-nucleotide dissociation inhibitors (GDIs) that inhibits both the exchange of GTP and the hydrolysis of bound GTP (Van Aelst and D'Souza-Schorey 1997). Although, platelets lack a complete enlisting and characterization of its GTPases, Rac, Cdc42 and Rho are the three important Rho-GTPase subfamilies that regulate different signalingpathways underlying distinct actin dynamics based processes in platelets (see section 1.4.4). Although the presence of different isoforms of Rho (RhoA, RhoB, and RhoC) and Cdc42 proteins in platelets are yet to be characterized, RhoA expression seems to be dominant in platelets (Nemoto et al. 1992). Recently, it was found that Rac1 but neither Rac2 nor Rac3 isoforms is expressed in human and murine platelets (McCarty et al. 2005). Apart from the Rho-GTPases family proteins, some members of other GTPase families such as Ras (e.g., Rap1B, Rap2B) and Arf (Arf6) have been suggested to regulate cytoskeletal dynamics in activated platelets (Torti et al. 1999; Chrzanowska-Wodnicka et al. 2005; Choi et al. 2006).
Endometriosis is characterized by growth of endometrial-like tissue outside the uterine cavity. Since its pathogenesis may involve epigenetic changes, we used Illumina 450K Methylation Beadchips to profile CpG methylation in endometriosis stromal cells compared to stromal cells from normal endometrium. We validated and extended the Beadchip data using bisulfite sequencing (bis-seq), and analyzed differential methylation (DM) at the CpG- level and by an element-level classification for groups of CpGs in chromatin domains. Gen- es found to have DM included examples encoding transporters (SLC22A23), signaling components (BDNF, DAPK1, ROR1, and WNT5A) and transcription factors (GATA family, HAND2, HOXA cluster, NR5A1, OSR2, TBX3). Intriguingly, among the TF genes with DM we also found JAZF1, a proto-oncogene affected by chromosomal translocations in endo- metrial stromal tumors. Using RNA-Seq we identified a subset of the DM genes showing dif- ferential expression (DE), with the likelihood of DE increasing with the extent of the DM and its location in enhancer elements. Supporting functional relevance, treatment of stromal cells with the hypomethylating drug 5aza-dC led to activation of DAPK1 and SLC22A23 and repression of HAND2, JAZF1, OSR2, and ROR1 mRNA expression. We found that global 5hmC is decreased in endometriotic versus normal epithelial but not stroma cells, and for JAZF1 and BDNF examined by oxidative bis-seq, found that when 5hmC is detected, pat- terns of 5hmC paralleled those of 5mC. Together with prior studies, these results define a consistent epigenetic signature in endometriosis stromal cells and nominate specific tran- scriptional and signalingpathways as therapeutic targets.
In recent years, evidence has emerged that the interaction between sugar and auxin signalingpathways regulates plant growth and development. Auxin biosynthesis in plants is quite complex and different pathways have been postulated, including the indole-3-acetamide (IAM) pathway, the indole-3-pyruvic acid (IPA) pathway and two pathways based on indole-3-acetaldoxime and indole- 3-acetonitrile found in the Brassicaceae (Mano and Nemoto, 2012). Several lines of evidence suggest that auxin biosynthesis, distribution and response are regulated by changes in sugar levels. Incubation of Arabidopsis seedlings on varying concentrations of glucose and sucrose increased the levels of IAA and those of the IAA precursor anthranilate and tryptophan (Sairanen et al., 2012). The sugar-induced IAA accumulation requires the induction of several genes associated to auxin biosynthesis. This involves the negative regulation by members of the PHYTOCHROME INTERACTING FACTOR (PIF) transcription factor family. It has been shown that the capacity of glucose to induce IAA biosynthesis was heightened in Arabidopsis pif1 pif3 pif4 pif5 quadruple mutant line, whereas the response to glucose was reduced in plants overexpressing PIF5. Besides its function in auxin synthesis, glucose also seems to influence IAA signaling and distribution. As revealed by microarray analysis, the presence of 3% glucose in growth media is sufficient to modulate the expression of 62% of IAA associated genes in Arabidopsis seedlings, including auxin receptor, transporter and auxin response factors (Mishra et al., 2009). Moreover, the Arabidopsis
In the field of endometriosis research, several studies have reported that the deregulated Ras/Raf/MEK/ERK, RhoA/ROCKII signalingpathways, and the abnormal E2 metabolism are involved in the occurrence and development of endometriosis. Targeting the abnormal activated Ras/Raf/MEK/ERK and RhoA/ROCKII signalingpathways with either ERK or ROCK inhibitor is efficient in the control of endometriosis progression. The suppression of E2 levels by using GnRH agonists or aromatase inhibitor has become a general therapy for endometriosis. Nevertheless, up to now to our knowledge, the detailed molecular mechanisms of the Ras/Raf/MEK/ERK and RhoA/ROCKII signalingpathways, as well as their interactions with E2 signaling in endometriosis pathogenesis and normal human endometrial physiology are still unclear. The present study is the first report to provide a comprehensive understanding of the molecular mechanisms of the Ras/Raf/MEK/ERK and RhoA/ROCKII signalingpathways involved in endometriosis pathogenesis, especially in the coordination between cell proliferation and migration. Additionally, it goes insights into the interactions of E2 signaling with these two pathways, wherein the roles of classical ER/ER and non-classical GPR30 are characterized in normal hESCs. This work not only increases the knowledge in endometriosis pathogenesis and normal human endometrial physiology, but also provides a molecular rationale of using specific inhibitors for the Ras/Raf/MEK/ERK and RhoA/ROCKII signalingpathways for endometriosis therapy. It also suggests a novel and efficient endometriosis therapy for the future, e.g. targeting both Ras/Raf/MEK/ERK and RhoA/ROCKII signalingpathways and/or in combination with hormone therapy.
] showed how to use ensem- bles to unravel operating principles in signalingpathways. They created an ensemble of plausible models of the target of rapamycin (TOR) pathway in S. cerevisiae, in which the different topologies accounted for uncertainties in network structure: each member of the ensemble extended a core model by including an additional reaction. By clustering the models according to their training errors, they determined the common features shared by those that better reproduced the experimentally observed behaviour. In this way, a new fac- tor was proposed as the key signaling mechanism. Ensembles of dynamic systems have been used for many years in weather forecasting. In that community, sets of simulations with different initial conditions (ensemble modeling) and/or models developed by different groups (multi-model ensemble) are combined to deliver improved forecasts [ 27 , 28 ]. In the context of metabolism, Lee et al [ 29 ] have shown how to use ensembles to assess the robust- ness of non-native engineered metabolic pathways. Using the ensemble generation method proposed in [ 18 ], a sampling scheme is used to generate representative sets of parameters/ fluxes vectors, compatible with a known stoichiometric matrix. This approach is based on the fact that this problem is typically underdetermined, i.e. there are more reactions/fluxes
] showed how to use ensem- bles to unravel operating principles in signalingpathways. They created an ensemble of plausible models of the target of rapamycin (TOR) pathway in S. cerevisiae, in which the different topologies accounted for uncertainties in network structure: each member of the ensemble extended a core model by including an additional reaction. By clustering the models according to their training errors, they determined the common features shared by those that better reproduced the experimentally observed behaviour. In this way, a new fac- tor was proposed as the key signaling mechanism. Ensembles of dynamic systems have been used for many years in weather forecasting. In that community, sets of simulations with different initial conditions (ensemble modeling) and/or models developed by different groups (multi-model ensemble) are combined to deliver improved forecasts [ 27 , 28 ]. In the context of metabolism, Lee et al [ 29 ] have shown how to use ensembles to assess the robust- ness of non-native engineered metabolic pathways. Using the ensemble generation method proposed in [ 18 ], a sampling scheme is used to generate representative sets of parameters/ fluxes vectors, compatible with a known stoichiometric matrix. This approach is based on the fact that this problem is typically underdetermined, i.e. there are more reactions/fluxes than metabolites. Thus, model ensembles may be generated by considering all theoretically possible models, or a representative sample of it. The use of an ensemble composed of all models compatible with the data has been applied to gene regulatory [ 15 ] and signal trans- duction networks [ 30 ].
To induce terminal differentiation and expression of β-casein a mixture of lactogenic hormones (DIP) was used. These lactogenic hormones (dexamethasone, insulin and prolactin) in turn activate three major signalingpathways regulating the transcription of target genes involved in differentiation, including the milk genes (Hennighausen, 1997). The prolactin pathway is a very important pathway in this respect and regulates the phosphorylation and activation of Stat5 (Gouilleux et al., 1994). Stat5 is the major player in prolactin-activated signaling governing proliferation and functional differentiation of alveolar epithelial cells (Miyoshi et al., 2001), and is also required for the maintenance of their secretory ability during lactation (Cui et al., 2004). From these data it can be surmised that rapamycin prevents β-casein expression by inhibiting the Stat5 signaling. However, western blot analysis showed that rapamycin has no influence either on phosphorylation of Stat5 (Figure 3.7) or on the nuclear translocation of Stat5 (Figure 3.8). Both experiments showed that there was no significant difference between treated and control cells. Finally, luciferase assays revealed that the transcriptional activation of the target gene by Stat5 was comparable in treated and control cells (Figure 3.9). From these results it can be concluded that mTOR mediates its effect on the differentiation of mammary epithelial cells independently of the Prolactin/Stat5 signaling.
The members of the JAK family associate constitutively with a variety of cytokine receptors. Cytokines have critical functions in regulating immune responses. A large number of these factors bind related receptors termed the typeⅠ and typeⅡ cytokine receptors (O'Shea et al. 2000). Upon binding of the specific ligand to these receptors, JAK are rapidly activated and their kinase activities induce tyrosine phosphorylation of various effectors and initiate activation of downstream signalingpathways (Verma et al. 2003). The receptors interacting with JAK family members are EPO-R (erythropoietin receptor), IL-6R, IL-2R, IFNγ-R and IFNα⁄β-R (Gauzzi et al. 1997). In addition to STAT JAK can activate other signalingpathways, i.e. MAP kinases. As JAK are essential elements in cytokine signaling, it has been widely recognized that these kinases might be reasonable targets for the development of novel immunosuppressants (Waldmann and O'Shea 1998). Various JAK-dependent pathways are also involved in malignancies, therefore these pathways gained recently more attention as putative antitumor targets (Nefedova and Gabrilovich 2007).
Upon activation of LT β R p52-RelB DNA binding complexes were formed while TNFR signaling induced negligible RelB DNA binding in three fibroblast systems used. In order to understand the mechanism underlying this qualitative and quantitative differences in NF- κ B response, protein levels for p100, RelB and p52 were analyzed by immunoblotting. Since p52 is generated from its precursor p100, one mechanism could be differential processing of the precursor downstream of these signalingpathways, making p52 levels the limiting factor. Indeed, while p100 was processed upon LT β R triggering and the generated p52 was partly nuclear, consistent with the observed increase in DNA binding activity, TNFR triggering resulted in massive production and accumulation of the p100 precursor both in cytoplasm and in nucleus (Fig. 3.17A, compare p100 levels in TNF and AC lanes). In addition, TNFR induced total cellular RelB levels more significantly than LT β R (Fig 3.17 A). Interestingly, nuclear RelB levels were significantly higher in TNF stimulated fibroblasts compared to LT β R activated ones. Despite this, no significant RelB DNA binding activity was detectable upon TNF induction (see Figures 3.13B and 3.15B).
Whereas conventional cAMP/PKA signalingpathways are found in several unicellular eukaryotes such as Saccharomyces cerevisiae (Santangelo, 2006) or Dictyostelium discoideum (Saran et al., 2002), kinetoplastid cAMP signaling seems to be significantly different (Figure 1.1, right). First evidence for the presence of cAMP in trypanosomes dates back to the year 1974 when Walter and co- workers reported AC and PDE activities in T. b. gambiense (Walter, 1974; Walter et al., 1974) followed by the discovery of changes in the intracellular cAMP concentration between different life cycle stages of T. lewisi by Strickler and Patton (1975). In the following decades, multiple reports analyzed putative components and functions of cAMP signaling in kinetoplastids (e.g. Naula and Seebeck, 2000; Seebeck et al., 2001; Seebeck et al., 2004; Laxman and Beavo, 2007; Oberholzer et al., 2007; Bhattacharya et al., 2008, 2009; Biswas et al., 2011; Gould and de Koning, 2011; Bhattacharya et al., 2012; de Koning et al., 2012; Salmon et al., 2012a; Salmon et al., 2012b; Gould et al., 2013; Lopez et al., 2014; Saada et al., 2014; Vij et al., 2014). Apart from the apparent absence of genes encoding GPCRs or heterotrimeric G proteins from kinetoplastid genomes (see chapter 1.1.1), the existence of a cAMP-dependent kinase activity in kinetoplastids is an open question. As in most other eukaryotes, cAMP is produced by ACs and degraded by PDEs. Kinetoplastid ACs, however, diverge from their mammalian orthologues in structure and sequence (see chapter 1.1.1) and were suggested to replace the receptor function of GPCRs. Yet, no ligand has been identified to date. Activation of trypanosomal ACs has been observed upon exposure to stress conditions such as acid treatment or osmotic lysis (Voorheis and Martin, 1980; Rolin et al., 1996; Nolan et al., 2000). Interestingly, there is a remarkable expansion of the AC family in the genomes of the African trypanosomes T. brucei and T. congolense (www.tritrypdb.org), which live extracellularly in the mammalian bloodstream, in contrast to T. cruzi or L. donovani, kinetoplastid parasites developing intracellularly in host cells. Four families of cAMP-specific class I PDEs have been characterized in T.
pathway had a protective effect against CPF-induced cytotoxicity, and the p38-MAPK inhibitor might influence the isoform of p38-MAPK and might contribute to the activation of the survival signalingpathways . CPF and CPF-oxone activated ERK44/42 signaling in both wild-type (CHOK1) and human muscarinic receptor-expressing ovarian cells of the Chinese hamster (CHO-M2). CPF-Oxone increased the activation of the ERK 44/42 pathway in CHOK1 2- to 3-fold control cells, depending on dose and time. Further, CPF-Oxon activated ERK 44/42 signaling in CHOK1 cells by P13-K, PKC, and MEK . The same group also showed that diacylglycerol (DAG) induced the activation of ERK 44/42 in the ovary of the Chinese hamster (CHOK1). Pretreatment of CHOK1 cells with CPF-oxone or a carbamate increased the effect of DAG on the activation of ERK44/42 in a dose- and time-dependent manner, suggesting that inhibition of DAG lipase activity by CPF-oxone altered DAG metabolism and induced activation of the ERK 44/42 signaling pathway . In another study by Lim et al., the effects of trichlorfon on the proliferation and apoptosis of porcine trophicotoderm (pTr) and uterine luminal epithelial cells (pLE) were investigated . Trichlorfon prevented the proliferation of pTr and pLE cells, which led to apoptosis, loss of mitochondrial membrane potential, and DNA fragmentation. In addition, trichlorfon reduced the phosphorylation of pTr and pLE cells. Trichlorfon temporarily stimulated several molecular signaling proteins such as P70S6K, P90RSK, S6, JNK, and p38-MAPK, and inhibition of the ERK1/2, JNK, and p38-MAPK signalingpathways by trichlorfon reduced proliferation compared to each inhibitor alone in pTr cells. In addition, inhibition of ERK1/2 and p38-MAPK reduced proliferation of pLE cells, and this reduction was more pronounced when trichlorfon was combined with U0126 (ERK-inhibitor) or p38-MAPK inhibitors. The results indicated that trichlorfon had a synergic antiproliferative effect with the MAPK inhibitor on pLE and pTr cells during early pregnancy .
The following sections describe crucial processes of neutrophil activation in more detail and relevant signalingpathways will additionally be explained.
3.2.1 Neutrophil migration
Invasion of pathogens and subsequent infection provoke proinflammatory responses of affected tissue cells and result in the release of host cell- as well as pathogen-derived chemoattractants . Those substances form a concentration gradient reaching from the site of infection to adjacent vessels and activate endothelial cells [94,95]. Activated endothelium itself presents chemotactic agents on the cell membrane and both processes generate a mixture of proinflammatory gradients consisting of several substances like formyl peptides, C5a, platelet-activating factor (PAF), leukotriene B4 (LTB4) or interleukin-8 (IL-8, CXCL8) [95,184,185]. Furthermore, endothelial cells initiate cell surface expression of P-selectin upon activation . Neutrophils express the counterreceptor P-selectin glycoprotein ligand-1 (PSGL-1) and are tethered to the induced endothelial P-selectin in proximity of the site of infection [187,188]. Further interactions are generated by endothelium expressed E-selectin and L-selectin ligands as well as according neutrophil receptors PSGL-1, E-selectin ligand 1 (ESL1) and L-selectin (CD62L) [180,187–191]. These interactions result in an ongoing movement of neutrophils on endothelial cells named “rolling” [94–96,110]. Rolling is driven by and dependent on the blood stream as selectin interactions are abrogated without shear force [94,94,192–194]. Of note, PSGL-1 is also able to bind L-selectin and respective interactions are thought to enable homotypic neutrophil adherence and therefore enhanced neutrophil recruitment [95,195,196]. The ligation of neutrophil adhesion receptors and the stimulation by aforementioned chemoattractants induce early degranulation of secretory vesicles which leads to upregulation and conformational activation of α M β 2 -integrin Mac-1 on the neutrophil surface
As being highly inducible on the transcriptional level by a multitude of stimuli and involved in diverse signalingpathways, HO-1 is a very important protective strategy of the cell against stress situations (Otterbein et al.; 2000). Consistent with the diversity of signaling cascades involved in HO-1 induction, the promotor region of HO-1 contains a variety of regulatory elements including binding sites for NF-κB and AP-1 (Lavrovsky et al.; 1994). In the past, the degradation products CO, free iron, and bilirubin were considered as toxic waste (Wagener et al.; 2003). Recent insights have provided evidence that this is not the case. Today, the vital function of HO-1 in maintaining cellular homeostasis is ascribed to its breakdown products. Although potentially harmful when present in excessive amounts, all three metabolites have important regulative and protective features:
Prerequisite for the investigation of apoptotic signaling in myocardial I/R was the development and characterization of a cell culture-based model thereof. Prior to this body of work no such model existed that closely reflected the in vivo situation: commonly used myocyte cell lines (H9c2, C2C12) and cultured neonatal cardiac myocytes require prolonged (from 8 to 24 h) hypoxia or simulated ischemia to activate death signalingpathways (Dougherty et al., 2002; Hollander et al., 2003; Hou and Hsu, 2005). Alternatively, oxidizing agents (e.g. hydrogen peroxide) or initiators of apoptosis (e.g. staurosporine) are employed as surrogates to explore I/R injury (Capano and Crompton, 2002; Akao et al., 2003), which do not necessarily recapitulate the events of I/R injury. We sought to establish an acute model which closely resembles the in vivo signalingpathways during I/R injury to the heart, using the recently established cardiac HL-1 cell line which has been described as a successful model for studying many aspects of cardiac cell physiology (White et al., 2004). As HL-1 cells are readily transfectable, as opposed to the adult cardiac cell which cannot be transfected using conventional means, we consider the HL-1 cell a unique system for investigating in a single cell resolution the pro-death and pro-survival pathways during I/R injury.
shown to inhibit migration of various types of tumor cells, including glioblastoma cells ([ 23 , 31 ]; this study), memantine constitutes a promising antagonist against “oncogenic NMDARs” [ 31 ], which is currently investigated in a phase I trial for post-radiation therapy in glioblastoma [ 46 ]. Interestingly, we further could show by colony formation assays and viability tests that blocking GluN2B-containing NMDARs by ifenprodil suppresses tumor cell survival more efficiently compared to the broad NMDAR antagonists MK801 and memantine. Based on these results we assume differential effects of NMDAR subunit-specific antagonists, indicating that subunit composition and/or membrane localization of the NMDAR subunits contribute different to glioblastoma malignancy. Indeed, we found a specific localization of the GluN2B-subunit in lamellipodia and a pronounced effect of the GluN2B-specific antagonist ifenprodil on migration of LN229 cells. Due to the high invasiveness of glioblastoma cells, our data are consistent with the idea of a specific role of GluN2B-NMDAR-signalingpathways at the invasive front of glioblastoma progression. This assumption is supported by the finding that grade 4 glioblastoma patients with high expression levels of GluN2B in the tumor have a worse prognosis [ 29 ] and underpin GluN2B-specific antagonists as an interesting therapeutic approach for treating brain tumors.
CSN5 knockdown was found to decrease the levels of Notch intracellular domain (NICD), the transcriptional activator of Notch target gene expression, in CRC cells. This promoted the transcription of ATOH-1 and DLL-1, two genes that are repressed by Notch signaling and known as markers for goblet cells. In a next step, an ex vivo small intestine organoid approach was used to further investigate Csn’s functional role in mouse gut. Induction of a homozygous Csn5 or Csn8 knockout resulted in the death of organoids within four days, whereas a heterozygous knockout of these Csn subunits did not apparently affect the organoids. Interestingly, only the heterozygous knockout of Csn5 or Csn8 resulted in enhanced mRNA expression of Atoh-1 and Dll-1, supporting the hypothesis of a shift of differentiation towards secretory cell types. Of note, the enterocyte marker ‘alkaline phosphatase’ was reduced in Csn subunit knockout organoids and the homozygous Csn5 knockout seemed to increase the number of enteroendocrine cells as evidenced by IHC staining. In contrast to my prediction, no goblet cell hyperplasia became visible in the knockout organoids, pointing to the importance of other cell types such as immune cells for epithelial cell differentiation in vivo. Organoid death observed following homozygous knockout of Csn5 or Csn8 was accompanied by an increased degree of apoptosis and a reduced proliferation rate. In addition, the homozygous knockout of the Csn subunits resulted in nuclear accumulation of β-catenin and increased Atoh-1 protein levels as measured by IHC, although it was not possible to confirm these results by immunoblotting. These results indicate that the integrity of the Csn complex is crucial for intestinal epithelium viability.
The discovery of caveolae is accredited to the Nobel laureate George Emil Palade (Palade, 1953), who called them plasmalemmal vesicles. Two years later, E. Yamada described similar structures in the gall bladder epithelium, naming them caveolae intracellulares due to their appearance (YAMADA, 1955). Caveolae are flask-shaped vesicular invaginations of the plasma membrane 50–100 nm in diameter. The main structural proteins of caveolae are the caveolins. The latter form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. In recent years interest in caveolae increased more and more due to improved insight in caveolin function and caveolae-associated signal transduction (Williams and Lisanti, 2004b). Caveolin proteins are expressed in most of the cell types that play a role in the development of atherosclerosis. These cells include endothelial cells, macrophages and smooth muscle cells (Frank and Lisanti, 2004). The next paragraphs will show recent findings essential for this study.
lation of intracellular signaling cascades that apply hydrogen peroxide as a second-messenger molecule, and regulation of cell proliferation and differentiation  . Mice with a targeted deletion of the Prx I gene have been reported to develop hemolytic anemia with an increase in reactive oxygen species in erythrocytes and malignancies such as sarcomas, lymphomas, and carcinomas, suggesting that Prx I may function as a tumor suppressor [8,9] . In contrast, it has also been demonstrated by others that Prx I is highly expressed in a number of malignant tumors and may promote an aggressive survival type of tumor cells and resistance to tumor therapy [10,11] . A number of stimuli, among them the phorbol ester 12-O-tetradecanoylphor- bol- 13-acetate (TPA), induce Prx I gene expression at the transcriptional level [12–15] . We have previously demonstrated that TPA-dependent Prx I gene expression is activated in mono- cytic cells via a protein kinase C (PKC)/Ras-dependent signaling pathway that targets the transcription factor (TF) activator protein-1 (AP-1)  .