Role of the small GTPase Rab7 in the late endocytic pathway.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic

LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes

<p>Abstract</p> <p>Background</p> <p>The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region.</p> <p>Results</p> <p>RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa.</p> <p>Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting.</p> <p>Conclusion</p> <p>The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.</p

TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters

We have recently reported that a novel MBD2 interactor (MBDin) has the capacity to reactivate transcription from MBD2-repressed methylated promoters even in the absence of demethylation events. Here we show that another unrelated protein, TACC3, displays a similar activity on methylated genes. In addition the data reported here provide possible molecular mechanisms for the observed phenomenon. Immunoprecipitation experiments showed that MBD2/TACC3 form a complex in vivo with the histone acetyltransferase pCAF. MBD2 could also associate with HDAC2, a component of MeCP1 repression complex. However, we found that the complexes formed by MBD2 with TACC3/pCAF and with HDAC2 were mutually exclusive. Moreover, HAT enzymatic assays demonstrated that HAT activity associates with MBD2 in vivo and that such association significantly increased when TACC3 was over-expressed. Overall our findings suggest that TACC3 can be recruited by MBD2 on methylated promoters and is able to reactivate transcription possibly by favoring the formation of an HAT-containing MBD2 complex and, thus, switching the repression potential of MBD2 in activation even prior to eventual demethylation

PATZ Attenuates the RNF4-mediated Enhancement of Androgen Receptor-dependent Transcription *

PATZ is a transcriptional repressor affecting the basal activity of different promoters, whereas RNF4 is a transcriptional activator. The association of PATZ with RNF4 switches the activation to repression of selected basal promoters. Because RNF4 interacts also with the androgen receptor (AR) functioning as a coactivator and, in turn, RNF4 associates with PATZ, we investigated whether PATZ functions as an AR coregulator. We demonstrate that PATZ does not influence directly the AR response but acts as an AR corepressor in the presence of RNF4. Such repression is not dependent on histone deacetylases. A mutant RNF4 that does not bind PATZ but enhances AR-dependent transcription is not influenced by PATZ, demonstrating that the repression by PATZ occurs only upon binding to RNF4. We also demonstrate that RNF4, AR, and PATZ belong to the same complex in vivo also in the presence of androgen, suggesting that repression is not mediated by the displacement of RNF4 from AR. Finally, we show that the repression of endogenous PATZ expression by antisense expression plasmids in LNCaP cells results in a stronger androgen response. Our findings demonstrate that PATZ is a novel AR coregulator that acts by modulating the effect of a coactivator. This could represent a novel and more general mechanism to finely tune the androgen response

The Platelet-derived Growth Factor Controls c-myc Expression through a JNK- and AP-1-dependent Signaling Pathway *

Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene

Helicobacter pylori Up-regulates Cyclooxygenase-2 mRNA Expression and Prostaglandin E2 Synthesis in MKN 28 Gastric Mucosal Cells in Vitro

Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pylori strain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity. This effect was specifically related to H. pylori because it was not observed with Escherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lacking picA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains

Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes

Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identification of a novel 45 kDa protein that specifically binds Rab7GTP at its C-terminus. This protein contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. We named it RILP (Rab-interacting lysosomal protein) since it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP. RILP-C33 (a truncated form of the protein lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant-negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant-negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traffic