Reciprocal regulation of the Cadherin-11/Stat3 axis by caveolin-1 in mouse fibroblasts and lung carcinoma cells.

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.

Classical cadherins control survival through the gp130/Stat3 axis.

Stat3 (Signal Transducer and Activator of Transcription-3) is activated by a number of receptor and nonreceptor tyrosine kinases. We recently demonstrated that engagement of E-cadherin, a calcium-dependent, cell to cell adhesion molecule which is often required for cells to remain tightly associated within the epithelium, also activates Stat3. We now examined the effect of two other classical cadherins, cadherin-11 and N-cadherin, whose expression often correlates with the epithelial to mesenchymal transition occurring in metastasis of carcinoma cells, upon Stat3 phosphorylation and activity. Our results indicate that engagement of these two cadherins also, can trigger a dramatic surge in Stat3 activity. This activation occurs through upregulation of members of the IL6 family of cytokines, and it is necessary for cell survival, proliferation and migration. Interestingly, our results also demonstrate for the first time that, in sharp contrast to Stat3, the activity of Erk (Extracellular Signal Regulated kinase) was unaffected by cadherin-11 engagement. Further examination indicated that, although IL6 was able to activate Erk in sparsely growing cells, IL6 could not induce an increase in Erk activity levels in densely growing cultures. Most importantly, cadherin-11 knock-down did allow Erk activation by IL6 at high densities, indicating that it is indeed cadherin engagement that prevents Erk activation by IL6. The fact that the three classical cadherins tested so far, E-cadherin, N-cadherin and cadherin11, which are present in essentially all tissues, actually activate Stat3 regardless of their role in metastasis, argues for Stat3 as a central survival, rather than invasion factor.

Elevated PIN1 expression by C/EBPalpha-p30 blocks C/EBPalpha-induced granulocytic differentiation through c-Jun in AML.

The transcription factor CCAAT enhancer-binding protein alpha (C/EBPalpha) has an important role in granulopoiesis. The tumor suppressor function of C/EBPalpha is shown by the findings that loss of expression or function of C/EBPalpha in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPalpha mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPalpha (C/EBPalpha-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPalpha-p30 in AML. In the present study, we show that C/EBPalpha-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPalpha-p30 results in recruitment of E2F1 in the PIN1 promoter. We show that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPalpha mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also show that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination, and c-Jun blocks granulocyte differentiation mediated by C/EBPalpha. Our data suggest that the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPalpha mutation.

Proteomic discovery of Max as a novel interacting partner of C/EBPalpha: a Myc/Max/Mad link.

The transcription factor CCAAT/enhancer binding protein a (C/EBPalpha) is important in the regulation of granulopoiesis and is disrupted in human acute myeloid leukemia. In the present study, we sought to identify novel C/EBPalpha interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPalpha in our screen. We confirmed the in vivo interaction of C/EBPalpha with Max and showed that this interaction involves the basic region of C/EBPalpha. Endogenous C/EBPalpha and Max, but not Myc and Max, colocalize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPalpha on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBPalpha promoter in vivo by Max and Myc under cellular settings and by C/EBPalpha and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPalpha results in granulocytic differentiation of the human hematopoietic CD34(+) cells, as evidenced by CD11b, CD15 and granulocyte colony-stimulating factor receptor expression. Silencing of Max by short hairpin RNA in CD34(+) and U937 cells strongly reduced the differentiation-inducing potential of C/EBPalpha, indicating the importance of C/EBPalpha-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPalpha functions, thereby suggesting a possible link between C/EBPalpha and Myc-Max-Mad network.

Proteomics of acute myeloid leukaemia: Cytogenetic risk groups differ specifically in their proteome, interactome and post-translational protein modifications.

Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of beta-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

Circulating TNF-alpha, TGF-beta, and IL-10 in tuberculosis patients and healthy contacts.

Levels of tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and interleukin (IL)-10 in plasma of pulmonary tuberculosis (TB) patients and healthy contacts and plasma and pleural fluid of patients with tuberculous pleuritis were examined by enzyme immunoassay. Plasma TNF-alpha and IL-10 were elevated to significant levels in healthy contacts. High levels of TGF-beta and IL-10 were also detected in plasma from TB patients and healthy contacts. Pleural fluid contained all three cytokines with the level of IL-10 being highest followed by TGF-beta and TNF-alpha. Plasma of tuberculous pleuritis patients also had detectable levels of the three cytokines. Increased levels of TNF-alpha in plasma of contacts and to some extent pleural fluid of pleuritis patients, is perhaps to limit the infection, while elevated IL-10 in plasma of TB patients and contacts and pleural fluid would perhaps modulate excess proinflammation. Elevated TGF-beta in TB patients suggests its role in the immunopathogenesis.

T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts.

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.

[Considerations on the crown restoration in youngsters and teenagers]. / Consideratii privind reconstituirea coronara la tineri si adolescent.

The crown restorations in young people and teenagers represents a priority in the current dental practice. After presenting plurifactorially the etiology of the odontal lesions, the different methods of treatment employed currently in our clinic are analysed. In order to avoid some confusion in the use of some already accepted terms and to select the best treatment, the most common therapeutic means of crown restoration are described and analysed.