MCPIP1 inhibits Wnt/ß-catenin signaling pathway activity and modulates epithelial-mesenchymal transition during clear cell renal cell carcinoma progression by targeting miRNAs.

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active ß-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/ß-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear ß-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active ß-catenin and EMT inducers.

Fatty Acids and a High-Fat Diet Induce Epithelial-Mesenchymal Transition by Activating TGFß and ß-Catenin in Liver Cells.

Nonalcoholic fatty liver disease is defined as the accumulation of excessive fat in the liver in the absence of excessive alcohol consumption or any secondary cause. Although the disease generally remains asymptomatic, chronic liver inflammation leads to fibrosis, liver cirrhosis, and even to the development of hepatocellular carcinoma (HCC). Fibrosis results from epithelial-mesenchymal transition (EMT), which leads to dedifferentiation of epithelial cells into cells with a mesenchymal-like phenotype. During EMT, epithelial cells with high expression of E-cadherin, influenced by growth factors, cytokines, and inflammatory processes, undergo morphological changes via enhanced expression of, e.g., vimentin, fibronectin, and N-cadherin. An inducer of EMT and, consequently, of fibrosis development is transforming growth factor beta (TGFß), a pleiotropic cytokine associated with the progression of hepatocarcinogenesis. However, the understanding of the molecular events that direct the development of steatosis into steatohepatitis and liver fibrosis remains incomplete. Our study revealed that both prolonged exposure of hepatocarcinoma cells to fatty acids in vitro and high-fat diet in mice (20 weeks) result in inflammation. Prolonged treatment with fatty acids increased the levels of TGFß, MMP9, and ß-catenin, important EMT inducers. Moreover, the livers of mice fed a high-fat diet exhibited features of liver fibrosis with increased TGFß and IL-1 levels. Increased expression of IL-1 correlated with a decrease in monocyte chemoattractant protein-induced protein 1 (MCPIP1), a negative regulator of the inflammatory response that regulates the stability of proinflammatory transcripts encoding IL-1. Our study showed that a high-fat diet induced EMT by increasing the levels of EMT-activating transcription factors, including Zeb1, Zeb2, and Snail and changed the protein profile to a profile characteristic of the mesenchymal phenotype.

Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors.

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.

Adipose-Derived Stromal Cells Seeded on Integra® Dermal Regeneration Template Improve Post-Burn Wound Reconstruction.

Fibrosis of burn-related wounds remains an unresolved clinical issue that leads to patient disability. The aim of this study was to assess the efficacy of the transplantation of adipose-derived stromal cells seeded onto a collagen-based matrix in the reconstruction of burn-related scars. Here, we characterized an in vitro interaction between adipose-derived stromal cells and a collagen-based matrix, Integra®DRT. Our results show that transcription of pro-angiogenic, remodeling, and immunomodulatory factors was more significant in adipose-derived stromal cells than in fibroblasts. Transcription of metalloproteinases 2 and 9 is positively correlated with the collagenolytic activity of the adipose-derived stromal cells seeded onto Integra®DRT. The increase in the enzymatic activity corresponds to the decrease in the elasticity of the whole construct. Finally, we validated the treatment of a post-excision wound using adipose-derived stromal cells and an Integra®DRT construct in a 25-year-old woman suffering from burn-related scars. Scarless healing was observed in the area treated by adipose-derived stromal cells and the Integra®DRT construct but not in the reference area where Integra®DRT was applied without cells. This clinical observation may be explained by in vitro findings: Enhanced transcription of the vascular endothelial growth factor as well as remodeling of the collagen-based matrix decreased mechanical stress. Our experimental treatment demonstrated that the adipose-derived stromal cells seeded onto Integra®DRT exhibit valuable properties that may improve post-excision wound healing and facilitate skin regeneration without scars.

Nuclear immunophilin FKBP39 from Drosophila melanogaster drives spontaneous liquid-liquid phase separation.

The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired us to investigate whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. We also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, we analysed it in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Inhibition of ERß induces resistance to cisplatin by enhancing Rad51-mediated DNA repair in human medulloblastoma cell lines.

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERß, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERß considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.

HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction.

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.

Solute-dependent activation of cell motility in strongly hypertonic solutions in Dictyostelium discoideum, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells.

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na(+) or K(+), sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na(+) ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.

Reversible inhibition of movement in the amoebae Dictyostelium discoideum and its effect on chemoattractant recognition.

The cell fixatives formaldehyde and KMnO4 at low concentrations reversibly inhibit the movement of D. discoideum amoebae without directly interfering with cell viability. This inhibition of cell movement is accompanied by the decreased attachment of cells to substratum. When the tenacity and attachment of immobilized cells are artificially increased by compressing cells between two glass surfaces, the amoebae begin to move even in the presence of the fixatives. Amoebae starved for 24 hours, subjected to fixatives and a mineral salt solution in which they remained motionless, maintained chemotactic responses to folic acid and only after a few hours of active locomotion became reactive to cAMP, in contrast to amoebae that reacted to cAMP after starvation in the absence of fixatives.

Some difficulties in research into cell motile activity under isotropic conditions.

Movement of Dictyostelium discoideum amoebae under isotropic and anisotropic conditions was recorded and analysed with computer-aided methods and the results are presented in various manners as described in the subject literature. Cell movement under isotropic conditions showed great diversity. Some cells moved almost in a straight path whereas others in close proximity turned around with little net translocation. When cell movement under isotropic conditions was observed, no direct correlation was found between the total length of cell trajectories and the length of final displacements of the cells. It was necessary to present the results in the form of histograms, circular diagrams of cell trajectories or in scatter correlation diagrams showing the motile behaviour of many individual cells. These methods of presentation are more informative than methods which present only average values, the "representative" behaviour of single cells, or start and end points of cell tracks. The latter methods can only illustrate but do not document the results of experiments. The use of statistical methods appears necessary in cases when it is difficult to monitor the same cells before and during experimental treatment. However, when cell movement under anisotropic conditions becomes oriented and ordered as during tactic cell movements, then the diversity in cell behaviour decreases and methods based on estimation of starting and end points of cell positions appear more credible.

Motile activities of Dictyostelium discoideum differ from those in Protista or vertebrate animal cells.

Cell movement in the amoebae Dictyostelium discoideum has been examined in media differing in monovalent cation concentration (i.e. Na+ and K+). Under isotonic or even slightly hypertonic conditions, the cells move equally well in solutions in which either potassium or sodium ions dominate. However, in strongly hypertonic solutions the amoebae showed motility in a 2% potassium chloride solution, but remained motionless in a hypertonic 2% sodium chloride solution. This inhibition of D. discoideum amoebae movement in a hypertonic sodium chloride solution was fully reversible. Such behaviour corresponds to that of plant, fungi, and some invertebrate animal cells rather than protozoan or vertebrate cells. These observations suggest that studies using D. discoideum as a model for cell motility in vertebrate animal tissue cells should be considered with caution, and would seem to confirm the classification of cellular slime moulds as related rather to Fungi than to Protista. This also shows that the cell membrane models should consider the asymmetry in sodium/potassium ion concentrations found in vertebrate animal cells as one of various possibilities.

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa.

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.