'Unpredictable chronic mild stress does not exacerbate memory impairment or altered neuronal and glial plasticity in the hippocampus of middle-aged vitamin D deficient mice'.

Vitamin D deficiency is a worldwide health concern, especially in the elderly population. Much remains unknown about the relationship between vitamin D deficiency (VDD), stress-induced cognitive dysfunctions and depressive-like behaviour. In this study, 4-month-old male C57Bl/6J mice were fed with control or vitamin D free diet for 6 months, followed by unpredictable chronic stress (UCMS) for 8 weeks. VDD induced cognitive impairment and reduced grooming behaviour, but did not induce depressive-like behaviour. While UCMS in vitamin D sufficient mice induced expected depressive-like phenotype and impairments in the contextual fear memory, chronic stress did not manifest as an additional risk factor for memory impairments and depressive-like behaviour in VDD mice. In fact, UCMS restored self-care behaviour in VDD mice. At the histopathological level, VDD mice exhibited cell loss in the granule cell layer, reduced survival of newly generated cells, accompanied with an increased number of apoptotic cells and alterations in glial morphology in the hippocampus; however, these effects were not exacerbated by UCMS. Interestingly, UCMS reversed VDD induced loss of microglial cells. Moreover, tyrosine hydroxylase levels decreased in the striatum of VDD mice, but not in stressed VDD mice. These findings indicate that long-term VDD in adulthood impairs cognition but does not augment behavioural response to UCMS in middle-aged mice. While VDD caused cell loss and altered glial response in the DG of the hippocampus, these effects were not exacerbated by UCMS and could contribute to mechanisms regulating altered stress response.

Characterization of IMG Microglial Cell Line as a Valuable In Vitro Tool for NLRP3 Inflammasome Studies.

Microglial cells constantly surveil the cerebral microenvironment and become activated following injury and disease to mediate inflammatory responses. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome, which is abundantly expressed in microglial cells, plays a key role in these responses as well as in the development of many neurological disorders. Microglial cell lines are a valuable tool to study the causes and possible treatments for neurological diseases which are linked to inflammation. Here, we investigated whether the mouse microglial cell line IMG is suitable to study NLRP3 inflammasome by incubating cells with different concentrations of NLRP3 inflammasome priming and activating agents lipopolysaccharide (LPS) and ATP, respectively, and applying short (4 h) or long (24 h) LPS incubation times. After short LPS incubation, the mRNA levels of most pro-inflammatory and NLRP3 inflammasome-associated genes were more upregulated than after long incubation. Moreover, the combination of higher LPS and ATP concentrations with short incubation time resulted in greater levels of active forms of caspase-1 and interleukin-1 beta (IL-1ß) proteins than low LPS and ATP concentrations or long incubation time. We also demonstrated that treatment with NLRP3 inflammasome inhibitor glibenclamide suppressed NLRP3 inflammasome activation in IMG cells, as illustrated by the downregulation of gasdermin D N-fragment and mature caspase-1 and IL-1ß protein levels. In addition, we conducted similar experiments with primary microglial cells and BV-2 cell line to determine the similarities and differences in their responses. Overall, our results indicate that IMG cell line could be a valuable tool for NLRP3 inflammasome studies. In IMG cells, 4-h incubation with lipopolysaccharide (LPS) induces a stronger upregulation of NLRP3 inflammasome-associated pro-inflammatory genes compared to 24-h incubation. NLRP3 inflammasome is robustly activated only after the addition of 3 mM of ATP following short LPS incubation time.

Enzyme-assisted extraction of anthocyanins and other phenolic compounds from blackcurrant (Ribes nigrum L.) press cake: From processing to bioactivities.

The effects of commercial enzymes (pectinases, cellulases, beta-1-3-glucanases, and pectin lyases) on the recovery of anthocyanins and polyphenols from blackcurrant press cake were studied considering two solid:solvent ratios (1:10 and 1:4 w/v). ß-glucanase enabled the recovery of the highest total phenolic content - 1142 mg/100 g, and the extraction of anthocyanins was similar using all enzymes (∼400 mg/100 g). The use of cellulases and pectinases enhanced the extraction of antioxidants (DPPH - 1080 mg/100 g; CUPRAC - 3697 mg/100 g). The freeze-dried extracts presented antioxidant potential (CUPRAC, DPPH), which was associated with their biological effects in different systems: antiviral activity against both non-enveloped viruses (enterovirus coxsackievirus A-9) and enveloped coronaviruses (HCoV-OC43), and cytotoxicity towards cancer cells (A549 and HCT8). No cytotoxic effects on normal human lung fibroblast (IMR90) were observed, and no anti-inflammatory activity was detected in lipopolysaccharides-treated murine immortalised microglial cells.

Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers.

Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inflammatory diseases with unresolved pathophysiological aspects. Extracellular vesicles (EVs) play an important role in intercellular communication. We compared the miRNA contents and surface proteome of the EVs in the blood serum of PsV and PsA patients to healthy controls. Size-exclusion chromatography was used to isolate EVs from the blood serum of 12 PsV patients, 12 PsA patients and 12 healthy control subjects. EV samples were characterized and RNA sequencing was used to identify differentially enriched EV-bound miRNAs. We found 212 differentially enriched EV-bound miRNAs present in both PsV and PsA groups-a total of 13 miRNAs at FDR ≤ 0.05. The predicted target genes of these miRNAs were significantly related to lesser known but potentially disease-relevant pathways. The EV array revealed that PsV patient EV samples were significantly enriched with CD9 EV-marker compared to controls. Analysis of EV-bound miRNAs suggests that signaling via EVs in the blood serum could play a role in the pathophysiological processes of PsV and PsA. EVs may be able to fill the void in clinically applicable diagnostic and prognostic biomarkers for PsV and PsA.

Bovine Follicular Fluid Derived Extracellular Vesicles Modulate the Viability, Capacitation and Acrosome Reaction of Bull Spermatozoa.

While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.

Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication.

BACKGROUND: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation-known as "window of implantation"-is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication. METHODS: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95-2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing. RESULTS: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors' signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs. CONCLUSION: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.

Oviduct as a sensor of embryo quality: deciphering the extracellular vesicle (EV)-mediated embryo-maternal dialogue.

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.

Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries.

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

Spermatozoa induce transcriptomic alterations in bovine oviductal epithelial cells prior to initial contact.

The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 µm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival.

Bovine Follicular Fluid and Extracellular Vesicles Derived from Follicular Fluid Alter the Bovine Oviductal Epithelial Cells Transcriptome.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability.

Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 µl droplets of culture media, and 50 µl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

Endogenous beta-galactosidase activity marks a TREM2-expressing Kupffer cell population in injured livers of Lgr5-LacZ and wild-type mice.

Lgr5-LacZ mice harbor the Escherichia coli LacZ gene encoding ß-galactosidase (ß-gal) under the control of the Lgr5 promoter, a stem/progenitor cell marker. In injured livers of Lgr5-LacZ mice, cells expressing ß-galactosidase (ß-gal) are considered as potential bipotent liver progenitors; however, their origin and identity remain unknown. Unexpectedly, using lineage tracing, we demonstrate that the ß-gal+ cells do not originate from liver parenchymal cells. Instead, ß-gal+ cells, isolated from injured livers of both Lgr5-LacZ and wild-type mice, are positive for markers of Kupffer cells, liver-resident macrophages. The ß-gal expression in these cells is a result of elevated expression of the endogenous beta-galactosidase Glb1. In injured livers of Lgr5-LacZ mice, bacterial ß-gal expression is very low, suggesting transgene silencing. The gene expression profile of the ß-gal+ Kupffer cells from injured livers suggests a role in liver regeneration.

Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk.

BACKGROUND: Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. METHODS: We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. RESULTS: We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. CONCLUSION: Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

A label-retaining but unipotent cell population resides in biliary compartment of mammalian liver.

Cells with slow proliferation kinetics that retain the nuclear label over long time periods-the label-retaining cells (LRCs)-represent multipotent stem cells in a number of adult tissues. Since the identity of liver LRCs (LLRCs) had remained elusive we utilized a genetic approach to reveal LLRCs in normal non-injured livers and characterized their regenerative properties in vivo and in culture. We found that LLRCs were located in biliary vessels and participated in the regeneration of biliary but not hepatocyte injury. In culture experiments the sorted LLRCs displayed an enhanced self-renewal capacity but a unipotent biliary differentiation potential. Transcriptome analysis revealed a unique set of tumorigenesis- and nervous system-related genes upregulated in LLRCs when compared to non-LRC cholangiocytes. We conclude that the LLRCs established during the normal morphogenesis of the liver do not represent a multipotent primitive somatic stem cell population but act as unipotent biliary progenitor cells.

The alterations in the extracellular matrix composition guide the repair of damaged liver tissue.

While the cellular mechanisms of liver regeneration have been thoroughly studied, the role of extracellular matrix (ECM) in liver regeneration is still poorly understood. We utilized a proteomics-based approach to identify the shifts in ECM composition after CCl4 or DDC treatment and studied their effect on the proliferation of liver cells by combining biophysical and cell culture methods. We identified notable alterations in the ECM structural components (eg collagens I, IV, V, fibronectin, elastin) as well as in non-structural proteins (eg olfactomedin-4, thrombospondin-4, armadillo repeat-containing x-linked protein 2 (Armcx2)). Comparable alterations in ECM composition were seen in damaged human livers. The increase in collagen content and decrease in elastic fibers resulted in rearrangement and increased stiffness of damaged liver ECM. Interestingly, the alterations in ECM components were nonhomogenous and differed between periportal and pericentral areas and thus our experiments demonstrated the differential ability of selected ECM components to regulate the proliferation of hepatocytes and biliary cells. We define for the first time the alterations in the ECM composition of livers recovering from damage and present functional evidence for a coordinated ECM remodelling that ensures an efficient restoration of liver tissue.

Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

A Novel Inhibitor of AKT1-PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth In Vivo.

The serine/threonine kinase AKT/PKB has a critical role in the regulation of cell proliferation. Because AKT signaling is deregulated in numerous human malignancies, it has become an attractive anticancer drug target. A number of small molecule AKT kinase inhibitors have been developed; however, severe side effects have prevented their use in clinical trials. To find inhibitors of AKT1 signaling with principally novel mechanism of action, we carried out a live cell-based screen for small molecule inhibitors of physical interaction between AKT1 and its primary activator PDPK1. The screen revealed one molecule-NSC156529, which downregulated AKT1 signaling, efficiently decreased the proliferation of human cancer cells in vitro, and substantially inhibited the growth of prostate tumor xenografts in vivo. Interestingly, the treated tumor xenografts exhibited higher expression level of normal prostate differentiation markers but did not show augmented cell death, suggesting that the identified compound primarily enhances the differentiation of malignant cells toward normal prostate epithelium and thus poses as an attractive lead compound for developing novel antitumor agents with less cytotoxic side effects.

Laminin-rich blood vessels display activated growth factor signaling and act as the proliferation centers in Dupuytren's contracture.

INTRODUCTION: Dupuytren's contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches. METHODS: We studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR). RESULTS: We found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types. CONCLUSIONS: Based on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis.

Leukocyte marker CD43 promotes cell growth in co-operation with ß-catenin in non-hematopoietic cancer cells.

The Wnt/ß-catenin pathway regulates key cellular processes such as differentiation, proliferation, apoptosis; and its activation promotes development of several cancer types. Expression of CD43 (leukosialin), the predominant leukocyte transmembrane sialoglycoprotein, has been detected in many tumors of non-hematopoietic origin. CD43 participates in cell adhesion and regulates intracellular signal transduction pathways involved in cell proliferation and survival. The cytoplasmic domain of CD43 has been reported to translocate to the nucleus, interact with ß-catenin and affect its target gene expression, but the impact of this action on cell fate is still unknown. We demonstrate, here, by colony formation assay and siRNA-mediated gene silencing that CD43 and ß-catenin co-operate in promoting cell growth. Moreover, in cells with down-regulated ß-catenin expression the activation of p53 in response to CD43 overexpression is significantly impaired. In addition, the presence of both CD43 and ß-catenin is required for the TCF/LEF-mediated transcription. Presumably, the full-length CD43 participates in this transcriptional regulation. We show that the mature CD43 localizes to the nucleus, where it binds chromatin, co-localizes and co-immunoprecipitates with ß-catenin, and enhances the reporter gene expression regulated by ß-catenin. These observations provide clear evidence linking CD43 to the Wnt/APC/ß-catenin signaling pathway and supporting our hypothesis according to which CD43 plays a role in tumor development.