Krassimira Todorova

Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

Background Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. Methods FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. Results PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Conclusion Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.

miR‐204 is Dysregulated in Metastatic Prostate Cancer In Vitro

During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro‐RNAs into pro‐oncogenic ones. We aimed to investigate miR‐204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR‐204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c‐MYB, ETS1 and RUNX2 transcript and protein expression and the miR‐204 affected global proteome. We further investigated if these transcription factors exert an effect on miR‐204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR‐204 effects, either acting as a tumor suppressor on c‐MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR‐204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR‐204 also differentially affected mRNA splicing and protein stability. miR‐204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR‐204, c‐MYB, RUNX2 and ETS1, the c‐MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro‐RNA loss, but also significant genome rearrangement‐driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro‐survival and metastatic potential.

Sertoli Cell Quiescence - New Insights

It is currently accepted that the Sertoli cells are proliferatively active only during the embryogenesis and early fetal development, seizing to divide after puberty, when the spermatogenic niche is prepared, and they become terminally differentiated. So far, only seasonal breeders from mammals have been reported as having season‐dependent variations in adult Sertoli cells number and proliferation activity. In this review, we will try to shed light on testis somatic cell plasticity and discuss new evidence for some unique proliferative features Sertoli cells harbor.

PIF direct immune regulation: Blocks mitogen-activated PBMCs proliferation, promotes TH2/TH1 bias, independent of Ca2+

PreImplantation Factor (PIF(9&15)) secreted by viable embryos exerts an essential transplant acceptance and immune regulatory role in pregnancy. Synthetic PIF replicates endogenous PIFs effect in pregnant and non-pregnant immune disorder models. PIF binds macrophages to regulate CD3/CD28-induced T-cell response. We present evidence that PIF regulates the co-stimulatory T-cell receptor, CD2, which binds to and is activated by phytohemagglutinin (PHA), a potent mitogen, confirming PIFs ability to systemically respond to diverse immune stimulants. PIFs effect on PHA-activated PBMC (male and non-pregnant females) proliferation and cytokine secretion was tested, showing that both PIF(9&15) block PHA-induced PBMC proliferation and promote anti-inflammatory IL10 secretion, while reducing pro-inflammatory IFNγ secretion. Thus favoring a T(H)2 cytokine bias. Surface plasmon resonance spectroscopy, immunocytochemistry and Flex station experiments reveal that PIF effect is direct. PIF targets intracellular targets but does not affect early Ca(2+) mobilization. By promoting the CD2 receptor in activated T-cells and through inhibition of co-ligand CD58 expression, PIF regulates antigen-presenting cell (APC)-T-cell interactions required for PHA action. Structure-based design demonstrated that PIF15 offers improved target specificity as compared to PIF9. Collectively, PIF directly regulates mitogen-induced PBMC activation. Results support PIF translation for therapy of immune disorders.

PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders.

Fundamental role of microRNAs in androgen-dependent male reproductive biology and prostate cancerogenesis.

Male reproductive failure has been linked to successive development of various urologic diseases including prostate cancer. There is strong epidemiologic data in support of this association, it is important therefore to identify the fundamental grounds that lay beneath such a connection. Male reproductive biology, as sex determined, is significantly dependent upon the hormonal regulation of androgens. With the advancement of knowledge on androgen receptivity and epigenetic regulation, the role of new regulatory factors such as microRNAs becomes essential. This review focuses on unraveling the role of microRNA tight incorporation in androgen‐dependent male reproductive biology in the context of recent prostate cancer data.

Key Cellular Components and Interactive Histocompatibility Molecules Regulating Tolerance to the Fetal Allograft

Implantation is a major landmark in life. It involves the correct apposition of the embryo in the maternal endometrium. The cellular environment influences placenta development, and direct contact of the fetus with maternal tissues is achieved through decidual cells. At the decidua, and at systemic level, the correct balance of cells potentially acting as antigen‐presenting cells and histocompatibility products play a pivotal role in achieving feto–maternal tolerance. Here, we review some of the current issues associated with the interplay between cells and molecules needed for pregnancy development.

Haberlea rHodopensis has potentIal as a new drug source based on Its broad bIologIcal modalItIes

ABSTRACT Haberlea rhodopensis—a Balkan endemite belonging to the group of resurrection plants, was studied as a potential source of novel cancer modulating drugs. Human embryonic (HEK293 p53++/+) and prostate cancer cell lines—LNCaP (p53+/+) and PC3 (p53−/-) were used as a model to follow the reaction to oxidative and genotoxic stress after pre-treatment with H. rhodopensis extract. Oxidative stress was estimated by flow cytometry (FCS) andfluorescent plate reader (FPR) using reactive oxygen speciesfluorescent dye (H2DCFDA). UV induced DNA damage was assessed by FCS PUMA (p53 upregulated modulator of apoptosis) expression. Inflammatory pathways were challenged using synthetic peptidoglycan by FCS Act1 expression and NFkB reporter stable cell line. Pre-treated-cells were assessed using DCF dye (FPR and FCS) for ROS (Reactive Oxygen Species) generation after dose-dependent H2O2 stress and decreased signal compared to non-treated cells. H. rhodopensis had cumulative effect on cell death in PC3 cells. UV-inducedgenotoxic stress resulted in FCS-detected PUMA upregulation only in PC3. Haberlea treated cells had better vitality and their challenge using a bacterial peptidoglycan resulted in an upregulation of Act1, but only in LNCaP cells. The NFkB reporter vector revealed transcription factor activation upon treatment with only peptidoglycan, while Haberlea pre-treatment resulted in negative modulation of the NFkB induction. Our data show that H. rhodopensis extracts have anti-oxidative effect in cancer vs. normal cell lines and differentially modulate distinct cell lines in genotoxic and inflammatory stress, favoring NFKB activation in p53+/+ cells, while suppressing its signaling in p53−/- cells.

Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1β secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1β restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1β secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1β expression, while NOD2 inversely promoted IL-1β. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction.

miR-204 Shifts the Epithelial to Mesenchymal Transition in Concert with the Transcription Factors RUNX2, ETS1, and cMYB in Prostate Cancer Cell Line Model

Epithelial to mesenchymal transition is an essential step in advanced cancer development. Many master transcription factors shift their expression to drive this process, while noncoding RNAs families like miR-200 are found to restrict it. In this study we investigated how the tumor suppressor miR-204 and several transcription factors modulate main markers of mesenchymal transformation like E- and N-cadherin, SLUG, VEGF, and SOX-9 in prostate cancer cell line model (LNCaP, PC3, VCaP, and NCI-H660). We found that SLUG, E-cadherin, and N-cadherin are differentially modulated by miR-204, using miR-204 specific mimics and inhibitors and siRNA gene silencing (RUNX2, ETS-1, and cMYB). The genome perturbation associated TMPRSS2-ERG fusion coincided with shift from tumor-suppressor to tumor-promoting activity of this miRNA. The ability of miR-204 to suppress cancer cell viability and migration was lost in the fusion harboring cell lines. We found differential E-cadherin splicing corroborating to miR-204 modulatory effects. RUNX2, ETS1, and cMYB are involved in the regulation of E-cadherin, N-cadherin, and VEGFA expression. RUNX2 knockdown results in SOX9 downregulation, while ETS1 and cMYB silencing result in SOX9 upregulation in VCaP cells. Their expression was found to be also methylation dependent. Our study provides means for understanding cancer heterogeneity in regard to adapted therapeutic approaches development.