Chromodomain Protein CDYL Acts as a Crotonyl-CoA Hydratase to Regulate Histone Crotonylation and Spermatogenesis.

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to ß-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.

Echo-Level SAR Imaging Simulation of Wakes Excited by a Submerged Body.

The paper introduces a numerical simulation method for Synthetic Aperture Radar (SAR) imaging of submerged body wakes by integrating hydrodynamics, electromagnetic scattering, and SAR imaging simulation. This work is helpful for better understanding SAR images of submerged body wakes. Among these, the hydrodynamic model consists of two sets of ocean dynamics closely related to SAR imaging, namely the wake of the submerged body and wind waves. For the wake, we simulated it using computational fluid dynamics (CFD) numerical methods. Furthermore, we compared and computed the electromagnetic scattering characteristics of wakes under various navigation parameters and sea surface conditions. Following that, based on the operational principles and imaging theory of synthetic aperture radar (SAR), we established the SAR raw echo signal of the wake. Employing a Range-Doppler (RD) algorithm, we generated simulated SAR images of the wake. The results indicate that utilizing Computational Fluid Dynamics (CFD) numerical methods enables the simulation of wake characteristics generated by the motion of a submerged body with different velocities. The backscattering features of wakes are closely associated with the relative orientation between the wake and the radar line of sight. Under specific wind speeds, the wake gets masked within the sea surface background, resulting in less discernible characteristics of the wake in SAR images. This suggests that at lower speeds of submerged body or under specific wind conditions, the detectability of the wake in SAR images significantly diminishes.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress.

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature-humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

Analysis of Onboard Verification Flight Test for the Salinity Satellite Scatterometer.

The upcoming Salinity Satellite, scheduled for launch in 2024, will feature the world's first phased array radar scatterometer. To validate its capability in measuring ocean surface backscatter coefficients, this paper conducts an in-depth analysis of the onboard verification flight test for the Salinity Satellite scatterometer. This paper provides a detailed introduction to the system design of the Salinity Satellite scatterometer, which utilizes phased array radar technology and digital beamforming techniques to achieve accurate measurements of sea surface scattering characteristics. The paper elaborates on the derivation of backscatter coefficients, system calibration, and phase amplitude correction for the phased array scatterometer. Furthermore, it describes the process of the onboard calibration flight test. By analyzing internal noise signals, onboard calibration signals, and external noise signals, the stability and reliability of the scatterometer system are validated. The experiment covers both land and ocean observations, with a particular focus on complex sea surface conditions in nearshore areas. Through the precise analysis of backscatter coefficients, the paper successfully distinguishes the different backscatter coefficient characteristics between ocean and land. The research results effectively demonstrate the feasibility of the Salinity Satellite scatterometer for measuring backscatter coefficients in a phased array configuration, as well as its outstanding performance in complex marine environments.

Rod photoreceptor clearance due to misfolded rhodopsin is linked to a DAMP-immune checkpoint switch.

Chronic endoplasmic reticulum stress resulting from misfolding of the visual pigment rhodopsin (RHO) can lead to loss of rod photoreceptors, which initiates retinitis pigmentosa, characterized initially by diminished nighttime and peripheral vision. Cone photoreceptors depend on rods for glucose transport, which the neurons use for assembly of visual pigment-rich structures; as such, loss of rods also leads to a secondary loss of cone function, diminishing high-resolution color vision utilized for tasks including reading, driving, and facial recognition. If dysfunctional rods could be maintained to continue to serve this secondary cone preservation function, it might benefit patients with retinitis pigmentosa, but the mechanisms by which rods are removed are not fully established. Using pigs expressing mutant RHO, we find that induction of a danger-associated molecular pattern (DAMP) "eat me" signal on the surface of mutant rods is correlated with targeting the live cells for (PrCR) by retinal myeloid cells. Glucocorticoid therapy leads to replacement of this DAMP with a "don't eat me" immune checkpoint on the rod surface and inhibition of PrCR. Surviving rods then continue to promote glucose transport to cones, maintaining their viability.

lncRNA Nuclear Factor of Activated T Cells Knockdown Alleviates Hypoxia/Reoxygenation-induced Cardiomyocyte Apoptosis by Upregulating HIF-1α Expression.

ABSTRACT: Acute myocardial infarction (AMI) has become the most common cause of death in the developed countries. However, its pathogenesis is poorly understood. Increasing studies have revealed that lncRNAs are important modulators of AMI development. This study aimed to explore the function of lncRNA noncoding repressor of nuclear factor of activated T cells (NRON) in hypoxia/reoxygenation (H/R)-stimulated H9c2 cells. NRON expression in peripheral blood of AMI patients and H/R-stimulated H9c2 cells was measured by quantitative real-time polymerase chain reaction. H9c2 cells were transfected with si-NRON or cotransfected with si-NRON and si-hypoxia-inducible factor-1 alpha (HIF-1α). The viability and apoptosis of these cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and flow cytometer, respectively. In addition, HIF-1α, AKT/mTOR signal pathways and ERK1/2 were detected by western blot. NRON knockdown in the myocardial infarction mouse model was conducted through adeno-associated virus injection, and cardiac function was evaluated by motion-mode echocardiography. The results showed that NRON was highly expressed in peripheral blood of AMI patients and H/R-stimulated H9c2 cells. NRON knockdown promoted cell viability and inhibited cell apoptosis of H/R-stimulated H9c2 cells. Meanwhile, NRON knockdown also significantly attenuated heart damage and improved cardiac function in an AMI mouse model. Furthermore, compared with si-normal control, NRON knockdown increased the levels of HIF-1α, p-AKT, p-mTOR, and p-ERK1/2. HIF-1α knockdown reversed the effects of NRON knockdown in H/R-stimulated-H9c2 cells damage. Overall, our study revealed that NRON knockdown alleviated H/R-induced cardiomyocyte apoptosis by upregulating HIF-1α expression, suggesting that NRON might be a novel therapeutic target for AMI.

Research on Ultra-Wideband NLFM Waveform Synthesis and Grating Lobe Suppression.

Ultra-wideband (UWB) nonlinear frequency modulation (NLFM) waveforms have the advantages of low sidelobes and high resolution. By extending the frequency domain wideband synthesis method to the NLFM waveform, the synthetic bandwidth will be limited, and the grating lobe will grow as the number of subpulses increases at a fixed synthetic bandwidth. Aiming for the highly periodic grating lobes caused by equally spaced splicing and small subpulse time-bandwidth products (TxBW), a multisubpulse UWB NLFM waveform synthesis method is proposed in this paper. Random frequency hopping and spectral correction are utilized to disperse the energy of periodic grating lobes and optimize the matched filter of the subpulse, thereby reducing notches and Fresnel ripples in the synthesized spectrum. The results of the hardware-in-the-loop simulation experiment show that the peak sidelobe ratio (PSLR) and the integral sidelobe ratio (ISLR) of the NLFM synthetic wideband waveform (SWW) obtained by 50 subpulses with a bandwidth of 36 MHz are improved by 4.8 dBs and 4.5 dBs, respectively, when compared to the frequency domain wideband synthesis method, and that the grating lobe is suppressed by an average of 10.6 dBs. It also performs well in terms of point target resolution, and it has potential for 2D radar super-resolution imaging.

EED, a member of the polycomb group, is required for nephron differentiation and the maintenance of nephron progenitor cells.

Epigenetic regulation of gene expression has a crucial role allowing for the self-renewal and differentiation of stem and progenitor populations during organogenesis. The mammalian kidney maintains a population of self-renewing stem cells that differentiate to give rise to thousands of nephrons, which are the functional units that carry out filtration to maintain physiological homeostasis. The polycomb repressive complex 2 (PRC2) epigenetically represses gene expression during development by placing the H3K27me3 mark on histone H3 at promoter and enhancer sites, resulting in gene silencing. To understand the role of PRC2 in nephron differentiation, we conditionally inactivated the Eed gene, which encodes a nonredundant component of the PRC2 complex, in nephron progenitor cells. Resultant kidneys were smaller and showed premature loss of progenitor cells. The progenitors in Eed mutant mice that were induced to differentiate did not develop into properly formed nephrons. Lhx1, normally expressed in the renal vesicle, was overexpressed in kidneys of Eed mutant mice. Thus, PRC2 has a crucial role in suppressing the expression of genes that maintain the progenitor state, allowing nephron differentiation to proceed.

Bursal peptide BP-IV as a novel immunoadjuvant enhances the protective efficacy of an epitope peptide vaccine containing T and B cell epitopes of the H9N2 avian influenza virus.

Short peptide antigens covering conserved T or B cell epitopes have been investigated in influenza vaccines. Bursal pentapeptide V (BPP-V) and bursal peptide IV (BP-IV) are small molecular peptides that were isolated and identified from the bursa of Fabricius (BF) and induce a strong immune response at both the humoural and cellular levels. To explore the molecular adjuvant potential of BPP-V and BP-IV with an epitope vaccine, an epitope peptide (HA284-298, GNCVVQCQTERGGLN) rich in T and B cell epitopes for the H9N2 avian influenza virus (AIV) haemagglutinin (HA) protein was selected. BPP-V and BP-IV were coupled with the epitope peptide sequence to form BPP-V and BP-IV-epitope vaccines, respectively. The immunoefficacy of BPP-V and BP-IV-epitope peptide vaccines was evaluated. The results showed that the epitope peptide had weak immunogenicity. The BPP-V-epitope peptide vaccine promoted only the secretion of anti-HA IgG and IgG1 antibodies. The BP-IV-epitope peptide vaccine not only promoted the production of anti-HA IgG and IgG1 antibodies but also significantly induced the production of the IgG2a antibody. The BP-IV-epitope peptide vaccine significantly promoted the production of interleukin (IL-4) and interferon-γ (IFN-γ) (the BPP-V epitope peptide vaccine promoted only the production of IL-4), enhanced the cytotoxic T lymphocyte (CTL) response, and increased the proportion of CD3+ T lymphocytes. Moreover, the BP-IV-epitope peptide vaccine promoted a cell-mediated immune response similar to that of the AIV vaccine group. Furthermore, BPP-V and BP-IV-epitope peptide vaccines could also accelerate the clearance of pulmonary virus and reduce pathological damage after the challenge with H9N2 AIV. This study demonstrates the potential of BP-IV as an effective adjuvant for the epitope peptide vaccine for the H9N2 AIV.

Enhancing the antibacterial activity of antimicrobial peptide PMAP-37(F34-R) by cholesterol modification.

BACKGROUND: The problem of increasing resistance against conventional antibiotics has drawn people's attention. Therefore, the development of novel antibacterial agents with effective and safe therapeutic effects is imminent. Antimicrobial peptides (AMPs) are considered a promising class of antibacterial agents due to their broad antibacterial spectrum. RESULTS: In this study, on the basis of our previously studied peptide PMAP-37(F34-R), a novel antimicrobial peptide Chol-37(F34-R) was developed by N-terminal cholesterol modification to increase hydrophobicity. We observed that the N-terminal cholesterol-modified Chol-37(F34-R) showed higher antimicrobial activity than PMAP-37(F34-R) in vitro. Chol-37(F34-R) also exhibited effective anti-biofilm activity and may kill bacteria by improving the permeability of their membranes. Chol-37(F34-R) exerted high stability in different pH, salt, serum, and boiling water environments. Chol-37(F34-R) also showed no hemolytic activity and substantially low toxicity. Furthermore, Chol-37(F34-R) exhibited good potency of bacteria eradication and promoted wound healing and abscess reduction in infected mice. Meanwhile, in S. aureus ATCC25923-infected peritonitis model, Chol-37(F34-R) exhibited an impressive therapeutic effect by reducing the decrease in systemic bacterial burden and alleviating organ damage. CONCLUSIONS: Our findings suggested that the N-terminal cholesterol modification of PMAP-37(F34-R) could improve antibacterial activity. Chol-37(F34-R) displayed excellent bactericidal efficacy and impressive therapeutic effect in vivo. Thus, Chol-37(F34-R) may be a candidate for antimicrobial agents against microbial infection in the clinic.

Antimicrobial activity of the antibacterial peptide PMAP-36 and its analogues.

The growing problem of antibiotic resistance has attracted people's attention; thus, the search for new antibacterial agents is imminent. In this study, a series of antimicrobial peptides (AMPs) based on the porcine antibacterial peptide PMAP-36 were designed by amino acid substitution to develop peptide analogues as new classes of antimicrobial agents. By extending the α-helix and increasing the positive charge, two peptide analogues, PMAP-36PW and PMAP-36PK, were synthesized. The antibacterial activities of PMAP-36 and its peptide analogues were detected in vitro and in vivo. The results showed that PMAP-36PW and PMAP-36PK had a broadened antibacterial spectrum compared to that of PMAP-36. After the modification, PMAP-36PW and PMAP-36PK exhibited antibacterial activities on swine Escherichia coli K88, while PMAP-36 did not. PMAP-36, PMAP-36PW and PMAP-36PK did not have antibacterial activities against Enterococcus faecium B21. PMAP-36 PW had significant antibacterial activity against seven bacterial strains compared to PMAP-36, and PMAP-36PK had significant antibacterial activity against five bacterial strains compared to PMAP-36. Furthermore, PMAP-36PW exhibited enhanced pH stability. Moreover, in the in vivo efficacy assessment of mice infected with Salmonella choleraesuis C78-1 and Listeria monocytogenes CICC 21533, the peptide analogues exhibited an impressive therapeutic effect by reducing bacterial gene copies and decreasing inflammatory damage in mouse livers and lungs, resulting in a reduction in mouse mortality. This study provides reference data for the design of clinically effective antibacterial peptides.

Antimicrobial peptide PMAP-37 analogs: Increasing the positive charge to enhance the antibacterial activity of PMAP-37.

Bacterial resistance induced by the use of antibiotics has provided a chance for the development of antimicrobial peptides (AMPs), and modification of AMPs to enhance the antibacterial activity or stability has become a research focus. PMAP-37 is an AMP isolated from porcine myeloid marrow, and studies on its modification have not yet been reported. In this study, three PMAP-37 analogs named PMAP-37(F9-R), PMAP-37(F34-R), and PMAP-37(F9/34-R) were designed by residue substitution to enhance the positive charge. The antimicrobial activity of PMAP-37 and its analogs in vitro and in vivo were detected. The results showed that compared with PMAP-37, PMAP-37(F9-R) and PMAP-37(F9/34-R) exhibited antibacterial activity against S. flexneri CICC21534. Although PMAP-37(F34-R) had no antibacterial activity against S. flexneri CICC21534, its minimal inhibitory concentrations (MICs) were significantly lower than those of PMAP-37 against most bacterial strains. Besides, all PMAP-37 analogs were pH stable, retaining stable antibacterial activity after treatment with solution from pH 2 to pH 8/9. In addition, the PMAP-37 analogs displayed increased thermal stability, and PMAP-37(F34-R) retained >60% antibacterial activity after boiling for 2 hours. Furthermore, the PMAP-37 analogs exhibited impressive therapeutic efficacy in bacterial infections by reducing bacterial burden and inflammatory damage in the lung and liver, resulting in a reduction in mortality. Notably, the therapeutic effect of PMAP-37(F34-R) was comparable to that of ceftiofur sodium, and even superior to antibiotics in L. monocytogenes CICC21533 infection model. In conclusion, the PMAP-37(F34-R) may be a candidate for the treatment of bacterial infections in the clinic.

Acetyl-11-keto-ß-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties.

Acquired docetaxel-resistance of prostate cancer (PCa) remains a clinical obstacle due to the lack of effective therapies. Acetyl-11-keto-ß-boswellic acid (AKBA) is a pentacyclic triterpenic acid isolated from the fragrant gum resin of the Boswellia serrata tree, which has shown intriguing antitumor activity against human cell lines established from PCa, colon cancer, malignant glioma, and leukemia. In this study, we examined the effects of AKBA against docetaxel-resistant PCa in vitro and in vivo as well as its anticancer mechanisms. We showed that AKBA dose-dependently inhibited cell proliferation and induced cell apoptosis in docetaxel-resistant PC3/Doc cells; its IC50 value in anti-proliferation was ∼17 µM. Furthermore, AKBA dose-dependently suppressed the chemoresistant stem cell-like properties of PC3/Doc cells, evidenced by significant decrease in the ability of mammosphere formation and down-regulated expression of a number of stemness-associated genes. The activation of Akt and Stat3 signaling pathways was remarkably enhanced in PC3/Doc cells, which contributed to their chemoresistant stem-like phenotype. AKBA (10-30 µM) dose-dependently suppressed the activation of Akt and Stat3 signaling pathways in PC3/Doc cells. In contrast, overexpression of Akt and Stat3 significantly attenuated the inhibition of AKBA on PC3/Doc cell proliferation. In docetaxel-resistant PCa homograft mice, treatment with AKBA significantly suppresses the growth of homograft RM-1/Doc, equivalent to its human PC3/Doc, but did not decrease their body weight. In summary, we demonstrate that AKBA inhibits the growth inhibition of docetaxel-resistant PCa cells in vitro and in vivo via blocking Akt and Stat3 signaling, thus suppressing their cancer stem cell-like properties.

Induction of ROS and DNA damage-dependent senescence by icaritin contributes to its antitumor activity in hepatocellular carcinoma cells.

Context: Icaritin (ICT), a prenylflavonoid derivative extracted from the Epimedium (Berberidaceae) genus, has been identified to exhibit antitumor effect in hepatocellular carcinoma (HCC) cells by inducing apoptosis. However, its effect on cellular senescence has not been elucidated. Objective: To investigate the mechanism for low concentrations of ICT exerting antitumor activity through induction of cellular senescence. Materials and methods: Human HepG2 and Huh7 cells were treated with low concentrations of ICT (1 and 2 µM) once per day for a week. Cellular senescence was evaluated through cell viability and senescence-associated-ß-galactosidase activity. Cell cycle distribution and ROS levels were measured with flow cytometry. Gene expression was detected using qRT-PCR and western blotting. Fluorescent punctuates formation of γH2AX was analyzed by immunofluorescence. Results: ICT (1 and 2 µM) promoted cellular senescence in HepG2 and Huh7 cells, as observed by enlarged and flattened morphology and increased senescence-associated-ß-galactosidase activity (∼7-8-fold and ∼11-12-fold of vehicle controls, respectively), accompanied by significant cell cycle arrest and decrease in DNA synthesis. Mechanistically, ICT-induced senescence occurred through accumulation of ROS (∼1.3-fold and ∼1.8-fold of vehicle controls in response to 1 and 2 µM ICT, respectively), which further resulted in DNA damage response, as evidenced by strong induction of γH2AX through immunofluorescence and western blotting assays. Pharmacological inhibition of ROS production with N-acetylcysteine attenuated ICT-induced γH2AX and senescence-associated-ß-galactosidase activity (∼0.28-0.30-fold decrease, p < 0.05). Discussion and conclusions: Induction of cellular senescence by ICT defines a novel anticancer mechanism of ICT and provides a rationale for generalizing the study design to a broader study population to further developing ICT as a novel therapeutic agent for treatment of HCC.

Sphere-Induced Rejuvenation of Swine and Human Müller Glia Is Primarily Caused by Telomere Elongation.

Müller cells are the major supportive and protective glial cells in the retina with important functions in histogenesis and synaptogenesis during development, and in maintenance of mature neurons as they show to secrete various cytokines and manifest potentials of self-renewal and transdifferentiation into retinal neurons following injury in the vertebrate retinas. The swine retina has a visual streak structure similar to the human macular where cone photoreceptors are highly concentrated, thereby can serve as a better model for studying retinal diseases and for formulating cell-based therapeutics than the rodent retinas. Like most differentiated somatic mammalian cells, the isolated swine and human Müller glia become senescent over passages in culture, which restricts their potential application in basic and clinic researches. Here, we demonstrate that the senescence of swine and human Müller cells is caused by telomere attrition upon multiplications in vitro; and the senescent cells can be rejuvenated by sphere suspension culture. We also provide evidence that sphere-induced extension of telomeres in swine and human Müller glia is achieved by alternative lengthening of telomeres or/and by telomerase activation. Stem Cells 2017;35:1579-1591.

Adipogenic niches for melanoma cell colonization and growth in bone marrow.

Bone marrow (BM) adipocytes are abundant in BM and may be involved in the process of bone metastasis. However, their behaviors in metastatic BM niches during bone metastasis have not been fully explored. In this study, intracardiac transplantation of B16-F10 melanoma cells into immunocompetent C57BL/6 mice was performed. Tibial marrow sections were stained with hematoxylin and eosin, Masson's trichrome, tartrate-resistant acid phosphatase, and fatty acid-binding protein 4 (FABP4) and analyzed using a histomorphometric system. The results showed that the number of BM adipocytes rapidly increased in melanoma metastatic BM niches, which were in direct contact with metastasizing melanoma cells and acted as a tumor stromal population in the BM-melanoma niche. Melanoma cell-derived factors could enhance BM adipogenesis, which promotes melanoma cell proliferation and cell cycle transitions. Moreover, BM adipocytes might aid in the modification of the osteolytic BM microenvironment. These results indicate that an increase in the number of BM adipocytes in a metastatic BM niche may facilitate melanoma cell colonization and growth in BM. BM adipocytes might therefore support the development of bone metastases.

The ZEB1 transcription factor acts in a negative feedback loop with miR200 downstream of Ras and Rb1 to regulate Bmi1 expression.

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

An Rb1-dependent amplification loop between Ets1 and Zeb1 is evident in thymocyte differentiation and invasive lung adenocarcinoma.

BACKGROUND: Ras pathway mutation leads to induction and Erk phosphorylation and activation of the Ets1 transcription factor. Ets1 in turn induces cyclin E and cyclin dependent kinase (cdk) 2 to drive cell cycle progression. Ets1 also induces expression of the epithelial-mesenchymal transition (EMT) transcription factor Zeb1, and thereby links Ras mutation to EMT, which is thought to drive tumor invasion. Ras pathway mutations are detected by the Rb1 tumor suppression pathway, and mutation or inactivation of the Rb1 pathway is required for EMT. RESULTS: We examined linkage between Rb1, Ets1 and Zeb1. We found that an Rb1-E2F complex binds the Ets1 promoter and constitutively limits Ets1 expression. But, Rb1 repression of Zeb1 provides the major impact of Rb1 on Ets1 expression. We link Rb1 repression of Zeb1 to induction of miR-200 family members, which in turn target Ets1 mRNA. These findings suggest that Ets1 and Zeb1 comprise an amplification loop that is dependent upon miR-200 and regulated by Rb1. Thus, induction of Ets1 when the Rb1 pathway is lost may contribute to deregulated cell cycle progression through Ets1 induction of cyclin E and cdk2. Consistent with such an amplification loop, we correlate expression of Ets1 and Zeb1 in mouse and human lung adenocarcinoma. In addition we demonstrate that Ets1 expression in thymocytes is also dependent upon Zeb1. CONCLUSIONS: Taken together, our results provide evidence of an Rb1-dependent Ets1-Zeb1 amplification loop in thymocyte differentiation and tumor invasion.

Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression.

Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.

Repression of Zeb1 and hypoxia cause sequential mesenchymal-to-epithelial transition and induction of aid, Oct4, and Dnmt1, leading to immortalization and multipotential reprogramming of fibroblasts in spheres.

In this study, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the epithelial-to-mesenchymal transition transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. Hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor.