Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation.

Peptidyl arginine deiminases (PADIs) catalyze protein citrullination, a post-translational conversion of arginine to citrulline. The most widely expressed member of this family, PADI2, regulates cellular processes that impact several diseases. We hypothesized that we could gain new insights into PADI2 function through a systematic evolutionary and structural analysis. Here, we identify 20 positively selected PADI2 residues, 16 of which are structurally exposed and maintain PADI2 interactions with cognate proteins. Many of these selected residues reside in non-catalytic regions of PADI2. We validate the importance of a prominent loop in the middle domain that encompasses PADI2 L162, a residue under positive selection. This site is essential for interaction with the transcription elongation factor (P-TEFb) and mediates the active transcription of the oncogenes c-MYC, and CCNB1, as well as impacting cellular proliferation. These insights could be key to understanding and addressing the role of the PADI2 c-MYC axis in cancer progression.

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

Endogenous Retroviral Sequences Behave as Putative Enhancers Controlling Gene Expression through HP1-Regulated Long-Range Chromatin Interactions.

About half of the mammalian genome is constituted of repeated elements, among which endogenous retroviruses (ERVs) are known to influence gene expression and cancer development. The HP1 (Heterochromatin Protein 1) proteins are known to be essential for heterochromatin establishment and function and its loss in hepatocytes leads to the reactivation of specific ERVs and to liver tumorigenesis. Here, by studying two ERVs located upstream of genes upregulated upon loss of HP1, Mbd1 and Trim24, we show that these HP1-dependent ERVs behave as either alternative promoters or as putative enhancers forming a loop with promoters of endogenous genes depending on the genomic context and HP1 expression level. These ERVs are characterised by a specific HP1-independent enrichment in heterochromatin-associated marks H3K9me3 and H4K20me3 as well as in the enhancer-specific mark H3K4me1, a combination that might represent a bookmark of putative ERV-derived enhancers. These ERVs are further enriched in a HP1-dependent manner in H3K27me3, suggesting a critical role of this mark together with HP1 in the silencing of the ERVs, as well as for the repression of the associated genes. Altogether, these results lead to the identification of a new regulatory hub involving the HP1-dependent formation of a physical loop between specific ERVs and endogenous genes.

RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells.

Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites. Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.

The Transcription Coregulator RIP140 Inhibits Cancer Cell Proliferation by Targeting the Pentose Phosphate Pathway.

Cancer cells switch their metabolism toward glucose metabolism to sustain their uncontrolled proliferation. Consequently, glycolytic intermediates are diverted into the pentose phosphate pathway (PPP) to produce macromolecules necessary for cell growth. The transcription regulator RIP140 controls glucose metabolism in tumor cells, but its role in cancer-associated reprogramming of cell metabolism remains poorly understood. Here, we show that, in human breast cancer cells and mouse embryonic fibroblasts, RIP140 inhibits the expression of the gene-encoding G6PD, the first enzyme of the PPP. RIP140 deficiency increases G6PD activity as well as the level of NADPH, a reducing cofactor essential for macromolecule synthesis. Moreover, G6PD knock-down inhibits the gain of proliferation observed when RIP140 expression is reduced. Importantly, RIP140-deficient cells are more sensitive to G6PD inhibition in cell proliferation assays and tumor growth experiments. Altogether, this study describes a novel role for RIP140 in regulating G6PD levels, which links its effect on breast cancer cell proliferation to metabolic rewiring.

RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53.

Glycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mammary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation.

Fabrication of Radio-Opaque and Macroporous Injectable Calcium Phosphate Cement.

The aim of this work was the development of injectable radio-opaque and macroporous calcium phosphate cement (CPC) to be used as a bone substitute for the treatment of pathologic vertebral fractures. A CPC was first rendered radio-opaque by the incorporation of zirconium dioxide (ZrO2). In order to create macroporosity, poly lactic-co-glycolic acid (PLGA) microspheres around 100 µm were homogeneously incorporated into the CPC as observed by scanning electron microscopy. Physicochemical analyses by X-ray diffraction and Fourier transform infrared spectroscopy confirmed the brushite phase of the cement. The mechanical properties of the CPC/PLGA cement containing 30% PLGA (wt/wt) were characterized by a compressive strength of 2 MPa and a Young's modulus of 1 GPa. The CPC/PLGA exhibited initial and final setting times of 7 and 12 min, respectively. Although the incorporation of PLGA microspheres increased the force necessary to inject the cement and decreased the percentage of injected mass as a function of time, the CPC/PLGA appeared fully injectable at 4 min. Moreover, in comparison with CPC, CPC/PLGA showed a full degradation in 6 weeks (with 100% mass loss), and this was associated with an acidification of the medium containing the CPC/PLGA sample (pH of 3.5 after 6 weeks). A cell viability test validated CPC/PLGA biocompatibility, and in vivo analyses using a bone defect assay in the caudal vertebrae of Wistar rats showed the good opacity of the CPC through the tail and a significant increased degradation of the CPC/PLGA cement a month after implantation. In conclusion, this injectable CPC scaffold appears to be an interesting material for bone substitution.

Fabrication of 3D printed antimicrobial polycaprolactone scaffolds for tissue engineering applications.

Synthetic polymers are widely employed for bone tissue engineering due to their tunable physical properties and biocompatibility. Inherently, most of these polymers display poor antimicrobial properties. Infection at the site of implantation is a major cause for failure or delay in bone healing process and the development of antimicrobial polymers is highly desired. In this study, silver nanoparticles (AgNps) were synthesized in polycaprolactone (PCL) solution by in-situ reduction and further extruded into PCL/AgNps filaments. Customized 3D structures were fabricated using the PCL/AgNps filaments through 3D printing technique. As demonstrated by scanning electron microscopy, the 3D printed scaffolds exhibited interconnected porous structures. Furthermore, X-ray photoelectron spectroscopy analysis revealed the reduction of silver ions. Transmission electron microscopy along with energy-dispersive X-ray spectroscopy analysis confirmed the formation of silver nanoparticles throughout the PCL matrix. In vitro enzymatic degradation studies showed that the PCL/AgNps scaffolds displayed 80% degradation in 20 days. The scaffolds were cytocompatible, as assessed using hFOB cells and their antibacterial activity was demonstrated on Escherichia coli. Due to their interconnected porous structure, mechanical and antibacterial properties, these cytocompatible multifunctional 3D printed PCL/AgNps scaffolds appear highly suitable for bone tissue engineering.

Development of new biocompatible 3D printed graphene oxide-based scaffolds.

The aim of this work was to develop a bioresorbable, biodegradable and biocompatible synthetic polymer with good mechanical properties for bone tissue engineering applications. Polylactic acid (PLA) scaffolds were generated by 3D printing using the fused deposition modelling method, and reinforced by incorporation of graphene oxide (GO). Morphological analysis by scanning electron microscopy indicated that the scaffold average pore size was between 400 and 500 µm. Topography imaging revealed a rougher surface upon GO incorporation (Sa = 5.8 µm for PLA scaffolds, and of 9.9 µm for PLA scaffolds with 0.2% GO), and contact angle measurements showed a transition from a hydrophobic surface (pure PLA scaffolds) to a hydrophilic surface after GO incorporation. PLA thermomechanical properties were enhanced by GO incorporation, as shown by the 70 °C increase of the degradation peak (thermal gravimetric analysis). However, GO incorporation did not change significantly the melting point assessed by differential scanning calorimetry. Physicochemical analyses by X-ray diffraction and Raman spectroscopy confirmed the filler presence. Tensile testing demonstrated that the mechanical properties were improved upon GO incorporation (30% increase of the Young's modulus with 0.3% GO). Cell viability, attachment, proliferation and differentiation assays using MG-63 osteosarcoma cells showed that PLA/GO scaffolds were biocompatible and that they promoted cell proliferation and mineralization more efficiently than pure PLA scaffolds. In conclusion, this new 3D printed nanocomposite is a promising scaffold with adequate mechanical properties and cytocompatibility which may allow bone formation.

Boron Nitride Based Nanobiocomposites: Design by 3D Printing for Bone Tissue Engineering.

Here, we produced a synthetic polymer having adequate biocompatibility, biodegradability, and bioresorbability, as well as mechanical properties for applications in bone tissue engineering. We used the fused deposition modeling (FDM) based 3D printing approach in order to produce biomimetic biodegradable scaffolds made of polylactic acid (PLA). We strengthened these scaffolds by addition of exfoliated boron nitride (EBN) as filler. We demonstrated the presence of EBN by physicochemical analysis using Raman spectroscopy and X-ray diffraction (XRD). Using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), we found that EBN incorporation did not influence the transition temperature, but reduced the polymer crystallinity. Scanning electron microscopy for morphology evaluation showed a mean scaffold pore size of 500 µm. EBN incorporation did not affect the scaffold mechanical properties (tensile test), but modified the surface roughness. Moreover, contact angle quantification indicated that the surface of PLA/EBN scaffolds was hydrophilic and that of PLA scaffolds hydrophobic. Finally, the results of the cytotoxicity, cell attachment, and proliferation experiments using MG-63 and MC3T3 cells indicated that PLA scaffolds filled with EBN were nontoxic and compatible with osteoblastic cells and also promoted the scaffold mineralization by MG-63 cells. Altogether, our results suggest that this 3D printed nanocomposite scaffold is suitable for tissue engineering.

Regulation of RXR-RAR Heterodimers by RXR- and RAR-Specific Ligands and Their Combinations.

The three subtypes (α, ß, and γ) of the retinoic acid receptor (RAR) are ligand-dependent transcription factors that mediate retinoic acid signaling by forming heterodimers with the retinoid X receptor (RXR). Heterodimers are functional units that bind ligands (retinoids), transcriptional co-regulators and DNA, to regulate gene networks controlling cell growth, differentiation, and death. Using biochemical, crystallographic, and cellular approaches, we have set out to explore the spectrum of possibilities to regulate RXR-RAR heterodimer-dependent transcription through various pharmacological classes of RAR- and RXR- specific ligands, alone or in combination. We reveal the molecular details by which these compounds direct specificity and functionality of RXR-RAR heterodimers. Among these ligands, we have reevaluated and improved the molecular and structural definition of compounds CD2665, Ro41-5253, LE135, or LG100754, highlighting novel functional features of these molecules. Our analysis reveals a model of RXR-RAR heterodimer action in which each subunit retains its intrinsic properties in terms of ligand and co-regulator binding. However, their interplay upon the combined action of RAR- and RXR-ligands allows for the fine tuning of heterodimer activity. It also stresses the importance of accurate ligand characterization to use synthetic selective retinoids appropriately and avoid data misinterpretations.

Design of Boron Nitride/Gelatin Electrospun Nanofibers for Bone Tissue Engineering.

Gelatin is a biodegradable biopolymer obtained by collagen denaturation, which shows poor mechanical properties. Hence, improving its mechanical properties is very essential toward the fabrication of efficient nontoxic material for biomedical applications. For this aim, various methods are employed using external fillers such as ceramics or bioglass. In this report, we introduce boron nitride (BN)-reinforced gelatin as a new class of two-dimensional biocompatible nanomaterials. The effect of the nanofiller on the mechanical behavior is analyzed. BN is efficiently exfoliated using the biopolymer gelatin as shown through Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The exfoliated BN reinforces gelatin electrospun fibers, which results in an increase in the Young's modulus. The Electrospun Mats (ESM) are stable after the glutaraldehyde cross-linking, and the fibrous morphology is preserved. The cross-linked gelatin/BN ESM is highly bioactive in forming bonelike hydroxyapatite as shown by scanning electron microscopy. Due to their enhanced mineralization ability, the cross-linked ESM have been tested on human bone cells (HOS osteosarcoma cell line). The cell attachment, proliferation, and biocompatibility results show that the ESM are nontoxic and biodegradable. The analysis of osteoblast gene expression and the measurement of alkaline phosphatase activity confirm that these materials are suitable for bone tissue engineering.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells.

Soft tissue sarcomas with complex genomics are very heterogeneous tumors lacking simple prognosis markers or targeted therapies. Overexpression of a subset of mitotic genes from a signature called CINSARC is of bad prognosis, but the significance of this signature remains elusive. Here we precisely measure the cell cycle and mitosis duration of sarcoma cell lines and we found that the mitotic gene products overexpression does not reflect variation in the time spent during mitosis or G2/M. We also found that the CINSARC cell lines, we studied, are composed of a mixture of aneuploid, diploid, and tetraploid cells that are highly motile in vitro. After sorting diploid and tetraploid cells, we showed that the tetraploid cell clones do not possess a proliferative advantage, but are strikingly more motile and invasive than their diploid counterparts. This is correlated with higher levels of mitotic proteins overexpression. Owing that mitotic proteins are almost systematically degraded at the end of mitosis, we propose that it is the abnormal activity of the mitotic proteins during interphase that boosts the sarcoma cells migratory properties by affecting their cytoskeleton. To test this hypothesis, we designed a screen for mitotic or cytoskeleton protein inhibitors affecting the sarcoma cell migration potential independently of cytotoxic activities. We found that inhibition of several mitotic kinases drastically impairs the CINSARC cell invasive and migratory properties. This finding could provide a handle by which to selectively inhibit the most invasive cells.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

Novel biocompatible electrospun gelatin fiber mats with antibiotic drug delivery properties.

The aim of this study was to synthesize stable gelatin electrospun mats (ESMs) (cross-linked by glutaraldehyde (GTA) vapors) with tunable drug release properties using pH as a stimulus. Gelatin ESMs loaded with rhodamine as a model drug were first synthesized. The in vitro release of rhodamine was characterized to understand the mechanisms of drug release and the effects of both cross-linker concentration and pH on drug release. An optimal cross-linker concentration of 5% was evidenced to provide ESMs allowing both sustainable release of drugs at pH 7 and burst release at pH 2. The release profiles were then fitted with a power law model to describe the release kinetics. The chlorhexidine antibiotic drug was finally loaded into the optimal electrospun mat and its bactericidal activity was demonstrated against Gram-negative (E. coli) and Gram-positive (S. epidermidis) bacteria by agar diffusion tests. This biocompatible material was shown to efficiently destroy bacterial biofilms and prevent bacterial growth within a short time (3 h), while maintaining its antibacterial activity up to at least 72 h. This study provides a promising material, which could treat infected sites and prevent infections, with tunable drug releasing properties using pH as a stimulus.

The emerging role of the transcriptional coregulator RIP140 in solid tumors.

RIP140 is a transcriptional coregulator (also known as NRIP1) which plays very important physiological roles by finely tuning the activity of a large number of transcription factors. Noticeably, the RIP140 gene has been shown to be involved in the regulation of energy expenditure, in mammary gland development and intestinal homeostasis as well as in behavior and cognition. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. This short review aims to summarize the role of this transcription factor on nuclear estrogen receptors, E2F and Wnt signaling pathways based on recent observations focusing on breast, ovary, liver and colon tumors.

An Unexpected Mode Of Binding Defines BMS948 as A Full Retinoic Acid Receptor ß (RARß, NR1B2) Selective Agonist.

Retinoic acid is an important regulator of cell differentiation which plays major roles in embryonic development and tissue remodeling. The biological action of retinoic acid is mediated by three nuclear receptors denoted RARα, ß and γ. Multiple studies support that RARß possesses functional characteristics of a tumor suppressor and indeed, its expression is frequently lost in neoplastic tissues. However, it has been recently reported that RARß could also play a role in mammary gland tumorigenesis, thus demonstrating the important but yet incompletely understood function of this receptor in cancer development. As a consequence, there is a great need for RARß-selective agonists and antagonists as tools to facilitate the pharmacological analysis of this protein in vitro and in vivo as well as for potential therapeutic interventions. Here we provide experimental evidences that the novel synthetic retinoid BMS948 is an RARß-selective ligand exhibiting a full transcriptional agonistic activity and activating RARß as efficiently as the reference agonist TTNPB. In addition, we solved the crystal structures of the RARß ligand-binding domain in complex with BMS948 and two related compounds, BMS641 and BMS411. These structures provided a rationale to explain how a single retinoid can be at the same time an RARα antagonist and an RARß full agonist, and revealed the structural basis of partial agonism. Finally, in addition to revealing that a flip by 180° of the amide linker, that usually confers RARα selectivity, accounts for the RARß selectivity of BMS948, the structural analysis uncovers guidelines for the rational design of RARß-selective antagonists.

Expression and role of RIP140/NRIP1 in chronic lymphocytic leukemia.

RIP140 is a transcriptional coregulator, (also known as NRIP1), which finely tunes the activity of various transcription factors and plays very important physiological roles. Noticeably, the RIP140 gene has been implicated in the control of energy expenditure, behavior, cognition, mammary gland development and intestinal homeostasis. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. During the past years, several papers have reported evidences linking RIP140 to hematologic malignancies. Among them, two recent studies with correlative data suggested that gene expression signatures including RIP140 can predict survival in chronic lymphocytic leukemia (CLL). This review aims to summarize the literature dealing with the expression of RIP140 in CLL and to explore the potential impact of this factor on transcription pathways which play key roles in this pathology.