Marianna Penzo

Inhibitor of NF-κB Kinases α and β Are Both Essential for High Mobility Group Box 1-Mediated Chemotaxis

Inhibitor of NF-κB kinases β (IKKβ) and α (IKKα) activate distinct NF-κB signaling modules. The IKKβ/canonical NF-κB pathway rapidly responds to stress-like conditions, whereas the IKKα/noncanonical pathway controls adaptive immunity. Moreover, IKKα can attenuate IKKβ-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKα and IKKβ are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKα and IKKβ are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKα-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKα and IKKβ, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKβ, but not IKKα, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKβ target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKβ, but not IKKα, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-κB signaling pathways.

Differential requirements for IKKα and IKKβ in the differentiation of primary human osteoarthritic chondrocytes

OBJECTIVE Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKalpha and IKKbeta are essential kinases that activate NF-kappaB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy. METHODS Expression of IKKalpha or IKKbeta was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed. RESULTS The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKalpha or IKKbeta. Silencing of IKKbeta markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKalpha dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKalpha-deficient cells retained the phenotype of cells in a pre-hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses. CONCLUSION IKKalpha and IKKbeta exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKalpha were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKalpha activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

The IKKα-Dependent NF-κB p52/RelB Noncanonical Pathway Is Essential To Sustain a CXCL12 Autocrine Loop in Cells Migrating in Response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12’s G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.

Matrix Metalloproteinase 13 Loss Associated With Impaired Extracellular Matrix Remodeling Disrupts Chondrocyte Differentiation by Concerted Effects on Multiple Regulatory Factors

OBJECTIVE To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. METHODS MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. RESULTS Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. CONCLUSION MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

Sustained NF-κB activation produces a short-term cell proliferation block in conjunction with repressing effectors of cell cycle progression controlled by E2F or FoxM1

NF‐κB transcription factors induce a host of genes involved in pro‐inflammatory/stress‐like responses; but the collateral effects and consequences of sustained NF‐κB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKβ T‐loop mutant (IKKβca) drove murine fibroblasts into transient growth arrest that subsided within 2–3 weeks of continuous culture. Proliferation arrest was associated with a G1/S phase block in immortalized and primary early passage MEFs. Molecular analysis in immortalized MEFs revealed that inhibition of cell proliferation in the initial 1–2 weeks after their IKKβca retroviral infection was linked to the transient, concerted repression of essential cell cycle effectors that are known targets of either E2F or FoxM1. Co‐expression of a phosphorylation resistant IκBα super repressor and IKKβca abrogated growth arrest and cell cycle effector repression, thereby linking IKKβcas effects to canonical NF‐κB activation. Transient growth arrest of IKKβca cells was associated with enhanced p21 (cyclin‐dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF‐κB and also likely due to strong repression of Skp2 and Csk1, both of which are FoxM1 direct targets mediating proteasomal dependent p21 turnover. Ablation of p21 in immortalized MEFs reduced their IKKβca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKβca, suggesting chromatin mediated gene silencing in IKKβcas short term repressive effects on E2F and FoxM1 target gene expression. J. Cell. Physiol. 218: 215–227, 2009.

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.

Human ribosomes from cells with reduced dyskerin levels are intrinsically altered in translation

Dyskerin is a pseudouridine (ψ)synthaseinvolved in fundamental cellular processes including uridine modification in rRNA and small nuclear RNA and telomere stabilization. Dyskerin functions are altered in X‐linked dyskeratosis congenita (X‐DC) and cancer. Dyskerins role in rRNA pseudouridylation has been suggested to underlie the alterations in mRNA translation described in cells lacking dyskerin function, although relevant direct evidences are currently lacking. Our purpose was to establish definitely whether defective dyskerin function might determine an intrinsic ribosomal defect leading to an altered synthetic activity. Therefore, ribosomes from dyskerin‐depleted human cells were purified and 1) added to a controlled reticulocyte cell‐free system devoid of ribosomes to study mRNA translation; 2) analyzed for protein contamination and composition by mass spectrometry, 3) analyzed for global pseudouridylation levels. Ribosomes purified from dyskerin‐depleted cells showed altered translational fidelity and internal ribosome entry site (IRES)‐mediated translation. These ribosomes displayed reduced uridine modification, whereas they were not different in terms of protein contamination or ribosomal protein composition with respect to ribosomes from matched control cells with full dyskerin activity. In conclusion, lack of dyskerin function in human cells induces a defect in rRNA uridine modification, which is sufficient to alter ribosome activity.—Penzo, M., Rocchi, L., Brugiere, S., Carnicelli, D., Onofrillo, C., Couté, Y., Brigotti, M., Montanaro, L. Human ribosomes from cells with reduced dyskerin levels are intrinsically altered in translation. FASEB J. 29, 3472‐3482 (2015). www.fasebj.org

The nucleolar size is associated to the methylation status of ribosomal DNA in breast carcinomas

BackgroundThere is a body of evidence that shows a link between tumorigenesis and ribosome biogenesis. The precursor of mature 18S, 28S and 5.8S ribosomal RNAs is transcribed from the ribosomal DNA gene (rDNA), which exists as 300–400 copies in the human diploid genome. Approximately one half of these copies are epigenetically silenced, but the exact role of epigenetic regulation on ribosome biogenesis is not completely understood. In this study we analyzed the methylation profiles of the rDNA promoter and of the 5’ regions of 18S and 28S in breast cancer.MethodsWe analyzed rDNA methylation in 68 breast cancer tissues of which the normal counterpart was partially available (45/68 samples) using the MassARRAY EpiTYPER assay, a sensitive and quantitative method with single base resolution.ResultsWe found that rDNA locus tended to be hypermethylated in tumor compared to matched normal breast tissues and that the DNA methylation level of several CpG units within the rDNA locus was associated to nuclear grade and to nucleolar size of tumor tissues. In addition we identified a subgroup of samples in which large nucleoli were associated with very limited or absent rDNA hypermethylation in tumor respect to matched normal tissue.ConclusionsIn conclusion, we suggest that rDNA is an important target of epigenetic regulation in breast tumors and that rDNA methylation level is associated to nucleolar size.

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling ☆

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1s receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.

The importance of being (slightly) modified: The role of rRNA editing on gene expression control and its connections with cancer

In human ribosomal RNAs, over 200 residues are modified by specific, RNA-driven enzymatic complexes or stand-alone, RNA-independent enzymes. In most cases, modification sites are placed in specific positions within important functional areas of the ribosome. Some evidence indicates that the altered control in ribosomal RNA modifications may affect ribosomal function during mRNA translation. Here we provide an overview of the connections linking ribosomal RNA modifications to ribosome function, and suggest how aberrant modifications may affect the control of the expression of key cancer genes, thus contributing to tumor development. In addition, the future perspectives in this field are discussed.