Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Ultrasound-guided hepatic portal vein injection is not a reproducible technique for delivery of cell therapies to the liver in mice.

AIMS: Our aim was to determine if ultrasound-guided HPV injection in mice would provide reproducible and reliable results, as is currently obtained via open laparotomy techniques, and offer a surgical refinement to emulate islet transplantation in humans. METHODS: Fluorescent-polymer microparticles (20 µm) were injected (27G-needle) into the HPV via open laparotomy (n = 4) or under ultrasound-guidance (n = 4) using an MX550D-transducer with a Vevo3100-scanner (FUJIFILM VisualSonics, Inc.). Mice were culled 24-h post injection; organs were frozen, step sectioned (10 µm-slices) and 10 sections/mouse (50 µm-spacing) were quantified for microparticles in the liver and other organs by fluorescent microscopy. RESULTS: Murine HPV injection, via open laparotomy-route, resulted in widespread distribution of microparticles in the liver, lungs and spleen; ultrasound-guided injection resulted in reduced microparticle delivery (p < 0.0001) and microparticle clustering in distinct areas of the liver at the site of needle penetration, with very few/no microparticles being seen in lung and spleen tissues, hypothesised to be due to flow into the body cavity: liver median (interquartile range) 4.15 (0.00-4.15) versus 0.00 (0.00-0.00) particle-count mm-2 , respectively. CONCLUSIONS: Ultrasound-guided injection results in microparticle clustering in the liver, with an overall reduction in microparticle number when compared to open laparotomy HPV injection, and high variability in microparticle-counts detected between mice. Ultrasound-guided injection is not currently a technique that can replace open laparotomy HPV of islet transplantation in mice.

Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.

MicroRNA-based therapeutics in cardiovascular disease: screening and delivery to the target.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼22 nucleotides, which have increasingly been recognized as potent post-transcriptional regulators of gene expression. MiRNA targeting is defined by the complementarities between positions 2-8 of miRNA 5'-end with generally the 3'-untranslated region of target mRNAs (messenger RNAs). The capacity of miRNAs to simultaneously inhibit many different mRNAs allows for an amplification of biological responses. Hence, miRNAs are extremely attractive targets for therapeutic regulation in several diseases, including cardiovascular. Novel approaches are emerging to identify the miRNA functions in cardiovascular biology processes and to improve miRNA delivery in the heart and vasculature. In the present study, we provide an overview of current studies of miRNA functions in cardiovascular cells by the use of high-content screening. We also discuss the challenge to achieve a safe and targeted delivery of miRNA therapeutics in cardiovascular cells.

EDD, a ubiquitin-protein ligase of the N-end rule pathway, associates with spindle assembly checkpoint components and regulates the mitotic response to nocodazole.

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G2/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.

Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations.

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.

Functional interaction between sequestosome-1/p62 and autophagy-linked FYVE-containing protein WDFY3 in human osteoclasts.

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype. Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates. Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.

miR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183-KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183-KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

MicroRNA regulation of vascular function.

MicroRNAs (miRNAs) are small non-coding RNAs that orchestrate genetic networks by modulating gene expression. Given their importance in vascular development, homeostasis and diseases, along with the technical feasibility in deploying their function in vivo, the so-called 'vascular miRNAs' have become key targets for therapeutic intervention. Herein, we have summarised the state-of-the-art on vascular miRNAs and we have discussed the role miRNA biogenesis and the extracellular vesicles (EVs) miRNA transport in vascular biology.

Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells.

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease.

Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development.

Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling.

Generation and culture of osteoclasts.

Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution in osteoclast biology began with the identification of macrophage-colony stimulating factor (M-CSF) and receptor activator NFκB-ligand (RANKL ) as the key regulators of osteoclast formation, fusion and function. The availability of recombinant human and mouse M-CSF and RANKL has enabled researchers to reliably generate osteoclasts from primary monocyte/macrophage cells as well as from cell lines such as RAW 264.7. This article summarizes the most commonly used procedures for the isolation, generation and characterization of human, rodent and chicken osteoclasts in vitro. Lists of further reading and recommendations are included to facilitate a successful application by the reader.

Transfection of osteoclasts and osteoclast precursors.

Osteoclasts and their precursors have traditionally been considered difficult cells to transfect using standard approaches. Here, we describe several methods for transfection of mature osteoclasts and their precursors using the Amaxa™ Nucleofector system, lentiviruses, and adenoviruses.

RANK-dependent autosomal recessive osteopetrosis: characterization of five new cases with novel mutations.

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis.

The skeleton: a multi-functional complex organ: the role of key signalling pathways in osteoclast differentiation and in bone resorption.

Osteoclasts are the specialised cells that resorb bone matrix and are important both for the growth and shaping of bones throughout development as well as during the process of bone remodelling that occurs throughout life to maintain a healthy skeleton. Osteoclast formation, function and survival are tightly regulated by a network of signalling pathways, many of which have been identified through the study of rare monogenic diseases, knockout mouse models and animal strains carrying naturally occurring mutations in key molecules. In this review, we describe the processes of osteoclast formation, activation and function and discuss the major transcription factors and signalling pathways (including those that control the cytoskeletal rearrangements) that are important at each stage.

Signal peptide mutations in RANK prevent downstream activation of NF-κB.

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins.