Engineering a Pro-Osteogenic Secretome through the Transient Silencing of the Gene Encoding Secreted Frizzled Related Protein 1.

The evidence sustaining the regenerative properties of mesenchymal stem cells' (MSCs) secretome has prompted a paradigm change, where MSCs have shifted from being considered direct contributors to tissue regeneration toward being seen as cell factories for producing biotech medicines. We have previously designed a method to prime MSCs towards osteogenic differentiation by silencing the Wnt/ß-Catenin inhibitor Sfpr1. This approach produces a significant increase in bone formation in osteoporotic mice. In this current work, we set to investigate the contribution of the secretome from the MSCs where Sfrp1 has been silenced, to the positive effect seen on bone regeneration in vivo. The conditioned media (CM) of the murine MSCs line C3H10T1/2, where Sfrp1 has been transiently silenced (CM-Sfrp1), was found to induce, in vitro, an increase in the osteogenic differentiation of this same cell line, as well as a decrease of the expression of the Wnt inhibitor Dkk1 in murine osteocytes ex vivo. A reduction in the RANKL/OPG ratio was also detected ex vivo, suggesting a negative effect of CM-Sfrp1 on osteoclastogenesis. Moreover, this CM significantly increases the mineralization of human primary MSCs isolated from osteoportotic patients in vitro. Proteomic analysis identified enrichment of proteins involved in osteogenesis within the soluble and vesicular fractions of this secretome. Altogether, we demonstrate the pro-osteogenic potential of the secretome of MSCs primmed in this fashion, suggesting that this is a valid approach to enhance the osteo-regenerative properties of MSCs' secretome.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning.

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling.

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly.

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans, the impairment of these motors through mutation results in the disease primary ciliary dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less-known assembly factor DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, The only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration.

Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear, whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.

Rare genetic variants with large effect on triglycerides in subjects with a clinical diagnosis of familial vs nonfamilial hypertriglyceridemia.

BACKGROUND: Most primary severe hypertriglyceridemias (HTGs) are diagnosed in adults, but their molecular foundations have not been completely elucidated. OBJECTIVE: We aimed to identify rare dysfunctional mutations in genes encoding regulators of lipoprotein lipase (LPL) function in patients with familial and non-familial primary HTG. METHODS: We sequenced promoters, exons, and exon-intron boundaries of LPL, APOA5, LMF1, and GPIHBP1 in 118 patients with severe primary HTG (triglycerides >500 mg/dL) and 53 normolipidemic controls. Variant functionality was analyzed using predictive software and functional assays for mutations in regulatory regions. RESULTS: We identified 29 rare variants, 10 of which had not been previously described: c.(-16A>G), c.(1018+2G>A), and p.(His80Arg) in LPL; p.(Arg143Alafs*57) in APOA5; p.(Val140Ile), p.(Leu235Ile), p.(Lys520*), and p.(Leu552Arg) in LMF1; and c.(-83G>A) and c.(-192A>G) in GPIHBP1. The c.(1018+2G>A) variant led to deletion of exon 6 in LPL cDNA, whereas the c.(-16A>G) analysis showed differences in the affinity for nuclear proteins. Overall, 20 (17.0%) of the patients carried at least one allele with a rare pathogenic variant in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant was not associated with lipid values, family history of HTG, clinical diagnosis, or previous pancreatitis. CONCLUSIONS: Less than one in five subjects with triglycerides >500 mg/dL and no major secondary cause for HTG may carry a rare pathogenic mutation in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant is not associated with a differential phenotype.

Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP).

Attempts to elucidate the cellular function of MTCH1 (mitochondrial carrier homolog 1) have not yet rendered a clear insight into the function of this outer mitochondrial membrane protein. Classical biochemical and cell biology approaches have not produced the expected outcome. In vitro experiments have indicated a likely role in the regulation of cell death by apoptosis, and its reported interaction with presenilin 1 suggests a role in the cellular pathways in which this membrane protease participates, nevertheless in vivo data are missing. In an attempt to identify cellular pathways in which this protein might participate, we have studied its promoter looking for transcriptional regulators. We have identified several putative binding sites for EGR-1 (Early growth response 1; a protein involved in growth, proliferation and differentiation), in the proximal region of the MTCH1 promoter. Chromatin immunoprecipitation showed an enrichment of these sequences in genomic DNA bound to EGR-1 and transient overexpression of EGR-1 in cultured HEK293T cells induces an increase of endogenous MTCH1 levels. We also show that MTCH1 levels increase in response to treatment of cells with doxorubicin, an apoptosis inducer through DNA damage. The endogenous levels of MTCH1 decrease when EGR-1 levels are lowered by RNA interference. Our results indicate that EGR-1 is a transcriptional regulator of MTCH1 and give some clues about the cellular processes in which MTCH1 might participate.

Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extracted from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account.

Characterization of variants in the glucosylceramide synthase gene and their association with type 1 Gaucher disease severity.

The extreme phenotypic variability of patients with Gaucher disease (GD) is not completely explained by glucocerebrosidase gene mutations. It has been proposed that genetic modifiers might influence GD phenotype. We examined seven polymorphisms of the glucosylceramide synthase gene (UGCG) and their correlation with severity of GD. Five UGCG variants were significantly associated with disease severity, according to the DS3 scoring system: c.-295C>T, c.-232_-241ins10, c.98+50A>G, c.98+68A>T, and c.861A>G. Heterozygous [N370S]+[L444P] patients with c.[-232_-241ins10;98+50G] haplotype had a significantly lower DS3 score in relation to patients carrying only one of these polymorphisms. Electrophoretic mobility shift assay analysis showed an increased nuclear protein binding ability for the G allele at the cDNA position c.98+50, as well as an altered pattern for the c.-232_-241ins10 allele. The promoter activity of the haplotypes decreased significantly with respect to wild type activity in HepG2 and COS-7 cells (-14% and -16% for the c.[-232_-241ins10;98+50A] haplotype, -44% and -25% for c.[-222nonins;98+50G] haplotypes, and -64% and -75% for c.[-232_-241ins10;98+50G] haplotype, respectively). These data indicate that the c.-232_-241ins10 and c.98+50A>G variants are modifying factors of GD severity, which can partly explain the variability in severity of the disease.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells.

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs). Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST. These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Association study of sirtuin 1 polymorphisms with bone mineral density and body mass index.

BACKGROUND AND AIMS: Sirtuin 1, encoded by the SIRT1 gene, is an emerging modulator of carbohydrate and lipid metabolism and may also influence the differentiation of bone cells. Our objective was to test the hypothesis that polymorphisms of SIRT1 are associated with body mass index (BMI) and bone mineral density (BMD). METHODS: We carried out a cross-sectional genetic association study with genotyping of ten single nucleotide polymorphisms of the SIRT1 region. The discovery cohort included 1394 individuals (342 males, 1052 females). Significant results were replicated in an independent cohort of 408 males. RESULTS: We did not find a significant association of genotypes with BMD. There were also no significant BMI differences across genotypes in females. However, in males, two polymorphisms tended to be associated with BMI in the discovery cohort (p 0.03 and 0.05). A similar trend was also observed in the replication cohort. Thus, in the combined analysis of both cohorts, males with C alleles at the rs12049646 locus had a lower BMI than TT homozygotes, with a mean difference of 0.82 kg/m(2) (95% confidence interval 0.15-1.48; p = 0.016). Differences in the DNA binding of nuclear proteins between C and T alleles were also observed in vitro. CONCLUSIONS: These results suggest that common variants of the SIRT1 gene influence BMI but not BMD.

Nuclear receptor NR5A2 and bone: gene expression and association with bone mineral density.

OBJECTIVE: There is growing evidence for a link between energy and bone metabolism. The nuclear receptor subfamily 5 member A2 (NR5A2) is involved in lipid metabolism and modulates the expression of estrogen-related genes in some tissues. The objective of this study was to explore the influence of NR5A2 on bone cells and to determine whether its allelic variations are associated with bone mineral density (BMD). DESIGN: Analyses of gene expression by quantitative PCR and inhibition of NR5A2 expression by siRNAs were used to explore the effects of NR5A2 in osteoblasts. Femoral neck BMD and 30 single nucleotide polymorphisms (SNPs) were first analyzed in 935 postmenopausal women and the association of NR5A2 genetic variants with BMD was explored in other 1284 women in replication cohorts. RESULTS: NR5A2 was highly expressed in bone. The inhibition of NR5A2 confirmed that it modulates the expression of osteocalcin, osteoprotegerin, and podoplanin in osteoblasts. Two SNPs were associated with BMD in the Spanish discovery cohort (rs6663479, P=0.0014, and rs2816948, P=0.0012). A similar trend was observed in another Spanish cohort, with statistically significant differences across genotypes in the combined analysis (P=0.03). However, the association in a cohort from the United States was rather weak. Electrophoretic mobility assays and studies with luciferase reporter vectors confirmed the existence of differences in the binding of nuclear proteins and the transcriptional activity of rs2816948 alleles. CONCLUSIONS: NR5A2 modulates gene expression in osteoblasts and some allelic variants are associated with bone mass in Spanish postmenopausal women.

Influence of aquaporin-1 gene polymorphism on water retention in liver cirrhosis.

UNLABELLED: Water retention is a major clinical problem in patients with liver cirrhosis. The factors that predispose to water retention are poorly understood but may involve genetic factors. Recent research suggests that renal aquaporins may be a pathophysiological factor involved in this condition. Aquaporin-1 (AQP1) is expressed in the proximal tubule and aquaporin-2 (AQP2) in the renal collecting duct cells. The aim of our study was to investigate the distribution of single nucleotide polymorphisms (SNPs) of AQP1: rs1049305 (C/G) and AQP2: rs3741559 (A/G) and rs467323 (C/T) in 100 cirrhotic patients with ascites and to analyze their relationship with dilutional hyponatremia. METHODS: Genomic DNA was extracted from peripheral blood. Genotyping for the presence of different polymorphisms was performed using the Custom Taqman SNP Genotyping Assays. The possible influence of rs1049305 (C/G) in AQP1 gene expression was evaluated by luciferase assays in vitro. RESULTS: The allelic frequencies of the AQP1 gene were the following: CC = 15%; CG = 49%; GG = 36%. Patients with CC genotype had significantly lower plasma sodium concentration than those with CG or GG genotype. Luciferase assays showed that the rs1049305 (C/G) in the AQP1 gene functionally affected the expression level in vitro. In addition, we did not find any relationship between AQP2 SNPs observed and plasma sodium concentration. CONCLUSIONS: Our results suggest that the rs1049305 (C/G, UTR3) AQP1 polymorphism could be involved in the genetic susceptibility to develop water retention in patients with liver cirrhosis.

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5' UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.-155_-150delACCCCinsTTCTGCAAACTCCTCCC, c.-136C>G, c.-140C>G, and c.-140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.-208T, c.-88A, and c.-36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.