Proteoglycan Biosynthesis Research Articles

Chondroitin - application in the treatment of degenerative joint disease.

Chondroitin is an organic compound, belonging to the group of glycosaminoglycans. In the treatment of degenerative joint disease, aka osteoarthritis, chondroitin sulphate is applied as a medicine or a dietary supplement. The biological importance of chondroitin sulphate has been already largely determined. The newest data on glycobiology research suggest that proteoglycans, as well as their complex polysaccharide macroparticles not only are the structural elements, but also they participate in multiple metabolic processes at a molecular level as well as in the physiological processes, regulating this type of mechanisms. The preparations applied in the treatment of degenerative joint disease, containing chondroitin sulphate, are attributed numerous therapeutic and chondroprotective properties including stabilizing synthesis processes and cartilage degradation through stimulation and inhibition of chondrocyte apoptosis (production of the elements of the intracellular substance and osteocyte stimulation), an increased proteoglycan and hyaluronic acid biosynthesis, inhibition of the activity of proteolytic enzymes and hyaluronidase, reduction of inflammatory mediators (prostaglandins and leukotrienes) and a decreased collagen II degradation. Based on the results of the multidirectional research available in the newest source literature, the analysis of the therapeutic efficacy and safety of chondroitin application in the treatment of degenerative joint disease was conducted.

Alteration of colonic epithelial cell differentiation in mice deficient for glucosaminyl N-deacetylase/N-sulfotransferase 4.

Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) are the first enzymes that mediate the initiation of heparan sulfate sulfation. We previously identified NDST4 as a putative tumor suppressor in human colorectal cancer. In the study, we generated an Ndst4 knockout (Ndst4−/−) mouse strain and explored its phenotypic characteristics, particularly in the development of colonic epithelial homeostasis. The Ndst4-deficient mice were viable and fertile, and their life spans were similar to those of wild-type littermates. No gross behavioral or morphological differences were observed between the Ndst4−/− and wild-type mice, and no significant changes were determined in the hematological or serum biochemical parameters of the Ndst4−/− mice. Ndst4 RNA transcripts were expressed in the brain, lung, gastrointestinal tract, pancreas, and ovary. However, Ndst4-null mice exhibited no gross or histological abnormalities in the studied organs, except for the colon. Although no alterations were observed in the crypt length or number of proliferating cells, the Ndst4−/− mice exhibited an increased number of goblet cells and a decreased number of colonocytes in the proximal colon compared with the wild-type mice. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic epithelium. Taken together, we established, for the first time, an Ndst4−/− mouse strain and revealed the involvement of Ndst4 in the development and homeostasis of colonic epithelium. Accordingly, NDST4 in human colon might direct the biosynthesis of specific heparan sulfate proteoglycans that are essential for the maintenance of colonic epithelial homeostasis. Thus, the loss of its function may result in the tumorigenesis and progression of colorectal cancer.

Novel mutation of EXT2 identified in a large family with multiple osteochondromas.

Multiple osteochondromas (MO), also known as hereditary multiple exostoses, is an autosomal dominant bone disorder. Mutations in exostosin glycosyl transferase-1 (EXT1) and exostosin glycosyl transferase-2 (EXT2), including missense, nonsense, frameshift and splice-site mutations, account for up to 80% of reported cases. The proteins EXT1 and EXT2 form a hetero-oligomeric complex that functions in heparan sulfate proteoglycan biosynthesis. A heterozygous EXT2 mutation, c.939+1G>T, was identified in a five-generation 33-member MO family, and was present in all 13 affected members. The mutation results in deletion of exon 5 in the mRNA, producing a frameshift that leads to a premature termination codon. The present study extends the mutational spectrum of EXT2.

Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa.

The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.

The plant secretory pathway for the trafficking of cell wall polysaccharides and glycoproteins.

Plant endomembranes are required for the biosynthesis and secretion of complex cell wall matrix polysaccharides, glycoproteins and proteoglycans. To define the biochemical roadmap that guides the synthesis and deposition of these cell wall components it is first necessary to outline the localization of the biosynthetic and modifying enzymes involved, as well as the distribution of the intermediate and final constituents of the cell wall. Thus far, a comprehensive understanding of cell wall matrix components has been hampered by the multiplicity of trafficking routes in the secretory pathway, and the diverse biosynthetic roles of the endomembrane organelles, which may exhibit tissue and development specific features. However, the recent identification of protein complexes producing matrix polysaccharides, and those supporting the synthesis and distribution of a grass-specific hemicellulose are advancing our understanding of the functional contribution of the plant secretory pathway in cell wall biosynthesis. In this review, we provide an overview of the plant membrane trafficking routes and report on recent exciting accomplishments in the understanding of the mechanisms underlying secretion with focus on cell wall synthesis in plants.

The Investigation of ADAMTS16 in Insulin-Induced Human Chondrosarcoma Cells.

A disintegrin-like metalloproteinase with thrombospondin motifs (ADAMTS) is a group of proteins that have enzymatic activity secreted by cells to the outside extracellular matrix. Insulin induces proteoglycan biosynthesis in chondrosarcoma chondrocytes. The purpose of the present in vitro study is to assess the time course effects of insulin on ADAMTS16 expression in OUMS-27 (human chondrosarcoma) cell line to examine whether insulin regulates ADAMTS16 expression as well as proteoglycan biosynthesis with multifaceted properties or not. Chondrosarcoma cells were cultured in Dulbecco's modified Eagle's medium having either 10 μg/mL insulin or not. While the experiment was going on, the medium containing insulin had been changed every other day. Cells were harvested at 1st, 3rd, 7th, and 11th days; subsequently, RNA and proteins were isolated in every experimental group according to their time interval. RNA expression of ADAMTS was estimated by quantitative real-time polymerase chain reaction (qRT-PCR) by using primers. Immunoreactive protein levels were encountered by the western blot protein detection technique by using proper anti-ADAMTS16 antibodies. ADAMTS16 mRNA expression level of chondrosarcoma cells was found to be insignificantly decreased in chondrosarcoma cells induced by insulin detected by the qRT-PCR instrument. On the other hand, there was a gradual decrease in immune-reactant ADAMTS16 protein amount by the time course in insulin-treated cell groups when compared with control cells. It has been suggested that insulin might possibly regulate ADAMTS16 levels/activities in OUMS-27 chondrosarcoma cells taking a role in extracellular matrix turnover.

Glycosylation Analysis for Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carbohydrate deficient transferrin (CDT) profile, N-glycan profile, and O-glycan profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The quantification of particular biomarkers, such as T antigens or sialylated T antigens, could also be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These techniques can be used to identify a majority of patients with defects in protein glycosylation, although different techniques, such as flow cytometry with immunostaining, are necessary to detect defects in glycolipid or proteoglycan biosynthesis which is not included in this unit.

Examining O‐xylosyltransferase in Drosophila

O‐xylosyltransferase (Oxt) is a transmembrane glycosyltransferase that initiates the first step in heperan and chrondroitin sulfate (HS and CS) proteoglycan biosynthesis. HSPGs and CSPGs are abundant in the extracellular environment of many tissues, such as articular cartilage in humans. In this tissue, HSPGs and CSPGs are essential regulation of signaling pathways that maintain tissue homeostasis. If homeostasis is not preserved, joint tissue will degrade and osteoarthritis (OA) can occur. Elevated levels of circulating Oxt have been detected in human OA patients suggesting shed Oxt may have an extracellular function. Little is known about Oxt behavior or function once it is released from the plasma membrane. Cell culture experiments have shown that Oxt, normally located in the Golgi and the endoplasmic reticulum (ER), can be shed by proteolysis activity and move into the media. It is likely that the same result will be observed in vivo. The overall aim of this project is to determine if Oxt released from expressing cells in vivo and what, if any, function extracellular Oxt has in organismal development. The data collected thus far shows localization of Oxt to the Golgi and the ER where it could have an active role in HSPG and CSPG biosynthesis. Movement of Oxt from expressing cells to non‐expressing cells is observed in the imaginal wing disc of Drosophila larva. Extracellular staining shows that Oxt is found predominantly on the apical surface and can be detected in the peripodial space suggesting it is released from expressing cells. Western analysis of Drosophila hemolymph indicated Oxt is released into hemolymph, as observed in humans. The results suggest that cleaved Oxt is found in the hemolymph of Drosophila and future studies will explore the potential function of shed Oxt.

Bradykinin release avoids high molecular weight kininogen endocytosis.

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.

Identification and characterization of human xylosyltransferase II promoter single nucleotide variants

The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity.XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry.Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant.This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong interindividual variabilities in serum XT activity, our data provide increasing evidence that XT-II activity is obviously regulated by hitherto unknown complex genetic pathways, such as cis- or trans-acting enhancers, silencers or miRNAs.

Man o' war mutation in UDP-α-D-xylose synthase favors the abortive catalytic cycle and uncovers a latent potential for hexamer formation.

The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.

Angelica sinensis and osteoarthritis: a natural therapeutic link?

The dried root of Angelica sinensis is widely used in Chinese traditional medicine for its beneficial effects against several diseases, including osteoarthritis. In order to understand the mechanism of action, two main components of the plant, the phytochemical, sodium ferulate, and a polysaccharidic fraction have been tested on osteoarthritis animal models or in human chondrocytes stimulated by the pro-inflammatory cytokine, Interleukine-1β. The results showed that sodium ferulate exhibited marked anti-inflammatory and anti-apoptotic properties by inhibiting the TNF/TNFR signal transduction pathway. On the other hand, the polysaccharidic fraction which contains a mixture of various carbohydrates was found to promote proteoglycan biosynthesis in cartilage matrix by stimulating the activity of the UDP-glycosyltransferases that synthesize the chondroitin sulfate chains of aggrecans. It is suggested that the combined action of sodium ferulate and polysaccharidic fraction would prevent cartilage destruction in osteoarthritis and favor cartilage repair.

Genome-Wide Association Study of Glomerular Filtration Rate in a Cohort of Sickle Cell Disease Patients

BACKGROUND: Nephropathy is a common and devastating complication of sickle cell disease (SCD) associated with mortality (Elmariah et al, 2014). However, early detection of SCD nephropathy (SCDN) has proven difficult. Discovery of genetic markers associated with SCDN could greatly improve our ability to identify patients at risk for renal decline. To that end, we have performed a genome-wide association study (GWAS) of glomerular filtration rate (GFR) in our adult SCD cohort.METHODS: Medical history, laboratory values and DNA for genotyping were collected as part of a multicenter study of outcome-modifying genes in SCD. Participating institutions included sickle cell centers at Duke University, University of North Carolina at Chapel Hill, Emory University, and East Carolina University. GFR was estimated using the 'Modification of Diet in Renal Disease’ (MDRD) study definition (Levey et al, 1999) and dichotomized at the clinically relevant threshold of 90 ml/min/1.73m2. 463 patients with complete data were included in the analysis (54.6% female, mean age 34.2 ± 12 years, 17.5% GFR < 90 ml/min/1.73m2). Genotyping was performed using the Illumina Human610-Quad BeadChip (Illumina, San Diego, CA), and a global reference panel from the 1000 Genomes project was used to impute genotypes not covered on the GWAS chip. Samples were pre-phased with SHAPEIT (Delaneau et al, 2012) and genotypes inferred using IMPUTE2 (Howie et al, 2009). After data cleaning and removal of single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) < 5%, 3,021,990 SNPs were available for analysis. Principal component (PC) analysis was performed to obtain measures of population stratification in our data set using EIGENSOFT (Patterson et al, 2006). Logistic regression was utilized to test for association between each SNP and dichotomized GFR, controlling for genome-wide PCs using PLINK (Purcell et al, 2008). False discovery rate (FDR) p-values were generated using PROC MULTTEST in SAS (SAS Systems, Cary, NC).RESULTS: Because GWAS chips capture common variation, several of the most highly associated SNPs were intergenic. The SNP with the most evidence for association that also lies within a gene was rs72777730 in XYLT1 (p=6.8E-6). For each additional C allele, the odds of having GFR < 90 ml/min/1.73m2 increased by 2.7. Other top hits included rs1990628 in ZNF423 (p=2.7E-5) and rs6449202 in SLC2A9 (p=3.8E-5). None of the findings met FDR significance. To further investigate our findings, we assessed the role of rare variants on renal function in our data set. We observed SNPs in XYLT1 from the Illumina HumanExome BeadChips associated with proteinuria (p=0.008) using the gene-based SKAT test (Wu et al, 2011). Further, we performed whole-exome sequencing of 15 patients with disparate GFR levels using VCRome 2.1 capture and 100bp paired-end reads generated from an Illumina HiSeq. One novel non-synonymous SNP (chr16:17353261, C>T) was identified in XYLT1 in 6 of 7 patients with low GFR (≤ 70 ml/min/1.73m2) but was absent in patients with normal GFR (> 90 ml/min/1.73m2). Additionally, four known non-synonymous SNPs were observed in low GFR patients but were absent in those with normal GFR levels.DISCUSSION: Xylosyltransferase 1 (XYLT1, also known as XT-1) encodes the enzyme responsible for biosynthesis of heparin sulfate proteoglycans, which affect permeability of the glomerular basement membrane. The c.343G>T polymorphism in XYLT1 has been implicated in diabetic nephropathy (Schön et al, 2006), and XT-1 is up-regulated in rats with adriamycin nephropathy (Sichuan et al, 2007). Zinc finger protein 423 (ZNF423) is involved in DNA damage response signaling, and mutations in ZNF423 have been identified in patients with nephronophthisis and Joubert syndrome (Chaki et al, 2012). Finally, solute carrier family 2 member 9 (SLC2A9, also known as GLUT9) is a transporter involved in urate reabsorption in the renal tubules. Mutations in this gene have been identified in patients with renal hypouricemia-2 (Matsuo et al, 2008). Thus, while none of the SNPs reached genome-wide significance, likely due to the modest size of our data set, we have identified several promising candidate genes nominally associated with renal function in our cohort of SCD patients and demonstrate that both common and rare variation in these genes likely contributes to risk for renal decline. DisclosuresNo relevant conflicts of interest to declare.

A computational analysis on the implications of age-related changes in the expression of cellular signals on the role of IGF-1 in intervertebral disc homeostasis

Insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent in intervertebral discs (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 plays a central role in IVD homeostasis. The IGF-mediated anabolic activity in IVD occurs when the growth factor, free from binding proteins (IGFBP), binds to IGF cell surface receptors (IGF-1R). Previous studies reported that, with aging, cellular expression of IGFBP increases, while that of IGF-1R decreases. Both changes in cellular signals are thought to be among the factors that are responsible for the age-related decline in IGF-mediated PG biosynthesis, which ultimately leads to disc degeneration.In this study, a computational model describing the role of IGF-1 in the homeostasis of IVD was deployed in a parametric analysis to investigate the effects of age-related changes in expression of IGF-1R and IGFBP on the IGF-mediated upregulation of PG biosynthesis and cellular proliferation.It was found that changes in the expression of IGF-1R and IGFBP mostly affected the nucleus pulposus, while in the most external disc regions (annulus fibrosus and cartilage endplates) the IVD homeostatic balance was unaltered. It was shown that a decrease of IGF-1R expression caused reduction of both PG levels and cell density in the tissue. In contrast, increase in IGFBP expression increased both PG and cell concentration, suggesting that such change in cellular signaling may be a plausible defense mechanism from age-related IVD degeneration.

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.

Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galβ1-4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.

Abstract 2604: Characterization of the activity and the mechanism of action of the new retinoid derivative ST5589 in pre-clinical models of lymphomas: involvement of MYC and cell cycle genes

Abstract Innovative and effective treatments are necessary for lymphomas that are common and still deadly cancers for many patients. Atypical retinoids show cytotoxic activity in different pre-clinical tumor models even though their mechanism of action is still largely unclear. Here, we report activity and mechanistic data with ST5589 (adarotene derivative), an active representative of this new class of agents (1), in pre-clinical lymphoma models. Methods. Cell lines from diffuse large B-cell lymphoma (DLBCL, n=17), mantle cell lymphoma (MCL, n=4), splenic marginal zone lymphoma (SMZL, n=3) underwent 72 hr exposure to increasing doses of ST5589 (Sigma Tau, Pomezia, IT) to assess anti-proliferative activity by MTT assay. Apoptosis was assessed by Annexin-V/7-AAD staining. Gene expression profiling (GEP) was done with Illumina HumanHT-12 Expression BeadChips at baseline on all cell lines and, in triplicate, on 1 ABC- (TMD8) and 1 GCB-DLBCL (DOHH2) cell line treated with 300 nM ST5589 or DMSO for 8 hrs. Cell lines with IC50 &amp;lt; than the median were defined as sensitive. GEP/IC50 correlation was assessed by Pearson correlation. Differential expression analysis was performed with LIMMA, GSEA, Metacore. Results. Median IC50 was 235 nM (91-2652) for all the cell lines without significant differences among histological subtypes or between GCB- and ABC-DLBCL: DLBCL, 269 (97-2652); GCB-DLBCL 250, (97-1684); ABC-DLBCL, 531 (181-2625); MCL, 237 (91-401); SMZL, 144 (135-203). ST5589 induced apoptosis in 4/5 DLBCL cell lines (DOHH2, TMD8, SU-DHL-2, VAL). GEP in two DLBCL treated with ST5589 or DMSO was performed, and ST5589 significantly affected transcripts involved in multiple cell cycle-related gene-sets. Aurora kinase A coding gene was one of the most down-regulated. MYC translocation was associated with lower IC50 (P 0.045) and higher number of responders (P 0.021). Transcripts associated with resistance to ST5589 were significantly enriched of genes involved in MYD88/TLR/STAT3 signaling and MAPK pathway, while transcripts associated with sensitivity were enriched of MYC target genes, genes high in Burkitt lymphoma, and involved in cellular respiration, and proteoglycan biosynthesis. Conclusions. ST5589 showed anti-proliferative and cytotoxic preclinical activity in lymphoma, affecting cell-cycle regulation. While MYD88, TLR, STAT3 and MAPK pathways activation correlated with reduced drug activity, MYC deregulation was associated with sensitivity to ST5589. Our data, together with direct cytotoxicity and immune-system mediated activity reported for the parent compound adarotene ST1926 (2), make ST5589 worth of further studies in MYC-driven lymphomas as a representative of the innovative therapeutic class of atypical retinoids. References 1 Giannini et al. Bioorganic and Med Chemistry 2012, 20: 2405-15 2 Pisano et al. Proceedings AACR 2004 Abstract #2075 Citation Format: Elena Bernasconi, Ivo Kwee, Andrea Rinaldi, Luciano Cascione, Maurilio Ponzoni, Loredana Vesci, Giuseppe Giannini, Anastasios Stathis, Emanuele Zucca, Eugenio Gaudio, Francesco Bertoni. Characterization of the activity and the mechanism of action of the new retinoid derivative ST5589 in pre-clinical models of lymphomas: involvement of MYC and cell cycle genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2604. doi:10.1158/1538-7445.AM2014-2604

EPO promotes bone repair through enhanced cartilaginous callus formation and angiogenesis.

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain.

The most popular strategy for normalization of RT-qPCR data involves presenting them in comparison with expression of "housekeeping" genes. However, the required stable expression of the control genes is not always achievable. As an alternative, we used ribonucleoprotein phage particles as an exogenous internal control and demonstrated that this type of normalization provides a simple and reliable method for quantification in RT-qPCR experiments. Using phage-based normalization, we analyzed mRNA levels of three popular housekeeping genes coding β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein L30 and showed high variability in their expression patterns during rat brain development, indicating that they should not be used as controls in gene expression studies of the developing brain either individually or in combination. Using phage-based controls, we showed interstrain differences and age-related changes in the expression of genes involved in proteoglycan biosynthesis and degradation in developing brain of senescence-accelerated OXYS rats and control Wistar rats.

ADAMTS13 expression in human chondrosarcoma cells induced by insulin

Objectives: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins is a proteinase enzyme group that primarily located in the extracellular matrix (ECM). Insulin has been known to stimulate proteoglycan biosynthesis in chondrosarcoma chondrocytes and thereby the levels of ADAMTS proteins. The aim of this study is to evaluate the time-dependent effects of insulin on the ADAMTS13 expression in OUMS-27 human chondrosarcoma cell line to test the hypothesis that insulin diminishes ADAMTS13 expression because of its anabolic effects. Methods: To test this hypothesis OUMS-27 cells were cultured in Dulbecco’s modified Eagle’ medium (DMEM) containing 10μg/mL insulin. The medium containing insulin was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and protein and RNA isolations were performed at the proper times. The levels of RNA expression of ADAMTS13 was quantified by qRT-PCR using appropriate primers while protein levels was detected by Western blot technique using anti-ADAMTS13 antibody. Results: Although there was a decrease in both RNA and protein levels in insulin-applied groups compared to the control cells, it was not statistically significant. Conclusion: Under the light of our findings, it is suggested that insulin does not participate in regulation of ADAMTS13 in OUMS-27 chondrosarcoma cells.

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.