Reduced cell proliferation and increased apoptosis are significant pathological mechanisms in a murine model of mild pseudoachondroplasia resulting from a mutation in the C-terminal domain of COMP

Pseudoachondroplasia (PSACH) is one of the more common skeletal dysplasias and results from mutations in cartilage oligomeric matrix protein (COMP). Most COMP mutations identified to date cluster in the TSP3 repeat region of COMP and the mutant protein is retained in the rough endoplasmic reticulum (rER) of chondrocytes and may result in increased cell death. In contrast, the pathomolecular mechanism of PSACH resulting from C-terminal domain COMP mutations remain largely unknown. This study describes the generation and analysis of a murine model of mild PSACH resulting from a p.Thr583Met mutation in the C-terminal globular domain (CTD) of COMP. Mutant animals are normal at birth, but grow slower than their wild-type littermates and by 9 weeks of age they have mild short-limb dwarfism. Furthermore, by 16 months of age mutant animals exhibit severe degeneration of articular cartilage, which is consistent with early onset osteoarthritis seen in PSACH patients. In the growth plates of mutant mice the chondrocyte columns are sparser and poorly organized. Mutant COMP is secreted into the extracellular matrix, but its localization is disrupted along with the distribution of several COMP-binding proteins. Although mutant COMP is not retained within the rER there is an unfolded protein/cell stress response and chondrocyte proliferation is significantly reduced, while apoptosis is both increased and spatially dysregulated. Overall, these data suggests a mutation in the CTD of COMP exerts a dominant-negative effect on both intra- and extracellular processes. This ultimately affects the morphology and proliferation of growth plate chondrocytes, eventually leading to chondrodysplasia and reduced long bone growth

A Novel Form of Chondrocyte Stress is Triggered by a COMP Mutation Causing Pseudoachondroplasia

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012. © 2011 Wiley Periodicals, Inc

Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization

SummaryObjectiveTo investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage.DesignXbp1flox/flox.Col2a1-Cre mice (Xbp1CartΔEx2), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation.ResultsXbp1CartΔEx2 displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1CartΔEx2 growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1CartΔEx2 embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1CartΔEx2 growth plate. IRE1 hyperactivation occurred in Xbp1CartΔEx2 chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR.ConclusionOur work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease

microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140- null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140- 3p.1 is more functional than original consensus miR-140-3p seed containing isomiR

Increased hippocampal excitability in miR-324-null mice

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3′UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease

Zawartość makro- i mikroelementów w środowisku korzeniowym pomidora szklarniowego uprawianego w wełnie mineralnej i włóknie drzewnym w zależności od zawartości azotu w pożywkach

The aim of investigations conducted in the years 2005–2007 was to determine the chemical composition of nutrient solutions in the root environment of tomato grown in wood fiber and rockwool, under the influence of diverse levels of nitrate nitrogen in the nutrient solution amounting 200, 220 and 240 mg N-NO3·dm-3. With an increase in nitrate nitrogen content in nutrient solutions used in plant fertigation a significant increase was observed in the contents of N-NO3 in nutrient solutions of the root environment, collected from wood fiber and rockwool. No such effect was found for contents of N-NH4, P, K, Ca, Mg, Fe, Mn, Zn, Cu, B, Na, Cl or pH and EC. In case of wood fiber the following nutrient concentration series was found in nutrient solutions of the root environment in relation to the nutrient solution flowing from the drippers: Na > Cu > Ca > Zn > K > Cl > B > N-NO3; the following were reduced Fe > Mg > P-PO4 > N-NH4 > Mn. Nutrients being concentrated in root environment solutions during tomato growing in rockwool were: Na > Ca > Cu > Fe > Cl > K > Zn > B > S-SO4 > N-NO3, while contents of Mg > P-PO4 > N-NH4 > Mn decreased. Despite of a wide range of carbon to nitrogen ratio (C:N) in wood fiber (123–127), no significant reduction of nitrates was shown in the root environment. It was a result of adequate application frequency of nutrient solutions during a day.Celem przeprowadzonych badań (2005–2007) było określenie zmian zawartości makro i mikroelementów oraz sodu w pożywkach pobieranych ze środowiska korzeniowego pomidora, uprawianego we włóknie drzewnym i wełnie mineralnej, pod wpływem zróżnicowanych poziomów azotu azotanowego w pożywkach, wynoszących 200, 220, 240 mg N-NO3·dm-3. Ze wzrostem zawartości azotu azotanowego w pożywkach stosowanych do fertygacji roślin stwierdzono istotny wzrost zawartości N-NO3 w pożywkach pobieranych ze środowiska korzeniowego roślin uprawianych we włóknie drzewnym i wełnie mineralnej. Wpływu takiego nie wykazano dla zawartości N-NH4, P, K, Ca, Mg, Fe, Mn, Zn, Cu, B, Na, Cl, oraz pH i EC. W przypadku włókna drzewnego stwierdzono następujący szereg zatężania składników w pożywkach pobieranych ze środowiska korzeniowego, w stosunku do pożywki dostarczanej roślinom: Na > Cu > Ca > Zn > K > Cl > B > N-NO3; obniżeniu ulegały zawartości Fe > Mg > P-PO4 > N-NH4 > Mn. Składnikami ulegającymi zatężaniu w pożywkach pobieranych ze strefy korzeniowej w uprawie pomidora w wełnie mineralnej były: Na > Ca > Cu > Fe > Cl > K > Zn > B > S-SO4 > N-NO3, natomiast obniżaniu ulegały Mg > P-PO4 > N-NH4 > Mn. Mimo szerokiego stosunku węgla do azotu we włóknie drzewnym, wynoszącym C:N = (123–127) i możliwości wystąpienia sorpcji biologicznej, nie wykazano istotnego obniżenia zawartości azotu azotanowego w pożywkach pobieranych ze strefy korzeniowej roślin. Było to wynikiem odpowiedniej częstotliwość dostarczania pożywki roślinom w ciągu doby