Fibrotic-like changes in degenerate human intervertebral discs revealed by quantitative proteomic analysis.

OBJECTIVE: Intervertebral disc degeneration (IDD) can lead to symptomatic conditions including sciatica and back pain. The purpose of this study is to understand the extracellular matrix (ECM) changes in disc biology through comparative proteomic analysis of degenerated and non-degenerated human intervertebral disc (IVD) tissues of different ages. DESIGN: Seven non-degenerated (11-46 years of age) and seven degenerated (16-53 years of age) annulus fibrosus (AF) and nucleus pulposus (NP) samples were used. Proteins were extracted using guanidine hydrochloride, separated from large proteoglycans (PGs) by caesium chloride (CsCl) density gradient ultracentrifugation, and identified using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). For quantitative comparison, proteins were labeled with iTRAQ reagents. Collagen fibrils in the NP were assessed using scanning electron microscopy (SEM). RESULTS: In the AF, quantitative analysis revealed increased levels of HTRA1, COMP and CILP in degeneration when compared with samples from older individuals. Fibronectin showed increment with age and degeneration. In the NP, more CILP and CILP2 were present in degenerated samples of younger individuals. Reduced protein solubility was observed in degenerated and older non-degenerated samples correlated with an accumulation of type I collagen in the insoluble fibers. Characterization of collagen fibrils in the NP revealed smaller mean fibril diameters and decreased porosity in the degenerated samples. CONCLUSIONS: Our study identified distinct matrix changes associated with aging and degeneration in the intervertebral discs (IVDs). The nature of the ECM changes, together with observed decreased in solubility and changes in fibril diameter is consistent with a fibrotic-like environment.

SOX9 directly regulates the type-II collagen gene.

Mutations in human SOX9 are associated with campomelic dysplasia (CD), characterised by skeletal malformation and XY sex reversal. During chondrogenesis in the mouse, Sox9 is co-expressed with Col2a1, the gene encoding type-II collagen, the major cartilage matrix protein. Col2a1 is therefore a candidate regulatory target of SOX9. Regulatory sequences required for chondrocyte-specific expression of the type-II collagen gene have been localized to conserved sequences in the first intron in rats, mice and humans. We show here that SOX9 protein binds specifically to sequences in the first intron of human COL2A1. Mutation of these sequences abolishes SOX9 binding and chondrocyte-specific expression of a COL2A1-driven reporter gene (COL2A1-lacZ) in transgenic mice. Furthermore, ectopic expression of Sox9 trans-activates both a COL2A1-driven reporter gene and the endogenous Col2a1 gene in transgenic mice. These results demonstrate that COL2A1 expression is directly regulated by SOX9 protein in vivo and implicate abnormal regulation of COL2A1 during, chondrogenesis as a cause of the skeletal abnormalities associated with campomelic dysplasia.

Different cis-regulatory DNA elements mediate developmental stage- and tissue-specific expression of the human COL2A1 gene in transgenic mice.

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5' flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2, 388 to +2,696) in intron 1 in conjunction with 6.1-kb 5' sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5' sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from -90 to -1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from -1,500 to -6,100 and +2,388 to +2,855.

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development.

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis-regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.

Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function.

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen alpha1 (X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen alpha1 (X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.

Phenotypic and population differences in the association between CILP and lumbar disc disease.

BACKGROUND: Lumbar disc disease (LDD) is one of the leading causes of disability in the working-age population. A functional single-nucleotide polymorphism (SNP), +1184T-->C, in exon 8 of the cartilage intermediate layer protein gene (CILP) was recently identified as a risk factor for LDD in the Japanese population (odds ratio (OR) 1.61, 95% CI 1.31 to 1.98), with implications for impaired transforming growth factorbeta1 signalling. AIM: To validate this finding in two different ethnic cohorts with LDD. METHODS: This SNP and flanking SNPs were analysed in 243 Finnish patients with symptoms of LDD and 259 controls, and in 348 Chinese subjects with MRI-defined LDD and 343 controls. RESULTS AND CONCLUSION: The results showed no evidence of association in the Finnish (OR = 1.35, 95% CI 0.97 to 1.87; p = 0.14) or the Chinese (OR = 1.05, 95% CI 0.77 to 1.43; p = 0.71) samples, suggesting that cartilage intermediate layer protein gene is not a major risk factor for symptoms of LDD in Caucasians or in the general population that included individuals with or without symptoms.

Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9.

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate delta-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.

The Mll-Een knockin fusion gene enhances proliferation of myeloid progenitors derived from mouse embryonic stem cells and causes myeloid leukaemia in chimeric mice.

Rearrangement of the mixed lineage leukaemia (MLL) gene with extra eleven nineteen (EEN) was previously identified in an infant with acute myeloid leukaemia. Using homologous recombination, we have created a mouse equivalent of the human MLL-EEN allele and showed that when Mll(Een/+) embryonic stem (ES) cells were induced to differentiate in vitro into haemopoietic cells, there was increased proliferation of myeloid progenitors with self-renewal property. We also generated Mll(Een/+) chimeric mice, which developed leukaemia displaying enlarged livers, spleens, thymuses and lymph nodes owing to infiltration of Mll(Een/+)-expressing leukemic cells. Immunophenotyping of cells from enlarged organs and bone marrow (BM) of the Mll(Een/+) chimeras revealed an accumulation of Mac-1+/Gr-1- immature myeloid cells and a reduction in normal B- and T-cell populations. We observed differential regulation of Hox genes between myeloid cells derived from Mll(Een/+) ES cells and mouse BM leukemic cells which suggested different waves of Hox expression may be activated by MLL fusion proteins for initiation (in ES cells) and maintenance (in leukemic cells) of the disease. We believe studies of MLL fusion proteins in ES cells combined with in vivo animal models offer new approaches to the dissection of molecular events in multistep pathogenesis of leukaemia.

Properties of Ascaris muscle mitochondria. 1. Cytochromes.

1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations. 2. Difference spectra (at 22 degrees C and -196 degrees C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N' ,N'-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c1, c and aa3, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component. 3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150mM KCl, leaving the membrane-bound cytochromes c1, b and aa3 in the KCl residue.

Effect of trifluoperazine on skeletal muscle mitochondrial respiration.

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca2+-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the ADP/O and Ca2+/O ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca2+-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c1 and cytochrome c1-aa3 segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c1 by 92% with succinate as substrate, and of cytochrome c and cytochrome aa3 by 50-60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.

Mitochondrial calcium transport and calcium-activated phospholipase in porcine malignant hyperthermia.

The interaction of Ca2+ with mitochondria isolated from longissismus dorsi, a predominantly white skeletal muscle, of normal and malignant hyperthermia pigs was investigated using tightly-coupled preparations. Arrhenius plots of mitochondrial Ca2+ -stimulated respiration for succinate oxidation of malignant hyperthermia pigs showed a transition temperature (Tt) of 26.31 +/- 0.80 degrees C (n = 5), which was decreased by spermine to 15.41 +/- 0.69 degrees C (n = 3), a value very similar to that for normal pigs. No difference in either the Tt or in the activation energy (Ea) was observed between the two types of pigs when ADP was used instead of Ca2+. Mitochondria of malignant hyperthermia pigs were uncoupled at 40 degrees C by exogenous Ca2+ at 1221 +/- 301 (n = 9) nmol Ca2+ per mg proteinn during succinate oxidation and the uncoupled mitochondria showed large amplitude swelling. Both the Ca2+ -induced uncoupling and swelling were prevented by bovine serum albumin and by the phospholipase inhibitors, spermine and tetracaine. In contrast, mitochondria of normal pigs were still tightly coupled even after a total addition of 2313 +/- 287 (n = 5) nmol Ca2+ per mg protein and retained the original condensed configuration in the presence or absence of spermine and tetracaine. Mitochondria of malignant hyperthermia pigs contained significantly (P less than 0.001) higher quantities of endogenous Ca2+ and showed a significantly (P less than 0.001) faster FCCP-induced endogenous Ca2+ efflux rate than normal when monitored spectroscopically with murexide. No significant difference was observed in either the rate of exogenous Ca2+ uptake or in the extent of Ca2+ accumulated in the aerobic steady state during succinate oxidation between the two types of pigs. The rate of mitochondrial Ca2+ efflux of malignant hyperthermia pigs during anaerobiosis was about twice that of normal. Experimental evidence suggests that mitochondria from musculi longissimus dorsi of malignant hyperthermia pigs contained a Ca2+ -stimulated phospholipase A2 (EC 3.1.1.4, phosphatide 2-acylhydrolase), and that this enzyme if present in mitochondria of normal pigs is either latent or in very low concentration. The significance of the Ca2+ -stimulated phospholipase A2 and its association with the enhanced rate of glycolysis in porcine malignant hyperthermia syndrome and in the post-mortem formation of the pale, soft and exudative conditions observed in white skeletal muscles of malignant hyperthermia pigs is discussed.

Identification and characterization of pigs prone to producing 'RSE' (reddish-pink, soft and exudative) meat in normal pigs.

RSE (reddish-pink, soft and exudative) meat was investigated using pigs of three different halothane genotypes. A significantly lower pH(1h), value was observed in RSE compared with that of RFN (red, firm and non-exudative) -meat, both of which have values higher than 6.0 at 1 hr post-mortem. Drip loss (%) in RSE-meat was ≥7%, which was twice that of RFN-meat. Normal values for fibre optic probe and Minolta L and a were observed for RSE-meat. RSE-meat could be derived from NN and Nn pigs, and its formation could be induced from RFN-prone pigs by poor post-slaughter management. Pigs expected to produce RSE-meat were identified using small biopsy samples of M. longissimus dorsi (LD). Predicted RSE-meat in live pigs was confirmed by post-mortem assessments of meat quality using LD muscle. With NN Landrace-Yorkshire × Duroc pigs, 15.6% were identified to be RSE-prone in live pigs, and a further 6.7% RSE was induced after slaughter from RFN pigs. The rate of glycolysis determined from biopsy LD samples and at 1 hr post-mortem (pH(1h)) were significantly (p < 0.001) faster in RSE than in RFN-prone pigs, but significantly slower than those of PSE-prone pigs. Good correlations (p < 0.001) were observed between biopsy fluid (F) values, an indicator of water-holding capacity (WHC), and drip loss (r = 0.652) from post-mortem LD muscle, and between biopsy pH (F), an indicator for the rate of glycolysis, and F (r = -0.828). These results show that the skeletal muscle test using biopsy LD muscle could be employed to reduce the incidence of RSE-meat.

Effect of dietary supplementation of vitamin E on pig meat quality.

The effect of vitamin E on pig meat quality was investigated using British Landrace (NN and nn), Landrace × Large White (NN and Nn) and Pietrain (nn) pigs. Daily dietary supplementation of 500 mg vitamin E/kg diet for 46 days could reduce drip loss in unfrozen longissimus thoracis (LT) by 45% and 54% in Nn and NN pigs, respectively. In PSE-prone Landrace × Large White Nn pigs, daily supplementation of 1000 mg vitamin E/kg diet for the same period could significantly (P < 0.001) reduce excess release of Ca(2+) and prevent the formation of PSE carcasses, evaluated from both biopsy and post- mortem LT samples. Experimental evidence from erythrocyte fragility and water-holding capacity of biopsy LT samples suggested that vitamin E improved pig meat quality by its ability to stabilize membranes. Stabilization of membrane integrity in isolated mitochondria of LT by vitamin E was associated with inhibition of phospholipase A(2) activity.

Variations in meat quality in live halothane heterozygotes identified by biopsy samples of M. longissimus dorsi.

The meat quality of halothane heterozygotes (Nn) was investigated using British Landrace (n = 18) and Landrace × Large White pigs (n = 67). Normal and PSE meat were identified in both breeds using M. longissimus dorsi (LD). In Landrace × Large White Nn pigs, the potential meat quality identified using small biopsy LD samples (500 mg) showed a wide spectrum in water-holding capacity (WHC), ranging from normal (43%) to PSE (57%). Predictions of meat quality in live pigs were confirmed from post-mortem assessments of LD samples based on pH(1) and fibre optic probe measurements. Our results show that Nn pigs have the propensity to produce a higher proportion of PSE than normal carcasses. The predictive meat quality test performed on small biopsy LD samples offers an opportunity not currently available to the pig industry, to select Nn pigs with the potential to produce pork of superior WHC.

Phospholipase A(2) activity, calmodulin, Ca(2+) and meat quality in young and adult halothane-sensitive and halothane-insensitive British Landrace pigs.

PSE (pale, soft and exudative) meat is the result of a developmental disorder and is not produced in meat from young (3-11 weeks) halothane-sensitive British Landrace pigs. Formation of the PSE condition is closely associated with the muscle mitochondrial phospholipase A(2) activity, which increases significantly from 12 weeks of age. The increase in phospholipase A(2) activity shows good correlation (r = 0·87) with the increase in post-mortem sarcoplasmic Ca(2+). In halothane-insensitive British Landrace both the phospholipase A(2) activity and sarcoplasmic Ca(2+) level remain constant throughout growth. Significant increases in the levels of endogenous mitochondrial calmodulin, fatty acids and phospholipase A(2) activity, and Ca(2+) (mitochondrial and sarcoplasmic) are observed in the M. longissimus dorsi of adult halothane-sensitive pigs when compared with either young halothane-sensitive or young and adult halothane-insensitive pigs. The enhanced mitochondrial phospholipase A(2) activity is calmodulin-dependent and the increase in activity is postulated to be due to increased endogenous calmodulin.

Prediction of meat quality in live pigs using stress-susceptible and stress-resistant animals.

A new in-vivo procedure for predicting the potential meat quality in live pigs was devised using stress-susceptible (halothane positive) and stress-resistant (halothone negative) pigs. The potential meat quality in live pigs was determined using small biopsy samples of M. longissimus dorsi (LD). Meat quality was assessed by the combined measurements of pH and water-holding capacity (WHC) on the 12 000 g supernatant after incubation of 500 mg biopsy LD muscle with an equal volume of 150 mm KCl at 39°C for 45 min. With the LD muscles of halothane positive (n = 37) and halothane negative (n = 55) pigs, high correlations (r = -0·854) were observed between the supernatant (i.e. fluid) pH and WHC of the biopsy samples, between fluid pH of the biopsy samples and 1-h post-mortem (pH(60)) LD muscles (r = 0·951), and between pH(60) and WHC (r = -0·956). The experimental data show that our in-vivo test can differentiate halothane positive from halothane negative pigs and can also predict the potential meat quality in live pigs. The test could be applied to select pigs with differences in WHC to improve meat quality.

Identification of halothane genotypes by calcium accumulation and their meat quality using live pigs.

The three halothane genotypes (NN, Nn, and nm) were identified by measuring the capacity for Ca(2+) accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of M. longissimus dorsi with a Ca(2+) specific electrode at 35°C. Significant differences (P < 0·001) in deterioration (%) of Ca(2+) accumulation, 12% for NN, 35% for Nn, and 81% for nn pigs, were observed after ageing the whole muscle homogenate preparations for 24 h in ice. Predictions of meat quality in live pigs (n = 34) based on the values for water-holding capacity, assessed as fluid (g/0·5 g wet wt LD), and pH (fluid) by using small biopsy LD samples (Cheah et al. 1993) were performed on all the halothane genotypes. The halothane genotype NN (n = 11) showed a fluid value of 0·37 ± 0·01 and a pH (fluid) value of 6·62 ± 0·03 as compared with 0·61 ± 0·02 and 5·84 ± 0·04, respectively, for the halothane genotype nn (n = 13). The Nn pigs (n = 10) showed fluid (0·49 ± 0·03) and pH (fluid) (6·19 ± 0·11) values between those values observed for the two homozygotes (NN and nn). Predictions of meat quality in live pigs from biopsy LD muscles were confirmed from assessments on post-mortem LD muscles based on pH(1) and fibre optic probe (FOP) measurements. The extent of deterioration (%) in Ca(2+) accumulation showed high correlations with fluid (r = -0·861) and pH (fluid) (r = -0·831) in the biopsy LD samples, and with pH(1) (r = 0·663), FOP (r = -0·812), and drip (%) loss (r = -0·777) in the post-mortem LD samples.

Relationship between Ca(2+) release, sarcoplasmic Ca(2+), glycolysis and meat quality in halothane-sensitive and halothane-insensitive pigs.

Post-mortem assessment of porcine stress susceptibility and meat quality by measurements of mitochondrial calcium (content and rate of release), water-holding capacity, pH at 1h post mortem and colour on M. longissimus dorsi confirm the usefulness of the halothane test for detecting stress susceptibility. Good correlations were observed between halothane sensitivity and meat quality. Post-mortem samples of M. longissimus dorsi of halothane-sensitive pigs showed significantly higher levels of sarcoplasmic calcium than similar muscle from halothane-insensitive pigs. This strongly suggests that elevated sarcoplasmic calcium levels are linked to the formation of PSE meat.