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.

Increased basal insulin secretion in Pdzd2-deficient mice.

Expression of the multi-PDZ protein Pdzd2 (PDZ domain-containing protein 2) is enriched in pancreatic islet beta cells, but not in exocrine or alpha cells, suggesting a role for Pdzd2 in the regulation of pancreatic beta-cell function. To explore the in vivo function of Pdzd2, Pdzd2-deficient mice were generated. Homozygous Pdzd2 mutant mice were viable and their gross morphology appeared normal. Interestingly, Pdzd2-deficient mice showed enhanced glucose tolerance in intraperitoneal glucose tolerance tests and their plasma insulin levels indicated increased basal insulin secretion after fasting. Moreover, insulin release from mutant pancreatic islets was found to be twofold higher than from normal islets. To verify the functional defect in vitro, Pdzd2 was depleted in INS-1E cells using two siRNA duplexes. Pdzd2-depleted INS-1E cells also displayed increased insulin secretion at low concentrations of glucose. Our results provide the first evidence that Pdzd2 is required for normal regulation of basal insulin secretion.

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.

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.

Expression of collagen and aggrecan genes in normal and osteoarthritic murine knee joints.

OBJECTIVE: The STR/ort mouse strain develops osteoarthritis (OA) of the medial tibial cartilage whilst CBA mice do not develop this disease. We investigated whether changes occur in the expression of genes encoding major extracellular matrix proteins in the connective tissue of the murine knee joint in OA. DESIGN: Expression of the genes encoding collagens II (Col2alpha1), X (Col10alpha1), alpha2(XI) (Col11alpha2) and aggrecan (Agc) was detected in skeletally mature and immature male mice of the CBA and STR/ort strains by in situ hybridization. RESULTS: Col2alpha1 was expressed by chondrocytes of the tibial and patella-femoral cartilage and by the meniscal cartilage in all young mice (4-9 weeks) but only in the patella-femoral cartilage in older mice of both strains (36-45 weeks). In contrast Col2alpha1 was expressed by growth plate chondrocytes of both species at all ages. Similarly, Col2alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 expression was evident in the hypertrophic chondrocytes in the growth plate of young CBA and STR mice, but was not active in these cells in mature animals. However, Col10alpha1 was transcribed in articular chondrocytes of the tibia, meniscal and patella-femoral cartilages of all ages, in normal and osteoarthritic mice. Transcripts were also present in ligament of some mature animals. Col11alpha2 followed a similar pattern of expression in CBA cartilages to Col2alpha1, being active in adult growth plate but generally inactive in adult articular cartilages. Young CBA and STR/ort mice expressed Col11alpha2 in articular cartilage and very strongly throughout the growth plate. Agc expression was detected in all articular cartilages at all ages in both strains. Interestingly, transcripts for all four genes were absent in tibial articular chondrocytes located close to osteoarthritic lesions in STR/ort mice, indicating that these cells are unable to synthesize matrix proteins. Adult STR/ort mice also showed evidence of tissue remodeling around the periphery of the knee joint. Cells in remodeling areas actively transcribed Col2alpha1, Col10alpha1, Col11alpha2 and Agc. CONCLUSION: It is unlikely that OA develops in STR/ort mice because of failure to express major proteins in joint tissue. However, once lesions develop in articular cartilage neighbouring chondrocytes fail to express genes encoding several matrix proteins.

Impact of mutations of cartilage matrix genes on matrix structure, gene activity and chondrogenesis.

OBJECTIVE: Chondrocytes in the growth plate at different stages of differentiation synthesize characteristic extracellular matrix (ECM) components. Mutations in some ECM genes result in chondrodysplasia in humans and mice. We aimed to evaluate the impact of loss- and gain-of-function mutations of ECM genes on matrix structure, gene expression and formation of the growth plate. DESIGN: We review information on the impact of deficiencies in proteoglycans, and types X and II collagens on skeletal development. Additionally, we compare the impact of a glycine904 to cysteine (G904C) mutation in the triple helical coding domain of mouse Col2a1 with two previously reported Col2a1 mutations (exon7 deletion (Del1) and G85C). The G904C Col2a1 gene was introduced as a transgene into mice. Transgenic newborn mice were examined for skeletal development. The histology of the epiphyseal cartilage and the growth plate, and the ultrastructure of chondrocytes and collagen fibrillar morphology in the ECM were studied in 18.5-day transgenic and wild-type fetuses. The distribution of the mRNAs for Col2a1, Col11a1, Col9a1, Matn1, Agc and Ihh in the growth plate of 18.5-day G904C/G904C and wild type fetuses were compared by in situ hybridization. RESULTS: Heterozygous transgenic mice harbouring five copies of the G904C Col2a1 transgene developed skeletal abnormalities and dwarfism. Homozygous G904C/G904C mice died at birth, showing cleft palate, disrupted zonation of chondrocytes and reduction of the zone of hypertrophic chondrocytes. Fewer collagen fibrils were found in ECM of the cartilage. Rough endoplasmic reticulum of the chondrocytes of G904C/+ and G904C/G904C mice was distended. In G904C/G904C mutant mice, Agc gene activity was extended to the hypertrophic zone. Expression of the other genes studied was unchanged. Calcified materials that were not found normally in the maturing and only at low abundance in the hypertrophic zones of the wild type growth plate, were present in these zones in G904C/G904C mice. Despite phenotypic similarities for the G904C and Del1 mice, reduced expression of types I, II, IX, X collagens and aggrecan were reported for the latter mutation. Changes in gene activity and matrix organization in the growth plate also accompanied deficiencies in aggrecan, perlecan and collagen II. CONCLUSIONS: The data suggest that a single amino acid alteration in collagen II could lead to skeletal abnormalities through multiple secondary effects on the synthesis and assembly of ECM components. The functional impact of mutations of ECM genes reveals that chondrodysplasia is caused not just by the formation of abnormal matrix molecules, but that the alteration of one ECM component may lead to a cascade of disruption of other gene activities in chondrocytes which collectively contribute to the pathological changes in the architecture of the growth plate.

Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation.

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.

Chondrocyte antigen expression, immune response and susceptibility to arthritis.

The association of HLA-B27 with certain forms of arthritis implies a role for MHC class I-restricted T cells in the arthritic process. Our aim was to study CD8(+) T cell responses towards specific antigens localized in joint tissue. Known determinants were introduced into chondrocytes of transgenic (TG) mice, under the control of the cis-regulatory sequences of the human type II collagen gene (COL2A1). Two Escherichia coli beta-galactosidase (beta-gal)-expressing lines were derived (CIIL73 and CIIL64) as well as two lines (CIINP) expressing influenza A virus nucleoprotein (NP). Expression of the antigens could be demonstrated in cartilaginous tissues. The TG lines showed variable degrees of responsiveness towards the transgene-introduced antigens; whilst 75% of CIIL73 mice had an impaired cytotoxic T lymphocyte (CTL) response towards beta-gal, the response in CIIL64 mice was essentially normal. However, both lines displayed normal proliferative and antibody responses to beta-gal. A reduced CTL response was seen to NP in the CIINP lines in approximately 65% of the animals. In spite of the persistence of T cell responses to the transgene antigens in these lines, induction of CTL responses alone has so far failed to induce clinical signs of arthritis. Interestingly, some animals expressing beta-gal were susceptible to arthritis following challenge with type II collagen alone, whilst their non-TG littermates and TG mice from other lines remained unaffected. As beta-gal is expressed by E. coli, a component of the normal gut flora, this suggests a possible role for gut-derived immune responses. We believe these lines could form the basis of a model for studying links between intestinal inflammation and arthritis.

Assessing clusters and motifs from gene expression data.

Large-scale gene expression studies and genomic sequencing projects are providing vast amounts of information that can be used to identify or predict cellular regulatory processes. Genes can be clustered on the basis of the similarity of their expression profiles or function and these clusters are likely to contain genes that are regulated by the same transcription factors. Searches for cis-regulatory elements can then be undertaken in the noncoding regions of the clustered genes. However, it is necessary to assess the efficiency of both the gene clustering and the postulated regulatory motifs, as there are many difficulties associated with clustering and determining the functional relevance of matches to sequence motifs. We have developed a method to assess the potential functional significance of clusters and motifs based on the probability of finding a certain number of matches to a motif in all of the gene clusters. To avoid problems with threshold scores for a match, the top matches to a motif are taken in several sample sizes. Genes from a sample are then counted by the cluster in which they appear. The probability of observing these counts by chance is calculated using the hypergeometric distribution. Because of the multiple sample sizes, strong and weak matching motifs can be detected and refined and significant matches to motifs across cluster boundaries are observed as all clusters are considered. By applying this method to many motifs and to a cluster set of yeast genes, we detected a similarity between Swi Five Factor and forkhead proteins and suggest that the currently unidentified Swi Five Factor is one of the yeast forkhead proteins.

Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease.

Schmid metaphyseal chondrodysplasia results from mutations in the collagen X (COL10A1) gene. With the exception of two cases, the known mutations are clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1 mutations result in impaired collagen X trimer assembly and secretion. In the two other cases, missense mutations that alter Gly(18) at the -1 position of the putative signal peptide cleavage site were identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro cell-free translation in the presence of microsomes, and cell transfection assays, these two mutations were created in COL10A1 by site-directed mutagenesis. The data suggest that translocation of the mutant pre-alpha1(X) chains into the microsomes is not affected, but cleavage of the signal peptide is inhibited, and the mutant chains remain anchored to the membrane of microsomes. Cell-free translation and transfection studies in cells showed that the mutant chains associate into trimers but cannot form a triple helix. The combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both result in impaired collagen X secretion, probably followed by intracellular retention and degradation of mutant chains, and causing the Schmid metaphyseal chondrodysplasia phenotype.

Tissue-specific expression and long-term deposition of human collagen VII in the skin of transgenic mice: implications for gene therapy.

We report the isolation of a cosmid clone containing the entire human COL7A1 gene in one piece. The ability of the genomic sequences within this clone to direct tissue-specific expression of human collagen VII in transgenic mice was tested. The data show that the gene construct is capable of directing expression of collagen VII in the skin of fetal and neonatal transgenic mice. Expression of COL7A1 in these mice was widespread, in a pattern consistent with that found in human tissues and was in parallel with that of the endogenous mouse gene. Immunostaining, using human-specific antibodies, showed that human collagen VII protein was present at the skin basement membrane zone of the transgenic mice. Dermal extracts from 19-month-old transgenic mice contained mature human collagen VII protein, and fibroblasts derived from skin biopsies of these mice actively synthesized human collagen VII. The demonstration of successful and stable expression of human collagen VII in in vivo gene transfer is the first step towards the future development of therapeutic protocols for the rescue of keratinocyte function in severe blistering diseases such as dystrophic epidermolysis bullosa.

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.

Disrupted expression of matrix genes in the growth plate of the mouse cartilage matrix deficiency (cmd) mutant.

Chondrodysplasia in the autosomal recessive cartilage matrix deficiency (cmd) mutant is caused by lack of the proteoglycan aggrecan arising from a mutation in the gene. Homozygous cmd/cmd mice are characterized by disorganisation of chondrocytes in the growth plate, disproportionate dwarfism, cleft palate, and perinatal lethality. We have studied the impact of the aggrecan deficiency on the expression of other matrix genes during the differentiation of chondrocytes in the growth plate of cmd/cmd 18.5 day fetuses. Compared with the wild-type, there are significant differences in the growth plates of cmd mutants in the combinations of co-expression of genes encoding the glycoprotein link protein, proteoglycan syndecan 3, collagens alpha 1 (X) [Col10a1], alpha 2(XI) [Col11a2], and the alternative transcripts of alpha 1 (II) [Col2a1 type IIA form], and alpha 1 (IX) [Col9a1 long and short forms]. The discordance of gene expression in cmd chondrocytes may be additional factors contributing to the disrupted cellular architecture of the growth plate resulting from the primary absence of aggrecan.

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.

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.

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.

Arthritis susceptibility in mice expressing human type II collagen in cartilage.

Collagen type II (CII) induced arthritis (CIA) in mice is an experimental model for rheumatoid arthritis. Induction with non-self (e.g. human) CII induces severe arthritis whereas the mice are less susceptible to induction with self CII (i.e. mouse). To analyse whether an autoimmune response to human CII can develop and is pathogenic the authors have established transgenic mice expressing human CII in cartilage and backcrossed them into two different gene backgrounds susceptible to CIA (DBA/1 and C3H.Q). The transgenic human CII expression was restricted to cartilage and did not disturb cartilage morphology or lead to chondrodystrophy. In addition, development of stress-induced arthritis was not affected by the transgene. The cartilage specific expression of human CII reduced, but did not eliminate, the susceptibility to CIA irrespective of the species source (human, bovine, chick, rat) of CII used for immunization. A common denominator between these heterologous CII in comparison with mouse CII is the previously defined CII 256-270 epitope. An expression level dependent T-cell tolerance was seen in this epitope as well as to the entire CII. However, all human transgenic mouse lines could still mount significant autoreactive T- and B-cell responses. Approximately 10% of the transgenic mice developed arthritis after immunization with human CII. These findings show, therefore, that cartilage-located human CII induce tolerance but can nevertheless be a target for development of arthritis.

SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse.

Two lines of evidence suggest that the Sry-related gene Sox9 is important for chondrogenesis in mammalian embryos. Sox9 mRNA is expressed in chondrogenic condensations in mice, and mutations in human SOX9 are known to cause skeletal dysplasia. We show here that mouse SOX9 protein is able to bind to a SOX/SRY consensus motif in DNA and contains a modular transcriptional activation domain, consistent with a role for SOX9 as a transcription factor acting on genes involved in cartilage development. One such gene is Col2a1, which encodes type II collagen, the major structural component of cartilage. We have compared, in detail, the expression of Sox9 and Col2a1 during mouse development. In chondrogenic tissues the expression profiles of the two genes were remarkably similar. Coexpression was detected in some nonchondrogenic tissues such as the notochord, otic vesicle, and neural tube, but others such as heart and lung differed in their expression of the two genes. Immunohistochemistry using an antibody specific for SOX9 revealed that expression of SOX9 protein mirrored the distribution of Sox9 mRNA. Our results suggest that SOX9 protein is involved in the regulation of Col2a1 during chondrogenesis, but that this regulation is likely to depend on additional cofactors.

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.

Extensive alternative splicing within the amino-propeptide coding domain of alpha2(XI) procollagen mRNAs. Expression of transcripts encoding truncated pro-alpha chains.

Heterogeneity in type XI procollagen structure is extensive because all three alpha(XI) collagen genes undergo complex alternative splicing within the amino-propeptide coding domain. Exon 7 of the human and exons 6-8 of the mouse alpha2(XI) collagen genes, encoding part of the amino-propeptide variable region, have recently been shown to be alternatively spliced. We show that exon 6-containing mRNAs for human alpha2(XI) procollagen are expressed at 28 weeks in fetal tendon and cartilage but not at 38-44 days or 11 weeks. In the mouse, exon 6 is expressed in chondrocytes from 13.5 days onward. We recently identified conserved sequences within intron 6 of the human and mouse alpha2(XI) collagen genes, containing additional consensus splice acceptor and donor sites that potentially increase the size of exon 7, dividing it into three parts, designated 7A, 7B, and 7C. We show by reverse transcription polymerase chain reaction and in situ hybridization that these potential splice sites are used to yield additional alpha2(XI) procollagen mRNA splice variants that are expressed in fetal tissues. In human, expression of exon 7B-containing transcripts may be developmental stage-specific. Interestingly, inclusion of exon 7A or exon 7B in human and mouse alpha2(XI) procollagen mRNAs, respectively, would result in the insertion of an in-frame termination codon, suggesting that some of the additional splice variants encode a truncated pro-alpha2(XI) chain.