COMP Expression Research Articles

Vitamin D and growth hormone regulate growth hormone/insulin-like growth factor (GH–IGF) axis gene expression in human fetal epiphyseal chondrocytes

Objective Cell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH–IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D 3 (VitD) and growth hormone (GH). Design Chondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10 −11 to 10 −6 M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed 3H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR. Results VitD dose-dependently and significantly inhibited 3H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1 ± 19.8 at VitD 10 −6 M), IGFBP-3 (2.6 ± 0.9), GHR (3.8 ± 2.8) and COMP (1.5 ± 0.6) expression whereas it inhibited SOX9 (0.7 ± 0.2), COL2A1 (0.6 ± 0.3) and aggrecan (0.6 ± 0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5 ± 7.4). Conclusions Our results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.

Cell therapy for tendinitis, experimental and clinical report

To compare cultured bone marrow mesenchymal cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo to repair collagenase-induced tissue damage in an equine model of experimental tendonitis, 6 Standardbred horses with no signs of previous SDF tendon injury have been recruited. Three weeks after collagenase treatment an average of either 5.5 x 10(6) cBMSCs or 122.3 x 10(6) BMMNCs, saline solution (placebo) or fibrin glue were injected intralesionally in random order. Horses were stall rested for 21 weeks, and tendon ultrasound scans performed before and during this period. Horses were euthanized and tendons harvested for histology and immunohistochemistry. Data observed in this study showed effectiveness of cBMSC and BMMNC in regenerating tendon tissue after collagenase -induced tendonitis. Both cBMSC and BMMNC transplantation resulted in qualitatively similar regeneration of tendon extracellular matrix in terms of type I/III collagen ratio, fiber orientation, and COMP expression. After this favourable results, 20 horses were recruited referred for spontaneous lesions of the flexor tendons or the suspensory ligament. Horses were treated with autologous graft of BMMNCs.After treatment the. the exercise program allowed was 8 weeks stall rest, 4 weeks hand walking, 4 weeks trotting, 4 weeks of gradually raising of exercise level then horses were gone back to race. US characteristics of lesions started to improve at T3. CSA-l, FPS and TLS were better in all patients, with an appreciable filling of lesions indicated by a decreasing of CSA-l and increasing of TLS. When horses started the exercise program T8 tendon architecture improved, demonstrated by their longitudinal alignment and length. At T6, and persistently in later follow-up, no lameness was evident by clinical examination. At time of writing 12 patients (60%) were go back to races, while other 8 (40%) are under controlled exercise program. Re-injury rate was assessed at 25%. All the owners judged good to excellent the outcome in term of athletic success.

Gene expression profile of rabbit cartilage by expressed sequence tag analysis

Although the rabbit is commonly used as an animal model for the in vivo study of cartilage formation or regeneration, genetic approaches to the rabbit cartilage are rare. We constructed an expressed sequence tag (EST) library from rabbit cartilage tissue for the first time to establish the foundations for genetic study on rabbit cartilage. From our results, we identified 2387 unique genes among 4885 clones, corresponding to 1839 matched to characterized genes including 1618 genes with known function and 548 uncharacterized and novel genes. Gene expression profiles based on EST frequency show that type II collagen (COL2A1) and type X collagen (COL10A1) among collagen clones, proteoglycan 4 (PRG4) and decorin (DCN) among proteoglycan clones, and cartilage oligomeric matrix protein (COMP) and matrix Gla protein (MGP) among other extracellular matrix clones, are highly expressed in rabbit cartilage. In addition, gene expression analysis based on real-time PCR of these major extracellular matrix constituents showed that expression of col2a1 and col10a1 remains constant whereas the expression of prg4, dcn, and comp reveals substantial change with rabbit age. This EST library will provide a valuable resource with which to identify genes involved in the biochemical and physiological functions of rabbit cartilage, and will contribute to establishing the rabbit as an animal model for cartilage research.

A30 HYPOXIC EXPANSION OF ARTICULAR CHONDROCYTES PROMOTES THE FORMATION OF A HYALINE CARTILAGE-LIKEMATRIX IN MICROMASS CULTURES

molecular events that drive this modified expression of COMP in OA cells. Methods: Human cartilage specimens were derived from the articular surface of patients undergoing total knee arthroplasty. Sampling was performed in different sites per each patient. Half of each sample was examined and graded histologically. Normal (N) or osteoarthritic (OA) cells were released from the remaining half by repeated enzymatic digestions. Cells were cultured in 10% serum-containing medium untill they reached 5 duplications. Cultures were included in the study when both N and OA cells were derived from the same subject. Four double cultures (N and OA) were obtained from four different donors. mRNA extraction, qualitative and Real Time RT-PCR were performed. A chromatin immunoprecipitation assay (ChIP) was used to detect the presence, on the COMP promoter, of the transcription factor Sox 9, a key regulator of chondrogenic differentiation. Maintenance of the chondrogenic potential was tested in vitro by means of high-cell density micromass culture; resulting pellets were processed for cytochemistry. Results: A marked reduction (48%) of the COMP mRNA level was evidenced in OA samples, (panels A and B). ChIP analysis, on the same cells, revealed that the transcription factor Sox 9 was binding the COMP promoter only in the extracts (E) derived from the N chondrocytes, whereas it was absent in those derived from the OA cells. On the contrary, the DNA inputs of N and OA chondrocytes, before immunoprecipitation, provided the proper amplified PCR products (T+) when used as control templates (panel C). Contamporarily, a Sox 9 transcript was PCR-amplified in N and OA cells, indicating the factor’s presence in both cell

Molecular characterization of spontaneous and growth-factor-augmented chondrogenesis in periosteum–bone tissue transferred into a joint

Multilineage potential of progenitor cells from periosteum is well established, but conditions for differentiation within their native niche are unclear. We evaluated at cellular and molecular levels whether chondrogenesis of periosteal progenitor cells is promoted spontaneously or by growth-factor mixture (GFM) application when transferring periosteum-bone cylinders into cartilage defects. Osteochondral defects in the patellar groove of minipigs were filled with periosteum-bone cylinders and randomly supplemented with GFM. Neochondrogenesis was characterized by histology, immunohistology, and quantitative gene expression analysis. According to morphology and glycosaminoglycan accumulation, spontaneous neocartilage formation occurred in the cambium layer already at 6 weeks, increased after 12 weeks, but declined until 52 weeks, independent of GFM. Multiple cartilage differentiation markers were induced after transfer. Expression of aggrecan, COMP, decorin, and Col10a1 increased significantly within 52 weeks. Sox 9 and Col2a1 mRNA levels were elevated at 6 versus 52 weeks in the GFM group and resulted in higher collagen type II protein accumulation. Neochondrogenesis was promoted in lower periosteum layers by transfer of periosteum-bone plugs into a joint, and collagen type II protein deposition was enhanced by GFM. The final tissue subsumed typical features of periosteum and fibrocartilage but lacked an intact tide mark and features of hyaline cartilage desired for cartilage repair.

An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype

Rat chondrosarcoma (RCS) cells are unusual in that they display a stable chondrocyte phenotype in monolayer culture. This phenotype is reflected by a rounded cellular morphology with few actin-containing stress fibers and production of an extracellular matrix rich in sulfated proteoglycans, with high-level expression of aggrecan, COMP, Sox9, and collagens type II, IX, and XI. Additionally, these cells do not express collagen type I. Here it is shown that in the absence of any mechanical stimulation, treatment of RCS cells with gadolinium chloride (Gd 3+), a stretch-activated cation channel blocker, caused the cells to undergo de-differentiation, adopting a flattened fibroblast phenotype with the marked appearance of actin stress fibers and vinculin-containing focal contacts. This change was accompanied by a dramatic reduction in the expression of aggrecan, Sox9, collagen types II, IX, and XI, with a corresponding increase in the expression of collagen type I and fibronectin. These effects were found to be reversible by simple removal of Gd 3+ from the medium. Gd 3+ also had a similar effect on expression of chondrocyte marker genes in freshly isolated human chondrocytes. These data suggest that mechanoreceptor signaling plays a key role in maintenance of the chondrocyte phenotype, even in the absence of mechanical stimulation. Further, treatment of RCS cells with Gd 3+ provides a tractable system for assessing the molecular events underlying the reversible differentiation of chondrocytes.

Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression.

The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA-binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.

Molecular cloning and expression patterns of mouse cartilage oligomeric matrix protein gene

Objective To develop transgenic mice harboring mutations in the COMP gene as animal models for pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), autosomal dominant disorders characterized by early onset osteoarthritis and epiphyseal abnormalities. As a first step in generating a mouse model for COMP mutations, we have cloned the cDNA of mouse COMP and examined its tissue expression pattern.Design Total mRNA was isolated from the skeletal tissues of newborn C57BL/6j mice and used as a template for oligo(dT) first-strand cDNA synthesis. The cDNA was used for PCR amplification of COMP using three oligonucleotide primer pairs designed from the published rat COMP cDNA sequence. Nested PCR was used to complete the sequence between the amplified fragments. The entire cDNA was sequenced and the expression pattern of the corresponding transcripts examined by Northern hybridizations.ResultsA full-length COMP cDNA was isolated. Analysis showed that the entire translated region of the mouse COMP gene is 2268bp and the derived amino acid sequence shows 90% homology to human COMP. Of eight adult mouse non-cartilage tissues tested, COMP expression was detected only in testis.

Tissue expression and calcium binding studies of human COMP

Tissue expression and calcium binding studies of human COMP

Effects of Seed Rate and Weed Control Methods on Yield Components of Rice Varieties

The effects of seed rates viz, 40, 60 and 80 kg/ha and chemical weed control with Stam-F.34 on the yield components of four rice varieties (Kanchi, Kannaki, Karuna and Cauveri) were studied. The panicle weight, 1000-grain weight and number of grains per panicle decreased with increasing seed rate while the number of empty spikelets increased with increasing seed rate. Chemical weed control in combination with lower seed rate resulted in better expression of yield comp:nents, The optimum seed rate was 40 kg/ha under chemical weed control and 60 kg under manual weeding.