Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during in vitro chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.     

Role of anchorin CII, a 31,000-mol-wt membrane protein, in the interaction of chondrocytes with type II collagen

We have previously reported the isolation of a hydrophobic, type-II collagen-binding glycoprotein of molecular weight 31,000 (31,000-mol-wt protein) from chick chondrocyte membranes (Mollenhauer, J., and K. von der Mark, EMBO Eur. Mol. Biol. Organ. J., 2:45-50). The function of this protein in anchoring pericellular type II collagen to the chondrocyte surface was inferred from its ability to bind native type-II collagen either when detergent solubilized or when inserted into liposomes. In the present study we have used specific antibodies to localize this protein, which we now call anchorin CII, to the surface of chondrocytes in both cartilage sections, and in cell culture. In immunofluorescence studies of isolated chondrocytes we observed a dense, punctate distribution of anchorin CII on the cell surface when chondrocytes were enclosed by a pericellular type II collagen matrix. Removal of the pericellular matrix with trypsin also removed anchorin CII. The membrane protein character of anchorin CII was indicated by the demonstration of antibody-induced patching and capping on the chondrocyte surface at 22 degrees C and 37 degrees C, respectively. In monolayer culture, the amount of anchorin CII appeared reduced on flattened chondrocytes lacking a pericellular type II collagen matrix but was prominent upon intercellular cell processes. Fab' fragments prepared from either anchorin CII antiserum or an antiserum directed against the entire chondrocyte membrane inhibited the attachment of chondrocytes to a type II collagen substrate. In each case, the inhibition of attachment was neutralized by preincubation of Fab' fragments with purified anchorin CII.     

Expression of anchorin CII, a collagen-binding protein of the annexin family, in the developing chick embryo.

Expression of anchorin CII, a collagen-binding protein of the annexin family, was followed in the developing chick embryo using Northern and in situ hybridization and Western blotting. During chick somite development, anchorin CII mRNA was detected by Northern blotting as early as stage 11. At stage 24, anchorin mRNA accumulated in the anterior part of the somite sclerotome near the resegmentation line, as shown by in situ hybridization. The presence of anchorin CII protein during stages 11 to 20 was confirmed by Western blotting. In situ hybridization identified anchorin CII also in the otic vesicle adjacent to the site of contact with the statoacoustic ganglion and in the mandibular mesenchyme. The level of anchorin CII mRNA in differentiated hyaline cartilage, exemplified by sternal cartilage, was lower than that in differentiating somites or cultured chondrocytes. These findings are consistent with our notion that anchorin CII may be involved in cell-matrix interactions preceding chondrogenic differentiation events in the chick embryo. A significant level of anchorin CII mRNA and protein synthesis was also found in cultured myoblasts, but less than that in chondroblasts. This distribution pattern is different from that reported for a related protein, p34, or calpactin, the major protein substrate for tyrosine kinase phosphorylation in chick chondrocytes and fibroblasts. The results confirm suggestions from previous sequencing studies that anchorin CII and p34 are different proteins of the annexin/calpactin family.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Biosynthesis, secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family.

The amino acid sequence of anchorin CII, a collagen-binding protein isolated originally from chondrocyte membranes, was previously determined by sequencing of cDNA and proteolytic fragments of the protein. Computer analysis of the protein sequence revealed four internal repeats of approximately 70-80 residues, each containing a highly conserved consensus sequence of 17 residues. These repeats show considerable homology with sequences in human and bovine calpactin, lipocortin, endonexin and protein II, which are members of a family of Ca2+- and phospholipid-binding proteins, as well as major substrates of tyrosine kinases. While these proteins have been located at the inner side of the plasma membrane of fibroblasts and epithelial cells, here we present experimental evidence that anchorin CII is at least partially released from cells and binds to the outer cell surface. Biosynthesis studies in cell-free systems and in cell culture indicate that anchorin CII is not processed, which is consistent with the absence of signal sequences from the protein. Yet, pulse-chase experiments show that anchorin is released into the culture medium of fibroblasts after 30 min, and in chondrocyte cultures after 20 h. Anchorin CII was located to the outer cell surface of chondrocytes by lactoperoxidase-catalyzed cell surface iodination as well as by antibody labeling both at light- and electron-microscopical level. The pericellular localization of anchorin CII is consistent with the notion that this protein is involved in the interaction of chondrocytes and fibroblasts with extracellular collagen.     

Two lipocortin-like proteins, endonexin II and anchorin CII, may be alternate splices of the same gene

The annexins are a family of phospholipid- and Ca2+-binding proteins that are structurally related. Two members of this family, human endonexin II and chicken anchorin CII, may arise from the same gene by alternative splicing of two structurally unrelated segments.     

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009. © 2009 Wiley‐Liss, Inc.     

The production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism .     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.     

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ${}^{14}\text{C}{}^3\text{H}$ ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ${}^{14}\text{C}{}^3\text{H}$ incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

UDP-glucuronate carboxy-lyase in cultured chondrocytes.

UDP-glucuronate carboxy-lyase has been demonstrated in chick chondrocytes in tissue culture. It occurs in the particulate fraction, and its activity is stimulated by exogenous NAD. The enzyme is allosterically activated by UDP-glucuronate and inhibited by UDP-xylose, n Values of 2.8 indicate positive cooperativity of at least three interacting sites on the enzyme. These data suggest that UDP-xylose concentration in chondrocytes is regulated by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. Activity levels of the enzyme during growth of the cells peak towards mid-log phase and decline thereafter, closely paralleling levels of chondroitin sulfate glycosyltransferases determined previously (Schwartz, N. B. (1976) J. Biol. Chem. 251, 3346-3351). Thus, it appears that during chondrocyte development a common mechanism governs induction of glycosyltransferases and of UDP-glucuronate carboxy-lyase.     

Apoptosis staining in cultured pseudoachondroplasia chondrocytes

Pseudoachondroplasia (PSACH) is a skeletal dysplasia caused by a mutation in cartilage oligomeric matrix protein (COMP), a glycoprotein of normal cartilage matrix. PSACH chondrocytes have a distinctive phenotype with enlarged rER cisternae containing COMP, aggrecan, type IX collagen, and chaperone proteins. Ultrastructural studies suggested that this accumulation compromises cell function, hastening cell death, and consequently reducing the number of cells in the growth plate contributing to linear bone growth. Using the alginate bead system, we cultured control and PSACH chondrocytes for twenty weeks and one year to determine the effect of the mutation on size and number of cartilage nodules; and the presence of apoptotic cell death (TUNEL assay). At 20 weeks, beads containing PSACH or control chondrocytes did not differ in size and number of cartilage nodules or number of TUNEL-positive cells. After one year, nodule number, size and percent cartilage per bead were significantly less in PSACH nodules, and the number of cells staining positive for apoptosis was significantly greater than in controls (71.8% vs. 44.6%). The increase in apoptosis in PSACH nodules correlates with a decrease in growth of cartilage, supporting our hypothesis that death of damaged cells contributes to the growth plate defects in PSACH.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.     

Levels of collagen mRNA in dedifferentiating chondrocytes

Chondrocytes liberated from chick embryo sterna were maintained in monolayer cultures and allowed to dedifferentiate. mRNA was prepared from these cultures at several intervals over a total period of 6 weeks. Levels of α1(I) and α2(I) procollagen mRNA were assayed by cell-free translation and by Northern blots using cDNA clones specific for the respective procollagen chains. Dedifferentiating chondrocytes first take up synthesis of α1(I) mRNA which is followed after several days by synthesis of α2(I) mRNA. This two-step mechanism for the onset of procollagen type I mRNA synthesis is accompanied by a proceeding loss of α1(II) procollagen gene expression.     

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.     

Expression of the mRNA (N6-adenosine)-methyltransferase S-adenosyl-L-methionine binding subunit mRNA in cultured cells

Ribonuclease protection assays (RPA) were used to detect and quantitate the amount of messenger RNA (mRNA) coding for the S-adenosyl-L-methionine binding subunit (MT-A70) of the mRNA (N6-adenosine)-methyltransferase from different types of cultured cells. HeLa cells cultured in suspension were analyzed at regular intervals along a normal growth curve. It was discovered that MT-A70 mRNA was transcribed constitutively across the time-course, irrespective of the rate of cellular proliferation. Further, 11 different cell lines representing non-tumorigenic, tumorigenic, and virally-transformed tumorigenic types from Homo sapiens, Mus musculus, and Rattus norvegicus were examined for MT-A70 mRNA expression. It was found that all the cell lines expressed a long and short splice-variant form of the gene. In general, the cell lines expressed a similar total amount of the MT-A70 mRNA while statistically significant differences existed between the quantity of the long and short forms among cell types. Tumorigenic cell lines synthesized as much as a 9-fold greater amount of long form versus short form MT-A70 mRNA. Comparatively, non-tumorigenic cell lines generally expressed only a 1.5-fold greater amount of long form versus short form MT-A70 mRNA.     

Studies of copper transport in cultured bovine chondrocytes

Copper serves as the cofactor for a number of important enzymes in cartillage, as well as in other tissues, including lysyl oxidase, superoxide dismutase, and cytochrome oxidase. Ceruloplasmin is resposible for the transport of approx. 95% of the copper in serum, but the mechanisms for intracellular copper transport are unknown. We have demonstrated recently that a high-molecular-weight cartilage glycoprotein, referred to as CMGP, has regions of sequence homology with ceruloplasmin. CMGP also binds copper and has at least some oxidase activity similar to that of ceruloplasmin. Other tissues synthesize intracellular ceruloplasmin-like proteins. The present report represents part of an effort examine the hypothesis the CMGP is a copper transport protein in chondrocytes and to characterize the enzymatic activities of CMGP. These studies demonstrate that CMGP is the principal chondrocyte protein labeled by ⁶⁷Cu in vitro and that the label is localized to the mitochondria, cytosol, and membrane fractions of sucrose gradients, suggesting copper transport through the cell. In parallel experiments, [³H]leucine was incorporated into proteins corresponding to the subunits and fragments of CMGP, as described previously, and in a similar distribution among the subcellular fractions as labeled copper. Additionally, CMGP has oxidase and ferroxidase activities similar to those of ceruloplasmin.