Activity of temozolomide in patients with advanced chemorefractory colorectal cancer and MGMT promoter methylation.

BACKGROUND: No evidence-based treatment options are available for patients with advanced colorectal cancer (CRC) progressing after standard therapies. MGMT is involved in repair of DNA damage and MGMT promoter methylation may predict benefit from alkylating agents such as temozolomide. The aim of our study was to evaluate the activity of temozolomide in terms of response rate in patients with metastatic CRC and MGMT methylation, after failure of approved treatments. PATIENTS AND METHODS: Patients were enrolled in a monocentre, open-label, phase II study and treated with temozolomide at a dose of 150 mg/m2/day for 5 consecutive days in 4-weekly cycles. The treatment was continued for at least six cycles or until progressive disease. RESULTS: Thirty-two patients were enrolled from August 2012 to July 2013. Treatment was well tolerated with one grade 4 thrombocytopenia and no other grade≥3 toxicities. No complete response occurred. The objective response rate was 12%, reaching the pre-specified level for promising activity. Median progression-free survival and overall survival were 1.8 and 8.4 months, respectively. Patients with KRAS, BRAF and NRAS wild-type CRC showed significantly higher response when compared with those with any RAS or BRAF mutation (44% versus 0%; P=0.004). TP53 status had no influence on the primary end point. CONCLUSIONS: Temozolomide is tolerable and active in heavily pre-treated patients with advanced CRC and MGMT promoter methylation. Further studies in biomolecularly enriched populations or in a randomized setting are necessary to demonstrate the efficacy of temozolomide after failure of standard treatments.

Distinct spatiotemporal roles of hedgehog signalling during chick and mouse cranial base and axial skeleton development.

The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches.

Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody.

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells--those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle-specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes.

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.

Modulation of fibronectin gene expression in chondrocytes by viral transformation and substrate attachment.

Chicken vertebral chondrocytes, which normally grow in suspension, synthesize large amounts of cartilage extracellular matrix proteins, but little fibronectin. We have analyzed the effects of both substrate attachment and transformation with a temperature-sensitive mutant of Rous sarcoma virus on fibronectin gene expression in these cells. Our experiments show that viral transformation increases fibronectin synthesis to a greater extent than substrate attachment. Furthermore, transformed chondrocytes have lost the ability to decrease fibronectin synthesis in response to suspension culture, suggesting that transformation alters the normal attachment-responsive control of fibronectin gene expression. Finally, infected substrate-attached chondrocytes shifted to the nonpermissive temperature for transformation use fibronectin RNA more efficiently in protein synthesis than cells grown under the other conditions, suggesting for the first time a role for translational control of fibronectin gene expression.

Multiple negative elements in a gene that codes for an extracellular matrix protein, collagen X, restrict expression to hypertrophic chondrocytes.

During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in areas of endochondral ossification. Mature hypertrophic chondrocytes differ from immature chondrocytes in that they become postmitotic, increase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein, collagen X. Previous studies have shown that collagen X is not expressed by other cell types and that its specific expression in hypertrophic chondrocytes is controlled by transcriptional mechanisms. To define these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chloramphenicol acetyl transferase, CAT) were transfected into primary cultures of collagen X-expressing and nonexpressing cells. A construct containing a short (558 bp) promoter exhibited high levels of CAT activity in all cell types (fibroblasts, immature, and hypertrophic chondrocytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and immature chondrocytes, but had no effect in hypertrophic chondrocytes. Addition of three subfragments of the 4.2-kb fragment to the initial construct, either individually or in various combinations, showed that all subfragments reduced CAT activity somewhat in non-collagen X-expressing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse upstream negative regulatory elements act in an additive manner to restrict transcription of the collagen X gene to hypertrophic chondrocytes.

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen.

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast-specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole-like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development.

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.

Wnt/beta-catenin signaling regulates cranial base development and growth.

Wnt proteins and beta-catenin signaling regulate major processes during embryonic development, and we hypothesized that they regulate cranial base synchondrosis development and growth. To address this issue, we analyzed cartilage-specific beta-catenin-deficient mice. Mutant synchondroses lacked typical growth plate zones, and endochondral ossification was delayed. In reciprocal transgenic experiments, cartilage overexpression of a constitutive active Lef1, a transcriptional mediator of Wnt/beta-catenin signaling, caused precocious chondrocyte hypertrophy and intermingling of immature and mature chondrocytes. The developmental changes seen in beta-catenin-deficient synchondroses were accompanied by marked reductions in Ihh and PTHrP as well as sFRP-1, an endogenous Wnt signaling antagonist and a potential Ihh signaling target. Thus, Wnt/beta-catenin signaling is essential for cranial base development and synchondrosis growth plate function. This pathway promotes chondrocyte maturation and ossification events, and may exert this important role by dampening the effects of Ihh-PTHrP together with sFRP-1.

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

Control of types I and II collagen and fibronectin gene expression in chondrocytes delineated by viral transformation.

We have analyzed the effects of transformation by Rous sarcoma virus on expression of types I and II collagen and fibronectin genes in vertebral chondrocytes and compared them with expression of these genes in skin fibroblasts. Transformed chondrocytes display a dramatically decreased amount of type II collagen RNA, which can account fully for the decreased synthetic rate of this protein. Paradoxically, these cells also display greatly increased amounts of type I collagen RNAs, which are translated efficiently in vitro, but not in the intact cells. We show here that the type I collagen RNAs in transformed chondrocytes are nearly indistinguishable from those found in skin fibroblasts, and that they clearly differ from the type I collagen RNAs found in normal chondrocytes. Transformed chondrocytes also display an increased amount of fibronectin RNAs, which can account fully for the increased synthetic rate of this protein. Thus, the effects of transformation by Rous sarcoma virus on type I collagen and fibronectin RNAs in chondrocytes are the opposite of those observed in fibroblasts, which display decreased amounts of these three RNAs. These data indicate that the effects of transformation on the genes encoding type I collagen and fibronectin must be modulated by host cell-specific factors. They also imply that the types I and II collagen genes may be regulated by different mechanisms, the type I genes being controlled at both transcriptional and posttranscriptional levels, and the type II gene being controlled primarily at the transcriptional level.

12-O-tetradecanoylphorbol-13-acetate-induced changes in sulfated proteoglycan synthesis in cultured chondroblasts.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly blocks the terminal differentiation of normal cultured chondroblasts. In TPA, the chondroblasts lose their characteristic polygonal morphology and initiate the synthesis of an atypical type IV sulfated proteoglycan. This atypical proteoglycan has lower molecular size, shorter polysaccharide chains, and different 4S-disaccharide:6S-disaccharide ratio in comparison with type IV sulfated proteoglycan from untreated chondroblasts. When removed from TPA, the cells reacquire their characteristic polygonal morphology and reinitiate the synthesis of their typical cartilage-characteristic type IV sulfated proteoglycan. TPA has no readily detectable effect on the type III sulfated proteoglycan synthesized by fibroblasts.

Effects of phorbol-12-myristate-13-acetate on the phenotypic program of cultured chondroblasts and fibroblasts.

Phorbol-12-myristate-13-acetate (PMA) has a prompt, differential, and partially reversible effect on cultured chick chondroblasts. Within 36 hr PMA transforms sessile, polygonal, epithelioid chondroblasts into motile, multilayered, fibroblastic cells. In PMA chick chondroblasts rapidly cease to synthesize two of their terminal luxury molecules, the type IV sulfated proteoglycan that characterizes the extracell matrix and a glycosylated protein with an apparent molecular weight of 180,000. This glycosylated protein constitutes approximately 5% of the total protein in normal chondroblasts. If returned to normal medium after 4 days in PMA, virtually 100% of the cells reinitiate the synthesis of their type IV sulfated proteoglycan, of the 180,000-dalton protein, and reacquire their polygonal, epithelioid morphology. If returned to normal medium after 12 days in PMA, the cells fail to synthesize their two characteristic luxury molecules, and 100% of the cells remain fibroblastic. PMA alters the morphology of chick fibroblasts but does not block synthesis of their characteristic type III sulfated proteoglycan. PMA proves to be a mitogen for chondroblasts but not for fibroblasts, in spite of the phenotypic similarities of these two cell types.

Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Articular chondrocytes produce factors that inhibit maturation of sternal chondrocytes in serum-free agarose cultures: a TGF-beta independent process.

Under normal conditions, articular chondrocytes persist throughout postnatal life, whereas "transient" chondrocytes, which constitute the bulk of prenatal and early postnatal cartilaginous skeleton, undergo maturation, hypertrophy, and replacement by bone cells. The mechanisms regulating the markedly different behavior and fate of articular and transient chondrocytes are largely unclear. In the present study, we asked whether articular chondrocytes possess dominant antimaturation properties which may subtend their ability to persist throughout life. Adult chicken articular chondrocytes and transient maturing chondrocytes from the core region of day 17, chick embryo cephalic sternum were cultured or cocultured in serum-free agarose conditions. When the sternal cells were grown by themselves, they quickly developed into hypertrophic type X collagen-synthesizing cells; however, when they were cocultured with as few as 10% articular chondrocytes or fed with articular chondrocyte-conditioned medium, their maturation was markedly impaired, as revealed by a sharp drop in type X collagen synthesis. A similar, albeit less potent, antimaturation activity characterized resting and proliferating immature chondrocytes isolated from other regions of embryonic sternum. Transforming growth factor-beta 2 (TGF-beta 2) was previously suggested to be an inhibitor of chondrocyte maturation. We found, however, that treatment with a neutralizing antiserum to TGF-beta did not counteract the inhibition of maturation in cocultures of articular and maturing core sternal chondrocytes. Indeed, articular chondrocytes produced and accumulated relatively low levels of TGF-beta in their culture medium, about 15 ng/ml/48 h, of which over 90% was latent; surprisingly, maturing sternal core chondrocytes accumulated over 10-fold more TGF-beta in the medium, about 150 ng/ml/48 h, of which over 20% was endogenously active. These results indicate that articular chondrocytes do possess dominant antimaturation properties which appear to be TGF-beta independent. The TGF-beta s may thus have a more prominent role in the terminal phases of chondrocyte maturation, as indicated by their abundance and greater activity in hypertrophic chondrocytes.

Retinoic acid induces a shift in the energetic state of hypertrophic chondrocytes.

In the epiphyseal growth plate, chondrocyte maturation is accompanied by dramatic alterations in energy metabolism. To explore the relationship between these two events, we used retinoic acid (RA) to promote chondrocyte maturation in culture. The specific question that was addressed was, does RA treatment of cultured chondrocytes in vitro induce a change in energy status similar to that seen in hypertrophic chondrocytes in vivo. Maturing chondrocytes isolated from the cephalic region of day 18 chick embryo sterna were allowed to grow for 7-14 days in monolayer until confluent and then treated with 10-300 nM RA. Immature chondrocytes from the caudal region of sternum were grown in parallel and served as control cells for the study. We found that in maturing cephalic cell cultures, RA had a rapid and profound effect on oxidative metabolism. The retinoid caused a reduction in the energy charge ratio (ECR) and the ATP/ADP ratio and a sharp decrease in cell ATP levels. Maximum inhibition was observed when the RA concentration was 10-35 nM. Compared with the adenine nucleotides, creatine phosphate levels were decreased to a lesser extent by RA, although there was substantial inhibition of creatine kinase activity. We expected to find a compensatory elevation in glycolytic activities; however, the lactate levels in the medium of the treated cells indicated that anaerobic glycolysis was depressed. In contrast to the cephalic chondrocytes, when caudal cell cultures were treated with RA, lactate formation was stimulated and there were minimal effects on oxidative metabolism. To determine the mechanism of inhibition of glycolysis, we measured the activity of pyruvate kinase in RA-treated cephalic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of transforming growth factor beta in chondrocytes undergoing endochondral ossification.

Transforming growth factor beta (TGF-beta) has well-documented roles in chondrocyte maturation and endochondral ossification, but the mechanisms of TGF-beta activation during these processes remain unclear. In this study, we analyzed TGF-beta activation in chick embryo resting, proliferating, and hypertrophic chondrocytes in culture. We found that both levels and activation of TGF-beta increased substantially with maturation. The majority of TGF-beta produced by resting cells over culture time remained latent, but a larger portion produced by proliferating and hypertrophic cells was activated with increasing maturation. Zymography of gelatin gels revealed that matrix metalloprotease 2 (MMP-2) and MMP-9 were expressed by each population and that MMP-13 characterized hypertrophic chondrocytes and to a lesser extent proliferating chondrocytes in late cultures. Treatment with pharmacologic agents revealed that both MMPs and serine proteases are involved in activation. However, because inhibition of MMPs almost completely prevented TGF-beta activation, MMPs appear crucial for activation. During culture, inclusion of the tetracycline-derived, collagenase/gelatinase inhibitor chemically modified nonantimicrobial tetracycline (CMT-8) at concentrations specific for MMP-13 inhibition resulted in complete inhibition of TGF-beta activation by proliferating and hypertrophic chondrocytes. These results show that TGF-beta production, release, and activation are regulated developmentally in chondrocytes. Our findings point to a strict mode of regulation of this potent factor to elicit diverse and highly specific effects during chondrocyte maturation and ossification.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes.

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of alpha 5 beta 1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on alpha 5 beta 1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against alpha 5 beta 1 or fibronectin, indicating that cell spreading involved alpha 5 beta 1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with alpha 5 beta 1 integrin-ligand interactions. Furthermore, DNA content was decreased by treatment with anti-fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the alpha 5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that alpha 5 beta 1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte-matrix interactions and proliferation.