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.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ.

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4(-/-) mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K-expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life.

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.

TMJ degeneration in SAMP8 mice is accompanied by deranged Ihh signaling.

The temporomandibular joint (TMJ) functions as a load-bearing diarthrodial joint during mastication, and its continuous use and stress can lead to degeneration over age. Using senescence-accelerated (SAMP8) mice that develop early osteoarthritis-like changes in synovial joints at high frequency, we analyzed possible molecular mechanisms of TMJ degeneration and tested whether and how malocclusion may accelerate it. Condylar articular cartilage in young SAMP8 mice displayed early-onset osteoarthritic changes that included reductions in superficial/chondroprogenitor cell number, proteoglycan/collagen content, and Indian hedgehog (Ihh)-expressing chondrocytes. Following malocclusion induced by tooth milling, the SAMP8 condyles became morphologically defective, displayed even lower proteoglycan levels, and underwent abnormal chondrocyte maturation compared with malocclusion-treated condyles in wild-type mice. Malocclusion also induced faster progression of pathologic changes with increasing age in SAMP8 condyles as indicated by decreased PCNA-positive proliferating chondroprogenitors and increased TUNEL-positive apoptotic cells. These changes were accompanied by steeper reductions in Ihh signaling and by expression of matrix metalloproteinase 13 at the chondro-osseous junction in SAMP8 articular cartilage. In sum, we show for the first time that precocious TMJ degeneration in SAMP8 mice is accompanied by--and possibly attributable to--altered Ihh signaling and that occlusal dysfunction accelerates progression toward degenerative TMJ disease in this model.

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.

Muenke syndrome mutation, FgfR3P²44R, causes TMJ defects.

Muenke syndrome is characterized by various craniofacial deformities and is caused by an autosomal-dominant activating mutation in fibroblast growth factor receptor 3 (FGFR3(P250R) ). Here, using mice carrying a corresponding mutation (FgfR3(P244R) ), we determined whether the mutation affects temporomandibular joint (TMJ) development and growth. In situ hybridization showed that FgfR3 was expressed in condylar chondroprogenitors and maturing chondrocytes that also expressed the Indian hedgehog (Ihh) receptor and transcriptional target Patched 1(Ptch1). In FgfR3(P244R) mutants, the condyles displayed reduced levels of Ihh expression, H4C-positive proliferating chondroprogenitors, and collagen type II- and type X-expressing chondrocytes. Primary bone spongiosa formation was also disturbed and was accompanied by increased osteoclastic activity and reduced trabecular bone formation. Treatment of wild-type condylar explants with recombinant FGF2/FGF9 decreased Ptch1 and PTHrP expression in superficial/polymorphic layers and proliferation in chondroprogenitors. We also observed early degenerative changes of condylar articular cartilage, abnormal development of the articular eminence/glenoid fossa in the TMJ, and fusion of the articular disc. Analysis of our data indicates that the activating FgfR3(P244R) mutation disturbs TMJ developmental processes, likely by reducing hedgehog signaling and endochondral ossification. We suggest that a balance between FGF and hedgehog signaling pathways is critical for the integrity of TMJ development and for the maintenance of cellular organization.

TMJ development and growth require primary cilia function.

Primary cilia regulate limb and axial skeletal formation and hedgehog signaling, but their roles in temporomandibular joint (TMJ) development are unknown. Thus, we created conditional mouse mutants deficient in ciliary transport protein Kif3a in cartilage. In post-natal wild-type mice, primary cilia were occasionally observed on the superior, inferior, or lateral side of condylar cells. Cilia were barely detectable in mutant chondrocytes but were evident in surrounding tissues, attesting to the specificity of chondrocyte Kif3a ablation. Mutant condyles from 3-month-old mice were narrow and flat along their antero-posterior and medio-lateral axes, were often fused with the articular disc, and displayed an irregular bony surface. The polymorphic layer in P15 mutants contained fewer Sox9-expressing chondroprogenitor cells because of reduced mitotic activity, and newly differentiated chondrocytes underwent precocious hypertrophic enlargement accompanied by early activation of Indian hedgehog (Ihh). Interestingly, there was excessive intramembranous ossification along the perichondrium, accompanied by local expression of the hedgehog receptor Patched-1 and up-regulation of Osterix and Collagen I. In summary, Kif3a and primary cilia are required for coordination of chondrocyte maturation, intramembranous bone formation, and chondrogenic condylar growth. Defects in these processes in Kif3a condylar cartilage are likely to reflect abnormal hedgehog signaling topography and dysfunction.

Ihh signaling regulates mandibular symphysis development and growth.

Symphyseal secondary cartilage is important for mandibular development, but the molecular mechanisms underlying its formation remain largely unknown. Here we asked whether Indian hedgehog (Ihh) regulates symphyseal cartilage development and growth. By embryonic days 16.5 to 18.5, Sox9-expressing chondrocytes formed within condensed Tgfß-1/Runx2-expressing mesenchymal cells at the prospective symphyseal joint site, and established a growth-plate-like structure with distinct Ihh, collagen X, and osteopontin expression patterns. In post-natal life, mesenchymal cells expressing the Ihh receptor Patched1 were present anterior to the Ihh-expressing secondary cartilage, proliferated, differentiated into chondrocytes, and contributed to anterior growth of alveolar bone. In Ihh-null mice, however, symphyseal development was defective, mainly because of enhanced chondrocyte maturation and reduced proliferation of chondroprogenitor cells. Proliferation was partially restored in dual Ihh;Gli3 mutants, suggesting that Gli3 is normally a negative regulator of symphyseal development. Thus, Ihh signaling is essential for symphyseal cartilage development and anterior mandibular growth.

Sulfotransferase Ndst1 is needed for mandibular and TMJ development.

Heparan sulfate proteoglycans (HS-PGs) regulate several developmental processes, but their possible roles in mandibular and TMJ formation are largely unclear. To uncover such roles, we generated mice lacking Golgi-associated N-sulfotransferase 1 (Ndst1) that catalyzes sulfation of HS-PG glycosaminoglycan chains. Ndst1-null mouse embryos exhibited different degrees of phenotypic penetrance. Severely affected mutants lacked the temporomandibular joint and condyle, but had a mandibular remnant that displayed abnormal tooth germs, substandard angiogenesis, and enhanced apoptosis. In mildly affected mutants, the condylar growth plate was dysfunctional and exhibited thicker superficial and polymorphic cell zones, a much wider distribution of Indian hedgehog signaling activity, and ectopic ossification along its lateral border. Interestingly, mildly affected mutants also exhibited facial asymmetry resembling that seen in individuals with hemifacial microsomia. Our findings indicate that Ndst1-dependent HS sulfation is critical for mandibular and TMJ development and allows HS-PGs to exert their roles via regulation of Ihh signaling topography and action.

Indian hedgehog roles in post-natal TMJ development and organization.

Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.

IART (Intra-Operative Avidination for Radionuclide Therapy) for accelerated radiotherapy in breast cancer patients. Technical aspects and preliminary results of a phase II study with 90Y-labelled biotin.

BACKGROUND: Breast conserving surgery (BCS) plus external beam radiotherapy (EBRT) is considered the standard treatment for early breast cancer. We have investigated the possibility of irradiating the residual gland, using an innovative nuclear medicine approach named IART(®) (Intra-operative Avidination for Radionuclide Therapy). AIM: The objective of this study was to determine the optimal dose of avidin with a fixed activity (3.7 GBq) of (90)Y-biotin, in order to provide a boost of 20 Gy, followed by EBRT to the whole breast (WB) at the reduced dose of 40 Gy. Local and systemic toxicity, patient's quality of life, including the cosmetic results after the combined treatment with IART(®) and EBRT, were assessed. METHODS: After tumour excision, the surgeon injected native avidin diluted in 30 ml of saline solution into and around the tumour bed (see video). Patients received one of three avidin dose levels: 50 mg (10 pts), 100 mg (15 pts) and 150 mg (10 pts). Between 12 to 24 h after surgery, 3.7 GBq (90)Y-biotin spiked with 185 MBq (111)In-biotin was administered intravenously (i.v.). Whole body scans and SPECT images were performed up to 30 h post-injection for dosimetric purposes. WB-EBRT was administered four weeks after the IART(®) boost. Local toxicity and quality of life were evaluated. RESULTS: Thirty-five patients were evaluated. No side effects were observed after avidin administration and (90)Y-biotin infusion. An avidin dose level of 100 mg resulted the most appropriate in order to deliver the required radiation dose (19.5 ± 4.0 Gy) to the surgical bed. At the end of IART(®), no local toxicity occurred and the overall cosmetic result was good. The tolerance to the reduced EBRT was also good. The highest grade of transient local toxicity was G3, which occurred in 3/32 pts following the completion of WB-EBRT. The combination of IART(®)+EBRT was well accepted by the patients, without any changes to their quality of life. CONCLUSIONS: These preliminary results support the hypothesis that IART(®) may represent a valid approach to accelerated WB irradiation after BCS. We hope that this nuclear medicine technique will contribute to a better management of breast cancer patients.

Roles of the primary cilium component Polaris in synchondrosis development.

Primary cilia regulate several developmental processes and mediate hedgehog signaling. To study their roles in cranial base development, we created conditional mouse mutants deficient in Polaris, a critical primary cilium component, in cartilage. Mutant post-natal cranial bases were deformed, and their synchondrosis growth plates were disorganized. Expression of Indian hedgehog, Patched-1, collagen X, and MMP-13 was reduced and accompanied by decreases in endochondral bone. Interestingly, there was excessive intramembranous ossification along the perichondrium, accompanied by excessive Patched-1 expression, suggesting that Ihh distribution was wider and responsible for such excessive response. Indeed, expression of heparan sulfate proteoglycans (HS-PGs), normally involved in restricting hedgehog distribution, was barely detectable in mutant synchondroses. Analyses of the data provides further evidence for the essential roles of primary cilia and hedgehog signaling in cranial base development and chondrocyte maturation, and point to a close interdependence between cilia and HS-PGs to delimit targets of hedgehog action in synchondroses.

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.

Roles of FGF-10 on the development of diathrodial limb joints.

OBJECTIVES: Members of the fibroblast growth (FGF) family of signaling proteins are known to play important roles in limb skeletal patterning and in chondrocyte proliferation and maturation. Recent work from this laboratory showed that FGF members are expressed in limb developing joints. Thus, the present project focused on what roles these proteins may have in joint development. METHODS: Heparin-coated beads precoated with recombinant FGF-10 or GDF-5 were implanted around incipient proximal and distal joints of digits 3 and 4 in Day 6-8 chick limb buds in organ culture. Specimens were processed for whole mount in situ hybridization using antisense riboprobes encoding chick GDF-5 and FGF-10 or for histology analysis at indicated time points. RESULTS: Whole mount in situ hybridization revealed that FGF-10 is expressed, and its transcripts are present, during interzone formation. Gain-of-function experiment revealed that exogenous FGF-10 caused down-regulation of expression of FGF-10 as well as GDF-5. In specimens continuously treated with exogenous FGF-10, joint formation was markedly impaired and often resulted in fusion of contiguous cartilaginous phalanges. CONCLUSIONS: The study provides evidence for the first time that FGF-10 is expressed during joint development in addition to FGF-2 and FGF-4. The precise roles of these signaling molecules will require further work. However, it is possible to speculate that these proteins, singly or in concert, may favor proliferation of mesenchymal cells during interzone formation. Our data also show that prolonged treatment with exogenous FGF-10 leads to joint impairment and fusion. Similar defects were observed previously when other joint-associated proteins were experimentally manipulated, indicating that a fine balance among distinct regulatory molecules is needed for normal joint formation.

Runx2 expression and action in chondrocytes are regulated by retinoid signaling and parathyroid hormone-related peptide (PTHrP).

OBJECTIVE: Runx2 (also known as Cbfa1) is a transcription factor required for chondrocyte maturation and osteoblast differentiation. While there is information on the regulation of its expression during osteogenesis, much less is known about it during cartilage maturation. Here we asked whether Runx2 expression and function are affected by retinoic acid (RA) and parathyroid hormone-related peptide (PTHrP), which represent an important stimulator and inhibitor of chondrocyte maturation, respectively. DESIGN: We first cloned and characterized Runx2 expressed by chick chondrocytes (cRunx2). We then constructed expression vectors of cRunx2 and a dominant-negative form (DN-cRunx2) and determined their effects on chondrocyte maturation in culture before and during retinoid and PTHrP treatment. RESULTS: cRunx2 showed similar transactivation activity to that of its mammalian counterparts although it has a very short QA domain and lacks a small portion of the PST domain. cRunx2 over-expression stimulated chondrocyte maturation, as indicated by increases in alkaline phosphatase activity (APase), mineralization, and type X collagen and MMP-13 expression, and by maintenance of Indian hedgehog (Ihh) expression. RA treatment stimulated cRunx2 gene expression and boosted its pro-maturation effects. PTHrP treatment blocked Runx2 expression and its pro-maturation effects. Over-expression of DN-cRunx2 inhibited maturation and even prevented RA from exerting its pro-maturation role. CONCLUSIONS: As previously indicated by mammalian studies, cRunx2 has chondrocyte pro-maturation activity. Its expression and roles are favorably modulated by retinoid signaling but are completely inhibited by PTHrP. A model integrating cRunx2 with PTHrP, Ihh and retinoid signaling and operating during skeletogenesis is proposed.

Vascular regression is required for mesenchymal condensation and chondrogenesis in the developing limb.

Vascular regression occurs during limb mesenchymal cell condensation and chondrogenesis, but it is unclear whether it is required for these processes or is a secondary phenomenon without major regulatory roles. To address this issue, beads presoaked with the potent angiogenic factor vascular endothelial growth factor (VEGF) were implanted in the vicinity of the prospective digit 2 in early chick embryo wing buds and the effects on angiogenesis and digit development were determined over time. We found that VEGF treatment caused a marked local increase in blood vessel number and density. Strikingly, this was accompanied by inhibition of digit 2 development as revealed by lack of expression of chondrogenic transcription factor Sox9 and absence of Alcian blue staining. Vascular distribution and skeletal development in adjacent areas remained largely unaffected. Inhibition of digit formation and excess vascularization were both reversible upon further embryonic growth and dissipation of VEGF activity. When supernumerary digits were induced at the anterior limb margin by retinoic acid treatment, their development was also preceded by vascular regression; interestingly, cotreatment with VEGF inhibited supernumerary digit development as well. Direct exposure of limb mesenchymal cells in micromass cultures to VEGF caused no obvious effects on condensation and chondrogenesis, indicating that VEGF effects are not due to direct action on skeletal cells. Our results are the first to provide evidence that vascular regression is required for mesenchymal condensation and chondrogenesis. A model of how patterning mechanisms and vascular regression may intersect and orchestrate limb skeletogenesis is proposed.

The role of ERG (ets related gene) in cartilage development.

OBJECTIVE: Based on function and developmental fate, cartilage tissue can be broadly classified into two types: transient (embryonic or growth-plate) cartilage and permanent cartilage. Chondrocytes in transient cartilage undergo terminal differentiation into hypertrophic cells, induce cartilage-matrix mineralization, and eventually disappear and are replaced by bone. On the other hand, chondrocytes in permanent cartilage do not differentiate further, do not become hypertrophic, and persist throughout life at specific sites, including joints and tracheal rings. While many studies have described differences in structure, matrix composition and biological characteristics between permanent and transient cartilage, it is poorly understood how the fates of permanent and transient cartilage are determined. Previous studies demonstrated that chondrocytes isolated from permanent cartilage have the potential to express markers of the mature hypertrophic phenotype once grown in culture, suggesting that cell hypertrophy is an intrinsic property of all chondrocytes and must be actively silenced in permanent cartilage in vivo. These silencing mechanisms, however, are largely unknown. In this paper, we first review nature of chondrocytes in transient and permanent cartilages and then report the cloning and characterization of a novel variant of ets transcription factor chERG, hereafter called C-1-1, which might be involved in regulation of permanent cartilage development. DESIGN: For cloning of a novel variant of chERG (C-1-1), we isolated RNA from the cartilaginous femur or tibiotarsus of Day 17 chick embryos and processed it for reverse transcription-polymerase chain reaction (RT-PCR) with the primers from sequences upstream and downstream of the 81 and 72 bp segments alternatively-spliced in mammals. For investigation of function of chERG and C-1-1, we over-expressed chERG or C-1-1 in cultured chick chondrocytes or the developing limb of chick embryo using a retrovirus (RCAS) system, and examined the phenotype changes in the infected chondrocytes or the infected limb elements. RESULTS: C-1-1 is an alternative and novel variant lacking the 27 amino acids segment of chERG that has been reported previously. C-1-1 is preferentially expressed in developing articular cartilage, whereas chERG is preferentially expressed in growth plate cartilage. Growth of articular chondrocytes in culture was accompanied by decreasing C-1-1 expression after several passages, while expression of hypertrophic markers increased. Expression of C-1-1 in cultured chondrocytes inhibited cell hypertrophy, alkaline phosphatase activity, and cartilage matrix mineralization. In contrast, over-expression of chERG promoted chondrocyte maturation and mineralization. CONCLUSION: Our data demonstrate for the first time that chERG and C-1-1 play distinct roles in skeletogenesis and may have crucial roles in the development and function of transient and permanent cartilages.

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Authentic matrix vesicles contain active metalloproteases (MMP). a role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta.

Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-beta (TGF-beta) and that MV-associated MMP-13 is capable of activating latent TGF-beta. To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-beta in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-beta. Since the amount of TGF-beta activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage.

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.