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Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component.
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BACKGROUND - Tibial eminence fractures occur most commonly in skeletally immature children. Several techniques using physeal-sparing fracture fixation have been described, but their structural properties have not been evaluated.
PURPOSE - To determine the strength and resistance to displacement of physeal-sparing techniques used to fix tibial eminence fractures.
STUDY DESIGN - Controlled laboratory study.
METHODS - Skeletally immature porcine knees were randomized into 4 treatment groups: (1) ultra-high molecular weight polyethylene suture-suture button (UHMWPE/SB), (2) suture anchor, (3) polydioxanone suture-suture button (PDS/SB), and (4) screw fixation. A prospective analysis of bone mineral density using dual-energy x-ray absorptiometry was performed on all specimens. Fracture fragments were created in a standardized manner and measured for size comparison. After fracture fixation, biomechanical testing was performed with cyclical and load-to-failure protocols by loading the tibia with an anterior shear force.
RESULTS - In load-to-failure testing, screw fixation had a significantly lower median peak failure load (186.4 N; lower quartile [LQ], 158.4 N; upper quartile [UQ], 232.6 N) than did UHMWPE/SB (465.8 N; LQ, 397.8 N; UQ, 527.8 N), suture anchors (440.5 N; LQ, 323.0 N; UQ, 562.3 N), and PDS/SB (404.3 N; LQ, 385.9 N; UQ, 415.6 N). UHMWPE/SB demonstrated a significantly higher median yield load (465.8 N; LQ, 397.8 N; UQ, 527.8 N) than did PDS/SB (306.7 N; LQ, 271.4, N; UQ, 405.7 N) and screw fixation (179.0 N; LQ, 120.2 N; UQ, 232.5 N). During cyclical testing, screw fixation demonstrated significantly lower percentage survival of specimens (0%) compared with the other groups (UHMWPE/SB, 100%; suture anchor, 78%; PDS/SB, 78%). After 1000 cycles of loading, PDS/SB fixation had significantly more median creep (6.76 mm; LQ, 6.34 mm; UQ, 8.28 mm) than did UHMWPE/SB (4.43 mm; LQ, 3.80 mm; UQ, 4.73 mm) and suture anchor fixation (3.06 mm; LQ, 2.59 mm; UQ, 4.28 mm). The lowest median stiffness was observed in the PDS/SB group (48.6 N/mm; LQ, 45.3 N/mm; UQ, 54.2 N/mm). UHMWPE/SB fixation demonstrated a significantly higher median peak failure load after cyclic testing (469.0 N; LQ, 380.6 N; UQ, 507.2 N) than did PDS/SB (237.7 N; LQ, 197.3 N; UQ, 298.3 N) and screw fixation (132.4 N; LQ, 123.7 N; UQ, 180.9 N). Suture anchor fixation had significantly more variance, as demonstrated by width of interquartile range, in peak failure load, yield load, and creep than did other techniques.
CONCLUSION - Physeal-sparing fixation of tibial eminence fractures with UHMWPE suture-suture button is biomechanically superior to both PDS suture-suture button and a single screw at the time of surgery and provides more consistent fixation than do suture anchors.
CLINICAL RELEVANCE - Suture anchors provide inconsistent fixation for tibial eminence fractures.
PURPOSE - To evaluate the feasibility of measuring T1ρ values in epiphyseal cartilage in children, we have conducted a novel study of spin locking techniques. Adult articular cartilage has been widely studied with spin locking techniques by magnetic resonance imaging. However, no results are available for in vivo T1ρ imaging of developing cartilage.
MATERIALS AND METHODS - Ten volunteers of age 6 ± 3 years were recruited to have T1ρ mapping performed on the knee at the conclusion of their clinical study. T1ρ maps were generated using a spin-lock cluster followed by a fast spin-echo imaging sequence. Regions of interest (ROIs) were placed in non-load-bearing (NLB), load-bearing (LB), and articular cartilage.
RESULTS - Student's t-tests were performed to compare means among the ROIs. Mean T1ρ for epiphyseal and articular cartilage was 49.8 ± 9 and 76.6 ± 7 ms, respectively. LB and NLB T1ρ vales were 47.1 ± 9.5 and 52.5 ± 9 ms, respectively. Significant differences were found in T1ρ values between epiphyseal and articular cartilage layers (P = 0.0001). No difference in T1ρ was observed between NLB and LB layers. A modest trend was also noted for epiphyseal and articular cartilage regions with age.
CONCLUSION - It is feasible to quantify differences in epiphyseal and articular cartilage layers with SL techniques. T1ρ holds promise as a noninvasive method of studying normal and abnormal developmental states of cartilage in children.
Copyright © 2012 Wiley Periodicals, Inc.
The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagen-expressing cells, (Nf1col2(-/-) mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1(-/-) chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1(-/-) chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1col2(-/-) mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.
BACKGROUND - Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted.
RESULTS - We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype.
CONCLUSIONS - We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation.
Copyright © 2012 Wiley Periodicals, Inc.
Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.
Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4(-/-)) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4(-/-) chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation.
The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1alpha (HIF1alpha), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL (Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1alpha leads to increased expression levels of HIF1alpha target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1a genes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1alpha. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.
Gamma-glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the gamma-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGT-/-) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGT-/- mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the observed defects are secondary to other abnormalities. N-acetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGT-/- mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGT-/- mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGT-/- N-acetylcysteine-treated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth.
Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.