Hyun-Duck Nah

The Roles of Annexins and Types II and X Collagen in Matrix Vesicle-mediated Mineralization of Growth Plate Cartilage

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca2+ uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca2+ influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca2+ channel blocker, K-201, not only inhibited Ca2+ influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca2+ uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca2+ channel activities, leading to an increased Ca2+ influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca2+ channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca2+ and initiate the formation of the first crystal phase.

Runx2 Regulates FGF2-Induced Bmp2 Expression During Cranial Bone Development

Calvarial bone is formed by the intramembranous bone‐forming process, which involves many signaling molecules. The constitutive activation of the fibroblast growth factor (FGF) signaling pathway accelerates osteoblast differentiation and results in premature cranial suture closure. Bone morphogenetic protein (BMP) signaling pathways, which involve the downstream transcription factors Dlx5 and Msx2, are also involved in the bone‐forming processes. However, the relationships between these two main signaling cascades are still unclear. We found that FGF2 treatment of developing bone fronts stimulated Bmp2 gene expression but that BMP2 treatment could not induce Fgf2 expression. Moreover, the disruption of the Runx2 gene completely eliminated the expression of Bmp2 and its downstream genes Dlx5 and Msx2 in the developing primordium of bone, while the expression of Fgf2 was maintained. In addition, cultured Runx2−/− cells expressed very low baseline levels of Bmp2 that were up‐regulated by transfection with a Runx2‐expressing plasmid. These levels in turn were markedly elevated by FGF2 treatment. FGF2 treatment also strongly enhanced the Bmp2 expression in MC3T3‐E1 cells, whose endogenous Runx2 gene is intact and which express Bmp2 at low baseline levels as well. These results indicate that Runx2 is an important mediator of the expression of Bmp2 in response to FGF stimulation in cranial bone development. Developmental Dynamics 233:115–121, 2005.

Type IIA procollagen: Expression in developing chicken limb cartilage and human osteoarthritic articular cartilage

Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)–rich globular domain in its amino (N)–propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5–19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys‐rich domain of the type IIA procollagen N‐propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre‐chondrogenic mesenchyme (day 5) and early cartilage (days 7–9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re‐expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N‐propeptide, but not the carboxyl (C)–propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N‐propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis.

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.

Metopic craniosynostosis due to mutations in GLI3: A novel association.

We report on the novel association of trigonocephaly and polysyndactyly in two unrelated patients due to mutations within the last third (exon 14) and first third (exon 6) of the GLI3 gene, respectively. GLI3 acts as a downstream mediator of the Sonic hedgehog signal‐transduction pathway which is essential for early development; and plays a role in cell growth, specialization, and patterning of structures such as the brain and limbs. GLI3 mutations have been identified in patients with Pallister–Hall, Grieg cephalopolysyndactyly syndrome (GCPS), postaxial polydactyly type A1, preaxial polydactyly type IV, and in one patient with acrocallosal syndrome (ACLS). Furthermore, deletions including the GLI3 gene have been reported in patients with features of GCPS and ACLS. To date, trigonocephaly has not been associated with abnormalities of GLI3 and craniosynostosis is not a feature of GCPS. However, Hootnick and Holmes reported on a father with polysyndactyly and son with trigonocephaly, polysyndactyly, and agenesis of the corpus callosum, considered GCPS thereafter. Guzzetta et al. subsequently described a patient with trigonocephaly, polysyndactyly, and agenesis of the corpus callosum postulating a diagnosis of GCPS, later considered ACLS. In retrospect, these two patients, evaluated prior to mutational analysis, and our patients, with confirmed mutations, likely fall within the GLI3 morphopathy spectrum and may provide a bridge to better understanding those patients with overlapping features of GCPS and ACLS. Based on this observation, we suggest GLI3 studies in patients presenting with this constellation of findings, specifically metopic craniosynostosis with polysyndactyly, in order to provide appropriate medical management and genetic counseling.

Dll3 and Notch1 genetic interactions model axial segmental and craniofacial malformations of human birth defects

Mutations in the Notch1 receptor and delta‐like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3‐Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3‐Notch1 double heterozygous animals exhibited localized, segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant reduction of mandibular height and elongation of maxillary hard palate. Examination of somite‐stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray‐based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch‐regulated segmental or craniofacial development. Thus, Dll3‐Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders. Developmental Dynamics 236:2943–2951, 2007.

The Muenke syndrome mutation (FgfR3P244R) causes cranial base shortening associated with growth plate dysfunction and premature perichondrial ossification in murine basicranial synchondroses.

Muenke syndrome caused by the FGFR3P250R mutation is an autosomal dominant disorder mostly identified with coronal suture synostosis, but it also presents with other craniofacial phenotypes that include mild to moderate midface hypoplasia. The Muenke syndrome mutation is thought to dysregulate intramembranous ossification at the cranial suture without disturbing endochondral bone formation in the skull. We show in this study that knock‐in mice harboring the mutation responsible for the Muenke syndrome (FgfR3P244R) display postnatal shortening of the cranial base along with synchondrosis growth plate dysfunction characterized by loss of resting, proliferating and hypertrophic chondrocyte zones and decreased Ihh expression. Furthermore, premature conversion of resting chondrocytes along the perichondrium into prehypertrophic chondrocytes leads to perichondrial bony bridge formation, effectively terminating the postnatal growth of the cranial base. Thus, we conclude that the Muenke syndrome mutation disturbs endochondral and perichondrial ossification in the cranial base, explaining the midface hypoplasia in patients. Developmental Dynamics 240:2584–2596, 2011.

Developmental restriction of embryonic calvarial cell populations as characterized by their in vitro potential for chondrogenic differentiation

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12‐day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17‐day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from ∼15 to ∼50% of the population, while the number of osteogenic cells remained almost constant at ∼35–40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.

Differential closure of the spheno-occipital synchondrosis in syndromic craniosynostosis

Background: The spheno-occipital synchondrosis is a driver of cranial base and facial growth. Its premature fusion has been associated with midface hypoplasia in animal models. The authors reviewed computed tomographic scans of patients with Apert and Muenke syndrome, craniosynostosis syndromes with midface hypoplasia, to assess premature fusion of the spheno-occipital synchondrosis when compared with normal controls. Methods: Ninety head computed tomographic scans of Apert syndrome patients and 31 head scans of Muenke syndrome patients were assessed, in addition to an equal number of control scans. Spheno-occipital synchondrosis fusion on axial images was graded as open, partially closed, or closed. Analysis focused on ages 7 to 14 years, as no control patient fused before age 7 or had failed to fuse after age 14. Results: All 38 Apert syndrome patients aged 7 to 14 had some degree of spheno-occipital synchondrosis closure, compared with 29 of 38 matched controls (p = 0.0023). Seventeen of 20 Muenke syndrome patients showed closure, compared with 14 of 20 matched controls (p = 0.4506). Partial fusion was seen as early as age 2 in Apert syndrome and age 6 in Muenke syndrome patients; the earliest fusion was seen at age 7 in the control group. Conclusions: Compared with matched controls, the spheno-occipital synchondrosis closes significantly earlier in patients with Apert syndrome but not Muenke syndrome. This correlates well to reported incidences of midface hypoplasia in these syndromes. Although causality cannot be concluded from this study, an association exists between midface phenotype and degree of spheno-occipital synchondrosis closure. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Activating (P253R, C278F) and dominant negative mutations of FGFR2: Differential effects on calvarial bone cell proliferation, differentiation, and mineralization

Various activating mutations of FgfR2 have been linked to a number of craniosynostosis syndromes, suggesting that FGFR2-mediated signaling plays significant roles in intramembranous bone formation. To define (i) the roles of FGFR2-mediated signaling in osteogenesis and (ii) bone cell functions affected by abnormal signaling induced by craniosynostosis mutations, chicken calvarial osteoblasts were infected with replication competent avian sarcoma viruses expressing FgfR2 with dominant negative (DN), P253R (Apert), or C278F (Pfeiffer and Crouzon) mutation. Analyses of the infected osteoblasts revealed that attenuated FGF/FGFR signaling by DN-FgfR2 resulted in a decrease in cell proliferation and accelerated mineralization. In contrast, the C278F mutation, which causes ligand-independent activation of the receptor, significantly stimulated cell proliferation and inhibited mineralization. Interestingly, the P253R mutation, which does not cause ligand-independent activation of the receptor, showed a weaker mitogenic effect than the C278F mutation and did not inhibit mineralization. Gene expression analysis also revealed diverse effects of C278F and P253R mutations on expression of several osteogenic genes. Based on these results, we conclude that one of the major functions of FGFR2 is to mediate mitogenic signals in osteoblasts and that distinctively different cellular mechanisms underlie the pathogenesis of craniosynostosis phenotypes resulting from P253R and C278F mutations of the FGFR2 gene.