SMOC can act as both an antagonist and an expander of BMP signaling.

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

Xenopus SMOC-1 Inhibits bone morphogenetic protein signaling downstream of receptor binding and is essential for postgastrulation development in Xenopus.

The bone morphogenetic protein (BMP) family of signaling molecules and their antagonists are involved in patterning of the body axis and numerous aspects of organogenesis. Classical biochemical purification and protein sequencing of highly purified fractions containing potent bone forming activity from bovine cartilage identified several BMPs together with a number of other proteins. One such protein was SMOC-2 (secreted modular calcium-binding protein-2), classified as belonging to the BM-40 family of modular extracellular proteins. Data regarding the biological function of SMOC-2 and closely related SMOC-1 remain limited, and their expression or function during embryological development is unknown. We therefore isolated the Xenopus ortholog of human SMOC-1 (XSMOC-1) and explored its function in Xenopus embryos. In gain-of-function assays, XSMOC-1 acted similarly to a BMP antagonist. However, in contrast to known extracellular ligand-binding BMP antagonists, such as noggin, SMOC antagonizes BMP activity in the presence of a constitutively active BMP receptor, indicating a mechanism of action downstream of the receptor. We provide several lines of evidence to suggest that SMOC acts downstream of the BMP receptor via MAPK-mediated phosphorylation of the Smad linker region. Loss-of-function studies, using antisense morpholino oligonucleotides, revealed XSMOC-1 to be essential for postgastrulation development. The catastrophic developmental failure observed following XSMOC knockdown resembles that observed following simultaneous depletion of three ligand-binding BMP antagonists described in prior studies. These findings provide a direct link between the extracellular matrix-associated protein SMOC and a signaling pathway of general importance in anatomic patterning and cell or tissue fate specification.

Establishment of retroviral pseudotypes with influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and specific detection of neutralizing antibodies.

Pseudotype reporter viruses provide a safe, quantitative, and high-throughput tool for assessing antibody neutralization for many viruses, including high pathogenicity H5 and H7 influenza A strains. However, adapting this system to other influenza subtypes has been hampered by variations in the protease cleavage site of hemagglutinin (HA) that make it less susceptible to the cleavage required for infectivity. In this report several proteases, reporter vectors, and cell substrates were evaluated while optimizing pseudovirus production, and robust methods were established for sensitive and specific neutralization of pseudotypes carrying influenza H1, H3, and H5 subtype HA that correlates well with titers obtained in microneutralization assays involving replicating influenza virus These findings should facilitate broad use of HA-pseudotypes that remove the need for infectious virus in a range of applications, including neutralization assays for serological surveys of viral infection and evaluations of vaccine sera.

Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice.

OBJECTIVE: Ligands and antagonists of the WNT pathway are linked to osteoporosis and osteoarthritis. In particular, polymorphisms in the FRZB gene, a secreted WNT antagonist, have been associated with osteoarthritis. The aim of this study was to examine cartilage and bone in Frzb(-/-) mice. METHODS: The Frzb gene in mice was inactivated using a Cre/loxP strategy. Three models of osteoarthritis were used: collagenase, papain, and methylated bovine serum albumin induced. Bone biology was studied using density measurements and microfocal computed tomography. Bone stiffness and mechanical loading-induced bone adaptation were studied by compression of the ulnae. RESULTS: Targeted deletion of the Frzb gene in mice increased articular cartilage loss during arthritis triggered by instability, enzymatic injury, or inflammation. Cartilage damage in Frzb(-/-) mice was associated with increased WNT signaling and matrix metalloproteinase 3 (MMP-3) expression and activity. Frzb(-/-) mice had increased cortical bone thickness and density, resulting in stiffer bones, as demonstrated by stress-strain relationship analyses. Moreover, Frzb(-/-) mice had an increased periosteal anabolic response to mechanical loading as compared with wild-type mice. CONCLUSION: The genetic association between osteoarthritis and FRZB polymorphisms is corroborated by increased cartilage proteoglycan loss in 3 different models of arthritis in Frzb(-/-) mice. Loss of Frzb may contribute to cartilage damage by increasing the expression and activity of MMPs, in a WNT-dependent and WNT-independent manner. FRZB deficiency also resulted in thicker cortical bone, with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to the development of osteoarthritis by producing increased strain on the articular cartilage during normal locomotion but may protect against osteoporotic fractures.

CDMP1/GDF5 has specific processing requirements that restrict its action to joint surfaces.

CDMP1/GDF5 has not demonstrated biological activity in Xenopus embryos when overexpressed by mRNA injection. We provide biological and biochemical evidence that to become active, the protein requires cleavage by two distinct proteolytic enzymes. We demonstrate a specific overlap in the expression patterns of CDMP1/GDF5 with the proteases required to release the mature peptide at the location of the future articular surface but not in the future joint space. Taken together, these observations provide a plausible mechanism for local action of CDMP1/GDF5 consistent with requirements imposed by current models of pattern formation in the developing limb.

Characterization of Frzb-Cre transgenic mouse.

The Wnt family of glycoproteins is involved in numerous developmental and disease processes in higher eukaryotes, exerting their action by binding to cell-surface receptors. In the extracellular space, Wnts are negatively regulated by secreted antagonists that either bind to the receptors directly (Dkk1) or to Wnt molecules themselves (Sfrp-FRZB family), preventing its subsequent binding to the receptor. Here we report on a transgenic mouse expressing Cre under the control of the mouse Frzb promoter element. Analysis of the Cre expression was carried out at 10.5 and 14.5 dpc using the ROSA26R mouse line. Expression of the transgenic construct was detected in the limbs, the heart, the nasal epithelium, bone, whiskers, and around the orbita of the eye. The mouse could be used for conditional gene modification in those tissues.

An enhancer complex confers both high-level and cell-specific expression of the human type X collagen gene.

Type X collagen expression is restricted to hypertrophic chondrocytes in the endochondral growth plate. Transient transfection of reporter constructs containing the human collagen X promoter into primary growth plate chondrocytes identified a cis-acting positive regulatory DNA element(s) that has cell-specific enhancer properties and binds a nuclear protein expressed specifically in growth plate chondrocytes. Functional disruption of this region results in a significant reduction in the activation of reporter gene transcription. The identified enhancer is a major element controlling both high-level and cell-specific expression of type X collagen gene.

The mutational spectrum of brachydactyly type C.

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5.