23 research outputs found
Nucleotide Polymorphisms in the Canine Noggin Gene and Their Distribution Among Dog (Canis lupus familiaris) Breeds
Noggin (NOG) is an important regulator for the signaling of bone morphogenetic proteins. In this study, we sequenced the complete coding sequence of the canine NOG gene and characterized the nucleotide polymorphisms. The sequence length varied from 717 to 729 bp, depending on the number of a 6-bp tandem repeat unit (GGCGCG), an insertion that has not been observed in other mammalian NOG genes investigated to date. It results in extensions of (Gly–Ala)3–5 in the putative NOG protein. To survey the distribution of these tandem repeat polymorphisms, we analyzed 126 individuals in seven dog breeds. We identified only three alleles: (GGCGCG)3, (GGCGCG)4, and (GGCGCG)5. Although the allele frequencies were remarkably different among the breeds, the three alleles were present in all seven of the breeds and did not show any deviation from Hardy–Weinberg equilibrium
Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling
Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIFâ 1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIFâ 1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIFâ 1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)â mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIFâ 1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1Q207D/+ mouse model of FOP. Thus, an inflammation and cellular oxygenâ sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIFâ 1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIFâ 1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO. © 2016 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134262/1/jbmr2848_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134262/2/jbmr2848.pd
Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN
Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling
Functional Analysis of Alleged NOGGIN Mutation G92E Disproves Its Pathogenic Relevance
We identified an amino acid change (p.G92E) in the Bone Morphogenetic Protein antagonist NOGGIN in a 22-month-old boy who presented with a unilateral brachydactyly type B phenotype. Brachydactyly type B is a skeletal malformation that has been associated with increased Bone Morphogenetic Protein pathway activation in other patients. Previously, the amino acid change p.G92E in NOGGIN was described as causing fibrodysplasia ossificans progressiva, a rare genetic disorder characterized by limb malformations and progressive heterotopic bone formation in soft tissues that, like Brachydactyly type B, is caused by increased activation of Bone Morphogenetic Protein signaling. To determine whether G92E-NOGGIN shows impaired antagonism that could lead to increased Bone Morphogenetic Protein signaling, we performed functional assays to evaluate inhibition of BMP signaling. Interestingly, wt-NOGGIN shows different inhibition efficacies towards various Bone Morphogenetic Proteins that are known to be essential in limb development. However, comparing the biological activity of G92E-NOGGIN with wt-NOGGIN, we observed that G92E-NOGGIN inhibits activation of bone morphogenetic protein signaling with equal efficiency as wt-NOGGIN, supporting that G92E-NOGGIN does not cause pathological effects. Genetic testing of the child's parents revealed the same amino acid change in the healthy father, further supporting that p.G92E is a neutral amino acid substitution in NOGGIN. We conclude that p.G92E represents a rare polymorphism of the NOGGIN gene - causing neither brachydactyly nor fibrodysplasia ossificans progressiva. This study highlights that a given genetic variation should not be considered pathogenic unless supported by functional analyses
Induced degradation of protein kinases by bifunctional small molecules: a next-generation strategy
In vitro Analyses of the Dysregulated R206H ALK2 Kinase-FKBP12 Interaction Associated with Heterotopic Ossification in FOP
Three-dimensional model of the NOG-BMP7 complex highlighting the unstructured polyglycine loop that harbors the substitution in p.G92E.
<p>NOG-BMP7 complex (PDB: 1M4U) is depicted as a cartoon structure, with monomers of the NOG homodimer in dark in light green and monomers of the BMP7 homodimer in red and orange with surfaces depicted (A). Labeled residues flank the polyglycine loop, which is unresolved due to an apparent high flexibility associated with the largely unrestricted chain of residues. The NOG monomers on the left are tilted slightly into, and the BMP monomers slightly out of, the image plane. The complex in the zoomed view (B) is tilted further in the same direction, as well as slightly counter-clockwise about the perpendicular axis.</p
Biological activity of wt-NOG and G92E-NOG and protein production in chicken micromass cells are comparable.
<p>Chicken micromass cells were infected with either wt-NOG or G92E-NOG from 1*10e07 viral particles/ml and decreasing to 0.05*10e07 viral particles/ml (A). At day 5, cells were stained with Alcian blue, and dye concentration quantified spectrophotometrically at 595 nm. Non-infected controls were normalized as 100% activity. Data shown are taken from a representative experiment performed with 3 replicates each. Error bars indicate standard deviation. Pellets from cells infected with 1*10e07 viral particles/ml were collected at day 3 from the same chicken micromass experiment to perform Western Blot analysis (B). Uninfected cells were used as a control. After SDS-PAGE under non-reducing conditions and subsequent Western Blot, NOG and β-Actin were detected with specific antibodies. For quantification, NOG was normalized to β-Actin. Wt-NOG and G92E-NOG are expressed in equal amounts in the micromass cultures.</p
wt-NOG and G92E-NOG show comparable ability to block BMP targets in the chicken Micromass system.
<p>Chicken micromass cells were infected with 1*10e07 viral particles/ml containing the gene for BMP2 (A), Bmp4 (B), Bmp7 (C) or GDF5 (D). Co-infection was performed with increasing virus titers of either wt-NOG or G92E-NOG as indicated. At day 5, cells were stained with Alcian blue, and dye concentration quantified spectrophotometrically at 595 nm. Controls infected exclusively with BMPs were normalized as 100% activity. Data shown are taken from a representative experiment performed with 3 replicates each. Error bars indicate standard deviation.</p
Chymotrypsin mRNA expression in digestive gland amoebocytes: cell specification occurs prior to metamorphosis and gut morphogenesis in the gastropod Haliotis rufescens
In the non-feeding larva of the marine gastropod, Haliotis rufescens, gut morphogenesis is initiated at metamorphosis. Intestine-specific chymotrypsin gene expression begins in amoebocytes located in the dorsoposterior region of the undifferentiated digestive gland prior to metamorphosis, 5 d post-fertilization. Transcript accumulates steadily in these cells over the next 6 d while the amoebocytes migrate slowly dorsally. Induction of metamorphosis dramatically accelerates the rates of chymotrypsin mRNA accumulation and amoebocyte migration, and is required for homing of the amoebocytes to the hindgut region. Induction of chymotrypsin gene expression occurs only in larvae that had developed competence to recognize an exogenous morphogenetic cue and initiate metamorphosis, with a more pronounced increase in chymotrypsin mRNA accumulation in occurring older larvae. Chymotrypsin mRNA accumulation patterns suggest that hindgut cell specification occurs prior to metamorphosis, but that completion of the morphogenetic program requires signaling events associated with metamorphosis