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

Gdf5 mutants N445T and N445K as well as Bmp9 cause strong chondrogenic differentiation when overexpressed in chicken limbs.

<p>Retroviral overexpression of indicated proteins after injection into chicken hind limbs at stage HH10. The contralateral hind limb was used as control. Embryos were harvested at day 7.5 and skeletal preparation stained with Alcian blue and Alizarin red. WtGdf5 lead to fusion of joints in the phalanges and metatarsals. In contrast, N445T and N455K Gdf5 as well as Bmp9 overexpression in vivo caused a severe phenotype with massive cartilage production leading to complete fusion of all skeletal elements of the limb and absence of interdigital mesenchyme. Scale: 1 mm.</p

Biacore interaction analysis (n = 6) showed no binding differences for rhGDF5 and rhN445T GDF5 with BMPR1Aecd and BMPR1Becd.

<p>Biacore interaction analysis (n = 6) showed no binding differences for rhGDF5 and rhN445T GDF5 with BMPR1Aecd and BMPR1Becd.</p

rhN445T GDF5 has a different signaling effect than rhGDF5.

<p>(A) Differentiation of myoblastic mouse cell line C2C12 was documented with alkaline phosphatase (ALP) staining (for osteoblasts) and immuno-myosin staining (for myoblasts) after five days of stimulation with 10 nM of the indicated recombinant proteins. RhBMP2 and rhBMP9 strongly induced ALP activity and inhibited myoblast differentiation, whereas rhGDF5 affected osteoblast differentiation only slightly while inhibiting myoblast differentiation. Interestingly rhN445T induced a moderate ALP activity and showed no detectable myosin staining. (B) ALP activity induced by stimulating the C2C12 with BMPs for three days. RhN445T, rhBMP2, and rhBMP9 showed dose-dependent induction of ALP activity whereas rhGDF5 had almost no ability to induce ALP activity. (E–G) Luciferase reporter assay of rhGDF5, rhN445T GDF5, rhBMP2, and rhBMP9 activity co-stimulated with soluble receptor extracellular domains (ecd) of the seven TGFβ superfamily type I receptors in C2C12 cells. rhGDF5, rhN445T GDF5 as well as rhBMP2 activity was competitively inhibited by BMPR1Aecd and BMPR1Becd, whereas luciferase activity induced by rhBMP9 was inhibited by ACVRL1ecd. Luciferase activity was normalized to Renilla. The lowest unstimulated value was determined as 0% and BMP stimulation without receptor ecd as 100%. Statistically relevant interactions were calculated with a two-tailed t-test and marked as: * p≤0.05, ** p≤0.01, *** p≤0.001.</p

Bmp9 and Bmp10 are not inhibited by Nog.

<p>Chicken limb bud micromass cultures were retrovirally infected to express the indicated proteins and stained with Alcian blue after five days of cultivation. (A) Schematic overview of the mature domain of mouse Bmp9. Conserved cysteines marked in black. (B) 3D surface model of a BMP9 dimer (light grey and dark grey; PDB∶1ZKZ) linked via a disulfide bridge. Mut2 (K371N) represents the homologous mutation site to GDF5 N445T and is indicated in red. Mut1 (K348L, yellow) and mut3 (YH415Q, blue) are additional sites, which were predicted to be important for the interaction of GDF5 with NOG. (C) Expression of wtBmp9 and wtBmp10 in chicken micromass strongly induce chondrogenesis. This effect cannot be inhibited by co-expression of Nog. The Bmp9 mutants mut1 (K348L), mut2 (K371N), mut3 (YH415Q), mut1+2, and mut2+3 are similar to wtBmp9. In contrast, the combination of K371N and YH415Q (mut2+3) results in partial inhibition by Nog. The combination of all three mutations (mut1+2+3) leads to a complete inhibition by Nog. (D) Quantification of Alcian blue staining after extraction and photometric measurement at OD₅₉₅ (n = 4).</p

NOG insensitivity of rhN445T GDF5 in vitro.

<p>(A) ALP activity induced in C2C12 myoblastic mouse cells overexpressing the Bmpr1b receptor (C2C12-Bmpr1b) after three days of stimulation. All BMPs show dose-dependent induction of ALP activity. (B) Inhibition of rhBMP-induced ALP activity in the C2C12-Bmpr1b cell line by rhNOG. ALP activity was measured after co-stimulation of 5 nM rhGDF5, rhN445T GDF5, rhBMP2, and 0.05 nM rhBMP9 with up to 40 nM rhNOG (rhBMP9 up to 0.4 nM). RhBMP2 is strongly inhibited by rhNOG. In contrast to rhGDF5 the mutant rhN445T GDF5 shows no response to rhNOG inhibition.</p

GDF5 mutant N445T has increased prochondrogenic activity.

<p>Stimulation of chicken micromass cultures with rhGDF5, rhN445T GDF5, rhBMP2, and rhBMP9 at day one for 72 h with the indicated concentrations. (A) Alcian blue staining of micromass cultures at day four. RhN445T showed a strong induction of chondrogenesis already at low concentrations. In comparison, rhBMP2 showed a strongly reduced and rhBMP9 the highest prochondrogenic activity. (B) Quantification of the Alcian blue staining, n = 3. (C) Semi-qRT-PCR for Nog after 12 h stimulation of one day old chicken micromass cultures with 5 nM rhGDF5, rhN445T GDF5, rhBMP2, and 0.5 nM rhBMP9. Stimulation with recombinant proteins induces a strong upregulation of endogenous Nog expression. (D) Whole mount in situ hybridizations showing <i>Nog</i> expression in chicken hind limbs after implantation of heparin beads soaked with 4 mM HCl + 0.2% BSA, 0.5 mg/ml rhGDF5, 0.5 mg/ml rhN445T GDF5, 0.5 mg/ml rhBMP2, and 0.25 mg/ml rhBMP9. Beads were implanted between HH stages 25–27 and HH29/30 (BMP9) and embryos were collected 20 h later. In contrast to the control limb, all beads soaked with BMP induced <i>Nog</i> expression in the periphery.</p