Single Application of Low-Dose, Hydroxyapatite-Bound BMP-2 or GDF-5 Induces Long-Term Bone Formation and Biomechanical Stabilization of a Bone Defect in a Senile Sheep Lumbar Osteopenia Model.

Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, osteopenic female sheep (n = 72; 9.00 ± 0.11 years; mean ± SEM). Treatment was: (a) HA particles (2.5 mg; L5); or (b) particles coated with BMP-2 (1 µg; 10 µg) or GDF-5 (5 µg; 50 µg; L4; all groups n = 6). Untouched vertebrae (L3) served as controls. Three and nine months post-therapy, bone formation was assessed by osteodensitometry, histomorphometry, and biomechanical testing. Cumulative 14-day BMP release was high in serum (76-100%), but max. 1.4% in PBS. In vivo induction of bone formation by HA particles with either growth factor was shown by: (i) significantly increased bone volume, trabecular and cortical thickness (overall increase HA + BMP vs. control close to the injection channel 71%, 110%, and 37%, respectively); (ii) partial significant effects for bone mineral density, bone formation, and compressive strength (increase 17%; 9 months; GDF-5). Treatment effects were not dose-dependent. Combined HA and BMPs (single low-dose) highly augment long-term bone formation and biomechanical stabilization in sheep lumbar osteopenia. Thus, carrier-bound BMP doses 20,000-fold to 1000-fold lower than previously applied appear suitable for spinal fusion/bone regeneration and improved treatment safety.

In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement.

Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide-co-glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11-34% for GDF5 and 6-17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Targeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose. PURPOSE: The aim of the current study was to test an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming CPC containing low-dose BB-1 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and were filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BB-1 (L5; CPC+fibers+BB-1; 5, 100, and 500 µg BB-1; n=6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BB-1) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (BV/TV) (assessed by micro-CT and histomorphometry), trabecular thickness (Tb.Th), and trabecular number (Tb.N); (3) bone formation, that is, osteoid volume/bone volume (OV/BV), osteoid surface/bone surface (OS/BS), osteoid thickness, mineralizing surface/bone surface (MS/BS), mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BB-1 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BB-1, additional significant effects of BB-1 were demonstrated for BMD, bone structure (BV/TV, Tb.Th, and Tb.N), and bone formation (OS/BS and MS/BS). The BB-1 effects on bone formation at 3 and 9 months were dose dependent, with 100 µg as the potentially optimal dosage. CONCLUSIONS: BB-1 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as 100 µg BB-1 was sufficient to augment middle- to long-term bone formation. A CPC containing the novel GDF5 mutant BB-1 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty and kyphoplasty.

Low-dose BMP-2 is sufficient to enhance the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Bioresorbable calcium phosphate cement (CPC) may be suitable for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures. However, additional targeted delivery of osteoinductive bone morphogenetic proteins (BMPs) in the CPC may be required to counteract the augmented local bone catabolism and support complete bone regeneration. PURPOSE: This study aimed at testing an injectable, poly (l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing low-dose bone morphogenetic protein BMP-2 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BMP-2 (L5; CPC+fibers+BMP-2; 1, 5, 100, and 500 µg BMP-2; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BMP-2) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography [micro-CT] and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralizing surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BMP-2 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BMP-2, additional significant effects of BMP-2 were demonstrated for bone structure (bone volume/total volume, trabecular thickness, trabecular number) and formation (osteoid surface/bone surface and mineralizing surface/bone surface), as well as for the compressive strength. The BMP-2 effects on bone formation at 3 and 9 months were dose-dependent, with 5-100 µg as the optimal dosage. CONCLUSIONS: BMP-2 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as ≤100 µg BMP-2 was sufficient to augment middle to long-term bone formation. The novel CPC+BMP-2 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty/kyphoplasty.

GDF5 significantly augments the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Biodegradable calcium phosphate cement (CPC) represents a promising option for the surgical treatment of osteoporotic vertebral fractures. Because of augmented local bone catabolism, however, additional targeted delivery of bone morphogenetic proteins with the CPC may be needed to promote rapid and complete bone regeneration. PURPOSE: In the present study, an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing the bone morphogenetic protein GDF5 was tested in a sheep lumbar osteopenia model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Defined bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep. Defects were treated with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of GDF5 (L5; CPC+fibers+GDF5; 1, 5, 100, and 500 µg GDF5; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months postoperation, structural and functional effects of the CPC (±GDF5) were assessed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralized surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), both CPC+fibers (L4) and CPC+fibers+GDF5 (L5) numerically or significantly improved all parameters of bone formation, bone resorption, and bone structure. These significant effects were observed both at 3 and 9 months, but for some parameters they were less pronounced at 9 months. Compared with CPC without GDF5, additional significant effects of CPC with GDF5 were demonstrated for BMD and parameters of bone formation and structure (bone volume/total volume, trabecular thickness, and trabecular number, as well as mineralized surface/bone surface). The GDF5 effects were dose-dependent (predominantly in the 5-100 µg range) at 3 and 9 months. CONCLUSIONS: GDF5 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. The results indicated that a local dose as low as ≤100 µg GDF5 may be sufficient to augment middle to long-term bone formation. The novel CPC+GDF5 combination may thus qualify as an alternative to the bioinert, supraphysiologically stiff poly(methyl methacrylate) cement currently applied for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures.

A consistent and potentially exploitable response during chondrogenesis of mesenchymal stem cells from osteoarthritis patients to the protein encoded by the susceptibility gene GDF5.

Osteoarthritis (OA) is a common joint disease characterised by the focal loss of the protective cartilage layer at the ends of the bones. It is painful, disabling, multifactorial and polygenic. The growth differentiation factor 5 gene GDF5 was one of the first reported OA susceptibility signals that showed consistent association to OA, with the transcript single nucleotide polymorphism (SNP) rs143383 demonstrating association in Asians and Europeans. The functional effect of the signal is reduced expression of the gene. The GDF5 protein is an extracellular matrix signalling molecule that is active during chondrogenesis and in mature chondrocytes. Due to the functional impact of the susceptibility, we previously assessed the effect of supplementing chondrocytes from OA patients with exogenous GDF5. Their response was highly discordant, precluding the application of GDF5 as a simple means of attenuating the genetic deficit. Since GDF5 is also active during development, we have now assessed the effect of exogenous GDF5 on bone marrow derived mesenchymal stem cells (MSCs) that are undergoing chondrogenesis during cartilage disc formation. MSCs from healthy donors and OA patients were studied and the effect of GDF5 was assessed by measuring the wet mass of the discs, by histological staining, and by monitoring the change in expression of anabolic, catabolic and hypertrophic protein-coding genes. The MSCs expressed the three principal GDF5 receptor genes and responded in a significantly anabolic manner (increase in wet mass, p = 0.0022; Bonferroni corrected p = 0.018) to a variant form of GDF5 that targets the most abundantly expressed receptor, BMPR-IA. GDF5 elicited significant (p < 0.05) changes in the expression of anabolic, catabolic and hypertrophic genes with several consistent effects in healthy donors and in OA patients. Our data implies that, unlike OA chondrocytes, OA MSCs do respond in a predictable, anabolic manner to GDF5, which could therefore provide a route to modulate the genetic deficit mediated by the rs143383 association signal.

Growth Differentiation Factor 5 Accelerates Wound Closure and Improves Skin Quality During Repair of Full-Thickness Skin Defects.

BACKGROUND: A fast and stable wound closure is important, especially for extended and unstable wounds found after burn injuries. Growth can regulate a variety of cellular processes, including those involved in wound healing. Growth differentiation factor 5 (GDF-5) can accelerate fibroblast cell migration, cell proliferation, and collagen synthesis, which are essential for wound healing. Nevertheless, no standardized evaluation of the effect of GDF-5 on the healing of full-thickness wounds has been published to date. METHODS: Five full-thickness skin defects were created on the backs of 6 minipigs. Three wounds were treated with GDF-5 in different concentrations with the help of a gelatin-collagen carrier, and 2 wounds served as control group. The first was treated with the gelatin carrier and an Opsite film (Smith & Nephew, Fort Worth, Texas), and the other was treated solely with an Opsite film that was placed above all wounds and renewed every second day. RESULTS: Growth differentiation factor 5 accelerates wound closure (10.91 [SD, 0.99] days) compared with treatment with the carrier alone (11.3 [SD, 1.49] days) and control wounds (13.3 [SD, 0.94] days). Epidermal cell count of wounds treated with GDF-5 revealed a higher number of cells compared with the control group. In addition, mean epidermal thickness was significantly increased in GDF-5-treated wounds compared with the control wounds. CONCLUSIONS: Because of its ability to improve skin quality, GDF-5 should be considered when developing composite biomaterials for wound healing.

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

Feasibility study of an active wound dressing based on hollow fiber membranes in a porcine wound model.

INVESTIGATION: A novel active wound dressing (AWD) concept based on a microporous hollow fiber membrane network was investigated in an animal model. It provides a local solution-perfused environment for regenerative cell nutrition, wound irrigation, debris removal, electrolyte balancing, pH regulation, and topical antibiosis. The device is capable of supplying soluble factors, as tested experimentally for the recombinant human growth and differentiation factor-5 (rhGDF-5). METHODS: Following in vitro studies for rhGDF-5 using primary human keratinocytes and dermal fibroblasts, we employed a porcine partial thickness wound model with five distinct wounds on each back of n=8 pigs. Four wound groups were perfused differently over 9 days and compared with a negative control wound without perfusion: (1) 1% trehalose solution, pH 5.5; (2) rhGDF-5 (150 ng/ml) in 1% trehalose solution, pH 5.5; (3) nutrition solution; and (4) rhGDF-5 (150 ng/ml) in nutrition solution with 1% trehalose, pH 5.5. RESULTS: Promoted wound healing was observed within group 1 and more pronounced within group 2. Groups 3 and 4, with nutrition solution, showed significant adverse effects on wound healing (p<0.05). CONCLUSIONS: The investigated AWD concept appears to be an interesting therapeutic tool to study further wound healing support. Additionally, topical application of rhGDF-5 could be promising.

Improved bone defect healing by a superagonistic GDF5 variant derived from a patient with multiple synostoses syndrome.

Multiple synostoses syndrome 2 (SYNS2) is a rare genetic disease characterized by multiple fusions of the joints of the extremities, like phalangeal joints, carpal and tarsal joints or the knee and elbows. SYNS2 is caused by point mutations in the Growth and Differentiation Factor 5 (GDF5), which plays an essential role during skeletal development and regeneration. We selected one of the SYNS2-causing GDF5 mutations, p.N445T, which is known to destabilize the interaction with the Bone Morphogenetic Protein (BMP) antagonist NOGGIN (NOG), in order to generate the superagonistic GDF5 variant GDF5(N445T). In this study, we tested its capacity to support regeneration in a rat critical-sized defect model in vivo. MicroCT and histological analyses indicate that GDF5(N445T)-treated defects show faster and more efficient healing compared to GDF5 wild type (GDF5(wt))-treated defects. Microarray-based gene expression and quantitative PCR analyses from callus tissue point to a specific acceleration of the early phases of bone healing, comprising the inflammation and chondrogenesis phase. These results support the concept that disease-deduced growth factor variants are promising lead structures for novel therapeutics with improved clinical activities.

Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.

A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-ß1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-ß1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.

A GDF5 point mutation strikes twice--causing BDA1 and SYNS2.

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R) variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

Generation of amyloid-ß is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-ß is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-ß42 levels in culture supernatants, while transfection with the P-domain increases amyloid-ß40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-ß40 and amyloid-ß42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.

The effect of two point mutations in GDF-5 on ectopic bone formation in a beta-tricalciumphosphate scaffold.

The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated beta-TCP carrier was compared to carriers coated with similar amounts (10 microg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.

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 and BMP2 inhibit apoptosis via activation of BMPR2 and subsequent stabilization of XIAP.

GDF5 and BMP2, members of the TGF-beta superfamily of growth factors, are known to regulate apoptosis in different cell types either positively or negatively. We wanted to investigate the effects of GDF5 and BMP2 on vascular smooth muscle cells and mouse embryonic fibroblasts and disclose the mechanism by which GDF5 and BMP2 might exert anti-apoptotic effects. The effect of GDF5 and BMP2 on proliferation and/or programmed cells death was assessed in isolated human vascular smooth muscle cells and mouse embryonic fibroblasts. We demonstrate that GDF5 and BMP2 prevent apoptosis induced by serum starvation in mouse embryonic fibroblasts but not in smooth muscle cells via the BMP receptor 2 (BMPR2), which is often mutated in hereditary cases of primary pulmonary hypertension. GDF5 and BMP2 stimulate the interaction of BMPR-2 with XIAP thereby reducing the ubiquitination of XIAP, which results in enhanced protein stability. The increased concentration of XIAP counteracts apoptosis by binding and inactivating activated caspases. We conclude that the inhibition of apoptosis in mouse embryonic fibroblasts by BMP2 and GDF5 does not depend on more complex signal transduction pathways such as smad and MAPK signaling but on direct stabilization of XIAP by BMPR2.

Brachydactyly type A2 associated with a defect in proGDF5 processing.

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the growth/differentiation factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in Escherichia coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin, and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity.

Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2.

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.