Sreedhara Sangadala

A 54-year-old man with a history of unimelic, post-traumatic multifocal heterotopic ossification (HO) and normal genetic analysis of ACVR1 and GNAS had variants of unknown significance (VUS) in PDLIM-7 (PDZ and LIM Domain Protein 7), the gene encoding LMP-1 (LIM Mineralization Protein-1), an intracellular protein involved in the bone morphogenetic protein (BMP) pathway signaling and ossification. In order to determine if the LMP-1 variants were plausibly responsible for the phenotype observed, a series of in vitro experiments were conducted. C2C12 cells were co-transfected with a BMP-responsive reporter as well as the LMP-1 wildtype (wt) construct or the LMP-1(T161I) or the LMP-1(D181G) constructs (herein designated as LMP-161 or LMP-181) corresponding to the coding variants detected in the patient. A significantly increased BMP-reporter activity was observed in LMP-161 or LMP-181 transfected cells compared to the wt cells. The LMP-181 variant exhibited BMP-reporter activity with a four-fold increase over the LMP-1 wt protein. Similarly, mouse pre-osteoblastic MC3T3 cells transfected with the patient's LMP-1 variants expressed higher levels of osteoblast markers both at mRNA and protein levels and preferentially mineralized when stimulated with recombinant BMP-2 compared to control cells. Presently, there are no pathogenic variants of LMP-1 known to induce HO in humans. Our findings suggest that the germline variants in LMP-1 detected in our patient are plausibly related to his multifocal HO (LMP1-related multifocal HO). Further observations will be required to firmly establish this gene-disease relationship.

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Esophageal atresia (EA/TEF) is a common congenital abnormality present in 1 of 4000 births. Here we show that atretic esophagi lack Noggin (NOG) expression, resulting in immature esophagus that contains respiratory glands. Moreover, when using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOUs) as a model of foregut development and differentiation in vitro, NOG determines whether foregut progenitors differentiate toward esophageal or tracheal epithelium. These results indicate that NOG is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis, and its lack of expression results in EA/TEF.

Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkaline phosphatase induction in C2C12 cells comparable to that seen with rhBMP-2. FK506 treatment activated the BMP receptor, as shown by increased pSmad1/5 levels, and produced significantly higher mRNA levels of the early response genes in BMP and TGF-β pathways. Additionally, the FK506 induction of alkaline phosphatase was shown to be resistant to Noggin treatment. In vivo osteogenic activity of FK506 was tested by local delivery on a collagen sponge in an ectopic subcutaneous implantation model in the rat. Dose responses of FK506 showed increasing levels of ectopic mineralization comparable to the mineral volume produced by BMP-2 delivery. These findings suggest that the use of FK506 can enhance osteoblastic differentiation in vitro and can induce mineralization when delivered locally in vivo.

Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding assays in vitro. The activities of various wild-type and mutant LMP-1 proteins were evaluated using a BMP-responsive luciferase reporter and alkaline phosphatase assay in mouse myoblastic cells that were differentiated toward the osteoblastic phenotype. Finally, to strengthen physiological relevance of LMP-1 and Jab1 interaction, we showed that overexpression of LMP-1 caused nuclear accumulation of Smad4 upon BMP treatment which is reflective of increased Smad signaling in cells.

Aims of this study were to test the ability of SVAK-12 to enhance bone formation in a rodent ectopic model and to test whether a single percutaneous injection of SVAK-12 can accelerate callus formation in a rodent femoral fracture model. Methods: Collagen disks with rhBMP-2 alone or with rhBMP-2 and SVAK-12 were implanted in a standard athymic rat chest ectopic model, and radiographic analysis was performed at four weeks. In a second set of rats (Sprague-Dawley), SVAK-12 was percutaneously injected into the site of a closed femoral fracture. The fractures were analyzed radiographically and biomechanically (with torsional testing) five weeks after surgery. Results: In the ectopic model, there was dose-dependent enhancement of rhBMP-2 activity with use of SVAK-12 at doses of 100 to 500 μg. In the fracture model, the SVAK-12-treated group had significantly higher radiographic healing scores than the untreated group (p = 0.028). Biomechanical testing revealed that the fractured femora in the 200 to 250-μg SVAK-12 group were 43% stronger (p = 0.008) and 93% stiffer (p = 0.014) than those in the control group. In summary, at five weeks the femoral fracture group injected with SVAK-12 showed significantly improved radiographic and biomechanical evidence of healing compared with the controls. Conclusions: A single local dose of a low-molecular-weight compound, SVAK-12, enhanced bone-healing in the presence of low-dose exogenous rhBMP-2 (in the ectopic model) and endogenous rhBMPs (in the femoral fracture model). Clinical Relevance: This study demonstrates that rhBMP-2 responsiveness can be enhanced by a novel small molecule, SVAK-12. Local application of anabolic small molecules has the potential for potentiating and accelerating fracture-healing. Use of this small molecule to lower required doses of rhBMPs might both decrease their cost and improve their safety profile....

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1−/− knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

The requirement of large amounts of the recombinant human bone morphogenetic protein‐2 (BMP‐2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP‐2. In this study, we describe the development and optimization of a cell‐based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP‐2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP‐responsive Smad1‐driven luciferase reporter gene. LIM mineralization protein‐1 (LMP‐1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP‐2. Our previous report elucidated that the binding of LMP‐1 with the WW2 domain in Smad ubiquitin regulatory factor‐1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell‐based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP‐1, and its mutant (LMP‐1ΔSmurf1) that lacks the Smurf1‐WW2 domain‐binding motif. Both the wild type and the mutant proteins were engineered to contain an 11‐amino acid HIV‐TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell‐based reporter assay confirmed that LMP‐1 potentiates the BMP‐induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP‐1 was significantly reduced when a specific‐motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell‐based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post‐translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression‐based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP‐1. Among them, we selected SVAK‐3, a compound that showed a dose‐dependent potentiation of BMP‐2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP‐1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP‐2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. Published in 2009 by John Wiley & Sons, Ltd.

LIM mineralization protein-1 (LMP-1) is a novel osteoin-ductive protein that has been cloned and shown to induce bone formation both in vitro and in