Yunshan Liu

Adenoviral Delivery of LIM Mineralization Protein-1 Induces New-Bone Formation in Vitro and in Vivo

Background: The LIM mineralization protein-1 (LMP-1) gene encodes for an intracellular protein that induces the expression of several bone growth factors. The purpose of the present study was to determine the feasibility and the optimal dose of adenoviral delivery of the LMP-1 cDNA to promote spinal fusion. Methods: A replication-deficient human recombinant adenovirus was constructed with the LMP-1 cDNA driven by a cytomegalovirus promoter. In phase 1, an in vitro dose-response experiment was performed to determine the optimal adenovirus-LMP-1 (AdLMP-1) concentration and infection time. In phase 2, nine rabbits had a single-level posterolateral arthrodesis of the lumbar spine with implantation of a carrier matrix loaded with bone-marrow-derived buffy-coat cells that had been infected for ten minutes with adenovirus containing the cDNA for LMP-1 (AdLMP-1) or b-galactosidase (AdBgal). In phase 3, posterolateral arthrodesis of the spine was performed with implantation of cells infected with AdLMP-1 (ten rabbits) or cells infected with an empty adenovirus that did not contain LMP-1 cDNA (ten rabbits) and the results were compared. In this phase, peripheral-blood-derived buffy-coat cells were used instead of bone-marrow-derived cells and a collagen-ceramic-composite sponge was used as the carrier. Results: In phase 1, the in vitro dose-response experiment showed that a multiplicity of infection of 0.25 plaque-forming units per cell was the most efficient dose. In phase 2, the implants that had received cells infected with AdLMP-1 induced a solid, continuous spinal fusion mass at five weeks. In contrast, the implants that had received cells infected with AdBgal or a lower dose of AdLMP-1 induced little or no bone formation. In phase 3, a solid spinal fusion was observed at four weeks in all ten rabbits that had received cells infected with AdLMP-1 and in none of the ten rabbits that had received cells infected with the empty adenovirus. Biomechanical and histological testing of the AdLMP-1-treated specimens revealed findings that were consistent with a high-quality spinal fusion. Conclusions: Adenoviral delivery of LMP-1 cDNA promotes spinal fusion in immune-competent rabbits. Clinical Relevance: The use of delivery cells that are readily available from peripheral blood and the short infection time should allow this technique to be performed in any operating room. The use of an ex vivo gene-transfer protocol with a very low dose of virus should minimize the immune response and toxicity seen in association with other adenoviral applications.

LIM Mineralization Protein-1 Potentiates Bone Morphogenetic Protein Responsiveness via a Novel Interaction with Smurf1 Resulting in Decreased Ubiquitination of Smads

Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. 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, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.

Overexpressed LIM mineralization proteins do not require LIM domains to induce bone.

Rat LIM mineralization protein 1 (LMP‐1, an LIM domain protein) mediates bone morphogenetic protein 6 (BMP‐6) induction of bone nodule formation in fetal rat calvarial osteoblast (ROB) cultures. We have isolated the complementary DNA (cDNA) for the human homologue of LMP‐1 from an adult human heart cDNA library and showed that when overexpressed it is osteoinductive in the same culture system. The recently revised cDNA sequence of Enigma, the protein product of which binds to the insulin receptor and the tyrosine kinase receptor ret, now matches the nucleotide sequence of human LMP‐1 (hLMP‐1). A truncated, 223 amino acid (AA) LMP‐1(t) protein has identical effects as the full‐length protein, despite the deletion of the LIM domains. Two splice variants of human LMP‐1 have been detected. Human LMP‐2 has a 119‐base pair (bp) deletion between bp 325 and 444 and a 17‐bp insertion at bp 444. The resulting derived protein contains 423 AA with the LIM domains intact and does not induce bone formation when overexpressed in ROB cultures. Human LMP‐3 has the same 17 nucleotide insertion at bp 444, resulting in a shift in the reading frame that causes a stop codon to occur at bp 505‐507. The resulting 153 AA protein does not have the LIM domains, but overexpression of hLMP‐3 induces bone formation in osteoblast cultures. These findings suggest that the LIM domains are not required for LMPs to induce bone formation. In addition, a small region (36 AA) of the LMP‐1 protein may be required for bone formation.

Overcoming the Immune Response to Permit Ex Vivo Gene Therapy for Spine Fusion With Human Type 5 Adenoviral Delivery of the LIM Mineralization Protein-1 cDNA

Study Design. An animal study in immune competent rabbits and athymic rats was conducted. Objectives. To develop an animal model for simulation of previous human Type 5 adenovirus (Ad5) exposure, to determine the impact of adenoviral pre-exposure on spine fusion induced with ex vivo Ad5-LMP-1, and to test strategies for overcoming any potential immune response. Summary of Background Data. Cells transduced with adenovirus containing the osteoinductive LMP-1 cDNA (Ad5-LMP-1) can induce spine fusion in rabbits. Because up to 80% of the human population has been exposed to adenovirus, immune responses to the vector may limit this strategy in humans. Few studies have modeled previous adenoviral exposure and tested strategies to circumvent it. Methods. Adult New Zealand white rabbits were injected with 108 or 109 viral particles of Ad5-LacZ. At 4 or 16 weeks after Ad5 injection, autologous buffy coats were prepared from peripheral blood, and 4 million cells per side were infected ex vivo for 10 minutes with Ad5-LMP-1 (multiplicity of infection = 4). Cells were implanted on a collagen matrix instead of an autograft for posterolateral lumbar arthrodesis. Unimmunized rabbits served as control subjects. Additional immunized rabbits underwent arthrodesis at 4 weeks with increased cell number (10 million) and viral dose (multiplicity of infection = 10), or with both parameters increased. The rabbits were killed at 4 weeks, and the spines were assessed by palpation and radiograph. A parallel study was performed in athymic rats using immunized rabbits for the donor cells. Results. All the unimmunized rabbits had solid spine fusions. None of the rabbits arthrodesed 4 weeks afterAd5 pre-exposure achieved fusion. At 4 weeks after Ad5 exposure, increasing the multiplicity of infection to 10 did not overcome the immune response (0/3 fused), but increasing the cell number to 10 million (2/3 fused) or increasing both cell number and multiplicity of infection (3/3 fused) did overcome the immune effects. Delaying arthrodesis until 16 weeks after Ad5 pre-exposure also overcame the immune response (3/3 fused). Similar results were seen in the athymic rat ectopic implant model, suggesting that the immune effect was mediated by humoral antibodies rather than a T-cell response. Conclusions. Two model systems were developed that simulate previous exposure to human Ad5 and could separate the cellular and humoral components of the response. There was a dose-dependent inhibition of ex vivo Ad5-LMP-1 gene transfer to cells from animals previously exposed to human Ad5. Data suggested that the inhibition of Ad5 infection was caused by humoral antibodies rather than a T-cell–based response. Minor modifications in the gene transfer protocol, such as doubling the viral dose or number of cells infected, or increasing the infection time, could overcome the immune response for an ex vivo approach.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity

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.

Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

Bone morphogenetic protein 2 (BMP‐2) plays a critical role in the differentiation of precursor cells and has been approved for clinical application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP‐2 have been required to induce bone healing in humans. Thus, enhancing cellular responsiveness to BMP‐2 potentially has critically important clinical implications. BMP responsiveness may be modulated in part by cross‐talk with other signaling pathways, including mitogen‐activated protein kinases (MAPKs). c‐Jun NH2‐terminal kinase (JNK) is a MAPK that has been reported to be required for late‐stage differentiation of preosteoblasts and BMP‐2‐induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3‐E1‐clone 24 cells (MC‐24) can be induced by BMP‐2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both JNK loss‐of‐function and gain‐of‐function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by BMP‐2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP‐2‐induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor (BMPR‐I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP‐2. Understanding this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost‐effective and safe clinical delivery of these important molecules.

Gene therapy for spine fusion

Spine fusion is a commonly performed yet often unsuccessful surgical procedure. As many as 40% of patients undergoing spine fusion may have a nonunion or failure to form a continuous bone bridge. This clinical challenge has focused much of the attention of osteoinductive bone growth factors toward spine applications. Clinical pilot and pivotal trials will show the feasibility of recombinant and purified bone growth factors to promote spine fusion in humans. Despite this, strategies of gene therapy for spine fusion and other bone healing applications are being pursued. This article reviews the state of the art of local gene therapy and highlights specific issues that must be addressed when pursuing a gene therapy program. Perhaps the most critical step in gene therapy for bone formation is choosing an appropriate osteoinductive gene. Such choices may be limited by differences in efficacy of the chosen gene and availability because of proprietary constraints. The choice of delivery vector is crucial and depends on the potency of the gene and the specific application intended. Establishing the effective dose, transduction time, and gene transfer method are important decisions. The choice of carrier material to form the scaffold for the new bone formation is paramount to successful bone formation. Finally, a strategy for in vitro and in vivo testing must be developed to maximize the chances of success in human trials.

Osteoinductive LIM mineralization protein-1 suppresses activation of NF-κB and selectively regulates MAPK pathways in pre-osteoclasts

LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible nitric oxide synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-kappaB), and prevented the phosphorylation of inhibitor of kappa B (IkappaB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-kappaB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-kappaB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways.

Natural antisense transcripts enhance bone formation by increasing sense IFITM5 transcription

Interferon induced transmembrane protein 5 (IFITM5) has been recognized as an osteoblast differentiation factor. Its regulation, however, is still unclear. In this report, four novel naturally occurring antisense transcripts of rat IFITM2 and IFITM5 transcribed from the opposite strand of the IFITM gene locus, were isolated and characterized. They are alternatively transcribed from rat chromosome 1 and expressed at relatively high levels during early differentiation of primary isolates of rat osteoblast cells. There are two common fragments in all of the isoform cDNA sequences that are complimentary to both IFITM2 and IFITM5 respectively. There is an additional unique region in one isoform, immediately downstream of the putative IFITM5 complimentary region, which is also complimentary to IFITM cDNA sequence. Reading frame analysis showed that these antisense transcripts are non protein coding mRNAs. We investigated the expression of these antisense transcripts and their effects on IFITM expression as well as osteoblast differentiation. All isoforms were positively correlated with IFITM5 expression and antisense specific siRNAs inhibited osteoblast differentiation significantly. In contrast, these antisense transcripts had no effect on the expression of IFITM2. We speculate that IFITM5 may be regulated by antisense transcripts.

Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain

Short peptide sequences known as protein transduction domains have become increasingly prevalent as tools to internalize molecules that would otherwise remain extracellular. Here, we determine whether a purified recombinant mammalian intracellular osteogenic factor delivered by a HIV-derived TAT-peptide tag is indeed capable of intracellular localization in a form accessible to interaction with other proteins. We engineered and bacterially expressed a TAT-fusion-cDNA construct of a known osteogenic factor, LIM mineralization protein-1 (LMP-1) involved in the bone morphogenetic protein (BMP) pathway that has the potential to serve as an enhancer of BMP-2 efficacy. The expressed recombinant protein contains an N-terminal (His)(6)-tag, a hemagglutinin(HA)-tag, and an 11-amino acid HIV-derived TAT-membrane transduction domain and was purified to homogeneity by Sephacryl S-100 molecular exclusion and Ni(2+)-affinity chromatography. The purified TAT-LMP-1 protein was chemically labeled with fluorescein, and its time and concentration dependent entry into rabbit blood cells was monitored by flow cytometry. We demonstrate the accumulation of TAT-tagged LMP-1 both in cytoplasmic and nuclear compartments. By performing affinity pull-down assays, we confirm our earlier findings that the recombinant TAT-LMP-1, when used as molecular bait to identify the intracellular binding proteins, interacts with Smurf1, a known binding partner of LMP-1. We also show potentiation of BMP-2 activity using the purified TAT-LMP-1 in mouse muscle C2C12 cells by assaying a heterologous luciferase-reporter construct containing multiple copies of a BMP-responsive sequence motif. Finally, we also confirm the biological activity of the purified TAT-LMP-1 by showing enhancement of BMP-2 induced increase of alkaline phosphatase mRNA and protein by RT-PCR and enzyme activity, respectively.