2001 — 2003 |
Safadi, Fayez F |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
The Role of Cdk4 in Bone Formation
For normal skeletal growth and development the relationship between cell proliferation and differentiation must be tightly regulated. Osteoblasts are committed to enter the cell cycle and proliferate under specific regulatory signals, then differentiate and product mineralized matrix. Various cyclins and cyclin dependent kinases (CDKs) have been postulated to play a role in osteoblast proliferation and differentiation. Among these kinases is CDK4, a cell cycle protein that plays a major role in the transition of cells from Go-phase (quiescent) to S-phase (DNA synthesis) of the cell cycle. The proposed studies will examine the role of CDK4 deficiency on osteoblast development and function using a mouse model null for CDK4. Previous studies showed that CDK4-/-mice compared to age-matched, wild-type (+/+) mice. Densitometry by peripheral quantitative computerized tomography (pQCT) showed a reduction in trabecular bone density in CDK4 -/- compared to +/+ mice. Aim 1 will confirm and extend these preliminary findings in CDK4 -/- and +/+ mice at different ages. Aim 2 will examine the expression of osteoblast-related genes and the role of CDK4 on cell proliferation and apoptosis in vivo and in primary osteoblast cultures derived from +/+ and -/- mice. Aim 3 will determine to what extent the diabetic condition exacerbates the bone abnormalities in CDK4 -/- mice, especially in older animals. Experiments will attempt to rescue the CDK4 -/- mice by treating them with insulin and also determine what role IGF-I may play in mediating these bone defects. We hypothesize that CDK4 plays a direct role in regulating osteoblast development and function, and the diabetes may accelerate the bone loss in older CDK4 -/- mice. Information generated from the proposed studies may be useful in developing novel therapeutic strategies to modulate or increase bone formation in certain osteopenic conditions or during fracture repair.
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1 |
2002 — 2006 |
Safadi, Fayez F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Osteoactivin in Osteoblast Development and Function
DESCRIPTION (provided by applicant): In a study examining differential gene expression in bone from normal and osteopetrotic (op) rats, we discovered a novel cDNA, termed osteoactivin (OA) that was over-expressed in op when compared to normal bone. Subsequent in situ hybridization and immunohistochemistry studies demonstrated that OA mRNA and protein are expressed by osteoblasts. In primary osteoblast cultures, OA mRNA levels exhibited a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization. Furthermore, the protein is synthesized by osteoblasts and secreted into the medium. Using an OA-anti-sense oligonucleotide, we were able to block OA expression. Down-regulation of OA expression inhibited osteoblast differentiation and function including decreased alkaline phosphatase activity, osteocalcin production and matrix mineralization. A CMV-rOA construct was generated and used to examine the effect of OA over-expression on osteoblast development and function in osteoblast cultures. OA over-expression increased nodule formation, alkaline phosphatase activity, osteocalcin production and matrix mineralization. Using a local injection model to test the anabolic effect of a small peptide of OA (OA-P) in vivo, OA-P induced a potent osteogenic response. We hypothesize that OA is an anabolic bone growth factor. We propose that OA is synthesized and secreted by osteoblasts, where it acts as an ECM-associated signaling molecule or a soluble factor to promote osteoblast differentiation and function Experiments proposed in this application will focus on the association between OA and bone with the following aims. Experiments in aim I will provide a more comprehensive evaluation on the effects of down-regulation of OA expression using an OA-anti-sense oligo transfection approach. OA over-expression will be achieved using a CMV-rOA construct and its effects on osteoblast development and function will be evaluated. Studies in aim 2 will examine the effects of recombinant OA (rOA) or OA-P on osteoblast development and function in vitro and bone formation in vivo. Studies in aim 3 will characterize rOA binding to osteoblasts and determine whether integrins serve as receptors for OA on osteoblasts. In aim 4, studies will examine transcriptional regulation of OA by three osteotropic factors, 1,25(OH)2D3, BMP-2 and TGF-beta1. Additional experiments will determine whether specific effects of BMP-2 on osteoblast differentiation are OA-dependent. Experiments in the first part of aim 5 will focus on the generation of OA null (-/-) mice and characterization of their skeletal phenotype. We will also examine the ability of OA -/- osteoblasts to differentiate in vitro. In the final part of this aim, we propose to generate transgenic mice expressing OA under control of the osteocalcin promoter and evaluate whether osteoblast-specific expression has an anabolic effect on bone. The identification of a novel anabolic agent in bone and elucidation of its mechanism(s) of action will eventually lead to the development of new therapeutic strategies to selectively stimulate osteogenesis in diseases associated with bone loss and in fracture repair.
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1 |
2009 — 2013 |
Safadi, Fayez F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Osteoactivin in Osteoblast Development and Function @ Temple Univ of the Commonwealth
DESCRIPTION (provided by applicant): From a study in which we discovered osteoactivin (OA) in bone, we demonstrated that OA mRNA and protein are expressed in osteoblasts and its expression exhibited a temporal pattern being at highest levels during the later stages of matrix maturation and mineralization. Furthermore, the protein is synthesized, processed, glycosylated and secreted by osteoblasts. Using gain-of-function and loss-of-function approaches in osteoblasts, we found that down-regulation of OA decreased osteoblast differentiation and function and over-expression increased osteoblast differentiation and function in vitro. We also demonstrated that the secreted form of OA regulates osteoblast differentiation and function. Treatment with recombinant OA promotes bone formation in vivo. The importance of OA in osteogenesis was confirmed in mice with the null allele for OA and in mice with a natural mutation in the OA gene caused a premature stop codon that results in the generation of a truncated OA protein. Both of these mice exhibit a skeletal phenotype associated with decreased bone mass. During the previous funding period and since the last submission, we established colonies of OA KO and OA mut. mice, and also generated transgenic (Tg) mice that over-express OA in bone. Preliminary data from OA KO, OA mut. and Tg mice support the hypothesis that OA is a novel bone anabolic factor which is synthesized and secreted by osteoblasts, and acts either as an ECM-associated signaling molecule or downstream of BMP2 to regulate osteoblast differentiation and function. In addition to its effects on osteoblasts, we present data showing that OA affects osteoclast differentiation, and we hypothesize that these abnormalities are secondary to altered production of osteoclastogenic factors (e.g. RANKL) by stromal cells/osteoblasts in the bone microenvironment. Studies proposed in aim 1 will evaluate the effects of OA deficiency (OA KO), truncated OA (OA mut.) or OA over-expression (Tg) on bone in vivo, and assess the differentiation and function of bone cells (osteoblasts and osteoclasts) derived from these mice in primary cultures. The presence of various domains in OA might reflect different functions, and evaluation the structure/function relationship and role of the various domains of OA on normal osteoblast differentiation will be investigated in aim 2 of this application. During the previous funding period, we also showed that the secreted isoform of OA can function as an ECM-associated (matricellular) protein and demonstrated that osteoblasts attach to OA via the av[unreadable]1 integrin, resulting in the formation of focal adhesions, cytoskeletal reorganization and the activation of FAK. Studies proposed in aim 3 will test the hypothesis that OA acts as a matricellular protein that binds to specific cell surface integrins on osteoblasts to initiate integrin-activated signaling, cytoskeletal reorganization, and regulate cell function. We recently demonstrated that BMP2 regulates OA expression and that OA is a downstream mediator of BMP2-induced osteoblast function, a response that is mediated by the Smad signaling pathway. We present preliminary data that BMP2 stimulates the recruitment of Smad1, Dlx5 and CBP to the OA promoter and this effect is dependent on the stage of osteoblast differentiation. Studies proposed in aim 4 will investigate the mechanism whereby OA acts as a downstream mediator of BMP2-induced osteoblast differentiation and function, and will evaluate the effects of BMP2 in stimulating the recruitment of Smad1, homeodomain proteins, and CBP co-activators to the OA promoter for transcriptional regulation during osteoblast differentiation. Proposed experiments are expected to generate novel information regarding the effects of OA deficiency or over-expression on bone formation/remodeling in vivo, its mechanisms of action in osteoblasts, and the molecular requirements for OA induction by BMP2 in osteoblasts. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs in the Untied States from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. Osteoactivin is a novel growth factor in bone and the proposed studies will generate new information regarding its effects and mechanisms of action on bone cell development and function. Once we understand its full effects on bone and how it works to promote bone formation, this information will be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with clinically significant bone loss.
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0.961 |
2020 — 2021 |
Safadi, Fayez F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Trappc9 in Osteoclast Differentiation and Function @ Northeast Ohio Medical University
Intellectual disability (ID) results from developmental conditions that are characterized by significant deficits in intellectual functioning with a prevalence of 1-3% in general population. In most cases, patients with ID also present with skeletal abnormalities. Recent studies identified several different nonsense mutations in the trafficking protein particle complex (TRAPPC9) gene in both consanguineous and non-consanguineous families. Patients with TRAPPC9 mutations share phenotypic features including unique facial appearance, skeletal abnormalities, moderate-to-severe ID, highlighting the importance of TRAPPC9 in both brain and skeletal development. However, its precise role in normal skeletogenesis remains obscure. TRAPPC9 is known as NIBP [NFkB inducing kinase (NIK) and IkB kinase 2 (IKK2) binding protein]. It enhances cytokine- induced NFkB activation by increasing the kinase activities of IKK2 and NIK, modulating both the canonical and non-canonical NFkB signaling. In addition, TRAPPC9 is also critical to intracellular vesicular trafficking. The relative importance of NFkB signaling and vesicular trafficking to the skeletal phenotype in TRAPPC9 patients is unknown. In this application, we propose to study the role of TRAPPC9 in normal bone physiology and its role in osteoclast (OC) differentiation and function. Our preliminary studies show that TRAPPC9 is expressed in bone cells and binds NIK and IKK2 in OC in vitro. Functional knockdown of TRAPPC9 in OC leads to defective OC differentiation and function. In addition, floxed-TRAPPC9 transgenic mice crossed with myeloid-specific Cre (LysM) mice, develop osteopetrosis. Together these results strongly suggest TRAPPC9 is a positive regulator of osteoclastogenesis. TRAPPC9 also mediates vesicular trafficking. Here we present data that TRAPPC9 binds to L-plastin and regulates actin-ring formation and OC function. These findings and others presented in this application, prompted us to hypothesize that TRAPPC9 regulates osteoclast differentiation and function via modulation of NFkB-dependent and unrelated signaling pathways. To test our hypothesis, we propose to examine the functional role of TRAPPC9 using conditional null mice in OC lineage. In addition, we will determine the mechanisms by which TRAPPC9 modulates NFkB-dependent signaling in OC. Aim 1 will focus on characterization of the skeletal phenotype of TRAPPC9 conditional knockout mice and will examine the impact of TRAPPC9 deficiency on OC differentiation and function. Aim 2 will elucidate the molecular mechanism by which TRAPPC9 regulates of NFkB signaling and determine the physiological role of TRAPPC9 in osteoporosis. Aim 3 will examine the role of TRAPPC9 in OC polarization and function. In sum, these experiments will decipher the role of TRAPPC9 in regulating bone mass and the mechanisms by which TRAPPC9 regulates NFkB signaling. The successful accomplishment of this project will generate new clues to enhance our knowledge of NFkB signaling in bone cells and the development of therapeutic modalities for bone loss diseases.
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0.961 |
2021 |
Ball, Hope Safadi, Fayez F |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
The Therapeutic Role of Gpnmb in Osteoarthritis
Osteoarthritis (OA) is a debilitating degenerative joint disease causing chronic joint pain and disability in 54 million adults in the US. OA can result from either chronic joint use (degenerative or age-related) or from trauma (post- traumatic). At present, there is no disease modifying agent to cure or treat the disease. Clinical management focuses on weight loss, NSAIDs, corticosteroids or HA injections, and other alternative therapies aimed at reducing joints pain and immobility. The final treatment, arthroplasty, is irreversible and requires revisional surgery in 10-15 years. Together, surgical and non-surgical treatments generate $27billion in healthcare costs per year. These socioeconomic burdens highlight the critical need for novel treatments to prevent or delay the cartilage damage caused by OA. Here, we propose a novel therapeutic candidate, osteoactivin (Gpnmb), a type I transmembrane glycoprotein expressed in various cell types with anti-inflammatory and chondroprotective properties. Preliminary studies presented in this application show Gpnmb is highly expressed in high-grade human osteoarthritic cartilage. When human HTB-94 chondrocytes were treated with recombinant Gpnmb protein (rGpnmb) followed by IL-1b stimulation, treated cells demonstrated reduced expression of catabolic markers MMP-9, MMP-13, and IL-6. Furthermore, rGpnmb treatment inhibited matrix degradation ex vivo in human cartilage explants. In vivo, intra-articular injection of rGpnmb in C57BL/6 mouse joints mitigated and prevented cartilage loss in an induced post-traumatic model of OA (destabilization of the medial meniscus, DMM). We determined that Gpnmb acts via interactions in the CD44 receptor in glial cells, macrophages and chondrocytes and that CD44-null mice (CD44-/-) developed severe joint damage using the DMM model compared to WT littermates. Finally, we present that Gpnmb assists in slowing the progression of age-related murine OA. Our lab recently identified a small Gpnmb peptide (Gpnmb-p) with the same anti-inflammatory and biologic properties as rGpnmb. This is significant since peptides are highly selective, potent, and cheaper to produce. Peptides also decrease the potential for toxicity and accumulative problems than the whole protein. Therefore, in this Phase-I SBIR, we propose to evaluate efficacy and safety of rGpnmb and Gpnmb-p to mitigate and treat inflammation and articular cartilage degradation and loss in OA. In aim one, we will assess the safety and efficacy of rGpnmb and Gpnmb- p for the treatment of induced post-traumatic osteoarthritis (PT-OA) induced via the DMM model. In aim two, we will assess the efficacy of rGpnmb and Gpnmb-p for the treatment of age-induced (degenerative) osteoarthritis in mice. For both aims, we will evaluate articular cartilage and matrix degradation using histological and imaging analyses. Successful completion of this work will demonstrate the potential therapeutic value of Gpnmb for the OA treatment with possible extension to other applications.
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0.907 |