Philip Trackman - US grants

Gingival fibrosis is a side effect of certain drugs. Fibrosis is characterized by excess extracellular matrix which is not resorbed. This condition, therefore, is treated by periodontal surgery. The biochemical composition of the fibrotic lesions varies, and depends on the drug that induced the pathology. Thus, cyclosporine A leads to collagen-rich fibrotic gingiva, whereas phenytoin and nifedipine lead to increased non- collagenous proteins. Histologic characterization of phenytoin- and nifedipine-induced lesions reveals increased group substance and oxytalan fibers. These fibers contain elastin. I hypothesize that drug induced gingival fibrosis is characterized by increased lysyl oxidase phenytoin and nifedipine-dependent lesions are likely to be elastin-rich. Lysyl oxidase catalyzes the oxidative deamination of lysine residues in collagen and elastin. This ultimately leads to crosslinking and insolubilization of both proteins, rendering them rendering them resistant to proteolysis and resorption. I propose to characterize the regulation of lysyl oxidase, collagen, and elastin mRNA and protein in gingival fibroblasts cultured from normal and drug-treated subjects. This will be accomplished by culturing early passage gingival fibroblasts in the presence and absence of fibrogenic drugs under optimized conditions. The changes in steady state levels of the mRNA's will be compared to changes in mRNA stability, rates of transcription, and rates of protein synthesis for all three gene products. Changes in lysyl oxidase enzyme activity will be measured. The role of prostaglandins in mediating the drug-dependent changes will be established. The ability of retinoic acid and vitamin D3 to restore normal regulation of lysyl oxidase, collagen and elastin will be evaluated. The content of lysyl oxidase, elastin, and collagen in the tissues of normal and drug-treated subjects will be determined to establish the link between in vitro studies and the in vivo pathology.

Lysyl oxidase catalyzes the final extracellular enzymatic step required for cross-linking collagen and elastin in the formation of a mature and functional extracellular matrix. Lysyl oxidase also has tumor suppressor activity in ras-transformed cells. The role of lysyl oxidase enzyme activity in tumor suppression has not been previously investigated. The hypothesis of this proposal is that lysyl oxidase gene expression is required for its tumor suppressor activity, but extracellular lysyl oxidase enzyme activity is not required. Preliminary studies in our laboratory show that phenotypic reversion of c-H-ras-transformed NIH3T3 cells (RS485 cells) by the anti-cancer drug suramin is accompanied by dramatically increased lysyl oxidase mRNA levels, but no increase in enzyme activity. Surprisingly, we found that suramin appears to be a potent inhibitor of lysyl oxidase enzyme activity. Three specific aims are proposed. Aim 1 will establish that lysyl oxidase gene expression is specifically required for phenotypic reversion of RS485 cells by suramin. RS485 cells will be stably transfected with an antisense lysyl oxidase expression vector, and the phenotype of transfected clones grown in the presence and absence of suramin will be determined. The hypothesis predicts that antisense- transfected RS485 cells grown in the presence of suramin will contain low lysyl oxidase mRNA levels and will resist phenotypic reversion. Cell phenotype will be characterized by cell morphology changes, growth rate changes, altered anchorage independent growth, and changes in cell cycle kinetics determined by flow cytometry. In addition, selected transformed oral human tumor cell lines, and selected murine tumor cell lines will be screened for suramin-induced phenotypic reversion, and the specific role of lysyl oxidase mRNA will be established using the same antisense approaches. Aim 2 will rigorously establish that suramin inhibits lysyl oxidase enzyme activity, thus supporting the hypothesis of this proposal. Lysyl oxidase will be purified and suramin inhibition of lysyl oxidase will be characterized as follows: the I50, the reversibility of inhibition, the K1 and kinetics type of inhibition, and whether substrates protect against suramin inhibition. Aim 3 will investigate the molecular mechanisms of tumor suppression by lysyl oxidase by determining whether inhibitors of pro-lysyl oxidase processing and activation prevent suramin-dependent phenotypic reversion of RS485 cells. The notion being tested is that proteolytic fragments of prolysyl oxidase such as the released propeptide, for example, may cause phenotypic changes by a feed-back mechanism. These studies will provide the novel approaches and new insights into mechanisms of tumor suppression relevant to oral cancer, will establish two separate activities for the lysyl oxidase gene, and will define a new class of lysyl oxidase inhibitor.

Significant progress in uncovering many of the molecular events with respect to control of bone formation has added considerably to our understanding of matrix mineralization. It is well established that insoluble collagen accumulation and cross-linking are essential for the development of a functional mineralized extracellular matrix. What is not known is how bone collagen maturation is regulated during development and whether cytokines modulate this process via the same mechanisms. Procollagen C-proteinase and lysyl oxidase have been identified as key players in the extracellular post-translational modification of collagen. In addition to processing procollagen, procollagen C-proteinase cleaves pro-lysyl oxidase into the 32 kDa active enzyme and the 18 kDa propeptide. Lysyl oxidase catalyzes the essential step necessary for collagen cross-linking. Recently, our laboratory has discovered a biological role for the 18 kDa propeptide in matrix mineralization. In Aim 1, the role of regulation of procollagen C-proteinase and lysyl oxidase in the formation of a mineralized extracellular matrix will be investigated in developing fetal rat calvaria osteoblastic cultures. Analyses will include a variety of molecular parameters including mRNA levels, protein levels, and enzyme activity, as well as assessment of extracellular matrix formation by measuring insoluble collagen and inorganic calcium accumulation. The recent development of knockout mice that lack the BPM-1 gene that encodes procollagen C-proteinase provides a supplementary approach to establish the role of procollagen C- proteinase and lysyl oxidase processing on in vitro bone formation. In Aim 2, the cytokine effects of TGF-beta and TNF-alpha on procollagen C- proteinase and lysyl oxidase, collagen synthesis, and collagen accumulation will be determined in developing osteoblastic cultures. In Aim 3, the osteogenic activity of the 18 kDa propeptide will be characterized. For this purpose recombinant lysyl oxidase propeptide will be prepared in a eukaryotic expression system and used to carry out functional studies and to explore receptor mediated mechanisms. The results obtained from these studies will add significantly to the understanding of how extracellular events lead to matrix maturation and mineralization. Once the molecular parameters addressed in this proposal are clearly defined, it may be possible to exploit the information gained for the development of therapeutic approaches in a variety of diseases affecting bone integrity.

[unreadable] DESCRIPTION (provided by applicant): Gingival overgrowth is a side effect of specific medications, and occurs as inherited and idiopathic forms (HGF). The condition impairs mastication, and predisposes affected individuals to systemic complications. Although gingival overgrowth lesions appear clinically similar, our studies have shown that the molecular and cellular features of gingival overgrowth vary as a function of the cause. Phenytoin-induced gingival overgrowth and HGF lesions are highly fibrotic and contain high levels of connective tissue growth factor (CTGF); whereas cyclosporin A induced overgrowth is less fibrotic, and contains low amounts of CTGF; and nifedipine-induced gingival overgrowth is intermediated in all respects. Recent studies have identified tissue specific pathways that provide the mechanism for elevated CTGF expression in fibrotic gingival tissues, and potential therapeutic strategies. In addition, we have published evidence that CTGF promotes extracellular matrix deposition via alpha-6 and beta-1 integrins. Preliminary data suggest that the process of epithelial- mesenchymal transition (EMT) contributes to all forms of gingival overgrowth; and that a critically important matrix metalloproteinase (MMP-13) is down regulated in fibrotic forms of gingival overgrowth. Thus, two aims are proposed. In Aim 1 we will establish that EMT occurs in all forms of human gingival overgrowth in vivo, and we will evaluate the mechanism of inhibitors of EMT to block progression of abnormalities in in vitro studies of primary gingival epithelial cells and fibroblasts. Aim 2 proposes to evaluate the hypothesis that CTGF regulates extracellular collagen processing enzymes and a matrix metalloproteinase (MMP-13), thereby increasing net extracellular matrix accumulation. Proposed studies will determine CTGF stimulated signal transduction pathways and regulated downstream genes that lead to increased extracellular matrix deposition. These studies take advantage of a peptide that inhibits CTGF-dependent extracellular matrix deposition that we have recently identified. The proposed experimental approach utilizes in situ analyses of human gingival overgrowth tissues, and primary cultured human gingival epithelial cells grown in monolayer and three dimensional configurations. Studies will identify novel cellular and molecular pathways that contribute to gingival overgrowth. Findings will have relevance to fibrosis in both oral and non-oral tissues in which CTGF is a contributing factor, thus potentially identifying new therapeutic strategies in various tissues. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Complications of diabetes result in part from elevated serum glucose levels. This leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGE's). AGE's play a significant role in many complications of diabetes. Osteopenia and low mineral density and weak bones is a complication of Type 1 diabetes and is known as "diabetic bone disease". Surprisingly little is known regarding the role of AGE's in modulating bone healing, bone formation, and osteoblast function. For example, the presence and activity of the receptor for advanced glycation end products (RAGE) has not been reported in osteoblasts. The principal hypothesis of the proposed research is that AGE's inhibit bone healing and formation by binding and activating RAGE in osteoblasts. We propose that this results in NFKB activation and transcriptional repression and down-regulation of key osteoblast growth factors and extracellular matrix genes. Aim 1 will measure in vivo the expression of selected growth factors and extracellular matrix products (BMP-1, BMP- 2, BMP-4, and type I collagen) in healing calvaria defects made in diabetic and non-diabetic mice. Diabetes will be induced by the multiple low dose streptozotocin protocol in Balb/c mice; selected studies will be performed in the nonchemically-induced murine diabetic model (NOD strain). The degree of inhibition of bone healing in diabetic animals and expression patterns of RAGE will be determined by quantitative histomorphometric and quantitative immunohistochemical measurements. Studies will directly determine the role of AGE's in diminished diabetic bone formation by local application of AGE's to calvaria defects in non-diabetic mice. The degree to which this mimics diabetic bone will be determined by measuring inhibition of healing and regulation of the same growth factors and extracellular matrix products. Aim 2 will determine in vitro in primary rat osteoblast cell cultures that AGE's inhibit production of osteoblast growth factors and type I collagen via RAGE activated NF-KB. RAGE function blocking antibody studies will identify the AGE/RAGE-dependent NF-KB activation mechanism in the regulation of the specified osteoblast genes. The role of NF-KB activation in down-regulating target osteoblast genes will be directly determined by transfection with the super-repressor 32A/36A IKB-alpha, a potent and specific inhibitor of NF-KB activation. These studies will identify a new mechanism that contributes to diabetic bone disease; and should lead to the identification of new therapeutic treatment targets for this increasingly prevalent clinical condition.

DESCRIPTION (provided by applicant): Diabetic bone disease is a condition in which bones become weak, resulting in diminished mobility, increased risk of foot and other fractures, more severe periodontal diseases, and a generally diminished quality of life. This condition is known to occur mainly in Type I diabetics, although poor bone quality is now understood to occur in Type II diabetics as well. Diabetic bone disease is characterized by diminished osteoblastic or bone synthetic activity. Advanced glycation endproducts (AGEs) accumulate at high levels in diabetic bone, and result from non-enzymatic reactions. TNF-a is a cytokine present at elevated levels in mineralized and non-mineralized tissues in diabetes. AGEs and TNF-a each contribute to complications of diabetes, and each interacts with osteoblast cell surface receptors resulting in increased levels of reactive oxygen species (ROS). Increased ROS stimulates synthesis and nuclear localization of FOXO1, a transcription factor that leads to cell cycle arrest and increased apoptosis. FOXO1 can bind to [unreadable]-catenin, a transcription cofactor that is regulated by the canonical Wnt pathway. The Wnt pathway promotes osteoblast proliferation and differentiation. The major hypothesis to be tested here is that AGEs and TNF-a each stimulate increased levels of active nuclear FOXO1, which competes with TCF/LEF transcription factors for the available pool of [unreadable]-catenin. This is proposed to effectively inhibit Wnt stimulated osteoblast differentiation, and contribute to diabetic osteopenia. Preliminary data supports that deficiency of the extracellular matrix enzyme lysyl oxidase results in poor bone structure that resembles osteopenic diabetic bone, and that lysyl oxidase is up-regulated by the Wnt canonical pathway in osteoblasts. Two specific aims are proposed. Aim 1 will investigate in vitro and in vivo the hypothesis that AGEs and TNF-a each inhibit the canonical Wnt pathway by competing for a limited [unreadable]-catenin pool. Aim 2 will determine the mechanism of Wnt regulation of lysyl oxidase, and the mechanism by which AGE's and TNF-a inhibit Wnt-stimulated lysyl oxidase production in vitro. Aim 2 will further determine in calvaria defects of diabetic mice the degree of down-regulation of the canonical Wnt pathway and LOX expression, and up-regulation of FOXO dependent gene expressions. In vivo studies will utilize analyses of calvaria defects made in non-diabetic and diabetic transgenic mice that express a canonical Wnt pathway responsive promoter that drives the expression of [unreadable]-galactosidase (TOPGAL mouse). This unique combination of calvaria defects made in the TOPGAL reporter mouse model with diabetes induction will provide a novel direct analysis of canonical Wnt pathway activity as a function of diabetes, and as a function of AGE and TNF-a treatments. In vitro studies will be performed using MC3T3 osteoblasts and primary calvaria osteoblasts. Results will provide information that may be ultimately be useful in preventing or reversing effects of diabetes on bone quality. PUBLIC HEALTH RELEVANCE: Osteopenia, or weak bones, occurs as a complication of diabetes. Advanced glycation endproducts (AGE's) and the cytokine TNF-a are elevated in diabetic tissues, and contribute to many of the complications of diabetes. The proposed research investigates the hypothesis that AGE's and TNF-a each inhibit the Wnt canonical pathway in osteoblasts (bone synthetic cells), and thereby inhibit differentiation and development of osteoblasts, and synthesis of lysyl oxidase, an important extracellular matrix enzyme needed for bone formation.

DESCRIPTION (provided by applicant): Oral cancer incidence is rising and is the now the 8th most common form of cancer. Prognosis is poor; at least 50% of patients die within five years of diagnosis. Lysyl oxidase (LOX) is a critically important extracellular matrix protein and enzyme. LOX is synthesized as a 50 kDa pre-proenzyme (Pro-LOX) that is secreted, and then processed extracellularly by procollagen C-proteinases to form active 30 kDa lysyl oxidase enzyme, and the 18 kDa lysyl oxidase propeptide (LOX-PP). The tumor suppressor activity originally attributed to LOX enzyme depends instead on LOX-PP. LOX-PP is a naturally occurring extracellular matrix protein, and likely has limited toxicity while having important anti-cancer activities. Different regions of LOX-PP bind to different targets in its inhibition of RAS-dependen cancer pathways. Some targets are intracellular (c-RAF and tubulin for example), while others are extracellular (FGFR1, for example). It is currently unknown which location and targets of LOX-PP are most important for its tumor suppressor activity. Knowledge regarding the functional location (intracellular or extracellular) is important for formulation of therapeutics based on rLOX-PP. The proposed research will establish which location of rLOX-PP is functional in its ability to inhibit oral cancer phenotype in vitro (Aim 1), and orthotopic oral tuor growth in mice in vivo (Aim 2). Particular attention will be paid to the HSP70 cancer cell marker which is a rLOX-PP binding partner and occurs both extracellularly and intracellularly and participates in RAS-dependent signal transduction and cell transformation. Fusion protein derivatives which are designed to either remain extracellular by fusion with the Fc-domain of IgG4, or are designed for increased cell uptake by fusion with cell penetrating peptide sequences, or palmitic acid, will be created and tested for anti-cancer activities and extracellular/intracellular location in vitro. These rLOX-PP derivatives are expected to have increased in vivo stability. The most active derivative will be systemically administered to mice i which tongue orthotopic tumors have been implanted using UMSCC2 (aggressive) and CAL 27 (less aggressive) oral cancer cell lines and tumor growth and expression of tumor markers and active RAS activity effectors will be determined. It is expected that at least one rLOX-PP derivative with both increased stability and anti-oral tumor growth effectiveness will be identifie. The importance of extracellular compared to intracellular interactions of the HSP70-binding domain of rLOX-PP in particular will be evaluated. Important information will be gained to further refine the design of anti-tumor molecules.

Osteosclerosis is a major complication of primary myelofibrosis (PMF) and is characterized, in addition, by excess proliferation of bone marrow myeloid cells, abnormal connective tissue and fibrosis. Although mutant megakaryocytes (MKs) play a major role in driving connective tissue abnormalities in PMF, molecular mediators which develop between abundant MKs in PMF and increased bone formation from mesenchymal progenitors have not been established. Here, we propose to examine an innovative concept involving the multifunctional enzyme known as lysyl oxidase (LOX) and other MK-derived growth factors as agents which directly promote osteosclerosis in PMF by stimulating the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs). Human PMF is most often caused by clonal expansion promoted by a hyper- activating causative mutation in the JAK2 gene. In Aim 1 we propose to use a mouse model of JAK2V617F? induced PMF to test the new contention that factors elaborated by JAK2V617F MKs impact bone marrow MSCs differentiation to osteoblasts. To this end, JAK2V617F MKs will be tested in co-cultures with wild type MSCs. Based on preliminary and other studies, levels of candidate factors (LOX, PDGF, TGF?, WNT3a) will first be assessed by ELISA assays, and functionality determined with blocking antibodies and proteins, and by LOX pharmacological inhibition. In the spirit of a relatively higher risk R21 grant mechanism, we also propose to measure 110 different cytokines via state-of-the-art Luminex assays, followed by blocking experiments of differentially regulated factors. In Aim 2, we will examine the novel hypothesis that LOX contributes to the pathological bone phenotype in PMF in vivo. To assess this, we will cross breed JAK2V617F mice with LOX+/- mice available to us, and then characterize the bone phenotype of compound mutant mice JAK2V617F/LOX+/- in vivo and in vitro. Taken together, data obtained in this project will determine factors elaborated by associations between JAK2V617F MKs and wild type MSCs which drive osteosclerosis, and which, therefore, will become potential therapeutic targets to address osteosclerosis in PMF.