Autophagy is the process of “cell self-eating” which has been implicated both in cell survival and cell death. Sphingosine kinase 1 (SphK1) regulates the intracellular balance between ceramide and sphingosine, bioactive lipids associated with cell death, and sphingosine-1-phosphate (S1P), whose actions are associated with survival and proliferation. Previous studies have implicated upregulation of SphK1 in the induction of autophagy. In this study, SK1-I, a SphK1 specific competitive inhibitor, induced autophagy in a concentration and time dependent manner in HCT116 colorectal carcinoma cells. This autophagic response was observed to be more intense in wild type p53 expressing HCT116 cells than in p53 null cells and ultimately led to non-apoptotic death in wild type and apoptotic death in p53 null cells. In agreement, cell death in wild type cells was not accompanied by cleavage of polyADP ribose polymerase, a hallmark of apoptosis. Knockdown of Beclin 1 demonstrated that it and its binding partners do not have a significant role in the induction of autophagy in response to SK1-I treatment. Similarly, mTORC1 signaling was not observed. In contrast, SK1-I markedly decreased Akt phosphorylation. However, this might not be the sole factor important for SK1-I induced autophagy, as pharmacological inhibition of Akt only led to a comparatively weak autophagic response. Indeed, phosphorylation of the endoplasmic reticulum (ER) stress marker eIF2 α, was greatly reduced, suggesting that an ER mediated mechanism also contributes to SK1-I induced autophagy. Thus, SK1-I induced autophagy was likely triggered by ER stress signaling and led to non-apoptotic cell death in the more highly autophagic wild type 53 expressing cells. These results suggest that an isotype specific SphK1 inhibitor might be a useful adjunct for the treatment of cancer or other diseases in which enhancement of cytotoxicity or autophagy is desirable.

The cardiovascular system develops through two distinct processes in embryogenesis: vasculogenesis, whereby the primary plexus in the heart is formed along with embryonic and extraembryonic vasculature, and angiogenesis, which begins after vasculogenesis and results in the refinement and maturation of the branched vessel system. In pathological angiogenesis, tumors expand by releasing pro-angiogenic factors in response to hypoxic conditions. The Tie receptors, Tie1 and Tie2, are receptor tyrosine kinases that are integral to angiogenic pathways. A family of Angiopoietins, Ang1-4, have been shown to act as ligands for Tie2, of which Ang1 and Ang2 are best characterized. Activation of the receptor causes dimerization and autophosphorylation, whereby adaptor proteins recognizing the phosphorylated tyrosine activate downstream signaling via their Src homology 2 (SH2) domains. Currently there are no phosphospecific antibodies for Tie2, therefore, identifying critical residues responsible for certain pathways remains difficult. In our study, we aim to use purified SH2 domains of known binding partners to Tie2 to assess the phosphorylation state of the receptor under various cellular conditions and settings, utilizing immunoprecipitation and western blotting. Unexpectedly, we found that Tie2 can bind non-specifically to nickel sepharose when the SH2 proteins were used as antibody mimetics, and was unable to be consistently precipitated in Protein A sepharose when used in conjunction with a monoclonal YFP antibody. Under the latter conditions however we were able to precipitate the SH2 protein itself. When immunoprecipitations were used with cobalt activated IMAC beads, we were able to precipitate Tie2 in overexpressed systems using the SH2 domains of Shp2 N-C and Grb2. As expected, phosphorylation of Tie2 in the presence of its orphan receptor Tie1 was attenuated compared to wild-type levels. Based upon available data, we anticipate this method as a useful tool to assess the phosphorylation state of Tie2 and its signaling pathways in the near future.

The interaction between low-density lipoprotein (LDL) and glycosaminoglycans is a key factor in atherosclerosis. The present study examines the characteristics of LDL and its binding properties with the main glycosaminoglycan of the vascular wall, chondroitin 6- sulphate (C6S). The compositional characteristics that were studied for each LDL sample were phase transition temperature, phospholipid content, free cholesterol content, cholesteryl ester content, triglyceride content, and size. Correlations of these characteristics with LDL-C6S binding were analyzed using a turbidity assay. Our results showed that there is no correlation between LDL-C6S binding and phase transition temperature, triglyceride content, or size. Strong correlations were present for LDL-C6S binding and phospholipid content (P < 0.0001, r2 = 0.4591), free cholesterol content (P < 0.01, r2 = 0.2495), and cholesteryl ester content (P < 0.005, r2 = 0.2952). When values for surface (phospholipids and free cholesterol) and core (cholesteryl esters and triglycerides) lipids were determined a positive correlation was also present with LDL-C6S binding (P < 0.0005, r2 = 0.4172; P < 0.0005, r2 = 0.4282; respectively). These results indicate that large, lipid-rich LDL particles have a higher capacity to bind C6S than smaller, lipid poor LDL. Possible implications for the atherogenicity of LDL are discussed.

KCl is traditionally used as a stimulus to examine the role of increases in cytosolic Ca2+ on the regulation of smooth muscle contraction. KCl bypasses GPCR activation, thereby avoiding activation of additional cell signaling systems, such as phospholipase C and PKC that are inherent to Gaq and Ga12/13 stimulation. GPCR activation causes Ca2+ sensitization (greater force for a given increase in Ca2+) by a ROCK- and PKC-dependent inhibition of myosin light chain (MLC) phosphatase. Recent studies have demonstrated that KCl can also produce Ca2+ sensitization, implying that Ca2+ itself may induce Ca2+ sensitization. To test the hypothesis that Ca2+ can induce Ca2+ sensitization, we permeabilized rabbit femoral artery rings with beta-escin and subjected the tissues to a Ca2+ concentration response curve in the absence (control) and presence of selective inhibitors of ROCK and PKC, the general ser/thr kinase inhibitor, staurosporine, and the MLCK inhibitor, wortmannin. For a comparison, tissues permeabilized with beta-escin were also contracted with the alpha-adrenergic agonist, phenylephrine (PE), and exposed to the same complement of inhibitors. Interestingly, Ca2+-induced contraction was inhibited by a PKC inhibitor in beta-escin-permeabilized tissue. Wortmannin and staurosporine nearly abolished Ca2+-induced contraction. The ROCK inhibitor, H-1152, significantly reduced Ca2+- and PE-induced contractions in beta-escin permeabilized tissues. Western blots showed significant decreases in basal MYPT1-pThr853 by ROCK inhibitors and reduction of MLC-p by ROCK and PKC inhibitors. These data support our hypothesis that elevations in Ca2+ or constitutive ROCK activity may cause ROCK-dependent Ca2+ sensitization. Moreover, data also suggest the possibility of PKC-directed activation of the RhoA/ROCK cascade. Once the mechanism for smooth muscle Ca2+ sensitization is understood, advanced treatments for vascular hyper-contraction disorders, such as vasospasm and hypertension, may be possible.

Previously, we have found that the co-administration of MEK1/2 inhibitors and Chk1 inhibitors synergistically induce multiple myeloma cell apoptosis through upregulation of the BH3-only pro-apoptotic protein Bim. However, these apoptotic events were largely blocked by the characteristic over-expression of Bcl-2 of Bcl-xL in multiple myeloma cells. HA14-1, a small molecule Bcl-2 inhibitor, may therefore circumvent this resistance to apoptosis by blocking Bcl-2 and Bcl-xL anti-apoptotic protein actions. In our project, we hypothesize that the co-administration of HA14-1 with MEK/Chk1 inhibitors will enhance apoptosis in multiple myeloma (MM) cells. To test this hypothesis, we exposed MM cells U266 and RPMI8226, or those cells with Bcl-2 over-expressing stable clones to minimally toxic concentrations of MEK1/2 inhibitor (PD184352) with Chk1 inhibitor (CEP3891) for 24 hours, followed by the Bcl-2 inhibitor (HA14-1). To date, our data indicates that co-administration of HA14-1 with the PD184352/CEP3891 regimen significantly enhances apoptotic death in U266/Bcl-2 multiple myeloma cells compared with the PD184352/CEP3891 regimen. Future studies are designed to elucidate mechanisms underlying Bcl-2 and Bcl-xL anti-apoptotic protein interactions with the Bak and Bim apoptotic proteins, focusing release of Bak and Bim from Bcl-2/Bcl-xL, and subsequent Bax/Bak activation.

Localized Aggressive Periodontitis (LAgP) is an aggressive, early onset form of periodontitis characterized by a unique myeloid cell phenotype. In addition to its bacterial origin, the unique phenotype of the myeloid cell contributes to disease pathogenesis and progression through mechanisms mediating host inflammatory and immune responses. LAgP monocytes synthesize increased levels of the potent proinflammatory lipid mediator, Prostaglandin E2 (PGE2), preferentially differentiate into dendritic cells, and lead to increased IgG2 production. In addition, levels of Platelet Activating Factor (PAF) have shown to be elevated in the gingival tissue and gingival crevicular fluid of subjects with periodontitis. The aim of this study was to further characterize the unique phenotype of the myeloid cell by investigating its role in the increased levels of PAF in periodontitis subjects, examining differences in gene expression of the immune response gene, STATl which is involved in IFN-γ signaling, and by examining the differential expression and function of the scavenger receptor CD36. LAgP monocytes have exhibited decreased activity of the PAF-acetylhydrolase (PAFAH), the catalytic enzyme that breaks down PAF. Since PAF levels are regulated by synthesis and degradation, we hypothesized that synthesis by myeloid cells, monocytes or PMN, also contribute to the increased PAF levels in LAgP. We also hypothesized, based on initial microarray data that myeloid cells have decreased gene expression of STATl and downstream IFNy related genes in LAgP. In addition, based on the initial microarray results, we hypothesized that LAgP monocytes have increased CD36 expression with increased capacity for the binding and uptake of chemically modified versions of LDL. Monocytes were isolated from the peripheral blood of LAgP and NP control subjects over a Ficoll gradient. A radiolabeled PAF assay was used to quantify total PAF synthesis in both resting monocytes and PMN, and in monocytes and PMN stimulated with calcium ionophore A23 187. Quantitative RT-PCR was used to quantify STATl and CD36 gene expression from RNA isolated from adherent monocytes, and CD36 expression and AcLDL (acetylated LDL) uptake was quantified using flow cytometry. Our results indicate that PAF synthesis is increased in LAgP PMN but not in monocytes. LAgP monocytes synthesize less PAF compared to NP control, and their response to calcium ionophore A23 187 (IoA), expressed as fold increase, was blunted. LAgP and NP monocytes did not differ in STATl gene expression as determined by quantitative RT-PCR, and CD36 experiments suggest the possibility that dendritic cells express increased scavenger receptor CD36 than macrophage cells. In conclusion, LAgP myeloid cells are unique in their response to A23 187, and LAgP PMN contribute to increased PAF primarily through synthesis, whereas the LAgP monocytes contributes to elevated PAF through decreased catabolism. STAT1 gene expression did not differ between LAgP and NP monocytes, however this does not rule out the possibility of differential STATl signaling in LAgP monocytes though inhibitory proteins or differential phosphorylation of STATl. Finally, CD36 expression appears from preliminary data to be increased in dendritic cells. These findings add to the current understanding of the unique phenotype of the LAgP monoctye and further experiments will continue to expand our understanding of how unique biology of myeloid cells and their ability to facilitate crosstalk between the innate and adaptive immune system, and the host inflammatory system.

Epithelial to mesenchymal transition occurs in the later stages of epithelial tumor progression, with cells expressing mesenchymal markers. Of these, the intermediate filament protein vimentin is frequently upregulated in metastatic carcinomas. Previously, microarray studies showed that the gene encoding vimentin is highly upregulated in metastatic HN12 cells compared to a related primary tumor cell line. In this study, we confirmed this difference using real-time quantitative PCR, western blot analysis, and immunostaining. Furthermore, EGF and TGF-β, growth factors that induce migration and invasion of HN12 cells, produced synergistic increases in vimentin expression. To assess the contribution of vimentin to the biological properties, HN12 cells were stably transfected with a plasmid that directs synthesis of vimentin shRNA. Clones expressing decreased amounts of vimentin were isolated and characterized. These cells showed significantly reduced proliferation compared to non-targeting controls. Moreover, downregulation of vimentin led to a decrease in cell motility, as well as reducing their ability to invade through a basement membrane substitute. Using transient transfection assays, vimentin promoter activity was determined in HN12 cells to define regulatory elements important for controlling vimentin upregulation in the absence or presence of EGF and TGF-β. Taken together, the data indicate that overexpression of vimentin is important for proliferation and invasion of metastatic HN12 cells, and suggest that EGF- dependent pathways target binding elements in the proximal vimentin promoter, while TGF-β is likely to act in an AP1-dependent manner. Furthermore, both growth factors appear to synergize by stimulating promoter activation through the ASE site, suggesting involvement of Stat-dependent pathways in regulation of vimentin expression in HN12 cells.

Much of our current understanding regarding trauma, mechanisms of healing, and treatment strategies have evolved as a result of injuries suffered during armed conflict. While our understanding of these processes has advanced during and since these conflicts, treatment methods of traumatic wound healing have failed to progress significantly in the last forty years. The overall objective of this research was to test the hypothesis that the immune regulating hormone Androstenediol (AED) modulates the host’s immune system to promote wound healing under conditions where it has been inhibited by stress and infection. In an effort to characterize the effects of Androstenediol on healing following trauma, this research focused on strategies to evaluate which levels of trauma, immunosuppressive agents, and Androstenediol are required to reverse inhibition of healing. Sprague-Dawley rats were assessed for their response to trauma and intervention through assessment of white blood cell levels, cytokine and chemokine expression, and quantification of wound closure. While these studies did provide some trends reflecting modulation of cell counts and protein expression following AED treatment in immune-suppressed animals, measurement of wound closure failed to reveal a significant response.

Musculoskeletal diseases, in particular osteoporosis, are increasingly becoming more prevalent in the U.S. due to the ageing population (Figure1). It is estimated that one-sixth of 300 million people in U.S. suffer from bone disorders or loss. About 10 million of those people above age 50 suffer from osteoporosis. Patients that suffer from osteoporosis have high morbidity and mortality rates. For instance, patients have decreased bone mineral density (BMD), a measurement of bone density that reflects the strength of bone as represented by calcium content. A decrease in BMD typically leads to an increased risk of bone fractures. In particular, hip fractures have an associated 20% mortality rate 1 year after injury among senior citizens 1. Patients that suffer from musculoskeletal diseases and from bone injuries, not associated with disease, account for 130 million hospital visit per year. Not to mention, 245 billion dollars of healthcare expenditure 2. Over that last 30 years, there has been much improvement in the field of bone research and its application to medicine. It has changed the quality of life and prolonged the life expectancy of patients suffering from bone disease. However, many details remain unknown about the underlying mechanism that control bone metabolism, formation, and healing. Furthermore, current effective therapies to combat bone disorders have limitations including unwanted side effects and prohibitive costs. For example, treatment with glucocorticoids which is a known inducer of osteoblastogenesis in vitro has been shown to produce an osteoporotic phenotype in vivo. Recognizing the importance of bone health and its affordability to the public makes the advancement of therapeutic targets work worth doing. Work in this field will eventually lead to the prevention, treatment, and cure for bone disease. A potential therapeutic candidate that maybe involved directly or indirectly with bone formation is secreted phosphoprotein-24 (Spp24). The following research aims to establish an importance and role for Spp24 in bone differentiation. A novel antibody that detects Spp24 which we have developed and characterized, has allowed us to feasibly study the protein. Our results demonstrate localization of Spp24 in different tissue, the processing of the protein during osteoblastogenesis, and have allowed us to conceptualize possible functions based on our data.

This dissertation sheds light on several novel mechanistic findings in astrocyte specific gene regulation and function by the NFI-X transcription factor which can be further extrapolated to astrocyte differentiation and glial tumor invasion. First, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including GFAP reporter, in astrocytes and glioma cells. The expression of NFI-X3 is dramatically upregulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers such as GFAP and SPARCL1. Overexpression of NFI-X3 dramatically upregulates GFAP and SPARCL1 expression in glioma cells, while the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, the TA domain of NFI-X3 activates GFAP expression by inducing alteration in the +1 nucleosome architecture that lead to the increased recruitment of RNA polymerase II. Thus, we propose that NFI-X3 is the major isoform of NFI-X regulating astrocyte specific gene expression during their differentiation, likely via nucleosomal remodeling of the astrocyte specific promoters. NFI-X knock-out animals display severe neuroanatomical defects including partial agenesis of the corpus callosum and hydrocephalus, however the target genes of NFI-X in the CNS remained elusive. Here, we show for the first time that YKL-40 is a novel target gene of NFI-X in astrocytes and controls their migration. In addition, we report that YKL-40 expression is activated during mouse brain development and also during the differentiation of neural progenitors into astrocytes in vitro. In primary astrocytes, YKL-40 expression is controlled by nuclear factor I-X (NFI-X) and signal transducer and activator of transcription 3 (STAT3), which are known to regulate gliogenesis. Indeed, knock-down of NFI-X and STAT3 significantly reduced YKL-40 expression in astrocytes, while overexpression of NFI-X3 (a splice isoform of NFI-X) dramatically induced YKL-40 expression in glioma cells. In addition, activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes. Interestingly, STAT3 activated YKL-40 expression via its binding site located in the YKL-40 proximal promoter, whereas direct NFI-X binding had not been detected. Surprisingly, NFI-X and STAT3 physically interact and this complex likely regulates YKL-40 expression in astrocytes. We further show that NFI-X controls migration and invasion of astrocytes and glioma cells, respectively, by regulating YKL-40 expression. These novel data suggest that YKL-40 is expressed by astrocytes during brain development and controls astrocyte migration. Since YKL-40 is used as a shared biomarker for ongoing inflammation and oncogenic transformation and its (high) levels correlate to the severity of disease, we have tested its expression in astrocytes and microglia (CNS macrophage) after treatment of various neuro-inflammatory cytokines. Here we report, that IL-1 and IL-6/OSM synergistically activate YKL-40 expression in astrocytes but not in microglia when added together. Furthermore, induced YKL-40 expression can be detected in the media from astrocytes but not from microglia. Since YKL-40 is a secreted molecule and is highly upregulated in CSF of multiple sclerosis patients, we have tested its role in oligodendrocyte differentiation. Preliminary observations clearly demonstrate that YKL-40 inhibits myelin basic protein (MBP) expression during the in-vitro differentiation of oligodendrocyte progenitor cells into myelin producing oligodendrocytes. Thus, we propose that YKL-40 is produced and secreted by reactive astrocytes during various CNS pathologies, and may inhibit MBP expression in MS. In summary, these studies have identified novel mechanisms in astrocyte gene regulation and functions, and provided new insights into astrocyte biology, with the implications for further understanding of the development and progression of CNS pathology.