• Altered cellular chemical environments in apoptosis and hypoxia induce matriptase zymogen activation

      Moore, Sean Michael; Lin, Chen-Yong (2012)
      Alterations in the cellular chemical environment, such as changes in pH, changes in ion concentrations, and changes in the oxidative state, are associated with both ongoing physiological activity and pathophysiological processes. This research focused on how such changes in the cellular chemical environment affect the activation of matriptase, a membrane-bound serine protease. Manipulating the cellular chemical environment of mammary epithelial cells revealed that the activation of matriptase from a zymogen to an active enzyme can be rapidly induced by a decreased pH level, or an increased oxidative state. Changing the pH from a physiological level (pH 7.4) to a mildly acidic level (pH 6.0) accelerated matriptase activation by shortening the time for the onset of activation from around 30-40 minutes to 2-3 minutes. Matriptase activation was significantly attenuated by the reductive state of the cytosol. These mechanisms likely prevent premature matriptase activation when trafficking through the increasingly acidified secretory pathway en route to the cell membrane. Similar attenuation was seen in the presence of extracellular physiological concentrations of chloride ions. This mechanism aids in matriptase regulation at the cell surface. The control of matriptase activation by acidity and redox state gives matriptase-expressing cells the ability to activate matriptase proteolysis when the cellular chemical environment changes. Shift to a more acidic and oxidized chemical environment are seen during stressful cellular events such as hypoxia and apoptosis. Therefore, it was hypothesized that these cellular events would induce matriptase activation. Cobalt chloride (CoCl2)-induced hypoxia and doxorubicin-mediated apoptosis each lead to increased matriptase activation. This activation was significantly suppressed by the antioxidant N-acetylcysteine (NAC), which further confirmed the stimulating role of an oxidized state in matriptase activation. The unique regulation of matriptase autoactivation by acidity and redox state makes matriptase a new candidate sensor for a variety of cellular processes under both physiologic and pathophysiological conditions.
    • A novel role for Semaphorin 4D in tumor metastasis

      Binmadi, Nada Omar; Basile, John R. (2011)
      The semaphorin proteins were originally identified as axonal guidance factors expressed during neuronal development. In addition to this function, several semaphorins are now known to play diverse roles outside of the nervous system. Semaphorin 4D (Sema4D/CD100), a transmembrane molecule that shares structural homology with the scatter factors, exerts important biological effects on a variety of cells, including neural, epithelial and immune cells. Interaction between Sema4D and its receptor, Plexin-B1, has proven to be important in many facets of tumor progression such as tumor angiogenesis, regulation of tumor-associated macrophages and control of invasive growth. Invasion and metastasis are key components of cancer progression. A distinct and largely forgotten path for tumor spread is perineural invasion (PNI), defined as the presence of cancer cells in the perineural space. PNI is frequently used by many human carcinomas, in particular by pancreatic, prostate, and oral squamous cell carcinoma (OSCC), and is associated with tumor recurrence and pain in patients with advanced disease. The work presented in this thesis shows that Sema4D and Plexin-B1 are involved in PNI in tumors and identifies Sema4D as a new player in the complex interaction between tumor cells, nerve cells and the tumor microenvironment. We have previously found that Sema4D is able to promote angiogenesis in several in vitro assays and tumor growth and vascularity of head and neck squamous cell carcinoma xenografts in vivo, but the intracellular signaling pathways engaged and the mechanisms of regulation of this pro-angiogenic function remained unclear. Results presented in the second part of this thesis demonstrate that ligation of Plexin-B1 by Sema4D activates a RhoA-dependent pro-angiogenic phenotype in endothelial cells that signals through phosphatidylinositol 4-phosphate 5-kinase (PI(4)P5K) and generates lipid second messengers. We also discuss the essential role of hypoxia inducible factor (HIF) in endothelial cell migration and tumor vascularity. In conclusion, deciphering the regulatory and signaling mechanisms of Sema4D could lead to the identification of new therapeutic targets for anti-angiogenic and anti-metastatic cancer therapy.
    • Regulation of vascular endothelial growth factor gene expression by hypoxia

      Forsythe, Jo Ann; Koos, Robert D. (1996)
      Vascular endothelial growth factor (VEGF), a specific endothelial cell mitogen and regulator of microvascular permeability plays a central role in angiogenesis. Hypoxia stimulates VEGF gene expression in a variety of cultured cell types. The ovary is one of the few sites where angiogenesis normally occurs. Granulosa cells are probably exposed to hypoxic conditions during follicular development; this environment likely leads to enhanced VEGF secretion. Increased VEGF expression may play a role in the processes of ovulation and corpus luteum development. This dissertation investigates the hypoxic induction of VEGF expression in granulosa cells and characterizes the hypoxia response element in the VEGF gene.Using reverse transcription-polymerase chain reaction (RT-PCR), we have shown that rat ovarian granulosa cells exposed to hypoxia respond by increasing steady-state levels of VEGF mRNA. This increase is apparent within 3 h and is maximal by 12 h. Reducing oxygen from 20% to just 15 or 10% induces an increase in VEGF message. Maximal induction is seen at 5, 2 or 1% O{dollar}\sb2{dollar}.;Apart from the female reproductive system, neovascularization in adults is essentially limited to disease states. Tumor growth, characterized by the development of hypoxic regions, is angiogenesis-dependent. VEGF is secreted at high levels by tumors of various origins. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric protein known to be a transcriptional activator of the human erythropoietin gene in hypoxic cells. We show the involvement of HIF-1 in activating VEGF transcription in Hep3B cells. VEGF sequences mediated transcriptional activation of luciferase (luc) reporter gene expression in response to hypoxia in Hep3B cells. A 47-base pair (bp) sequence, located 985 to 939 bp upstream of the VEGF transcription initiation site, mediated hypoxia-inducible gene expression. Reporters containing VEGF sequences, in the context of either the native VEGF or the heterologous SV40 promoter, were co-transfected with expression vectors encoding HIF-1{dollar}\alpha{dollar} and HIF-1{dollar}\beta{dollar}. Luc expression was significantly increased in both hypoxic and non-hypoxic cells relative to cells transfected with reporters alone. A HIF-1 binding site was identified in the 47-bp hypoxia response element. A 3-bp substitution eliminated HIF-1 mediated transcriptional activation in response to hypoxia and/or recombinant HIF-1. Co-transfection of a luc reporter containing the 47-bp element with a vector expressing a dominant negative form of HIF-1{dollar}\alpha{dollar} inhibited transcriptional activation in hypoxic cells in a dose-dependent manner. These data indicate that VEGF expression responds rapidly to a reduction in oxygen and that this response is partly due to an increase in transcription mediated by HIF-1.