Yan Zhu - US grants

Nitric oxide is an endogenous vasodilator with an important role in the control of vascular smooth muscle cell tone. Nitric oxide and related nitrovasodilators act by stimulating guanylate cyclase and elevating cyclic GMP (cGMP), but the mechanism by which increased cGMP leads to vasodilation is not fully understood. To identify potential cGMP- binding proteins in VSMC, I have constructed and screened a human coronary artery smooth muscle cell cDNA library and isolated a novel 3.7 Kb cDNA clone that encodes a novel cDNA for a putative cGMP-gated ion channel. This proposal seeks to test the hypothesis that this cDNA encodes a functional ion channel important to the physiology of cGMP- mediated VSMC relaxation. Aim I. Functional expression and characterization of the Human VSMC cGMP-gated channel cDNA : The electrophysiological properties of the cloned channel will be studied by patch-clamp recordings of transfected cells to define the ligand specificity, ion selectivity, and pharmacologic response to known channel blockers. In addition, mutant channels with altered cGMP- binding domain and pore region will be generated by site-directed mutagenesis and antibodies to the CASMC channel will be prepared for use in subsequent functional studies. Aim II. Identification and characterization of the native cGMP-gated channel in VSMC: Northern blot analysis and in situ hybridization will be used to determine both the pattern of channel expression in tissues and its cellular localization. Patch-clamp recordings will then be used to characterize the native channel in freshly isolated vascular smooth muscle cells and to understand any functional differences between the recombinantly expressed channel and the channel in its native milieu. Aim III. Physiological role of the cGMP-gated channel in VSMC function: The physiological role of the cGMP-gated channel in vascular tone will be explored by (i) electrophysiologic studies of vascular smooth muscle cells in which the cloned CNG channel or mutants of the channel are overexpressed; and (ii) studies using an ex vivo vascular ring model and channel-specific inhibitors, as well as antibodies and channel- derived peptides. Finally, wild-type and dominant-negative mutant forms of the channel will be introduced into vessels of intact animals by adenoviral methods and the relaxation properties of blood vessel then will be studied ex vivo with the vascular ring model. These studies will explore a new pathway for cGMP regulation of vascular tone and are thus directly relevant to the study of vascular diseases and their treatment.

DESCRIPTION: (Adapted from investigator's abstract) Ischemic vascular diseases are the leading cause of morbidity and mortality in women in our society, but are uncommon prior to menopause. In addition, post-menopausal estrogen replacement decreases vascular events in women. It is now appreciated that estrogen has direct effects on the blood vessel wall that likely contribute to the differences in vascular diseases and their treatment between men and women. Our laboratory has recently (i) demonstrated human vascular smooth muscle cells contain a functional estrogen receptor; (ii) identified novel estrogen-regulated genes in vascular smooth muscle cells; and (iii) shown that physiologic estrogen replacement markedly suppresses the vascular response-to-injury in a mouse carotid artery model. These data support the hypothesis that estrogen inhibits smooth muscle cell proliferation following vascular injury via estrogen receptor-mediated changes in endothelial cell and/or vascular smooth muscle cell gene expression. The Specific Aims of this proposal include: (1) Investigation of the mechanism by which estrogen inhibits the response-to injury in the mouse carotid model, using morphometric, immunohistochemical, biochemical and molecular methods to study (a) the effects of estrogen on endothelial cell growth; (b) the carotid response-to-injury in transgenic mice in which the estrogen receptor has been disrupted; (c) the effect of estrogen receptor antagonists on estrogen inhibition of vascular injury; and (d) the effects of estrogen on carotid injury in normal and transgenic male mice; (2) Investigation of the mechanisms by which estrogen inhibits the response-to-injury in the porcine femoral injury model using morphometric, immunohistochemical, and molecular methods to study the effects of estrogen on vascular cell growth and the injury response; and (3) Further investigation of the molecular mechanisms of estrogen's inhibitory effects in cells and vascular tissue from these animal studies, including (a) study of vascular endothelial growth factor (VEGF) in vascular tissue from the murine and porcine studies, using immunohistochemistry and in situ hybridization; (b) study of both hormone-independent (TAF-1 domain) and hormone-dependent (TAF-2 domain) estrogen receptor activation of the VEGF gene in vascular cells; and (c) use of subtractive hybridization methods to identify novel estrogen-regulated genes from cells and tissues obtained in the animal experiments. These studies will contribute to our understanding of molecular mechanisms important in the vascular injury response, and to our understanding of the pathophysiology and treatment of vascular disorders in both women and men.

ABSTRACT The p53 tumor suppressor serves as one of the major cellular barriers against cancer development. p53 is controlled by its negative regulator MDM2, an E3 ubiquitin ligase. Amplification of mdm2 has been observed in many human cancers and is sufficient to induce tumorigenesis. Although only a limited number of cancer- associated mdm2 mutations have been reported, their functional studies have provided valuable information regarding the oncogenic functions of MDM2. Our previous studies have identified several novel MDM2- interacting proteins and revealed the mechanistic basis of their roles in the regulation of MDM2 function. In this proposal, we will focus on several mutant forms of MDM2 identified in tumor samples that contain a wild type p53 gene. These mutants carry MDM2 mutations within MDM2 domains previously shown to be critical for MDM2 functions. Our preliminary results indicate that these MDM2 variants have distinct functions and vary in their ability to degrade p53. In vitro transformation assays have further revealed that some MDM2 mutants have higher transformation potential than others. Moreover, we have established p53 wild-type cell lines with mutations at the endogenous mdm2 locus using TALEN-based genome editing method. Our central hypothesis is that tumor- derived MDM2 mutations deregulate the p53 function by altering p53 transcriptomes important for tumor suppression, and/or modifying MDM2 binding to its interacting proteins, which are important for the regulation of MDM2 function. This hypothesis will be tested in two independent aims using our established p53 wild-type cell lines with mutations at the endogenous mdm2 locus. In Aim 1, we will test the hypothesis that the tumor-derived MDM2 mutants alter p53 transcriptomes that are important for tumor suppression. In Aim 2, we will test the hypothesis that the MDM2 mutations modify MDM2 binding to its interacting proteins, resulting in deregulation of MDM2 function. These studies will provide a better understanding of the oncogenic activities of MDM2 and a mechanistic basis for development of novel anti-cancer strategies. In addition, this project will enhance the research environment at St. John?s University by providing undergraduate and graduate students with numerous opportunities to learn the fundamentals of biomedical research.