This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In the primitive heart tube, the cardiac jelly forms the extracellular matrix that is sandwiched between myocardial and endothelial layers. Hyaluronan, versican and other extracellular materials are constituents of the cardiac jelly. Expansion of cardiac jelly results in the formation of endocardial cushions unique to the atrioventricular canal (AVC) and outflow tract (OFT) regions but not in the adjacent atrial or ventricular endothelia. At a precise time of development the myocardium sends signals to a subset of endothelial / endocardial cells of the AVC and OFT, which respond by losing their adhesive properties and extending filopodia, thus transforming into mesenchymal cushion cells (EMT) and invading the underlying cardiac jelly. These mesenchymal cells contribute to the formation of mitral and tricuspid valve leaflets and atrial and ventricular septa (Markwald et al 1977, Potts et al 1991, Eisenberg and Markwald, 1995). So, this transformation process is critical for the development of a full four-chambered heart. Atrioventricular septal malformation is the most common of all anatomical birth defect found within the first year of life (Ferencz and Boughman 1993). The mechanism of septal malformation remains elusive. Hyaluronan (HA) is a chemically homogeneous linear mega-dalton polymer, which consists of repeating disaccharide units of [?1?3]D- glucuronate- [?1?4]N-Acetyl-D-glucosamine. HA is synthesized by plasma membrane enzymes, HA synthases (HAS1, 2 and 3), their active sites being situated on the cytoplasmic side of the membrane, and the elongated molecule is extruded into the extracellular matrix concomitantly with synthesis. In vivo HA binds: (1) to its receptor CD44 on the cell surface via its HA binding domain and, when activated, sends intracellular signals; (2) to other extracellular matrix molecules thus creating a network of macromolecules, e.g., HA, link protein, versican, and aggrecan. HA is always present at sites of cell migration, including within the developing cardiovascular system. Hyaluronan, the ErbB family of receptors and the TGF? family are important in the cushion developmental process. ErbB2 and 3 are important in differentiation of cardiac ventricular myocytes, as seen in heregulin -/- and ErbB2 -/- mice (Erickson et al 1977). ErbB3-/- embryos exhibit cardiac cushion abnormalities and cause blood reflux through defective valves. HAS-/- mice embryos lack HA at ED9.5, impeding cell migration and EMT in AV canal and cushion cell formation (Camenisch et al 2000, 2002). These defects are corrected ex-vivo by up-regulating HA production experimentally or adding exogenous HA to the culture. TGF??and BMP families are important for cellular growth and development. Depending on the species, TGF??BMP requirements vary. For example, TGF???signals to avian AVC?endocardial cells for activation and separation whereas TGF?3?is essential for mesenchymal cell formation and invasion. TGF?? and BMP2 seem to be important in mouse AV endocardial cushion morphogenesis (Sugi et al 2004). Endogenous hyaluronan-CD44 interaction sends signals for cell survival and proliferation. In cancer cells, I have found that hyaluronan induces a constitutive signaling complex consisting of CD44-erbB2/3-PI3 kinase-ezrin-HSP90-cdc37; perturbation of HA-CD44 interaction disrupts the complex and attenuates HA-mediated signaling (Ghatak et al 2005). Other investigators have found that HA-CD44 induces TGF? and BMP2 signaling and that CD44 interacts with Smads 1/2 (Bourguignon et al 2002, Peterson et al 2004). I hypothesize that HA-CD44 interaction is of paramount importance in TGFbeta/BMP2 signaling during cardiac cushion cell morphogenesis. Thus I propose: Hypothesis1: HA-CD44 interaction induces signals for cardiac EMT. Following experiments are designed to address this hypothesis. H1.1. HA-CD44 interaction is required for endocardial EMT AV explants from 16-18 somite stage of mouse embryo (ED 9.25) will be treated with the HA antagonists, CD44 RNAi or HA oligomers, on collagen gels (Sugi et al 2004). Parallel experiments will be carried out with nonspecific control RNA or with control chitin oligomers. Ten explant cultures for each treatment group will be set up. The number of mesenchyme cells that invade the collagen gels will be counted after 48 hr. The collagen gel cultures will be processed for the detection of PECAM (endothelial marker), smooth muscle a-actin (mesenchymal marker) and nuclear ?-catenin (EMT marker) by immunostaining. H1.2. HA-CD44 interaction is required for TGF- or BMP2-induced EMT Endocardial cells will be prepared from AV explants on collagen gels (Yukiko et al 2004). The cells will be treated with TGF? (1-2 ng/ml) or BMP2 (200 ng/ml) in the presence and absence of HA antagonists, HA oligomers and CD44 siRNA on collagen gels as above. Additional two explant groups will be set up for treatment with anti-TGF??antibody and noggin (500 ng/ml)antibodies as negative control. The collagen gels will be processed as above for the detection of PECAM (endothelial marker), and smooth muscle a-actin (mesenchymal marker) and for nuclear ?-catenin (EMT marker). H.1.3.Up-regulation of HA synthesis is sufficient to induce EMT in endocardium Endocardial cells from AV explants on the collagen gel will be collected. The cells will be infected with adenovirus driving HAS2 expression or a control beta-galactosidase adenovirus. The cells will be re-plated on collagen gels and assessed for EMT. Number of invaded mesenchyme will be counted after 48 hr. The PECAM, smooth muscle a-actin and nuclear ?-catenin will be measured by immunostaining as before. H1.4. Up-regulation of HA in endocardial cells induces CD44-Smad interaction Endocardial cells from AV explants on the collagen gel will be collected. The cells will be infected with adenovirus driving HAS2 or beta-gal expression as above. I will examine the interaction of CD44 and Smad1/2 in the Has2-treated endocardium (+/- HA antagonists). The cells will be lysed in 1% NP-40 buffer containing protease and phosphatase inhibitors. Immunoprecipitation (IP) will be done with anti-CD44 antibody. The IP will be western blotted and probed for Smad1/2 and phospho-Smad1/2 (Peterson et al 2004). Nuclear localization of Smads will be assessed by immunostaining (Peterson et al 2004). Hypothesis 2: BMP2/TGF? and ?? form a positive feedback loop. Experiments below are designed to address the above hypothesis: H.2.1. BMP2 and/or TGF- regulate HA production Endocardial cells from mouse AV explants will be treated with BMP2 or TGF?? Has2 and Hyal-1/2 expression will be assessed by RT-PCR and HA synthesis will be measured by ELISA-like method (ref Leslie). H2.2. HA regulates BMP-2/ TGF- activity I will examine the up regulation of BMP-2/ TGF- activity in the endocardial cells in presence and absence of HA antagonists, by measuring Smad 1/2 phosphorylation and nuclear localization, as described in H.1.4.
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