Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading

AbstractVitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin αvβ3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.Structured summaryMINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071

The mitogenic potential of heparin-binding epidermal growth factor in the human endometrium is mediated by the epidermal growth factor receptor and is modulated by tumor necrosis factor-alpha

Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFalpha has a cooperative effect on HB-EGF, EGF, TGFalpha, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFalpha in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFalpha in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFalpha, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation

Structure-function studies of fibronectin domains in the human endometrium

The function of the endometrium is to mediate implantation of the embryo. During the early stages of implantation, the endometrial stroma undergoes differentiation known as decidualization, a process critical for successful embryo implantation. The precise mechanisms involved are not clearly understood but extracellular matrix (ECM) remodelling is a key feature. Fibronectins (FNs) are large glycoproteins abundant in the ECM of the human endometrium. Up to twenty isoforms of FNs are generated from alternative splicing, including the EDIIIA+ and EDIIIB+ variants. This thesis investigated changes in endometrial stromal ECM levels, in particular FN and its splice variants, during decidualization and in response to the endometrial cytokines and growth factors that drive the implantation process. Furthermore, the influence of these splice variants on the functional properties of FN was explored, including cell attachment, spreading and proliferation, integrin binding and focal adhesion kinase activation. Structural studies including crystallization trials were carried out to investigate how the insertion of EDIIIA modulates the conformation of FN and accessibility of its integrin binding sites. These combined studies allow us to test the hypothesis that the regulation of alternative splicing provides a biological mechanism for modulating function of FN in the endometrium. The main findings from this study can be summarized as follows: Immunocytochemistry and immunoblotting demonstrated reduced endometrial stromal levels of EDIIIA+FN, total FN and tenascin during in vitro decidualization. Substrate-associated FN production by endometrial stromal cells was reduced in response to the endometrial cytokine TNFalpha as detected by ELISA. Recombinant FIII7-12A±B± fragments were expressed, purified and mediated endometrial stromal cell adhesion. Inclusion of EDIIIA in the recombinant FIII7-12 fragment decreased binding affinities to integrin alpha5beta1. These findings suggest that production of FNs in the endometrial stroma is modified during in vitro decidualization and in response to endometrial TNFalpha. This modification in ECM composition is likely to result in modulation of cellular processes, perhaps to allow for cellular differentiation and migration that is required for invasion of the implanting embryo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structure-function studies of fibronectin domains in the human endometrium

The function of the endometrium is to mediate implantation of the embryo. During the early stages of implantation, the endometrial stroma undergoes differentiation known as decidualization, a process critical for successful embryo implantation. The precise mechanisms involved are not clearly understood but extracellular matrix (ECM) remodelling is a key feature. Fibronectins (FNs) are large glycoproteins abundant in the ECM of the human endometrium. Up to twenty isoforms of FNs are generated from alternative splicing, including the EDIIIA+ and EDIIIB+ variants. This thesis investigated changes in endometrial stromal ECM levels, in particular FN and its splice variants, during decidualization and in response to the endometrial cytokines and growth factors that drive the implantation process. Furthermore, the influence of these splice variants on the functional properties of FN was explored, including cell attachment, spreading and proliferation, integrin binding and focal adhesion kinase activation. Structural studies including crystallization trials were carried out to investigate how the insertion of EDIIIA modulates the conformation of FN and accessibility of its integrin binding sites. These combined studies allow us to test the hypothesis that the regulation of alternative splicing provides a biological mechanism for modulating function of FN in the endometrium. The main findings from this study can be summarized as follows: Immunocytochemistry and immunoblotting demonstrated reduced endometrial stromal levels of EDIIIA+FN, total FN and tenascin during in vitro decidualization. Substrate-associated FN production by endometrial stromal cells was reduced in response to the endometrial cytokine TNFalpha as detected by ELISA. Recombinant FIII7-12A±B± fragments were expressed, purified and mediated endometrial stromal cell adhesion. Inclusion of EDIIIA in the recombinant FIII7-12 fragment decreased binding affinities to integrin alpha5beta1. These findings suggest that production of FNs in the endometrial stroma is modified during in vitro decidualization and in response to endometrial TNFalpha. This modification in ECM composition is likely to result in modulation of cellular processes, perhaps to allow for cellular differentiation and migration that is required for invasion of the implanting embryo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Human Endometrial Stromal Cell Rho GTPases Have Opposing Roles in Regulating Focal Adhesion Turnover and Embryo Invasion In Vitro

Implantation of the embryo into the uterine compartment is a multistep event involving attachment of the embryo to the endometrial epithelia, followed by invasion of the embryo through the endometrial stroma. RHOA, RAC1, and CDC42 are members of the Rho GTPase family of proteins, which control cell functions such as cell migration and cytoskeletal reorgani-zation. Herein, using a heterologous in vitro coculture model, we show that implantation of mouse blastocysts into human endometrial stromal cells (hESCs) is regulated by Rho GTPase activity in hESCs. Whereas iRNA-mediated silencing of RAC1 expression in hESCs led to inhibition of embryo implantation, silencing of either RHOA or CDC42 in hESCs promoted embryo implantation in coculture assays. Analysis of downstream signaling pathways demonstrated that RAC1 silencing was associated with decreased focal adhesion disassembly and resulted in large focal adhesion complexes in hESCs. In contrast, RHOA or CDC42 silencing resulted in perturbed focal adhesion assembly, leading to a decrease in the number of focal adhesions observed. Furthermore, inhibition of Rho signaling using a Rho kinase inhibitor, Y27632, led to decreased activation of protein tyrosine kinase 2 (PTK2, also called focal adhesion kinase) and decreased focal adhesion assembly. Importantly, perturbation of focal adhesion turnover in hESCs, mediated by PTK2 silencing, resulted in inhibition of embryo implantation into hESC monolayers. These findings suggest that Rho GTPase-PTK2-dependent remodeling of the endometrial stromal cell compart-ment may be critical for successful embryo implantation. developmental biology, early development, embryo, endometrium, implantation, pregnancy, Rho GTPase

Clustered integrin α5β1 ligand displays model fibronectin-mediated adhesion of human endometrial stromal cells

Progress towards endometrial tissue engineering for modelling endometrial diseases and infertility is frustrated by the inability to mimic the fibronectin (FN) extracellular matrix required by human endometrial stromal cells (EnSCs). Here we show that this is because of the requirement to present integrin α5β1 (the FN receptor) ligands in specifically oriented, polyvalent displays; by engineering controlled self-assembly of the 9th-10th type III FN domain pair (FIII9-10, the minimal integrin α5β1 ligand) immobilised in a specific orientation to cell culture surfaces. The fraction of adherent EnSCs seen to spread increased significantly for the multimeric ligand surfaces in the order: tetramer>trimer>dimer>monomer. The extent of EnSC spread morphology also increased in the same order, with the tetrameric ligand supporting a morphology most similar to that supported by FN. Our data suggest that only higher-order multimers of FIII9-10 will fully promote cell spreading mediated through integrin α5β1 binding

Heparin-binding epidermal growth factor and its receptors mediate decidualization and potentiate survival of human endometrial stromal cells

Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNF alpha and TGF beta. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNF alpha- and TGF beta-induced apoptosis of endometrial stromal cells