CCAAT/enhancer binding protein beta is expressed in satellite cells and controls myogenesis.

Upon injury, muscle satellite cells become activated and produce skeletal muscle precursors that engage in myogenesis. We demonstrate that the transcription factor CCAAT/enhancer binding protein beta (C/EBPß) is expressed in the satellite cells of healthy muscle. C/EBPß expression is regulated during myogenesis such that C/EBPß is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPß in myoblasts potently inhibits differentiation at least in part through the inhibition of MyoD protein function and stability. As a consequence, myogenic factor expression, myosin heavy chain expression, and fusogenic activity were reduced in C/EBPß-overexpressing cells. Using knockout models, we demonstrate that loss of Cebpb expression in satellite cells results in precocious differentiation of myoblasts in growth conditions and greater cell fusion upon differentiation. In vivo, loss of Cebpb expression in satellite cells resulted in larger muscle fiber cross-sectional area and improved repair after muscle injury. Our results support the notion that C/EBPß inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation.

Mechanisms in decorin regulation of vascular endothelial growth factor-induced human trophoblast migration and acquisition of endothelial phenotype.

Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.

Decorin is a novel VEGFR-2-binding antagonist for the human extravillous trophoblast.

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-ß and a TGF-ß-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.