MECHANISMS OF DECORIN INHIBITION OF VEGF-STIMULATED HUMAN TROPHOBLAST MIGRATION AND ACQUISITION OF AN ENDOVASCULAR PHENOTYPE

The human placenta is a highly invasive tumour-like organ in which fetal-derived trophoblast cells constitute the major cell type. A subpopulation of trophoblast cells, known as extravillous trophoblast, invades the uterine decidua and maternal arteries to establish sufficient fetal-maternal exchange to maintain healthy utero-placental homeostasis. Trophoblast invasion is highly regulated by a variety of factors at the fetal-maternal interface. Decorin is a member of the small leucine-rich proteoglycan family, produced by the decidua, and is a negative regulator of trophoblast invasiveness. I hypothesized that decorin inhibits vascular endothelial growth factor (VEGF)-stimulated endovascular differentiation and migration by interfering with signalling pathways downstream of VEGF Receptor (VEGFR)-2. Using in vitro migration and endothelial-like tube formation assays, I determined that decorin inhibits trophoblast migration and endovascular differentiation by interfering with VEGF-induced p38 and p44/p42 mitogen-activated protein kinase (MAPK) activation. These results have implications for the pathobiology of preeclampsia, a hypo-invasive trophoblast disorder in pregnancy

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 TGFbeta.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. © 2012 by the Society for the Study of Reproduction, Inc

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-β and a TGF-β-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 toVEGFR-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 125I-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. © 2011 by The Endocrine Society

Expression of CCAAT/Enhancer Binding Protein Beta in Muscle Satellite Cells Inhibits Myogenesis in Cancer Cachexia.

Cancer cachexia is a paraneoplastic syndrome that causes profound weight loss and muscle mass atrophy and is estimated to be the cause of up to 30% of cancer deaths. Though the exact cause is unknown, patients with cancer cachexia have increased muscle protein catabolism. In healthy muscle, injury activates skeletal muscle stem cells, called satellite cells, to differentiate and promote regeneration. Here, we provide evidence that this mechanism is inhibited in cancer cachexia due to persistent expression of CCAAT/Enhancer Binding Protein beta (C/EBPβ) in muscle myoblasts. C/EBPβ is a bzip transcription factor that is expressed in muscle satellite cells and is normally downregulated upon differentiation. However, in myoblasts exposed to a cachectic milieu, C/EBPβ expression remains elevated, despite activation to differentiate, resulting in the inhibition of myogenin expression and myogenesis. In vivo, cancer cachexia results in increased number of Pax7+ cells that also express C/EBPβ and the inhibition of normal repair mechanisms. Loss of C/EBPβ expression in primary myoblasts rescues differentiation under cachectic conditions without restoring myotube size, indicating that C/EBPβ is an important inhibitor of myogenesis in cancer cachexia

Inhibition of myogenesis correlates with induction of C/EBPβ expression in myoblasts.

<p><b>(A)</b> Immunocytochemistry staining of myosin heavy chain expression in C2C12 myoblasts pre-treated with conditioned media from SKOV3 or PC-3 cells mixed 1:1 with fresh media, for 48hrs, and induced to differentiate for an additional 5 days. DAPI stains nuclei blue. <b>(B)</b> C2C12 cultures were induced to differentiate as in (A) and the differentiation and fusion indices were calculated as relative to SKOV3-treated cells. Actual values are shown in the respective bars. *p<0.05, n = 5. <b>(C)</b> C/EBPβ, and myogenic marker protein expression in C2C12 myoblasts treated as in (A). Cyclophilin B (CyPB) is shown as a loading control. <b>(D)</b> Immunocytochemistry staining of myosin heavy chain (MYH) expression in primary myoblasts pre-treated with conditioned media from SKOV3 or PC-3 cells mixed 1:1 with fresh media, for 48hrs, and induced to differentiate for an additional 48 hours in DMEM containing 10% horse serum. DAPI stains nuclei blue. <b>(E)</b> Primary myoblast cultures were induced to differentiate as in <b>(D)</b> and the differentiation and fusion indices were calculated as relative to SKOV3-treated cells. Actual values are shown in the respective bars. **p<0.01, n = 4. <b>(F)</b> Percentage of Pax7+ cells relative to total nuclei remaining in C2C12 cells cultured in conditioned medium and differentiated as in (D) as determined by immunocytochemistry. *p<0.05, n = 4. <b>(G)</b> C/EBPβ, and myogenic marker protein expression in primary myoblasts treated as in (D). Cyclophilin B (CyPB) is shown as a loading control.</p

Pre-treatment with conditioned media from a prostate tumor inhibits skeletal muscle differentiation and upregulates C/EBPβ expression.

<p><b>(A)</b> Schematic of the treatment procedure for the tissue culture model of cachexia. Conditioned media from PC-3 cells was mixed 1:1 with fresh media, and was added onto proliferating C2C12 myoblasts for 48 hours after which cells were induced to differentiate in fresh DMEM containing 2% horse serum for 5 days. <b>(B)</b> Immunocytochemistry staining for myosin heavy chain expression in C2C12 cultures treated with conditioned media from PC-3 or DU145 prostate cancers or unconditioned media (UM) as in (A). DAPI stains nuclei blue. <b>(C)</b> C2C12 cultures were induced to differentiate as in (A) and the fusion index (#myonuclei/myotube) and differentiation index (#myonuclei/# total nuclei) was calculated, and shown relative to UM. Actual values are shown in the bars. *p<0.05, n = 7. <b>(D)</b> Western blot analysis of C/EBPβ, Pax7, and MyoD expression in proliferating C2C12 cells on day 2 after incubation with conditioned medium. Actin is a loading control. <b>(E)</b> Western blot analysis of myogenic marker expression in differentiated C2C12 cells on day 7. Actin is a loading control. <b>(F)</b> qRT-PCR analysis of <i>Myod1</i>, <i>Myog</i> and neonatal myosin heavy chain (<i>Myh1</i>, <i>Myh2</i>, <i>Myh8</i>, <i>Myh13</i>) expression, shown relative to cells treated with unconditioned medium (UM) on day 7. *p<0.05, n = 4.</p

Conditioned medium from human cancers stimulate C/EBPβ expression in myoblasts.

<p><b>(A)</b> C2C12 myoblasts were incubated with conditioned medium from indicated human cancers or unconditioned medium (UM) mixed 1:1 with fresh myoblast medium for 48 hours. C/EBPβ expression was assessed by western blot. β-actin is a loading control. <b>(B)</b><i>Cebpb</i> mRNA expression in myoblasts treated with PC-3 medium or unconditioned medium for 48 hours. *p<0.05, n = 5. <b>(C)</b><i>Il1b</i> expression in SKOV3 and PC-3 cancer cells. *p<0.05, n = 5.</p

LLC tumor graft increases C/EBPβ expression in myoblasts and prevents myogenesis.

<p><b>(A)</b> 5x10⁵ Lewis Lung Carcinoma (LLC) cells or PBS (Sham) was injected into the flank of C57BL/6 mice and allowed to engraft for 3 weeks to induce cachexia. <b>(B)</b> Immunocytochemistry for myosin heavy chain expression in primary myoblasts isolated from sham-injected and LLC-injected mice and differentiated for 2 days. <b>(C)</b> Differentiation (DI) and fusion (FI) indices from cultures isolated and differentiated as in (B). *p<0.05, n = 5. <b>(D)</b> Western analysis of C/EBPβ and myogenic marker expression in cells isolated from healthy or cachectic mice as in (A) and after culture expansion for 5 days, differentiated for 2 days. Cyclophilin B (CyPB) is a loading control.</p

SOX7 Is Required for Muscle Satellite Cell Development and Maintenance

Satellite cells are skeletal-muscle-specific stem cells that are activated by injury to proliferate, differentiate, and fuse to enable repair. SOX7, a member of the SRY-related HMG-box family of transcription factors is expressed in quiescent satellite cells. To elucidate SOX7 function in skeletal muscle, we knocked down Sox7 expression in embryonic stem cells and primary myoblasts and generated a conditional knockout mouse in which Sox7 is excised in PAX3+ cells. Loss of Sox7 in embryonic stem cells reduced Pax3 and Pax7 expression. In vivo, conditional knockdown of Sox7 reduced the satellite cell population from birth, reduced myofiber caliber, and impaired regeneration after acute injury. Although Sox7-deficient primary myoblasts differentiated normally, impaired myoblast fusion and increased sensitivity to apoptosis in culture and in vivo were observed. Taken together, these results indicate that SOX7 is dispensable for myogenesis but is necessary to promote satellite cell development and survival