QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 30, 2006
doi: 10.1242/10.1242/jcs.03232

This Article Summary Full Text Full Text (PDF) Supplemental Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spessotto, P. Articles by Colombatti, A. Search for Related Content PubMed PubMed Citation Articles by Spessotto, P. Articles by Colombatti, A. Social Bookmarking                         What's this?

EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall

Paola Spessotto^1,*, Roberta Bulla^2,*, Carla Danussi¹, Oriano Radillo³, Marta Cervi¹, Giada Monami^1,, Fleur Bossi², Francesco Tedesco³, Roberto Doliana¹ and Alfonso Colombatti^1,4,5,

¹ Divisione di Oncologia Sperimentale 2, CRO-IRCCS, 33081 Aviano, Italy
² Dipartimento di Fisiologia e Patologia, University of Trieste, Trieste, Italy
³ Laboratorio di Analisi, IRCCS Burlo Garofalo, University of Trieste, Trieste, Italy
⁴ Dipartimento di Scienze e Tecnologie Biomediche, University of Udine, Udine, Italy
⁵ MATI Center of Excellence, University of Udine, 35100 Udine, Italy

View larger version (72K): [in a new window]   Fig. 1. Localization of EMILIN1 in first-trimester human placenta. (A) Cryostat sections of first-trimester placenta were stained with antibodies specific for EMILIN1 followed by secondary HRP-labeled antibodies and developed with DAB. The sections were counterstained with hemallum. (B) This section was immunostained for EMILIN1 (in brown) and for the presence of vWF, visualized with an alkaline-phosphatase-labeled secondary antibody, and it was not counterstained. Asterisk indicates a modified vessel; lower panel: triple staining for EMILIN1 (in brown), and cytokeratin (in green fluorescence) and vWF (in red) to visualize EVT and endothelial cells, respectively. (C) Cryostat sections of first-trimester placenta were immunostained for EMILIN1 and for the presence of vWF. Upper panel: EMILIN1 (in brown) and vWF (in pink); lower panel: EMILIN1 (in pink) and vWF (in brown). Arrowheads indicate spiral arteries and asterisks large modified vessels where the gradient distribution of EMILIN1 seems more evident. Bars, 50 µm.

View larger version (12K): [in a new window]   Fig. 2. Cell adhesion to ECM substrates. (A) Levels of HTR-8/SVneo cell adhesion to a selected panel of purified ECM proteins. Substrate proteins were coated at 10 µg/ml and cell adhesion was performed in the presence of 1.0 mM Mg2+ and 1.0 mM Ca2+. Statistical tests were not performed for this descriptive adhesive analysis. (B) Adhesion of HTR-8/SVneo and freshly isolated EVT cells to EMILIN1 and FN. Four experiments were performed and mean ± s.d. reported. No significative difference was seen between EVT and HTR-8/SVneo adhesion capability on EMILIN1 and FN substrates. (C) Perturbation of HTR-8/SVneo cell attachment to gC1q1. Wells were coated with gC1q1 (10 µg/ml). The anti-EMILIN1 mAb 1H2G8 was added to the coated wells just before addition of the cells. In other instances the cells were preincubated with the anti-4 integrin subunit mAb P1H4 for 15 minutes at 37°C and were then allowed to adhere at 37°C for 20 minutes. Data are expressed as mean ± s.d. Post-hoc comparison of control mAb versus test samples after significant ANOVA for repeated-measures main effect, Dunnet's test (n=3). *P<0.01.

View larger version (21K): [in a new window]   Fig. 3. Cell migration towards ECM substrates. (A) Haptotactic migration of HTR-8/SVneo towards various purified ECM substrates. The filters were coated on the lower surface with 20 µg/ml of the substrates. Data are expressed as mean ± s.d. under baseline conditions (BSA). Post-hoc comparison of BSA versus test substrates after significant ANOVA for repeated-measures main effect, Dunnet's test (n=4). *P<0.05. (B) Migration versus haptotaxis of HTR-8/SVneo cells. Filters were coated with purified FN or EMILIN-1 on their upper (migration) or lower (haptotaxis) surfaces and the cells were allowed to migrate for 5 hours. The values shown represent the mean ± s.d. of five experiments. P was calculated for values obtained in haptotactic versus migration conditions. (C) Haptotactic migration of HTR-8/SVneo and fresh EVT cells in response to FN or EMILIN1 was determined by FATIMA assay at one time point (5 hours). The mean ± s.d. of three experiments is reported. (D) The effect of the 4 function-blocking mAb P1H4 is shown. For EMILIN1 and gC1q1 P values are reported. The values shown represent the mean ± s.d. of three experiments.

View larger version (80K): [in a new window]   Fig. 4. (A) Expression and deposition of EMILIN1 and FN in vitro. Cells were grown in vitro for 48-72 hours, fixed and stained with anti-EMILIN1 or anti-FN antibodies. Bar, 25 µm. (B). Confocal images obtained from cryostat sections of first-trimester placenta using triple-labeling staining. Nuclei are visualized with Sytox Green and pseudocoloured in blue, EMILIN1 is detected with Alexa Fluor® 633 and pseudocolored in green, trophoblast cells are stained with anti-cytokeratin 7 (red, upper panel), and stromal cells with anti-vimentin (red, lower panel). EMILIN1 is deposited in proximity to stromal cells, whereas it seems absent near to trophoblast cells (magnification). Images were acquired with a Leica TCS SP2 confocal system (Leica Microsystems Heidelberg).

View larger version (55K): [in a new window]   Fig. 5. Transmigration of HTR-8/SVneo through a stromal cell layer. (A) DiI-labeled DSC were plated on the upper side of the filters and allowed to attach. Their spontaneous migration was evaluated before (left side) or after (right side) the addition of unstained HTR-8/SVneo cells. The migration was stopped after 5 hours and the filters were stained with the nuclear dye Hoechst 33342. DSC did not migrate at all (note the absence of red color on the lower part of the filters). The white arrows indicate the nuclei of migrated HTR-8/SVneo cells. Bar, 75 µm. (B) DSC were plated on the lower side of an inverted FluoBlok insert and incubated for 72 hours. The insert was then placed in the chamber and HTR-8/SVneo cells labeled with DiI were added on the upper side. The chambers for migration are schematically represented in the figure. Quantification of HTR-8/SVneo cells migrated through DSC grown on the upper or lower side of the filter is reported in the graph. When DSC were grown on the lower side of the filter, HTR-8/SVneo cell migration was significantly higher than that observed with DSC monolayer in the upper side of the insert. Bar, 75 µm. (C) Inhibition of HTR-8/SVneo and EVT cell transmigration. In some instances, anti-gC1q1 or anti-4 integrin subunit antibodies were added alone or together to block cell transmigration with DSC monolayer on the lower side of the filter. Anti-1 integrin subunit antibody was used as negative inhibition control. Data are expressed as mean ± s.d. of four experiments under baseline conditions (mAb anti-1). Post-hoc comparison of mAb anti-1 versus specific antibodies after significant ANOVA for repeated-measures main effect, Dunnet's test. *P<0.05, **P<0.01. (D) HTR-8/SVneo cell transmigration under EMILIN1 gene silencing conditions. Upper panel: decidual stromal cell EMILIN1 mRNA levels (30 amplification cycles) at 48, 72 and 96 hours after transfection with the siRNA-specific SMARTpool. Lane 1: control, lipofectamine-treated cells; lane 2: scrambled siRNA; lane 3: EMILIN1 siRNA. RT-PCR analysis reveals that the levels of EMILIN1 mRNA decrease only at 48 hours. Lower panel: evaluation of HTR-8/SVneo cell transmigration after the EMILIN1 gene silencing in decidual stromal cell. The three confocal images, concerning different fields of FluoroBlok inserts, represent the staining of EMILIN1 (in green) produced by decidual stromal cells treated with lipofectamine, transfected with scrambled siRNA or with EMILIN1 siRNA and cultured on the lower side of a FluoBlok filter for 48 hours. Fast DiI-labeled HTR-8/SVneo (in red) were fixed after 7 hours of migration. Bars, 100 µm.

View larger version (21K): [in a new window]   Fig. 6. Production of MMPs in co-cultures. (A) Zymography of conditioned media obtained from co-cultures of DSC and HTR-8/SVneo cells. HTR-8/SVneo cells were added to wells of DSC monolayers. The conditioned media were collected after 24 hours and analysed by zymography. The cells were lysed and loaded for zymography or assayed for MT1-MMP activity. Quantitative determination of total MT1-MMP was performed using the Biotrak MT1-MMP activity assay system (Amersham Biosciences Europe, Orsay, France) according to the manufacturer's instructions. Equal amounts of HTR-8/SVneo and DSC cells were cultivated alone and then processed for zymography and MT1-MMP activity. The increase in MT1-MMP activity in co-cultures was significantly higher (P=0.002) than the summed activity of the enzyme produced by DSC and HTR-8/SVneo cells. (B) Inhibition of haptotaxis. GM6001 (10 µM) was added to block migration of DiI-labeled HTR-8/SVneo cells in the presence of an equal number of DSC. HTS FluoroBlokTM inserts were previously coated underside with gC1q1 or FN. The migratory process was evaluated at different times (2, 4, 6 and 18 hours). P values were determined versus control samples. Open squares, P>0.05; asterisks, P<0.01.

View larger version (28K): [in a new window]   Fig. 7. Schematic representation for the proposed mechanism in the migratory process involving trophoblast cells. The interaction of the gC1q1 domain of EMILIN1, produced by decidual stromal cells, with 4ß1 promotes trophoblast migration towards the vessels. The engagement of 4ß1 also promotes the release from trophoblast cells of MT1-MMP (60 kDa form) that in turn activates MMP-2 on the membrane of stromal cells. The activated enzyme could digest the ECM component of blood vessels (i.e. laminins), exert an improved efficiency in binding capability or signaling functions of the 4ß1 receptor, or unmask EMILIN1 cryptic sites in order to facilitate the migratory phenotype of trophoblast cells. The potential role of FN and other ECM molecules and of integrins other than 4ß1 is not represented for clarity.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter