Fig 1.
Zyxin localization in endothelial cells in response to endoglin and BMP9.
(A) Western blot analysis of the QProteome-derived cytoskeletal fraction, including focal adhesion-associated proteins, and cytosolic fraction of wild type and eng+/- mouse lungs. (B) Double immunofluorescence confocal microscopy of wild type and eng+/- mouse lung tissue sections using α-zyxin and α-SMA antibodies. (C) Western blot of HUVEC focal adhesion and total cell lysate subcellular fractions (representative example of three replicates) following treatment with BMP9 or TGFβ (5ng/ml, Annexin A2; AnxA2). (D) Lentivirus-transduced control (shNT) and endoglin-targeting (shENG) short hairpin RNAs were used to suppress HUVEC endoglin expression; focal adhesion subcellular fractions were analyzed by Western blot. (E) Immunofluorescence analysis of zyxin localization using confocal microscopy. Anti-zyxin antibody and DAPI staining are in green and purple, respectively. Arrows indicate sites of focal adhesion (shENG) and the plasma membrane (shENG+BMP9), respectively. Scale bars indicate 15 μm.
Fig 2.
Endoglin- and BMP9-dependent localization of SMAD1/5 and YAP1.
ShNT and shENG were used to suppress HUVEC endoglin expression. (A-B, E) immunofluorescence analysis using confocal microscopy shows that suppression of endoglin expression results in the loss of BMP9 dependent SMAD1/5 (A) and YAP1 (B) localization in the nucleus and increases their expression in the cytoplasm (arrows). The ratios of the immunofluorescence intensities measured in the cell nucleus versus cytoplasm are shown below panels A and B. The asterisk indicates a p value < 0.05. (C) Western blot analysis of YAP1 localization in HUVEC nuclear and cytoplasmic subcellular fractions, and total cell lysate (TCL). (D) SMAD1/5 and YAP1 coimmunoprecipitation show BMP9 dependent nuclear localization of the bound complex and cytoplasmic retention when endoglin is suppressed. (E) SMAD2/3 expression and localization appear unaffected in response to BMP9 and endoglin suppression.
Fig 3.
YAP1 targets CYR61 and connective tissue growth factor (CTGF) expression and secretion are regulated by BMP9 and endoglin.
All cells were plated on fibronectin. (A-B) Quantitative real-time RT-PCR (qRT-PCR) of (A) CTGF and (B) CYR61 following BMP9 treatment and endoglin suppression using shRNA. (C) Immunofluorescence confocal microscopy analysis of CTGF in response to BMP9 treatment and endoglin suppression. (D) qRT-PCR of CTGF (gray) and CYR61 (black) mRNA from wild type and eng+/- mouse lung mRNA preparations. (E) qRT-PCR of CTGF following BMP9 treatment and Alk1 suppression using shRNA. (F-G) qRT-PCR of (F) CTGF and (G) CYR61 following BMP9 treatment and YAP1 suppression using shRNA. Human cyclophilin primers were used for normalization.
Fig 4.
Mass spectrometric analysis suggests BMP9-dependent CTGF and CYR61 secretion and focal adhesion localization.
(A-B) Survey spectra of BMP9-treated HUVEC focal adhesion preparations indicate the presence of CYR61 (A) and CTGF (B) proteins in focal adhesion fractions. MS identification and MSMS collision-induced decay sequence spectra are shown in the top and bottom spectra panels, respectively. Sequence data are presented in the table showing expected and observed ions (shaded) for this protein. (C) Isotope-coded affinity tag (ICAT) mass spectrometry of control and BMP9-treated and control HUVEC conditioned medium samples. Heavy isotope BMP9-treated and light isotope-tagged control peptide spectra are indicated with h or l, respectively. Left panels (A-C) are MS-TOF analyses for identification (A-C) and quantitation (C). (C) Left panel shows an example of heavy- (BMP9-treated) and light-isotope-tagged (control) CTGF peptide ICAT-labeled ions (arrows indicate ICAT-ions); right panels are MSMS-TOF collision-induced decay-based protein sequence analyses, used to identify the indicated peptides for CTGF. (D) Western blot analysis of HUVEC focal adhesion subcellular fraction for CYR61, CTGF, and integrins β1 and α5 proteins with or without BMP9 treatment. (E) Mass spectrometry ICAT analysis of the HUVEC focal adhesion subcellular fraction for integrin β1.
Table 1.
Proteins identified as up-regulated and down-regulated in CM from HUVEC +/- BMP9 ICAT data.
Fig 5.
Altered wound healing patterns in endoglin-deficient HUVEC and extracellular matrix (ECM) remodeling in eng+/- mouse lungs.
(A) In vitro scratch assay with HUVEC and endoglin deficiency obtained at 24 and 48 hrs after “wounding”. Insets exemplify altered morphology seen with endoglin knockdown (24 hr shENG, arrows). (B) Immunofluorescent visualization of collagen exposure using the collagen cryptic epitope-specific antibody, HU177 [81]. Accompanying ImageJ color histograms show total pixel counts for the green HU177 channel. Horizontal bar indicates value differences with p<0.05. (C) Dual immunofluorescence microscopy obtained from wild type and eng+/- mouse lungs using HU177 and smooth muscle alpha actin (SMA) antibodies. Arrows indicate regions of exposed collagen cryptic epitope (α-HU177), shown predominately in the eng+/- mouse lung. (D) Dual immunofluorescence analysis of eng+/- mouse lung sections for HU177 (green) and MMP9 (red) Arrow exemplifies adjacent staining).
Fig 6.
Inflammatory cytokines are regulated by BMP9 and endoglin expression and signaling.
(A) CCL2 and CCR2 levels of expression in HUVEC following treatment with TGFβ or BMP9, as compared to control (NT). (B) qRT-PCR analysis of time course of BMP9-dependent changes in mRNA levels for CCL2, CCR2, and CCL5. (C) Relative to control lentivirus (NT), qRT-PCR indicates shRNA for endoglin or ALK1 (e, a, respectively) rescues BMP9-dependent repression of CCL2 expression. (D) CCL2 and CCR2 expression in WT (n = 3) and eng+/- (n = 3) mouse lung mRNA preparations. *, p<0.025; **, p<0.001. Human and mouse β2-microglobulin was used for normalization. (E) Monocyte infiltration and α-CCL2 expression in eng+/- lungs, as compared to wild type, eng+/+. (F) qRT-PCR analysis of BMP9-dependent CCL2 expression with YAP1 knockdown.
Fig 7.
Integration of BMP and Hippo signaling in endothelial cells.
We hypothesize that BMP9 regulates the expression and secretion of matricellular proteins, CYR61 and CTGF, via crosstalk between the BMP (endoglin, ALK1, and SMAD1/5) and Hippo (Zyxin, YAP1, SMAD) signaling pathways. We suggest that the secretion of CYR61/CTGF regulates the composition and function of focal adhesion proteins, cell migration and ECM remodeling, and acts as a key effector of BMP9 signaling via the HHT target genes, ALK1 and endoglin.