Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

<div><p>Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII’s Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.</p></div

ActRIIA and BMPRII physically interact

<p>. Coimmunoprecipitation experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g007" target="_blank">Figure 7</a>. In all experiments, cells were transfected, cell surface proteins crosslinked, immunoprecipitation (IP) performed, crosslinks reversed, and Western blot (IB) performed as indicated. In some studies, cells were transfected with FLAG-endoglin as a positive control. Input lysate, post IP lysate, and IP samples are loaded as indicated. (<b>A and B</b>) ActRIIA precipitates with BMPRII. (<b>C and D</b>) ActRIIA kinase domain is dispensable for interaction with BMPRII. (<b>E and F</b>) BMPRII kinase activity and tail domain are dispensable for interaction with BMPRII. All data are from a representative experiment, repeated at least N = 5 separate times.</p

Smad1 is the main downstream target of endoglin.

<p>PC3-M cells were transfected with endoglin, vector (Vec), or with siRNA to Smad1, (siSm1), Smad5 (siSm5), Smad8 (siSm8), BMPRII (siRII) or non-targeting (siNeg), and processed 48 hrs later as indicated. <b>A</b>) Smad-targeting siRNA suppresses transcript in a Smad-specific fashion. Smad1, -5, and -8 mRNA expression was assessed via qRT-PCR, normalized to GAPDH, and expressed relative to siNeg-transfected cells (normalized to 1.0). Data represent mean ± SD from a single experiment conducted in replicates of N = 2, that was repeated 3 separate times (also in replicates of N = 2) with similar results. *, p≤0.05 compared to siNeg. <b>B</b>) Effect of siRNA on phospho-Smad1/5/8, phospho-Smad1/5, and total Smad1 protein levels. Cell lysates were probed by antibody directed towards phospho-Smad1/5/8 (pSmad1/5/8) and total Smad1 protein by Western blot. The non-specific band (*) immediately under the pSmad1/5/8 band (arrow) confirms even loading. Negative control cells (Neg Ctl) were transfected with vector and serum starved overnight. Positive control cells (Pos Ctl) were transfected with endoglin, not serum starved and were treated with TGFβ for 30 min. Separate samples were similarly transfected and treated, and cell lysates were probed for phospho-Smad1/5 (pSmad1/5). Data are from one representative experiment in each case, repeated 3 separate times with similar results. <b>C</b>) Endoglin-mediated BRE₂-luciferase activity is largely mediated by Smad1. Cells were transfected with endoglin and were additionally co-transfected with BRE₂- and <i>Renilla</i> luciferase construct, and luciferase assays performed. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 3 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg. <b>D</b>) BMPRII-mediated suppression of BRE₂-luciferase activity is largely mediated by Smad1. Cells were transfected as in (C) with addition of indicated siRNA and luciferase activity as assessed as above. Data represent mean ± SD of a single representative experiment conducted in replicates of N = 2, repeated 2 separate times (replicates of N = 2) with similar results. *, p≤0.05 compared to Endoglin/siNeg/siBMPRII.</p

BMPRII suppresses Smad1 signaling in a dose- and tail domain-dependent manner.

<p><b>A)</b> BMPRII suppresses BRE₂-luciferase activity in a dose-dependent manner. PC3-M cells were transfected with indicated siRNA and BRE₂-luciferase and <i>Renilla</i> luciferase constructs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g002" target="_blank">Figure 2B</a>. Wild type BMPRII was co-transfected over a range of concentrations; x-axis displays ng of plasmid. Luciferase assay performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g002" target="_blank">Figure 2B</a>. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated 3 times (N = 2 replicates) with similar results. *, p≤0.05 compared to 0/siNeg. # p≤0.05 compared to 0/siBMPR2. <b>B</b>) Experiment performed as in A, except that tail domain deleted (Δtail) – not wild type – BMPRII was expressed over a range of concentrations. Data represent mean ± SD from one representative of two experiments, each in replicates of N = 2. *, p≤0.05 compared to 0/siNeg.</p

Endoglin physically interacts with ActRIIA and BMPRII.

<p>After transient transfection, the surface proteins of PC3-M cells were crosslinked, cells lysed, immunoprecipitation performed, crosslinking reversed, and Western blot performed. <b>A</b>) ActRIIA coprecipitates with endoglin. Cells were transfected with Myc-ActRIIA and FLAG-endoglin, FLAG (endoglin) immunoprecipitated, and Western blots probed for ActRIIA (with anti-Myc) and endoglin. Controls for immunoprecipitation included agarose beads alone (no Ig) and nonspecific isotype control IgG (IgG isotype). Input lysate, lysate post-immunoprecipitation (i.e. supernatant), and immunoprecipitation (IP) samples were loaded as indicated. Data are from a representative experiment (N = 2 experiments). <b>B</b>) The kinase domain of ActRIIA is dispensable for interaction with endoglin. Cells transfected with Myc-WT or -ΔKD-ActRIIA and FLAG-endoglin as indicated, FLAG or Myc was immunoprecipitated as indicated, and Western blots probed as indicated. Data are from a representative experiment (N = 4 experiments). (<b>C</b>) BMPRII precipitates with endoglin. Cells were transfected with FLAG-BMPRII and untagged endoglin, FLAG immunoprecipitated, and Western blots probed as indicated. Data are from a representative experiment (N = 2 experiments). (<b>D</b>) The kinase activity and tail domain of BMPRII are dispensable for interaction with endoglin. Cells transfected with FLAG-WT, -KI, or -Δtail-BMPRII and untagged endoglin as indicated, FLAG-BMPRII immunoprecipitated, and Western blots probed as indicated. In some instances surface proteins were crosslinked (+), which was reversed after immunoprecipitation, while in other instances proteins were not crosslinked (-) Data are from a representative experiment (N = 3 experiments).</p

BMPRII suppresses ActRIIA-mediated Smad1 activity.

<p>PC3-M cells were transfected with BRE₂-luciferase, <i>Renilla</i> luciferase, and indicated plasmid DNA with or without indicated siRNA. siRNA lanes marked with a hyphen were transfected with non-targeting siRNA. Two days later cells were lysed and luciferase activity was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g002" target="_blank">Figure 2B</a>. <b>A)</b> Increased BRE₂-luciferase activity upon silencing BMPRII is mediated by ActRIIA. Neg  =  non-targeting siRNA. 2A  =  siActRIIA. 2B  =  siActRIIB. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. <b>B</b>) BMPRII-mediated suppression of BRE₂-luciferase activity is dependent on ActRIIA expression. BMPRII construct abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g004" target="_blank">Figure 4</a>. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated 3 times (N = 2 replicates) with similar results. *, p≤0.05 for indicated comparison. <b>C</b>) BMPRII suppresses signaling from ActRIIA. Data represent mean ± SD from one representative experiment (N = 2 replicates) of three repeated separately (N = 2). *, p<0.05 for indicated comparison.</p

ActRIIA promotion of Smad1 signaling is kinase dependent, while BMPRII inhibition occurs via the tail domain.

<p><b>A)</b> Schematic depiction of ActRIIA and BMPRII constructs. Signal peptide (hatched), transmembrane domain (light gray), kinase domain (black), and BMPRII tail domain (dark gray) are indicated with the amino acid position that begins each portion. Also indicated are five sequential Myc tags or single FLAG tag at the C-terminus of ΔKD ActRIIA and BMPRII constructs, respectively (checkered). The small white stripe in KI BMPRII’s kinase domain represents the site of kinase-inactivating mutation. Segment lengths are to scale. <b>B</b>) ActRIIA promotes Smad1 signaling dependent on the kinase domain. PC3-M cells were transfected with BRE₂-luciferase and <i>Renilla</i> luciferase, endoglin, wild type (WT) or kinase domain deletion (ΔKD) ActRIIA constructs, and siRNA to ActRIIA or non-targeting as indicated. Luciferase assay performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g002" target="_blank">Figure 2B</a>. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 compared to Eng/siNeg. <b>C</b>) BMPRII suppresses Smad1 signaling independent of kinase function but dependent on the tail domain. PC3-M cells were transfected as above except that BMPRII constructs and siRNA were used. WT  =  wild type; KI  =  kinase inactive; Δtail  =  tail domain deleted. Luciferase assay performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g002" target="_blank">Figure 2B</a>. Data represent mean ± SD of a single representative experiment (N = 2 replicates), repeated twice (N = 2 replicates) with similar results. *, p≤0.05 compared to Eng/siBMPRII.</p

Endoglin requires ActRIIA and BMPRII to suppress invasion.

<p><b>A)</b> The effect of type II receptors on endoglin mediated suppression of invasion (EMSI). PC3-M (left) or DU-145 (right) cells were transiently transfected with empty vector (Vec) or with endoglin along with siRNA, as indicated. The following siRNAs were used: siNeg – non-targeting negative control, si2A - targets ActRIIA, si2B - targets ActRIIB, siBMP - targets BMPRII, siTGF - targets TGFβRII. After 48 hours, cell invasion was measured. Data represent the mean ± SEM of 3 independent experiments, each in replicates of 3. *, p≤0.05 compared to Vec/siNeg. Micrographs are representative images of cells that have invaded through Matrigel, were stained for βgal, and imaged (magnification 100X). <b>B</b>) ActRIIA and BMPRII siRNA is specific. PC3-M cells were transiently transfected with endoglin along with the indicated siRNAs as in (A). After 48 hours mRNA expression was assessed via qRT-PCR, normalized to GAPDH, and expressed relative to siNeg-transfected cells (normalized to 1.0). Data represent the mean ± SD of a single experiment, performed in replicates of N = 2; similar results were seen in an independent experiment (N = 2 replicates). *, p≤0.05 compared to siNeg. <b>C</b>) ActRIIA and BMPRII siRNA suppresses target protein. PC3-M cells were transfected with ActRIIA-myc (upper panels) or BMPRII-flag (lower panels), as well as with the indicated siRNAs, followed by Western blot for indicated proteins. Data are from a representative experiment (N = 2 separate experiments). <b>D</b>) Blocking ActRIIA or BMPRII ligand binding inhibits EMSI. PC3-M cells were transiently transfected with empty vector or endoglin as above. After 2 days, cells were pretreated for 5 hrs ligand traps comprised of ActRIIA or BMPRII extracellular domain fused to immunoglobulin Fc region (Fc-A2 or Fc-B2 respectively). Treatment continued during the subsequent conduct of cell invasion assays. Data represent mean ± SEM of 3 independent experiments, each in replicates of N = 3. *, p≤0.05 compared to Vec/-.</p

Endoglin-Mediated Suppression of Invasion is Dependent on ActRIIA Kinase Domain and Independent of BMPRII Kinase Activity or Tail Domain.

<p>Cells were transfected with endoglin, ActRIIA (A) or BMPRII constructs (B), and ActRIIA or BMPRII directed siRNA, as indicated, and cell invasion assays conducted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072407#pone-0072407-g001" target="_blank">Figure 1</a>. Data represent the mean ± SEM of N = 2 independent experiments (A) or N = 3 independent experiments (B), each in replicates of 3. * denotes p≤0.05 compared to cells transfected with empty vector and non-targeting siRNA.</p

Proposed model for the regulation of endoglin-mediated suppression of invasion by ActRIIA and BMPRII.

<p>Based upon our current and prior findings, we propose the model depicted in this schema. A ligand-stimulated endoglin-ActRIIA-ALK2 signaling axis promotes Smad1 signaling to decrease the invasiveness of PCa cells. BMPRII is simultaneously required through additional, noncanonical regulatory elements (depicted as a dashed arrow). See text for expanded discussion. BMPRII plays a bimodal role in Smad1 signaling, promoting it via the kinase domain while inhibiting it in a tail-domain-dependent manner, potentially through a tail-domain-interacting protein or by direct interaction with ActRIIA. Endoglin physically interacts with both ActRIIA and BMPRII, and BMPRII interacts with ActRIIA in the absence of endoglin (bidirectional arrows). Previous work from our group has demonstrated that TGFβ signals through Smad3 to promote PCa invasion, that the balance between Smad3 and Smad1 regulates motility and invasion, and that endoglin acts as a gatekeeper in this regard.</p