Large-scale phosphoproteomics reveals activation of the MAPK/GADD45ß/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells.

BACKGROUND: BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. METHODS: To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. RESULTS: Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45ß. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. CONCLUSIONS: Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly.

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of ß-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

The alternative matrisome: Alternative splicing of ECM proteins in development, homeostasis and tumor progression.

The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized "alternative" matrisome in physiological and pathological states such as cancer.

Shaping Up the Tumor Microenvironment With Cellular Fibronectin.

Normal tissue homeostasis and architecture restrain tumor growth. Thus, for a tumor to develop and spread, malignant cells must overcome growth-repressive inputs from surrounding tissue and escape immune surveillance mechanisms that curb cancer progression. This is achieved by promoting the conversion of a physiological microenvironment to a pro-tumoral state and it requires a constant dialog between malignant cells and ostensibly normal cells of adjacent tissue. Pro-tumoral reprogramming of the stroma is accompanied by an upregulation of certain extracellular matrix (ECM) proteins and their cognate receptors. Fibronectin (FN) is one such component of the tumor matrisome. This large multidomain glycoprotein dimer expressed over a wide range of human cancers is assembled by cell-driven forces into a fibrillar array that provides an obligate scaffold for the deposition of other matrix proteins and binding sites for functionalization by soluble factors in the tumor microenvironment. Encoded by a single gene, FN regulates the proliferation, motile behavior and fate of multiple cell types, largely through mechanisms that involve integrin-mediated signaling. These processes are coordinated by distinct isoforms of FN, collectively known as cellular FN (as opposed to circulating plasma FN) that arise through alternative splicing of the FN1 gene. Cellular FN isoforms differ in their solubility, receptor binding ability and spatiotemporal expression, and functions that have yet to be fully defined. FN induction at tumor sites constitutes an important step in the acquisition of biological capabilities required for several cancer hallmarks such as sustaining proliferative signaling, promoting angiogenesis, facilitating invasion and metastasis, modulating growth suppressor activity and regulating anti-tumoral immunity. In this review, we will first provide an overview of ECM reprogramming through tumor-stroma crosstalk, then focus on the role of cellular FN in tumor progression with respect to these hallmarks. Last, we will discuss the impact of dysregulated ECM on clinical efficacy of classical (radio-/chemo-) therapies and emerging treatments that target immune checkpoints and explore how our expanding knowledge of the tumor ECM and the central role of FN can be leveraged for therapeutic benefit.

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus.

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFß family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation.

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.

Multiple functions of tristetraprolin/TIS11 RNA-binding proteins in the regulation of mRNA biogenesis and degradation.

Members of the tristetraprolin (TTP/TIS11) family are important RNA-binding proteins initially characterized as mediators of mRNA degradation. They act via their interaction with AU-rich elements present in the 3'UTR of regulated transcripts. However, it is progressively appearing that the different steps of mRNA processing and fate including transcription, splicing, polyadenylation, translation, and degradation are coordinately regulated by multifunctional integrator proteins that possess a larger panel of functions than originally anticipated. Tristetraprolin and related proteins are very good examples of such integrators. This review gathers the present knowledge on the functions of this family of RNA-binding proteins, including their role in AU-rich element-mediated mRNA decay and focuses on recent advances that support the concept of their broader involvement in distinct steps of mRNA biogenesis and degradation.

DSIR: assessing the design of highly potent siRNA by testing a set of cancer-relevant target genes.

Chemically synthesized small interfering RNA (siRNA) is a widespread molecular tool used to knock down genes in mammalian cells. However, designing potent siRNA remains challenging. Among tools predicting siRNA efficacy, very few have been validated on endogenous targets in realistic experimental conditions. We previously described a tool to assist efficient siRNA design (DSIR, Designer of siRNA), which focuses on intrinsic features of the siRNA sequence. Here, we evaluated DSIR's performance by systematically investigating the potency of the siRNA it designs to target ten cancer-related genes. mRNA knockdown was measured by quantitative RT-PCR in cell-based assays, revealing that over 60% of siRNA sequences designed by DSIR silenced their target genes by at least 70%. Silencing efficacy was sustained even when low siRNA concentrations were used. This systematic analysis revealed in particular that, for a subset of genes, the efficiency of siRNA constructs significantly increases when the sequence is located closer to the 5'-end of the target gene coding sequence, suggesting the distance to the 5'-end as a new feature for siRNA potency prediction. A new version of DSIR incorporating these new findings, as well as the list of validated siRNA against the tested cancer genes, has been made available on the web (http://biodev.extra.cea.fr/DSIR).

BMP9 and BMP10 are critical for postnatal retinal vascular remodeling.

ALK1 is a type I receptor of the TGF-ß family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

A novel function of Tis11b/BRF1 as a regulator of Dll4 mRNA 3'-end processing.

Tis11b/BRF1 belongs to the tristetraprolin family, the members of which are involved in AU-rich-dependent regulation of mRNA stability/degradation. Mouse inactivation of the Tis11b gene has revealed disorganization of the vascular network and up-regulation of the proangiogenic factor VEGF. However, the VEGF deregulation alone cannot explain the phenotype of Tis11b knockouts. Therefore we investigated the role of Tis11b in expression of Dll4, another angiogenic gene for which haploinsufficiency is lethal. In this paper, we show that Tis11b silencing in endothelial cells leads to up-regulation of Dll4 protein and mRNA expressions, indicating that Dll4 is a physiological target of Tis11b. Tis11b protein binds to endogenous Dll4 mRNA, and represses mRNA expression without affecting its stability. In the Dll4 mRNA 3' untranslated region, we identified one particular AUUUA motif embedded in a weak noncanonical polyadenylation (poly(A)) signal as the major Tis11b-binding site. Moreover, we observed that inhibition of Tis11b expression changes the ratio between mRNAs that are cleaved or read through at the poly(A) signal position, suggesting that Tis11b can interfere with mRNA cleavage and poly(A) efficiency. Last, we report that this Tis11b-mediated mechanism is used by endothelial cells under hypoxia for controlling Dll4 mRNA levels. This work constitutes the first description of a new function for Tis11b in mammalian cell mRNA 3'-end maturation.

Regulation of epithelial to mesenchymal transition: CK2ß on stage.

Protein kinase CK2 participates in the regulation of fundamental cellular processes. Among these processes, cell polarity and cell morphology are controlled by this enzyme probably through the phosphorylation of key proteins. To further study the involvement of CK2 in these processes, we showed that in epithelial cells, the regulatory CK2ß subunit was required for LKB1-dependent polarization and cell adhesion. Moreover, CK2ß silencing in MCF10A mammary epithelial cells triggered changes in their morphology correlated with the acquisition of mesenchymal phenotype, which were reminiscent to TGFß-induced epithelial-to-mesenchymal-transition (EMT). TGFß has emerged as a major inducer of EMT both in vitro and in vivo. We found that among the TGFß isoforms, TGFß2 expression was strongly induced in CK2ß-knockdown cells. However, the EMT phenotype induced in response to CK2ß silencing was not abolished by blocking the TGFß signaling pathway at TGFß receptor level, suggesting that alternative pathways might be involved. Given the importance of CK2 in tumorigenesis, a dysregulation of CK2ß expression might contribute to EMT induction during cancer progression.

Hypoxia-inducible factor-1α mRNA: a new target for destabilization by tristetraprolin in endothelial cells.

Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3'UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3'UTR; and 5) the most distal AU-rich elements present in HIF-1α 3'UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.

The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3' processing pathway.

Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3'-extended transcript at the expense of the mature CTH2 mRNA. The 3' ends of the CTH2 mRNA were mapped to a [GU(3-5)](5) repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3'-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3'-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

Destabilization of vascular endothelial growth factor mRNA by the zinc-finger protein TIS11b.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine, which plays a major role in tumor angiogenesis. VEGF mRNA expression is controlled by hypoxia, growth factors and hormones through both transcriptional and post-transcriptional mechanisms. VEGF mRNA has a short half-life and its abundance is regulated by the binding of stabilizing (HuR, hRNP-L) and still uncharacterised destabilizing proteins to its 3'-untranslated region. Here, we report that the ACTH-regulated zinc-finger protein TIS11b and its homologs TIS11 and TIS11d interact with the 3'-untranslated region of VEGF mRNA and decrease its stability (half-life reduced from 130 to 60 min). Within the 2201 bp 3'-untranslated region of VEGF mRNA, we identified a 75 bp domain, containing two consensus AU-rich motifs, which binds TIS11b and mediates its destabilizing activity. Ribonucleoprotein (RNP) complex immunoprecipitation experiments allowed us to demonstrate that the interaction between TIS11b and VEGF 3'-untranslated region occurs in live cells. Knocking down TIS11b expression in primary adrenocortical cells with small interfering (si)RNAs clearly indicated that TIS11b participates in the control of both basal and, to a larger extent, ACTH-induced VEGF mRNA expression levels. TIS11b is the first VEGF mRNA-destabilizing protein identified so far and therefore appears as a new potential target in antiangiogenic therapies.

Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b].

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3'-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3'-untranslated region.