Cyclic Adenosine Monophosphate-Response Element–Binding Protein Mediates the Proangiogenic or Proinflammatory Activity of Gremlin

Abstract

Objective-Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs).Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin. Approach and Results-Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Corsini et al CREB in Gremlin Activity 137diseases 24 and cancer. [25][26][27] Gremlin acts as a bone morphogenic protein (BMP) antagonist by binding BMP2, BMP4, and BMP7. 28 Also, gremlin stimulates EC intracellular signaling and motility in a BMP-independent manner, leading to a potent angiogenic response in vivo. 27,29 This is because of the capacity of gremlin to bind and activate VEGF receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals. 30,31 Also, gremlin interacts with heparan-sulfate proteoglycans that act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2.32 However, at variance with heparin-binding VEGFs, gremlin does not interact with the coreceptor neuropilin-1. 32 Thus, gremlin is a novel proangiogenic VEGFR2 agonist distinct from canonical VEGFs.Here, we demonstrate that gremlin induces a proinflammatory response in ECs by inducing the expression of various chemokines and cell adhesion molecules, causing a VEGFR2-mediated activation of CREB. In turn, CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to leukocyte-adhesion to ECs and their extravasation. In keeping with these observations, gremlin and gremlin-expressing tumor cells induce a proinflammatory or proangiogenic response in vivo that is suppressed by the anti-inflammatory drug hydrocortisone. These data point to a nonredundant role of CRE...

Tubular overexpression of Gremlin in transgenic mice aggravates renal damage in diabetic nephropathy

“…Gremlin AGGRAVATES RENAL DAMAGE IN DIABETIC NEPHROPATHY Gremlin administration to mice induces the presence of inflammatory cells in the kidney and upregulates proinflammatory genes, including MCP-1 (20), extending previous in vitro studies in endothelial cells showing proinflammatory gene upregulation, closely related to its proangiogenic response (9).…”

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist

“…Cells were alternatively analysed using Axiovert 200M epifluorescence microscope equipped with Plan-Apochromat 63X/1.4 NA oil objective (Zeiss) or subjected to FACS analysis ( MACSQuant, Miltenyi Biotec) respectively. Intracellular ROS levels were assessed by measuring fluorescence intensity in HUVEC cells suspended in serum-free medium and loaded with the redox-sensitive dye DCFH-DA (5 μM, Molecular Probe, Life Technologies), as previously described [16]. Cells were then washed, stimulated with gremlin WT or gremlin C141A and finally subjected to FACS analysis.…”

“…Alginate beads containing PBS or gremlin WT or gremlin C141A were placed on the CAM of fertilized white Leghorn chicken eggs at day 11 of incubation [16]. After 72 hours, newly formed blood vessels converging toward the implant were counted at X5 magnification using a STEMI SR stereo-microscope equipped with an objective f of 100 mm with adapter ring 475070 (Zeiss).…”

“…Also, gremlin, once released by tumor cells, stimulates neovascular and pro-inflammatory responses in a BMP-independent manner [14] due to its capacity to bind and activate vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals [15–17], and to bind heparan-sulphate proteoglycans (HSPGs) [18, 19] on endothelial cells (ECs). Interestingly, gremlin is overexpressed in several human cancers [12], where it may neutralize the negative regulatory role of BMPs on cell proliferation, modulate EMT [13], and enhance tumor growth by stimulating tumor angiogenesis [14, 15] and inflammation [16]. …”

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