Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2-dependent signalling

Abstract

13 páginas, 8 figuras -- PAGS nros. 456-468The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF-β) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium-derived nitric oxide in endoglin+/− (Eng+/−) mice and cultured endothelial cells. Here, we find that TGF-β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/− mice, suggesting the involvement of endoglin in the TGF-β regulated vascular homeostasis. The endoglin-dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF-β receptor ALK5 and its downstream substrate Smad2. In addition, Smad2-specific signaling is upregulated in endoglin-induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin-dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin-inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome-dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF-β signaling, and leading to an increased eNOS expression in endothelial cells. J. Cell. Physiol. 210: 456–468, 2007. © 2006 Wiley-Liss, Inc.ALK, activin receptor-like kinase; Dox, doxycycline; eNOS, endothelial nitric oxide synthase; MEFs, murine embryonic fibroblasts; Pp, perfusion pressure; siRNA, small interference RNA; TGF-β, transforming growth factor-β; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; HHT, hereditary hemorrhagic telangiectasia. The endothelial nitric oxide synthase (eNOS or NOS3) is a critical regulator of cardiovascular homeostasis, vascular remodeling, and angiogenesis, and whose dysregulation leads to different types of vascular pathology (Kawashima and Yokoyama, 2004; Sessa, 2004; Tai et al., 2004). eNOS-derived NO is an endogenous vasodilatory molecule that regulates the tone of blood vessels and maintains an anti-thrombotic, anti-proliferative, and anti-apoptotic environment in the vessel wall (Sessa, 2004). Elucidation of the mechanisms and factors determining the expression and activity of eNOS under different physiological and pathophysiological conditions has long been considered central in order to understand the alterations in vascular NO production (Tai et al., 2004). The expression of eNOS is regulated by extracellular stimuli such as shear stress, estrogens, hypoxia, or growth factors (Davis et al., 2001; Simoncini et al., 2002; Tai et al., 2004). Among these, TGF-β1 has been shown to increase bovine aortic endothelial cell (BAEC) and human umbilical vein endothelial cell (HUVEC) steady-state eNOS mRNA expression (Inoue et al., 1995). Interestingly, eNOS expression is modulated not only by transcriptional, but also by post-transcriptional mechanisms (Sessa, 2004; Tai et al., 2004). At the transcriptional level, the eNOS promoter lacks a TATA box, but exhibits proximal elements such as Sp1 and GATA motifs, which are common characteristics of constitutively expressed genes (Zhang et al., 1995; Tai et al., 2004). The 5′-flanking region also contains many putative sites for further transcriptional regulation of eNOS. However, only a few of these sites have been formally shown to regulate eNOS transcription, including PEA3 (Cieslik et al., 1998) and AP-1 binding sites (Navarro-Antolin et al., 2000). Also, a region of the eNOS promoter extending from −1269 and −935 that contains Smad binding sites and mediates TGF-β induction of eNOS transcription, has been identified (Saura et al.,2002)Endoglin (CD105), is a 180-kDa homodimeric membrane glycoprotein which is strongly expressed by human endothelial cells and is involved in angiogenesis, cardiovascular development, and vascular homeostasis (Gougos and Letarte, 1990; Duff et al., 2003; Lebrin et al., 2005). The gene encoding endoglin has been identified as the target for the dominant vascular disorder known as hereditary hemorrhagic telangiectasia type 1 (HHT1) (Guttmacher et al., 1995). HHT is a highly penetrant autosomal dominant vascular dysplasia associated with frequent epistaxis, gastrointestinal bleedings, telangiectases, and arteriovenous malformations in brain, lung, and liver (Shovlin and Letarte, 1999; Marchuk and Lux, 2001). The mechanistic role of endoglin in angiogenesis and vascular remodeling is not known, but it is likely related to the transforming growth factor-β (TGF-β) system, as endoglin is a functional component of the membrane TGF-β receptor complex (Lastres et al., 1996) and is a substrate for TGF-β receptor-mediated phosphorylation (Guerrero-Esteo et al., 2002; Koleva et al., 2006). Signaling induction by TGF-β and related members of this superfamily regulate a variety of human diseases including cancer, fibrosis, developmental disorders, or cardiovascular pathology (Blobe et al., 2000; Siegel and Massague, 2003; Waite and Eng, 2003; Lebrin et al., 2005). TGF-β signaling is initiated when ligand induces formation of heteromeric complexes of type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively), which in turn activate and induce translocation of the Smad family of proteins to the nucleus (Attisano and Wrana, 2002; Miyazawa et al., 2002; Shi and Massague, 2003; Feng and Derynck, 2005). The association of endoglin with TβRII and TβRI (ALK1 and ALK5) regulates Smad signaling and cellular responses to TGF-β (Lastres et al., 1996; Letamendia et al., 1998; Li et al., 2000; Guerrero-Esteo et al., 2002). Endoglin regulates signal transduction of TGF-β1 by potentiating ALK1/Smad1 pathway and repressing the ALK5/Smad3 (Lebrin et al., 2004; Blanco et al., 2005), whereas it enhances ALK5/Smad2 signaling (Guerrero-Esteo et al., 2002). Conversely, in endoglin-deficient mice it has been shown that TGF-β/ALK5 signaling from endothelial cells to adjacent mesothelial cells is defective, as evidenced by reduced phosphorylation of Smad2 (Carvalho et al., 2004).Recently, it has been shown that endoglin regulates nitric oxide-dependent vasodilatation, as well as eNOS expression and activity (Jerkic et al., 2004; Toporsian et al., 2005). In addition, eNOS derived NO seems to play a major role in endoglin-dependent angiogenesis (Jerkic et al., 2006a) and COX-2 regulated expression (Jerkic et al., 2006b). The role of endoglin in the control of vascular tone was examined by measuring NO-dependent vasodilation in haploinsufficient mice (Eng+/−) and their Eng+/+ littermates (Jerkic et al., 2004). Urinary and plasma concentrations of nitrites were lower in Eng+/− than in Eng+/+ mice. The levels of eNOS in kidneys and femoral arteries were about half in Eng+/− than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/− compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively (Jerkic et al., 2004). However, the mechanisms by which endoglin regulates eNOS expression are poorly understood. In the present work we show that endoglin enhances eNOS expression by increasing Smad2 protein levels and thereby enhancing TGF-β-dependent induction of eNOS in endothelial cellsThis work was supported by Ministerio de Educacion y Ciencia, Spain; grant numbers: SAF2004-01390 to C.B. and BFU2004-00285/BFI to J.M.L.-N.; Fondo de Investigaciœn Sanitaria, Spain; grant number: PI020200 to C.B.; COBRE Program, National Center for Research Resources, NIH, USA; grant number: P20-RR15555 to C.P.H.V.; Fondo Nacional de Ciencia y Tecnologia, Chile; grant number: FONDECYT-1050476 to J.F.S.; HHT Foundation International, Inc.; grant number: 3 to C.B.; Canadian Institute of Health Research (CIHR) to L.A.Peer reviewe