LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRβ-Dependent Phosphatidylinositol 3-Kinase Activation

Herz J (2009) LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRb-Dependent Phosphatidylinositol 3-Kinase Activation. PLoS ONE 4(9): e6922. doi:10.1371/journal.pone.0006922 LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRb-Dependent Phosphatidylinositol 3-Kinase Activation Li Zhou 0 Yoshiharu Takayama 0 Philippe Boucher 0 Michelle D. Tallquist 0 Joachim Herz 0 Harald HHW Schmidt, Monash University, Australia 0 1 Department of Molecular Genetics, UT Southwestern Medical Center , Dallas, Texas , United States of America, 2 Molecular Biology, UT Southwestern Medical Center , Dallas, Texas , United States of America, 3 Department of Pharmacology, University of Strasbourg , Strasbourg , France Background: Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor b (PDGFRb) in vascular smooth muscle cells (SMCs). Activated PDGFRb undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement. Methods and Principal Findings: In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP12/2) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor b (TGFb) signaling are present in smooth muscle-specific LRP1 knockout (smLRP12/2) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRb in atherogenesis, we generated a strain of smLRP12/2 mice in which tyrosine 739/750 of the PDGFRb had been mutated to phenylalanines (PDGFRb F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP12/2 animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFb signaling, as indicated by high levels of nuclear phospho-Smad2. Conclusions and Significance: Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRb-dependent activation of PI3K. TGFb activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRb is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome. - Funding: This study was supported by grants from NIH (HL20948, HL63762, and NS43408), the Alzheimers Association, the Humboldt Foundation and the Perot Family Foundation for J.H., by NIH (HL074257) and American Heart Association (AHA) Scientific Development Grant (0330351) to M.D.T., and by a Postdoctoral Fellowship (Grant 0825316F) from the South Central Affiliate of the AHA to L.Z.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Low density lipoprotein receptor related protein 1 (LRP1) is a multifunctional member of the LDL receptor (LDLR) gene family with a unique capacity of binding over 40 distinct ligands [1]. It plays diverse roles in a variety of biological processes including lipoprotein metabolism, protease degradation, activation of lysosomal enzymes, and endocytosis of bacterial toxins and viruses [1,2]. Binding of apolipoprotein E (apoE) to the extracellular domain of LRP1 removes apoE-containing lipoprotein remnants from the circulation into the liver by endocytosis [3,4,5]. By contrast, in the smooth muscle cells (SMCs) of the arterial wall, apoE-lipoprotein binding inhibits platelet-derived growth factor (PDGF)-directed SMC migration [6]. Studies from our laboratory have shown that LRP1 suppresses PDGF receptor b (PDGFRb) activation and protects against atherosclerosis [7]. Activated PDGFRb undergoes tyrosine phosphorylation and subsequently interacts with a variety of SH2 domain-containing signaling molecules including phosphatidylinositol 3-kinase (PI3K), phospholipase Cc (PLCc), Src family kinase, and phosphotyrosine phosphatase SHP-2 [8]. Among these interacting proteins, PI3K which binds to the phosphorylated tyrosine 740/751 residues (739/750 in the mouse) of PDGFRb through its p85 regulatory subunit [9], is particularly important for regulating actin organization [10,11], cell growth and migration [12]. LRP1 is also known as transforming growth factor b (TGFb) receptor V (TbR-V) and appears to be required for mediating the growth inhibitory response of TGFb, in conjunction with Smad signaling through TbR-II and I [13,14]. TGFb signaling is abnormally elevated in the absence of LRP1 in vivo, where analysis of SMC-specific LRP1 knockout (smLRP12/2) mice revealed a Marfan syndrome-like phenotype with nuclear accumulation of phosphorylated Smad2 (p-Smad2) and disruption of elastic layers in the vessel wall [15]. For the present study we have generated a new, genetically complex strain of compound mutant mice that are LDL receptordeficient (LDLR2/2), lack LRP1 only in their vascular smooth muscle cells, and express an endogenous, crippled form of the PDGFRb that is incapable of activating PI3K. Our goal was to test, whether increased PDGFRb signaling through PI3K is the primary cause for the increased susceptibility to atherosclerotic lesion development in LDLR 2/2 mice lacking LRP1 in their SMCs, and whether PDGFRb-dependent PI3K signaling is required for the expression of the Marfan syndrome-like phenotype in smLRP1-deficient mice. Lack of LRP1 expression in the SMCs results in cell hypertrophy and vessel elongation Earlier data from our laboratory showed that smLRP12/2; LDLR2/2 mice are highly susceptible to atherosclerosis when fed a high-cholesterol diet [7]. To determine if this increased susceptibility to atherosclerosis is preserved in smLRP12/2 mice in the absence of hypercholesterolemia, smLRP12/2 mice either expressing or lacking LDLR were maintained on a standard low-fat rodent chow. Determination of plasma total cholesterol confirmed that smLRP12/2 mice did not develop hypercholesterolemia (129.965.3 mg/dl, Table 1). Whereas, smLRP12/2; LDLR2/2 mice had high total cholesterol levels of 246.5661.0 mg/dl with a major increase in LDL (Figure S1). Elongated aortas were present in the absence of hypercholesterolemia in smLRP12/2 mice (Figure 1c, h). However, atherosclerotic lesions were only visible in smLRP12/2; LDLR2/2 mice (Figure 1e, j). To study structural changes in the vascular wall of these elongated aortas, H&E, trichrome and elastin staining were performed. Thickened aortic walls with intima thickening, disarranged and hypertro (...truncated)