No abstract

Background-Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor ␤ (PDGFR␤), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. Methods and Results-In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFR␤ signaling pathway, as shown by PDGFR␤ tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFR␤ activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFR␤ activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFR␤ (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFR␤ adduct formation and subsequent PDGFR␤ activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H 2 O 2 (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFR␤ adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFR␤ activation may occur during atherogenesis. Conclusions-MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFR␤ acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFR␤ activation in atherosclerotic areas.

Abstract-Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time-and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR-deficient B82L and EGFR-transduced B82LK ϩ cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology. (Circ Res. 1999;85:892-899.)

In order to gain insight into the biological role of receptor protein tyrosine phosphatase ␥ (RPTP␥), we have generated RPTP␥-null mice. RPTP␥ was disrupted by insertion of the ␤-galactosidase gene under the control of the RPTP␥ promoter. As the RPTP␥-null mice did not exhibit any obvious phenotype, we made use of these mice to study RPTP␥ expression and thus shed light on potential biological functions of this phosphatase. Inspection of mouse embryos shows that RPTP␥ is expressed in a variety of tissues during embryogenesis. RPTP␥ is expressed in both embryonic and adult brains. Specifically, we detected RPTP␥ expression in cortical layers II and V and in the stratum pyramidale of the hippocampus, indicating that RPTP␥ is a marker for pyramidal neurons. Mixed primary culture of glial cells showed a lack of expression of RPTP␥ in astrocytes and a low expression of RPTP␥ in oligodendrocytes and in microglia. Interestingly, RPTP␥ expression was detected in all sensory organs, including the ear, nose, tongue, eye, and vibrissa follicles, suggesting a potential role of RPTP␥ in sensory neurons. An initial behavioral analysis showed minor changes in the RPTP␥-null mice.The phosphorylation of proteins on tyrosine residues is essential for transmission of signals for cell growth, proliferation, and differentiation. Phosphorylation depends on a regulated balance between the activities of protein tyrosine kinases and protein tyrosine phosphatases (PTP). While the roles and the mechanisms of action of tyrosine kinases are well characterized, our present understanding of tyrosine phosphatases is less developed.PTP are classified into two groups, one comprising cytoplasmic and the other transmembrane, or receptor type (receptor protein tyrosine phosphatases [RPTP]), proteins. Seven different classes of RPTP have been defined, based primarily upon variations in the extracellular domain (reviewed in references 2 and 9). RPTP have one or more intracellular catalytic domains with a conserved cysteine at the active site, a transmembrane region, and an extracellular domain. We have focused our studies on RPTP␥, a receptor-type PTP containing an extracellular carbonic anhydrase domain, a fibronectin type III domain, and a spacer domain (1). RPTP␥ is representative of a subfamily of RPTP that includes RPTP (also designated RPTP␤). As is the case for RPTP, several isoforms have been described for RPTP␥ (19). RPTP␥-B lacks 29 amino acids in a cytosolic helix-turn-helix like motif in the juxtamembrane position compared to the full-length RPTP␥-A isoform. RPTP␥-C contains only one phosphatase domain, while RPTP␥-D is a soluble isoform and has lost all phosphatase activity.Very few studies have reported on the biological function of RPTP␥. Overexpression of RPTP␥-A in the neuronal PC12D cell line prevented neurite outgrowth upon nerve growth factor (NGF) treatment (20). Both interleukin 1␤ and tumor necrosis factor upregulate RPTP␥ mRNA in astrocytoma cell lines, suggesting a potential role for RPTP␥ in the development of inflammatory d...