Kai Kappert

Preferential oxidation of the second phosphatase domain of receptor-like PTP-α revealed by an antibody against oxidized protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) α after treatment of cells with H2O2 were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTPα was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTPα demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.

Liver X Receptor Agonists Suppress Vascular Smooth Muscle Cell Proliferation and Inhibit Neointima Formation in Balloon-Injured Rat Carotid Arteries

The liver X receptors α and β (LXRα and LXRβ) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1→S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1→S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase–associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1→S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand–mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org.

Redox-Sensitive Signaling by Angiotensin II Involves Oxidative Inactivation and Blunted Phosphorylation of Protein Tyrosine Phosphatase SHP-2 in Vascular Smooth Muscle Cells From SHR

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II—induced SHP-2 effects were inhibited by valsartan (AT1R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II–mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT1R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II–stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II–induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2–dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II–AKT signaling.

Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47phox Is Critical for α-Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47phox, and we systematically evaluated the signal relay mechanisms by which the αPDGF receptor (αPDGFR) induces ROS liberation. Stimulation of the αPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. αPDGFR activation is rapidly followed by the translocation of p47phox and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47phox(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant αPDGFRs that lack specific binding sites for αPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cγ, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cγ, or SHP-2) completely abolished PDGF-dependent p47phox and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant αPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110α (but not p110β) was identified as the crucial isoform that elicits αPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110α mediates growth factor-dependent ROS production by recruiting p47phox and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

Antioxidants Relieve Phosphatase Inhibition and Reduce PDGF Signaling in Cultured VSMCs and in Restenosis

Objective—Growth factor– and reactive oxygen species (ROS)-induced activation of VSMCs is involved in vascular disease. This study investigates whether inhibitory oxidation of protein tyrosine phosphatases (PTPs) contributes to signaling in VSMCs in vitro and in vivo, and analyzes whether ROS- and growth factor–dependent vascular smooth muscle cell (VSMC) signaling is blunted by antioxidants that are able to activate oxidized PTPs. Methods and Results—Signaling induced by H2O2 and platelet-derived growth factor (PDGF) was analyzed in VSMCs with or without the antioxidants N-acetyl-cysteine (NAC) and tempol. Effects of antioxidants on PDGF-stimulated chemotaxis and proliferation were determined. In vivo effects of antioxidants were analyzed in the rat carotid balloon-injury model, by analyzing neointima formation, cell proliferation, PDGF &bgr;-receptor status, and PTP expression and activity. NAC treatment prevented H2O2-induced PTP inhibition, and reduced H2O2- and ligand-induced PDGF &bgr;-receptor phosphorylation, PDGF-induced proliferation, and chemotaxis of VSMCs. Antioxidants inhibited neointima formation and reduced PDGF receptor phosphorylation in the neointima and also increased PTP activity. Conclusion—PTP-inhibition was identified as an intrinsic component of H2O2- and PDGF-induced signaling in cultured VSMCs. The reduction in PDGF &bgr;-receptor phosphorylation in vivo, and the increase in PTP activity, by antioxidants indicate activation of oxidized PTPs as a previously unrecognized mechanism for the antirestenotic effects of antioxidants. The findings thus suggest, in general terms, reactivation of oxidized PTPs as a novel antirestenotic strategy.

Histone Deacetylase 6 (HDAC6) Is an Essential Modifier of Glucocorticoid-Induced Hepatic Gluconeogenesis

In the current study, we investigated the importance of histone deacetylase (HDAC)6 for glucocorticoid receptor–mediated effects on glucose metabolism and its potential as a therapeutic target for the prevention of glucocorticoid-induced diabetes. Dexamethasone-induced hepatic glucose output and glucocorticoid receptor translocation were analyzed in wild-type (wt) and HDAC6-deficient (HDAC6KO) mice. The effect of the specific HDAC6 inhibitor tubacin was analyzed in vitro. wt and HDAC6KO mice were subjected to 3 weeks’ dexamethasone treatment before analysis of glucose and insulin tolerance. HDAC6KO mice showed impaired dexamethasone-induced hepatic glucocorticoid receptor translocation. Accordingly, dexamethasone-induced expression of a large number of hepatic genes was significantly attenuated in mice lacking HDAC6 and by tubacin in vitro. Glucose output of primary hepatocytes from HDAC6KO mice was diminished. A significant improvement of dexamethasone-induced whole-body glucose intolerance as well as insulin resistance in HDAC6KO mice compared with wt littermates was observed. This study demonstrates that HDAC6 is an essential regulator of hepatic glucocorticoid-stimulated gluconeogenesis and impairment of whole-body glucose metabolism through modification of glucocorticoid receptor nuclear translocation. Selective pharmacological inhibition of HDAC6 may provide a future therapeutic option against the prodiabetogenic actions of glucocorticoids.

Integrins alphavbeta3 and alphavbeta5 mediate VSMC migration and are elevated during neointima formation in the rat aorta.

Abstract Neointima formation involves tissue expression of matrix proteins and growth factors. The role of αvβ3, but not αvβ5 integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of αvβ3 and αvβ5 integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express αvβ3 and αvβ5 integrin subunits. The αvβ5 integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the αvβ3 integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both αvβ3 and αvβ5 integrins. Selective blocking antibodies for αvβ3 and αvβ5 inhibited migration of RASMC and HCSMC by more than 60% (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for αvβ3 and β5 mRNA in uninjuried aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of αvβ3 and β5 mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased inegrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that αvβ3 and αvβ5 are elevated during neointima formation in the rat and indicates a novel role for αvβ5 participating in mechanisms regulating smooth muscle cell migration.

Cardiac c‐kit+AT2+ Cell Population is Increased in Response to Ischemic Injury and Supports Cardiomyocyte Performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia‐induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c‐kit+ cells accumulating in peri‐infarct zone and c‐kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c‐kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c‐kit+AT2+ cells, represented ∼0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata‐4, Notch‐2, Nkx‐2.5) and genes required for self‐renewal (Tbx‐3, c‐Myc, Akt). When adult cardiomyocytes and cardiac c‐kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c‐kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c‐kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c‐kit+AT2+ cell population. STEM CELLS 2009;27:2488–2497

Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation

Growth factor‐dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor‐tyrosine‐kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein‐tyrosine‐phosphatases (PTPs) are additional candidate regulators of these processes. Using laser‐capture‐microdissection and quantitative RT‐polymerase chain reaction (qRT‐PCR), we investigated the layer‐specific expression of the four platelet‐derived growth factor (PDGF) isoforms, the PDGF‐ and receptors, and five PTPs implied in control of PDGF‐receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF‐ receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression—particularly, DEP‐1 μlevels—appeared to be the dominating factor determining receptor‐phosphorylation and VSMC proliferation. In support of these findings, cultured DEP‐1–/–cells displayed increased PDGF‐dependent cell signaling. Hyperactivation of PDGF‐induced signaling was also observed after siRNA‐down‐regulation of DEP‐1 in VSMCs. The results indicate a previously unrecognized role of PDGF‐receptor‐targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK‐dependent tissue remodeling.—FASEB J. 21, 523–534 (2007)

Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

RATIONALE Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia. OBJECTIVES To investigate whether hypoxia alters PDGF β receptor (βPDGFR) signaling in the pulmonary vasculature. METHODS The impact of chronic hypoxia on signal transduction by the βPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo. MEASUREMENTS AND MAIN RESULTS Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect βPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated βPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced βPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. βPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH. CONCLUSIONS Hypoxia reduces expression and activity of βPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the βPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.