Metalloprotease-dependent ErbB ligand shedding in mediating EGFR transactivation and vascular remodelling.

AngII (angiotensin II) and its G-protein-coupled AT(1) receptor play critical roles in mediating cardiovascular diseases such as hypertension, atherosclerosis and restenosis after vascular injury. It is widely believed that AngII promotes these diseases by inducing vascular remodelling that involves hypertrophy, hyperplasia and migration of VSMCs (vascular smooth muscle cells). We have shown that transactivation of an ErbB family receptor, EGFR (epidermal growth factor receptor; ErbB1), is essential for VSMC hypertrophy and migration induced by AngII. However, the precise signal transduction mechanism by which AngII transactivates EGFR/ErbB1 and whether other ErbBs are also required for AngII function remains unclear. Recent studies suggest an involvement of a metalloprotease-dependent ErbB family ligand production in the transactivation. Here, we will discuss the roles and mechanisms of AngII/AT(1) receptor in promoting ErbB receptors transactivation in VSMCs. Further elucidation of this ErbB activation machinery not only will give us a better understanding of the critical molecular mechanism underlying vascular remodelling stimulated by AngII, but will also contribute to development of novel treatment strategies for cardiovascular diseases.

Unique regulation of c-Jun N-terminal kinase by PYK2/CAK-beta in angiotensin II-stimulated vascular smooth muscle cells.

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.

N-acetylcysteine inhibits angiotensin ii-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor.

Angiotensin II (Ang II) is known to stimulate reactive oxygen species (ROS) generation and epidermal growth factor (EGF) receptor transactivation to mediate growth-promoting signals such as extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). However, how ROS and EGF receptor interact to orchestrate these signals in VSMCs remains unclear. Here we found that an antioxidant, N-acetylcysteine, inhibited ERK activation and EGF receptor tyrosine phosphorylation induced by Ang II. Moreover, H(2)O(2) stimulates EGF receptor tyrosine phosphorylation and EGF receptor inhibitors attenuated H(2)O(2)-induced ERK activation. These data indicate that ROS mediate Ang II-induced EGF receptor transactivation, a critical mechanism for ERK-dependent growth in VSMCs.

Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK.

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.

Involvement of reactive oxygen species in the activation of tyrosine kinase and extracellular signal-regulated kinase by angiotensin II.

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant, alpha-tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of p120 and p70, and ERK activation in VSMCs. H2O2 also enhanced PTK activity. From these data, we conclude that ROS play a critical role in the Ang II-induced PTK and ERK activation in VSMCs, thereby contributing to vascular growth associated with enhanced Ang II activity.

PYK2/CAKbeta represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells.

In vascular smooth muscle cells (VSMCs), the focal adhesion kinase-related tyrosine kinase PYK2/CAKbeta is activated by vascular mitogens. Because reactive oxygen species (ROS) are assumed to mediate mitogenic signals by these agonists, we examined the possible link between ROS and PYK2 in cultured rat VSMCs. Here we present several lines of evidence showing that PYK2 is activated by ROS in VSMCs. The inhibitory effect of an antioxidant, N-acetyl-cysteine, on PYK2 activation by its specific agonists further suggests the pivotal role of PYK2 in vascular remodeling associated with enhanced ROS production.

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt.

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.

Epidermal growth factor receptor is indispensable for c-Fos expression and protein synthesis by angiotensin II.

We have reported that angiotensin II induces the epidermal growth factor (EGF) receptor transactivation leading to extracellular signal-regulated kinase (ERK) activation in rat vascular smooth muscle cells. Here, we report that the EGF receptor kinase inhibitor AG1478 and the ERK kinase inhibitor PD98059 markedly inhibited angiotensin II-induced c-Fos expression and protein synthesis but not c-Jun expression in these cells. These data suggest that the EGF receptor transactivation and subsequent ERK activation are indispensable for angiotensin II-mediated growth promotion of vascular smooth muscle cells providing a new mechanistic insight whereby angiotensin II contributes abnormal vascular remodeling.

The level of CD4 surface protein influences T cell selection in the thymus.

During T cell development thymocytes are subjected to positive and negative selection criteria to ensure that the mature T cell repertoire is MHC restricted, yet self tolerant at the same time. The CD4 and CD8 coreceptors are thought to play a crucial role in this developmental process. To elucidate the role of CD4 in T cell selection, we have produced a mouse strain that expresses CD4 at a reduced level. We used homologous recombination in embryonic stem cells to insert neo into the 3' untranslated region of CD4. The resulting mice have a reduction in the percentage of CD4+ cells in the thymus and a concomitant increase in CD8+ cells. In addition, breeding two individual class II-restricted TCR transgenic mice onto the CD4low (low level of CD4) mutant background affects the selection of each TCR differentially. In one case (AND TCR transgenic), significantly fewer CD4+ cells with the transgenic TCR develop on the CD4low mutant background, whereas in the other (5C.C7 TCR transgenic), selection to the CD4 lineage is only slightly reduced. These data support the differential avidity model of positive and negative selection. With little or no avidity, the cell succumbs to programmed cell death, low to moderate avidity leads to positive selection, and an avidity above a certain threshold, presumably above one that would lead to autoreactivity in the periphery results in clonal deletion. These data also support the idea that a minimum avidity threshold for selection exists and that CD4 plays a crucial role in determining this avidity.

Content and binding characteristics of the mitochondrial ATPase inhibitor, IF1, in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms.

We determined the IF1 contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF1-mediated inhibition of the ATPase activity of IF1-depleted rabbit heart mitochondrial particles (RHMP) by IF1-containing extracts from these same eight species. The IF1 binding experiments were run at both species-endogenous IF1 levels and at an IF1 level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF1 or either species-endogenous or normalized levels of pig heart IF1 were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF1-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF1 were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF1-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF1 inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF1 inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF1, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF1, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF1.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of MHC gene dosage and allelic variation on T cell receptor selection.

In a T cell receptor transgenic mouse model of thymic selection, the efficiency of selection of the transgenic alpha beta heterodimer is significantly enhanced in animals that express higher densities of the relevant major histocompatibility complex molecule (I-Ek/b). These results imply that there is a stochastic component to positive selection in the thymus. Allelic variants of the original selecting I-Ek molecule are either less efficient (E alpha k:E beta b) or incapable (E alpha k:E beta s and I-Ed) of mediating the selection of transgenic alpha beta + T cells. Two of these three I-E variants appear to differ from I-Ek in amino acid residues of the peptide binding site and not in residues capable of contacting the T cell receptor, suggesting that specific peptides, or conformations of peptides, play a role in positive selection. In contrast, mice transgenic for only the beta chain of this T cell receptor show selection for CD4+ T cells in the presence of all four I-E variants tested.