Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor

Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.     

A disintegrin and metalloproteinase 17 (ADAM17) mediates epidermal growth factor receptor transactivation by angiotensin II on hepatic stellate cells

Aims

Epidermal growth factor receptor (EGFR) transactivation induced by angiotensin II (Ang II) participates in the progression of various diseases. A disintegrin and metalloproteinase 17 (ADAM17) is thought to promote renal fibrosis, cardiac hypertrophy with fibrosis and atherosclerosis by activation of the EGFR through secretion of EGFR ligands. The purpose of this study was to investigate whether Ang II-induced EGFR transactivation occurs on hepatic stellate cells (HSCs) and whether the reaction is mediated via ADAM17.

Main methods

Ang II-induced EGFR transactivation and cellular proliferation of the human HSC line LI90 were investigated using Western blotting and ATP assay, respectively. Ang II-induced secretion of mature amphiregulin into the cell culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA).

Key findings

An inhibitor of ADAM17, TAPI-1, as well as antagonists of EGFR and angiotensin II type-1 receptor (AT1), attenuated Ang II-induced EGFR transactivation and proliferation of LI90 cells. Furthermore, silencing of ADAM17 inhibited Ang II-induced secretion of mature amphiregulin in addition to EGFR transactivation.

Significance

These results indicate that ADAM17 mediates Ang II-induced EGFR transactivation on HSCs, and that this process may participate in the progression of liver fibrosis.     

A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR₁), and by PAR₁ inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR₁-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.     

Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars

Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and production of extracellular matrix, which are very important process in skin wound healing and scarring; however, the signaling pathways involved in this process, especially in humans, are less explored. In the present study, we used skin fibroblasts of human hypertrophic scar, which expressed both AT1 and AT2 receptors, and observed that Ang II increased Akt phosphorylation and phosphoinositide 3 kinase (PI 3-K) activity. In addition, the Ang II-induced Akt phosphorylation was blocked by wortmannin, a PI 3-K inhibitor. This Ang II-activated PI 3-K/Akt cascade was markedly inhibited by valsartan, an AT(1) receptor-specific blocker, whereas it was enhanced by PD123319, an AT(2) receptor antagonist. On the other hand, the Ang II- or EGF-induced activation of PI 3-K/Akt was strongly attenuated by AG1478, an inhibitor of epidermal growth factor (EGF) receptor kinase. Moreover, Ang II stimulated tyrosine phosphorylation of EGF receptor and p85alpha subunit of PI 3-K accompanied by an increase in their association, which was inhibited by valsartan, and enhanced by PD123319. The Ang II-induced transactivation of EGF receptor resulted in activation of extracellular signal-regulated kinase (ERK) that was also inhibited by valsartan, and enhanced by PD123319. Taken together, our results showed that AT(1) receptor-mediated activation of PI 3-K/Akt cascades occurs at least partially via the transactivation of EGF receptor, which is under a negative control by AT(2) receptor in hypertrophic scar fibroblasts. These findings contribute to understanding the molecular mechanism of human hypertrophic scar formation.     

Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor

Stimulation of the angiotensin II (Ang II) type 1 receptor (AT1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.     

G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor

A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of G(q) markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks G(q) protein coupling. However, in cells expressing AT1 mutants that retain G(q) protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/G(q)/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation.     

The mechanism of angiotensin II-induced extracellular signal-regulated kinase-1/2 activation is independent of angiotensin AT(1A) receptor internalisation

The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.     

Angiotensin II promotes smooth muscle cell proliferation and migration through release of heparin-binding epidermal growth factor and activation of EGF-receptor pathway

Transactivation of EGF-receptor (EGFR) by G-protein coupled receptors (GPCRs) is emerging as an important pathway in cell proliferation, which plays a crucial role in the development of atherosclerotic lesion. Angiotensin II (Ang II) has been identified to have a major role in the formation of atherosclerotic lesions, although the underlying mechanisms remain largely unclear. We hypothesize that Ang II promotes the proliferation and migration of smooth muscle cells through the release of heparin-binding epidermal growth factor like growth factor (HB-EGF), transactivation of EGFR and activation of Akt and Erk 1/2, with matrix metalloproteases (MMPs) playing a dispensable role. Primary rat aortic smooth muscle cells were used in this study. Smooth muscle cells rendered quiescent by serum deprivation for 12 h were treated with Ang II (100 nM) in the presence of either GM6001 (20 microM), a specific inhibitor of MMPs or AG1478 (10 microM), an inhibitor of EGFR. The levels of phosphorylation of EGFR, Akt and Erk 1/2 were assessed in the cell lysates. Inhibition of MMPs by GM6001 significantly attenuated Ang II-stimulated phosphorylation of EGFR, suggesting that MMPs may be involved in the transactivation of EGFR by Ang II receptor. Furthermore Ang II-stimulated proliferation and migration of smooth muscle cells were significantly blunted by inhibiting MMPs and EGFR and applying HB-EGF neutralization antibody, indicating that MMPs, HB-EGF and EGFR activation is necessary for Ang-II stimulated migration and proliferation of smooth muscle cells. Our results suggest that inhibition of MMPs may represent one of the strategies to counter the mitogenic and motogenic effects of Ang II on smooth muscle cells and thereby prevent the formation and development of atherosclerotic lesions.     

Independence of angiotensin II-induced MAP kinase activation from angiotensin type 1 receptor internalization in clone 9 hepatocytes

The agonist-induced internalization of several G protein-coupled receptors is an obligatory requirement for their activation of MAPKs. Studies on the relationship between endocytosis of the angiotensin II (Ang II) type 1 receptor (AT1-R) and Ang II-induced ERK1/2 activation were performed in clone 9 (C9) rat hepatic cells treated with inhibitors of endocytosis [sucrose, phenylarsine oxide (PAO), and concanavalin A]. Although Ang II-induced endocytosis of the AT1-R was prevented by sucrose and PAO, and was partially inhibited by concanavalin A, there was no impairment of Ang II-induced ERK activation. However, the specific epidermal growth factor receptor (EGF-R) kinase inhibitor, AG1478, abolished Ang II-induced activation of ERK1/2. Sucrose and PAO also inhibited EGFinduced internalization of the EGF-R in C9 cells, and the inability of these agents to impair EGF-induced ERK activation suggested that the latter is also independent of receptor endocytosis. In COS-7 cells transiently expressing the rat AT1A-R, Ang II also caused ERK activation through EGF-R transactivation. Furthermore, a mutant AT1A-R with truncated carboxyl terminus and impaired internalization retained full ability to activate ERK1/2 in response to Ang II stimulation. These findings demonstrate that Ang II-induced ERK1/2 activation in C9 hepatocytes is independent of both AT1-R and EGF-R endocytosis and is mediated by transactivation of the EGF-R.     

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.     

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.     

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.     

Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.     

Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th) to 15(th) day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.     

Angiotensin II downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway

Angiotensin II (Ang II) plays a profound regulatory effect on NADPH oxidase and the functional features of vascular adventitial fibroblasts, but its role in antioxidant enzyme defense remains unclear. This study investigated the effect of Ang II on expressions and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in adventitial fibroblasts and the possible mechanism involved. Ang II decreased the expression and activity of CAT in a dose- and time-dependent manner, but not that of SOD and GPx. The effects were abolished by the angiotensin II type 1 receptor (AT1R) blocker losartan and AT1R small-interfering RNA (siRNA). Incubation with polyethylene glycol-CAT prevented the Ang II-induced effects on reactive oxygen species (ROS) generation and myofibroblast differentiation. Moreover, Ang II rapidly induced phosphorylation of ERK1/2, which was reversed by losartan and AT1R siRNA. Pharmacological blockade of ERK1/2 improved Ang II-induced decrease in CAT protein expression. These in vitro results indicate that Ang II induces ERK1/2 activation, contributing to the downregulation of CAT as well as promoting oxidative stress and adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.     

Angiotensin-II mediates nonmuscle myosin II activation and expression and contributes to human keloid disease progression

Aberrant fibroblast migration in response to fibrogenic peptides plays a significant role in keloid pathogenesis. Angiotensin II (Ang II) is an octapeptide hormone recently implicated as a mediator of organ fibrosis and cutaneous repair. Ang II promotes cell migration but its role in keloid fibroblast phenotypic behavior has not been studied. We investigated Ang II signaling in keloid fibroblast behavior as a potential mechanism of disease. Primary human keloid fibroblasts were stimulated to migrate in the presence of Ang II and Ang II receptor 1 (AT?), Ang II receptor 2 (AT?) or nonmuscle myosin II (NMM II) antagonists. Keloid and the surrounding normal dermis were immunostained for NMM IIA, NMM IIB, AT? and AT? expression. Primary human keloid fibroblasts were stimulated to migrate with Ang II and the increased migration was inhibited by the AT? antagonist EMD66684, but not the AT? antagonist PD123319. Inhibition of the promigratory motor protein NMM II by addition of the specific NMM II antagonist blebbistatin inhibited Ang II-stimulated migration. Ang II stimulation of NMM II protein expression was prevented by AT? blockade but not by AT? antagonists. Immunostaining demonstrated increased NMM IIA, NMM IIB and AT? expression in keloid fibroblasts compared with scant staining in normal surrounding dermis. AT? immunostaining was absent in keloid and normal human dermal fibroblasts. These results indicate that Ang II mediates keloid fibroblast migration and possibly pathogenesis through AT? activation and upregulation of NMM II.     

Involvement of the platelet-derived growth factor receptor in angiotensin II-induced activation of extracellular regulated kinases 1 and 2 in human mesangial cells

In mesangial cells angiotensin II (Ang II) has been shown to activate extracellular regulated kinases 1 and 2 (ERK1/2). Here, we studied the role of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) in Ang II-induced ERK1/2 activation in human mesangial cells. Ang II induced activation of ERK1/2 via the AT(1) receptor, and this response was blocked by the PDGFR-selective tyrosine kinase inhibitor AG1295, but not by AG1478, an EGFR-selective tyrosine kinase inhibitor, indicating participation of the PDGFR, but not of the EGFR in Ang II-induced ERK1/2 activation. In agreement with this assumption, Ang II caused tyrosine phosphorylation of the PDGFR and the adapter protein Shc in an AG1295-sensitive fashion. In conclusion, our data show that Ang II-induced activation of mitogenic signalling cascade in human mesangial cells involves ligand-independent activation of the PDGFR, but not of the coexpressed EGFR.     

Regulation of muscle satellite cell activation and chemotaxis by angiotensin II

The role of angiotensin II (Ang II) in skeletal muscle is poorly understood. We report that pharmacological inhibition of Ang II signaling or ablation of the AT1a receptor significantly impaired skeletal muscle growth following myotrauma, in vivo, likely due to impaired satellite cell activation and chemotaxis. In vitro experiments demonstrated that Ang II treatment activated quiescent myoblasts as evidenced by the upregulation of myogenic regulatory factors, increased number of β-gal+, Myf5-LacZ myoblasts and the acquisition of cellular motility. Furthermore, exogenous treatment with Ang II significantly increased the chemotactic capacity of C2C12 and primary cells while AT1a(-/-) myoblasts demonstrated a severe impairment in basal migration and were not responsive to Ang II treatment. Additionally, Ang II interacted with myoblasts in a paracrine-mediated fashion as 4 h of cyclic mechanical stimulation resulted in Ang II-induced migration of cocultured myoblasts. Ang II-induced chemotaxis appeared to be regulated by multiple mechanisms including reorganization of the actin cytoskeleton and augmentation of MMP2 activity. Collectively, these results highlight a novel role for Ang II and ACE inhibitors in the regulation of skeletal muscle growth and satellite cell function.