Effects of Angiotensin II Receptor Signaling during Skin Wound Healing
2004; Elsevier BV; Volume: 165; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63422-0
ISSN1525-2191
AutoresHikaru Takeda, Yohtaro Katagata, Yutaka Hozumi, Shigeo Kondô,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe tissue angiotensin (Ang) system, which acts independently of the circulating renin Ang system, is supposed to play an important role in tissue repair in the heart and kidney. In the skin, the role of the system for wound healing has remained to be ascertained. Our study demonstrated that oral administration of selective AngII type-1 receptor (AT1) blocker suppressed keratinocyte re-epithelization and angiogenesis during skin wound healing in rats. Immunoprecipitation and Western blot analysis indicated the existence of AT1 and AngII type-2 receptor (AT2) in cultured keratinocytes and myofibroblasts. In a bromodeoxyuridine incorporation study, induction of AT1 signaling enhanced the incorporation into keratinocytes and myofibroblasts. Wound healing migration assays revealed that induction of AT1 signaling accelerated keratinocyte re-epithelization and myofibroblasts recovering. In these experiments, induction of AT2 signaling acted vice versa. Taken together, our study suggests that skin wound healing is regulated by balance of opposing signals between AT1 and AT2. The tissue angiotensin (Ang) system, which acts independently of the circulating renin Ang system, is supposed to play an important role in tissue repair in the heart and kidney. In the skin, the role of the system for wound healing has remained to be ascertained. Our study demonstrated that oral administration of selective AngII type-1 receptor (AT1) blocker suppressed keratinocyte re-epithelization and angiogenesis during skin wound healing in rats. Immunoprecipitation and Western blot analysis indicated the existence of AT1 and AngII type-2 receptor (AT2) in cultured keratinocytes and myofibroblasts. In a bromodeoxyuridine incorporation study, induction of AT1 signaling enhanced the incorporation into keratinocytes and myofibroblasts. Wound healing migration assays revealed that induction of AT1 signaling accelerated keratinocyte re-epithelization and myofibroblasts recovering. In these experiments, induction of AT2 signaling acted vice versa. Taken together, our study suggests that skin wound healing is regulated by balance of opposing signals between AT1 and AT2. Angiotensin (Ang) II is the active biological octapeptide of the systemic renin Ang system, which is a circulating humoral system responsible for blood pressure regulation and salt-water homeostasis. AngII exerts its effects through binding the specific receptors. There are two major subtypes of AngII receptors: type 1 (AT1) and type 2 (AT2).1Gyurko R Kimura B Kurian P Crews FT Philips MI Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices.Biochem Biophys Res Commun. 1992; 186: 285-292Crossref PubMed Scopus (37) Google Scholar Most of the physiological actions of AngII are regulated through AT1. Recently, in addition to the classical role, AngII has attracted attention for its novel role as a growth factor. Growth-modulating effects of these receptors were reported in cardiac vascular endothelial cells, smooth muscle cells, and fibroblasts, where AT1 caused cell proliferation and synthesis of extracellular matrix proteins, whereas AT2 acted in an anti-proliferative manner.2Fujiyama S Matsubara H Nozawa Y Maruyama K Mori Y Tsutsumi Y Masaki H Uchiyama Y Koyama Y Nose A Iba O Tateishi E Ogata N Jyo N Higashiyama S Iwasaka T Angiotensin AT1 and AT2 receptors differentially regulate angiopoietin-2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation.Circ Res. 2001; 88: 22-29Crossref PubMed Scopus (206) Google Scholar, 3Shibasaki Y Matsubara H Nozawa Y Mori Y Masaki H Kosaki A Tsutsumi Y Uchiyama Y Fujiyama S Nose A Iba O Tateishi E Hasegawa T Horiuchi M Nahmias C Iwasaka T Angiotensin II type 2 receptor inhibits epidermal growth factor receptor transactivation by increasing association of SHP-1 tyrosine phosphatase.Hypertension. 2001; 38: 367-372Crossref PubMed Scopus (45) Google Scholar, 4Murasawa S Mori Y Nozawa Y Gotoh N Shibuya M Masaki H Maruyama K Tsutsumi Y Moriguchi Y Shibasaki Y Tanaka Y Iwasaka T Inada M Matsubara H Angiotensin II type 1 receptor-induced extracellular signal-related protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor.Circ Res. 1998; 82: 1338-1348Crossref PubMed Scopus (181) Google Scholar, 5Eguchi S Numaguchi K Iwasaki H Matsumoto T Yamakawa T Utsumonoa H Motley ED Kawakatsu H Owada KM Hirata Y Marumo F Inagami T Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells.J Biol Chem. 1998; 273: 8890-8896Crossref PubMed Scopus (507) Google Scholar Despite the fact that the role of AngII and its receptors has been well discussed in the cardiovascular field, a few reports can be found in the dermatological field. Literature has shown that the topical administration of AngII accelerated skin wound healing,6Rodgers KE Abiko M Giegis W St. Amand K Campeau J diZerega G Acceleration of dermal tissue repair by angiotensin II.Wound Repair Regen. 1997; 5: 175-183Crossref PubMed Scopus (45) Google Scholar, 7Rodgers KE Xiong S Felix JC Roda R Espinosa T Maldonado S diZerega G Development of angiotensin (1-7) as an agent to accelerate dermal regeneration.Wound Repair Regen. 2001; 9: 241-250Crossref Google Scholar, 8Rodgers KE Roda N Felix JC Espinosa T Maldonado S diZerega G Histological evaluation of the effects of angiotensin peptides on wound repair in diabetic mice.Exp Dermatol. 1997; 5: 175-183Google Scholar an increased level of AngII or Ang-converting enzyme in the rat skin1Gyurko R Kimura B Kurian P Crews FT Philips MI Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices.Biochem Biophys Res Commun. 1992; 186: 285-292Crossref PubMed Scopus (37) Google Scholar, 9Sun Y Weber KT Angiotensin-converting enzyme and wound healing in diverse tissue of the rat.J Lab Clin Med. 1996; 127: 94-101Abstract Full Text PDF PubMed Scopus (64) Google Scholar during wound healing, and the existence of AngII receptors by harvesting whole wounded skin.10Abiko M Rodgers KE Campeau JD Nakamura R Dizerega GS Alterations of angiotensin II receptor levels in sutured wounds in rat skin.J Invest Surg. 1996; 9: 447-453Crossref PubMed Scopus (28) Google Scholar, 11Kimura B Sumners C Philips MI Changes in skin angiotensin II receptors in rats during wound healing.Biochem Biophys Res Commun. 1992; 187: 1087-1090Crossref Scopus (82) Google Scholar, 12Viswanathan M Saavedra JM Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing.Peptides. 1992; 13: 783-786Crossref PubMed Scopus (166) Google Scholar, 13Gyurko R Kimura B Kurian P Crews FT Philips MI Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices.Biochem Biophys Res Commun. 1992; 186: 285-292Crossref PubMed Google Scholar However, the mechanism of AngII on skin wound healing has remained unknown. In this report, we studied the mechanism of AngII receptor signaling using selective AT1 or AT2 blockers to assess the effects of AngII receptor signaling during skin wound healing. Candesartan (CV-11974) and Candesartan cilexetil (TCV-116) were kind gifts from Takeda Chemical Industries (Osaka, Japan). Candesartan is a potent and selective AT1 blocker that binds tightly to and dissociates slowly from AT1. Candesartan cilexetil is a prodrug, and is administered orally and converted rapidly and completely to its active form, candesartan, by the enzyme esterase at the time of intestinal absorption.14Yoshimura M Yasue H Hemodynamic and humoral effects of angiotensin II antagonist, candesartan cilexetil, in patients with congestive heart failure.Cardiolgy. 2000; 93: 175-182Crossref PubMed Scopus (9) Google Scholar PD-123319 ditrifluoroacetate (Sigma Japan, Tokyo, Japan) is a selective AT2 blocker and used in experiments in vitro.2Fujiyama S Matsubara H Nozawa Y Maruyama K Mori Y Tsutsumi Y Masaki H Uchiyama Y Koyama Y Nose A Iba O Tateishi E Ogata N Jyo N Higashiyama S Iwasaka T Angiotensin AT1 and AT2 receptors differentially regulate angiopoietin-2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation.Circ Res. 2001; 88: 22-29Crossref PubMed Scopus (206) Google Scholar, 3Shibasaki Y Matsubara H Nozawa Y Mori Y Masaki H Kosaki A Tsutsumi Y Uchiyama Y Fujiyama S Nose A Iba O Tateishi E Hasegawa T Horiuchi M Nahmias C Iwasaka T Angiotensin II type 2 receptor inhibits epidermal growth factor receptor transactivation by increasing association of SHP-1 tyrosine phosphatase.Hypertension. 2001; 38: 367-372Crossref PubMed Scopus (45) Google Scholar Sprague-Dawley male rats (250 to 300 g, ∼10 weeks of age) were purchased from SLC Inc., Japan and housed on a 12:12-hour light:dark cycle. Food and water were available ad libitum. The experiments were performed according to the institutional guidelines for care and use of laboratory animals. TCV-116, 1 mg/kg/day (n = 5) or 10 mg/kg/day (n = 5), suspended with gum arabic in a volume of 2 ml/kg water or vehicle (n = 5) were orally administered to the rats once a day starting day 0 until the end of the experiment. On day 0, a full-thickness skin wound was made on the dorsal site of each rat using 8-mm biopsy trepan (Kai Industries, Seki, Japan). The wounds had 50 μl of 70% ethanol once a day starting day 0 until day 5 after injury without bandaging. No wounds had infections. The wounds were totally excised at each selected time point (day 3, 6, 9, 12, and 15 after wounding), and were fixed in 10% formalin, embedded in paraffin, and offered for hematoxylin and eosin (H&E) or immunohistochemical staining. Rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.) during wounding and tissue collection. Using the H&E sections, the extent of re-epithelization of wounds was evaluated by measuring the epidermal migration from the normal wound margin to the point where the migrating epithelium stopped processing.6Rodgers KE Abiko M Giegis W St. Amand K Campeau J diZerega G Acceleration of dermal tissue repair by angiotensin II.Wound Repair Regen. 1997; 5: 175-183Crossref PubMed Scopus (45) Google Scholar The re-epithelization index was determined by the percentage of the new epithelium present in the total wound. To detect the blood vessels, immunohistochemical staining using the rabbit anti-von Willebrand factor polyclonal antibody (DAKO Japan, Kyoto, Japan) was performed on the sections by the labeled streptavidin-biotin method.15Giorno R A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction.Diagn Immunol. 1984; 2: 161-166PubMed Google Scholar The reaction was visualized using 3-amino-9-ethylcarbazole (DAKO Japan) as chromogen and the slides were counterstained with hematoxylin and mounted in aqueous mounting medium for examination. The average number of blood vessels with a diameter of more than 4 μm per square millimeter in the wound site was calculated as vascular density. Two of the authors independently counted the number of blood vessels without any information on each section. Keratinocytes were cultured as previously described.16Kondo S Hozumi Y Mitsuhashi Y Comparative inhibitory effects of vitamin D3 and an analogue on normal psoriatic epidermis in organ culture.Arch Dermatol Res. 2000; 292: 550-555Crossref PubMed Scopus (8) Google Scholar In brief, epidermal strips were isolated from the surgically removed rat dorsal skin using dispase (1.2 U/ml, Sigma Japan, Tokyo, Japan) for 24 hours at 4°C. After incubation of epidermal strips in 0.05% trypsin/0.02% ethylenediaminetetraacetic acid for 25 minutes at room temperature, keratinocytes were plated on culture dishes (78.5 cm2; Falcon Plastics, Becton-Dickinson Labware, Franklin Lakes, NJ) at ∼4.0 × 104 cells/cm2 in Defined Keratinocyte Serum-Free Medium (Life Technologies, Inc., Grand Island, NY) with the addition of the growth supplement included with the medium. Calcium level, as stated by the manufacturer, was lower than 0.1 mmol/L. The cells were grown as monolayers at 37°C in a humidified incubator with 5% CO2/95% air. Myofibroblasts from rat cutaneous granulation tissue were obtained as previously described.17Oda D Gown AM Vande Berg JS Stern R Instability of the myofibroblast phenotype in culture.Exp Mol Pathol. 1990; 52: 221-234Crossref PubMed Scopus (34) Google Scholar The cells within 17 passages were used because myofibroblasts were maintained in culture for more than 17 passages. Myofibroblasts were grown to confluence and subcultured on culture dishes (78.5 cm2, Falcon Plastics) at 37°C in a humidified incubator with 5% CO2/95% air in Dulbecco's modified medium with 10% fetal bovine serum (Life Technologies, Inc.), penicillin (100 U/ml), streptomycin (50 μg/ml), kanamycin (50 μg/ml), and hydrocortisone (0.4 μg/ml). The epidermal strips as a source of keratinocytes, cultured keratinocytes, and myofibroblasts were washed twice with ice-cold phosphate-buffered saline, and harvested and lysed in lysis buffer containing 10 mmol/L Tris-HCl, 10 mmol/L ethylenediaminetetraacetic acid, 0.4 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml leupeptin, and 1% Triton X-100. After they were incubated for 1 hour at 4°C, the cell lysates were centrifuged at 12,000 × g for 10 minutes, and the supernatant was collected.18Murasawa S Mori Y Nozawa Y Gotoh N Shibuya M Masaki H Maruyama K Tsutsumi Y Moriguchi Y Shibasaki Y Tanaka Y Iwasaka T Inada M Matsubara H Angiotensin II type 1 receptor-induced extracellular signal-related protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor.Circ Res. 1998; 82: 1338-1348Crossref PubMed Google Scholar Proteins were precleared with protein G agarose for 30 minutes at 4°C. Then anti-AT1 (affinity-purified rabbit polyclonal anti-AT1; Santa Cruz Biotechnology, Santa Cruz, CA) or AT2 antibodies (affinity-purified goat polyclonal anti-AT2, Santa Cruz) were added to the precleared samples and incubated for 1 hour at 4°C. Immune complexes were boiled in sodium dodecyl sulfate-sample buffer, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,19Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (218087) Google Scholar transferred to polyvinylidene difluoride membrane,20Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1973; 76: 4350-4354Crossref Scopus (47750) Google Scholar and immunoblotted with anti-AT1 or AT2 antibodies, respectively. After incubation with secondary antibodies, immunoreactive proteins were detected by the enhanced chemiluminescence reaction (Amersham Bioscience Japan, Tokyo, Japan). Staining of standard proteins on the polyvinylidene difluoride membranes was performed with 0.04% Coomassie brilliant blue R-250. Keratinocytes and myofibroblasts were plated on culture disks (0.79 cm2; Sumitomo Chemical, Tokyo, Japan) at ∼1.5 × 102 cells/cm2. Subsequently, keratinocytes and myofibroblasts were exposed to AngII (100 pg/ml; Wako, Tokyo, Japan), CV-11974 (10−7 mol/L), CV-11974 (10−7 mol/L with AngII 100 pg/ml), PD-123319 (10−5 mol/L, dissolved in 0.9% saline), PD-123319 (10−5 mol/L with AngII 100 pg/ml), or vehicle (0.9% saline with 0.1 N Na2CO3), respectively, with BrdU (15 μg/ml) in the medium for 24 hours and fixed in 100% ethanol. The BrdU immunohistochemistry was performed using a cell proliferation kit (Amersham Bioscience Japan). After quenching endogenous peroxidase with 3% H2O2, the DNA in cells was subsequently denatured with denaturing solution for 30 minutes, followed by a 1-hour incubation with biotinylated mouse monoclonal anti-BrdU antibody at 37°C. Disks were incubated with streptavidin-peroxidase for 10 minutes at room temperature. The incorporation of BrdU into the genomic DNA of proliferating cells was detected by staining with 3-amino-9-ethylcarbazole. Finally, the disks were counterstained with hematoxylin and mounted in aqueous mounting medium. Negative disks were not incubated with anti-BrdU antibody. Cells with red-stained nuclei by 3-amino-9-ethylcarbazole were identified as cells with BrdU incorporation. The BrdU labeling index (percentage) was determined by the number of BrdU-positive nuclei in cells and analyzed and scored by two different observers under light microscopy. The experiments were done in triplicate. Wound healing migration assays were performed as previously described.21Penas PF Gomez M Buezo GF Rios L Yanez-Mo M Cabanas C Sanchez-Madrid F Garcia-Diez A Differential expression of activation epitopes of beta1 integrins in psoriasis and normal skin.J Invest Dermatol. 1998; 111: 19-24Crossref PubMed Scopus (26) Google Scholar In brief, keratinocytes or myofibroblasts were seeded on 24-well multiplates and grown to confluence. After 60 minutes of incubation with AngII (100 pg/ml), CV-11974 (10−7 mol/L), CV-11974 (10−7 mol/L with AngII 100 pg/ml), PD-123319 (10−5 mol/L), PD-123319 (10−5 mol/L with AngII 100 pg/ml), or vehicle in the medium, respectively, the monolayers of keratinocytes or myofibroblasts were wounded with a cell scraper and photographed at different times by a phase contrast microscope equipped with Olympus Camedia C-3030ZOOM digital camera (Olympus, Tokyo, Japan). The re-epithelization index (percentage) was determined by the percentage of new epithelium present in the initial wound using image analysis software program Analytical Imaging Station for Windows (Imaging Research Inc., Willowdale, Canada). The experiments were done in triplicate. Statistical significance of the quantitative measurement was assessed by Turkey's multiple range test. Each time point was analyzed separately, and two-tailed α-levels of P ≤ 0.05 are significant. The effect of AT1 blocker on re-epithelization in vivo was evaluated kinetically (Figure 1, A and B). On day 12, the control group and the group with TCV-116, 1 mg/kg/day, showed 100% re-epithelization of the wound (Figure 1A). However, the group with TCV-116, 10 mg/kg/day, showed delayed re-epithelization and delayed dermal repair. At all time points, administration of TCV-116 suppressed the keratinocyte re-epithelization dose dependently (Figure 1B). The effect of AT1 blocker on vascular growth at the wound bed was evaluated kinetically (Figure 1, C and D). On day 9 after wounding, the control group showed many blood vessels in the dermis, however, the administration of TCV-116 suppressed angiogenesis dose dependently (Figure 1C). Administration of TCV-116, 1 mg/kg/day, slightly suppressed angiogenesis at all time points except for the significant suppression on day 9 (Figure 1D). Administration of TCV-116, 10 mg/kg/day, significantly suppressed angiogenesis at all time points. To analyze the effects of AngII receptor signaling on keratinocytes and myofibroblasts, we examined the expression of AngII receptors using immunoprecipitation and Western blot analysis (Figure 2). The results showed the signals of both AT1 and AT2 in the epidermal strips, cultured keratinocytes, and myofibroblasts. The signal of AT1 is prominent compared to that of AT2 in the epidermal strips, keratinocytes, and myofibroblasts. We investigated the level of cell proliferation by using BrdU labeling index. BrdU, a thymidine analog, can be incorporated into DNA in the S phase of cell cycle.16Kondo S Hozumi Y Mitsuhashi Y Comparative inhibitory effects of vitamin D3 and an analogue on normal psoriatic epidermis in organ culture.Arch Dermatol Res. 2000; 292: 550-555Crossref PubMed Scopus (8) Google Scholar Hence, the cells that traversed S phase can be detected with an anti-BrdU antibody. Based on the analysis of AngII receptor expression, keratinocytes were exposed to AngII, CV-11974, CV-11974 with AngII (selective AT2 signaling), PD-123319, PD-123319 with AngII (selective AT1 signaling), or vehicle, respectively, for 24 hours and offered for analyzing BrdU labeling index (percentage) (Figure 3, A and B). The presence of AngII significantly enhanced (P < 0.05) the BrdU incorporation into keratinocytes compared to the control. The similar enhancing effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed (P < 0.05) the BrdU incorporation compared to the control or the group with AngII. These results suggest that AT1 signaling accelerates and AT2 signaling inhibits BrdU incorporation into keratinocytes, respectively. The re-epithelizing movements of keratinocytes were recorded starting from wounding until one of the wounds was re-epithelized (40 hours later) (Figure 3, C and D). At all time points except for 16 hours after scraping, the presence of AngII accelerated re-epithelization compared to the control. The similar accelerating effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed re-epithelization compared to the control or the group with AngII. The results suggest that AT1 signaling accelerates and AT2 signaling inhibits the re-epithelization of keratinocytes, respectively. Based on the analysis of AngII receptor expression, myofibroblasts were exposed to AngII, CV-11974, CV-11974 with AngII (selective AT2 signaling), PD-123319, PD-123319 with AngII (selective AT1 signaling), or vehicle, respectively, for 24 hours and offered for analyzing BrdU labeling index (percentage) (Figure 4, A and B). The presence of AngII significantly enhanced (P < 0.05) the BrdU incorporation into myofibroblasts compared to the control. The similar enhancing effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed (P < 0.05) the BrdU incorporation compared to the control group or the group with AngII. The results suggest that AT1 signaling enhances and AT2 signaling suppresses the BrdU incorporation into myofibroblasts, respectively. The recovering movements of myofibroblasts were recorded starting from wounding until one of the wounds was recovered (38 hours later) (Figure 4, C and D). At all of the time points except for 12 hours after scraping, the presence of AngII significantly accelerated (P < 0.05) recovering compared to the control. The similar accelerating effect was recognized in the group of PD-123319 with AngII. On the contrary, the presence of CV-11974 with AngII significantly inhibited (P < 0.05) recovering compared to the control or the group with AngII. The results suggest that AT1 signaling accelerates and AT2 signaling inhibits the recovering of myofibroblasts, respectively. It has been proposed that exogenous administration of AngII is a potent accelerator of cutaneous wound repair.7Rodgers KE Xiong S Felix JC Roda R Espinosa T Maldonado S diZerega G Development of angiotensin (1-7) as an agent to accelerate dermal regeneration.Wound Repair Regen. 2001; 9: 241-250Crossref Google Scholar, 8Rodgers KE Roda N Felix JC Espinosa T Maldonado S diZerega G Histological evaluation of the effects of angiotensin peptides on wound repair in diabetic mice.Exp Dermatol. 1997; 5: 175-183Google Scholar These reports showed that topical administration of AngII could accelerate skin wound healing by stimulating dermal repair including angiogenesis and epidermal repair in vivo. However, the mechanism of AngII-induced acceleration of skin wound healing is poorly understood. In the present report, we studied the effects of AngII on skin wound healing focusing on AngII receptor-mediated signaling. AngII, the effector peptide of the renin-Ang system, is now known to have growth regulating properties in various cell types.22Timmermans PB Wong PC Chiu AT Herblin WF Benfield P Carini DJ Lee RJ Wexler RR Saye JA Smith RD Angiotensin II receptors and angiotensin II receptor antagonists.Pharmacol Rev. 1993; 45: 205-251PubMed Google Scholar AngII exerts its actions by binding the specific receptors. Two major AngII receptors with the seven transmembrane domains, namely, AT1 and AT2, have been identified. AT1 is expressed in the brain, kidney, liver, heart, adrenal gland, ovary, testis, and vascular smooth muscle cells.23Burnier M Brunnerr HR Angiotensin II receptor antagonists.Lancet. 2000; 355: 637-645Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar AT2 is abundantly and widely expressed in fetal tissues. Most of the AngII-induced actions are mediated by AT1. AT1 activates many signaling pathways and can stimulate tyrosine phosphorylation [through c-Src, Pyk2, and transactivation of epidermal growth factor (EGF) receptor (EGFR)] and activation of phospholipases and protein kinase C.24Berk BC Angiotensin type 2 receptor (AT2R): a challenging twin.Sci STKE. 2003; 181: 16Google Scholar A recent study showed that AngII-induced extracellular signal-regulated kinase (ERK) activation was mediated by EGFR transactivated via AT1.18Murasawa S Mori Y Nozawa Y Gotoh N Shibuya M Masaki H Maruyama K Tsutsumi Y Moriguchi Y Shibasaki Y Tanaka Y Iwasaka T Inada M Matsubara H Angiotensin II type 1 receptor-induced extracellular signal-related protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor.Circ Res. 1998; 82: 1338-1348Crossref PubMed Google Scholar In contrast, AT2 stimulates none of these (except p38 MAPK) and appears to involve protein tyrosine phosphatases (SHP-1), MAP kinases (p38 and ERK1/2), and Gαs (independent of βγ G proteins). EGFR is an 1186-amino acid glycoprotein containing a single transmembrane domain, an extracellular portion involved in ligand binding, and an intracellular portion harboring the tyrosine kinase domain.25Wells A EGF receptor.Int J Biochem. 1999; 31: 637-643Crossref Scopus (912) Google Scholar The C-terminal sequences harbor autophosphorylation sites that are important for the initiation of downstream signaling.26Gschwind A Zwick E Prenzel N Lessser M Ullrich A Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission.Oncogene. 2001; 20: 1594-1600Crossref PubMed Scopus (415) Google Scholar Stimulation of EGFR results in the simultaneous activation of multiple signaling pathways including the RAS-RAF-MEK-MAPK, the PLC-γ/PKC, the PI-3K/AKT cascades, and the JAK and STATS pathways, and these pathways are often functionally interlinked. The known mammalian ligands that act as direct agonists for EGFR include transforming growth factor (TGF)-α, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, epiregulin, and epigen.27Harris RC Chung E Coffey RJ EGF receptor ligands.Exp Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (630) Google Scholar These ligands are synthesized as transmembrane precursors and must therefore be proteolytically cleaved by metalloproteases to release the mature growth factor. The agonist-occupied EGFR undergoes gene transcription and mitogenesis. In addition to its cognate ligands, EGFR is activated by stimuli that do not directly interact with EGFR ectodomain including G protein-coupled receptor (GPCR) ligands, other receptor tyrosine kinase agonists, cytokines, chemokines, and cell adhesion elements.26Gschwind A Zwick E Prenzel N Lessser M Ullrich A Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission.Oncogene. 2001; 20: 1594-1600Crossref PubMed Scopus (415) Google Scholar This process is termed EGFR transactivation and the GPCR-induced EGFR transactivation is stimulated by bradykinin, endothelin (ET)-1, lysophosphatidic acid, interleukin-8, carbachol stimulation, or AngII. Literature has shown that AT1 stimulates processing of pro-HB-EGF by metalloproteinases including a disintegrin and metalloprotease 12 (ADAM 12), and the released HB-EGF transactivates EGFR.28Asakura M Kitakaze M Takashima S Liao Y Ishikura F Yoshinaka T Ohmoto H Node K Yoshino K Ishiguro H Asanuma H Sanada S Matsumura Y Takeda H Beppu S Tada M Hori M Higashiyama S Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy.Nat Med. 2002; 8: 35-40Crossref PubMed Scopus (647) Google Scholar AngII-induced transactivation of EGFR is essential for the activation of downstream Sr/Thr kinases such as ERK and subsequent protein synthesis.29Eguchi S Inagami T Signal transduction of angiotensin II type1 receptor through receptor tyrosine kinase.Regul Peptides. 2000; 91: 13-20Crossref PubMed Scopus (130) Google Scholar Our in vivo study showed that administration of AT1 blocker suppressed rat skin wound healing by inhibiting re-epithelization, dermal repair, and angiogenesis. The process of re-epithelization consists of keratinocyte migration and proliferation. Keratinocytes undergo profound changes in morphology as they migrate.30Falanga V Mechanisms of cutaneous wound repair.in: Freedberg IM Eizen AZ Wolff K Austen KF Goldsmith LA Katz SI Fitzpatrick's Dermatology in General Medicine. McGraw-Hill, New York2003: 236-246Google Scholar The cytoskeleton is critical to these changes and cell migration. EGF-induced activation of PLC-γ stimulates cell motility by releasing PIP2-bound gelsolin from the membrane, thereby restoring its ability to bind, sever, and cap polymerized actin filaments, a process required for filopodia/lamellipodia extension and retraction in motile cells.31Chen
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