Epidermal Growth Factor Receptor Signaling Is Partially Responsible for the Increased Matrix Metalloproteinase-1 Expression in Ocular Epithelial Cells after UVB Radiation
2005; Elsevier BV; Volume: 167; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62992-6
ISSN1525-2191
AutoresNick Di Girolamo, Minas T. Coroneo, Denis Wakefield,
Tópico(s)Corneal surgery and disorders
ResumoPterygia are inflammatory, invasive, and proliferative lesions of the human ocular surface in which the matrix metalloproteinase (MMP) collagenase-1 (MMP-1) is highly expressed. Pterygia development may involve MMP-1 activity against interstitial fibrillar collagen, an abundant extracellular matrix component of the cornea, and its induction by ultraviolet light (UVB). We examined the pathways responsible for enhanced expression of MMP-1 in pterygium epithelial cells after UVB exposure and/or treatment with chemical inhibitors of mitogen-activated protein kinases or epidermal growth factor receptor. The induction of MMP-1 by UVB was comparable to that mediated by heparin-binding epidermal growth factor-like growth factor and epidermal growth factor. The epidermal growth factor receptor inhibitor PD153035 partially blocked the UVB-mediated induction of MMP-1 and totally abrogated its production after stimulation with either heparin-binding epidermal growth factor-like growth factor or epidermal growth factor. UVB exposure enhanced the phosphorylated form of ERK1/2 in a time-dependent manner whereas the ERK1/2 inhibitor PD98059 decreased this induction by at least fivefold. Transcripts for c-jun and c-fos were detected as early as 2 hours after UVB exposure and were suppressed by PD98059. The identification of a specific intracellular signaling pathway responsible for the enhanced production of a key enzyme that denatures intact fibrillar collagen has important implications for understanding the pathophysiology and future therapy for pterygia. Pterygia are inflammatory, invasive, and proliferative lesions of the human ocular surface in which the matrix metalloproteinase (MMP) collagenase-1 (MMP-1) is highly expressed. Pterygia development may involve MMP-1 activity against interstitial fibrillar collagen, an abundant extracellular matrix component of the cornea, and its induction by ultraviolet light (UVB). We examined the pathways responsible for enhanced expression of MMP-1 in pterygium epithelial cells after UVB exposure and/or treatment with chemical inhibitors of mitogen-activated protein kinases or epidermal growth factor receptor. The induction of MMP-1 by UVB was comparable to that mediated by heparin-binding epidermal growth factor-like growth factor and epidermal growth factor. The epidermal growth factor receptor inhibitor PD153035 partially blocked the UVB-mediated induction of MMP-1 and totally abrogated its production after stimulation with either heparin-binding epidermal growth factor-like growth factor or epidermal growth factor. UVB exposure enhanced the phosphorylated form of ERK1/2 in a time-dependent manner whereas the ERK1/2 inhibitor PD98059 decreased this induction by at least fivefold. Transcripts for c-jun and c-fos were detected as early as 2 hours after UVB exposure and were suppressed by PD98059. The identification of a specific intracellular signaling pathway responsible for the enhanced production of a key enzyme that denatures intact fibrillar collagen has important implications for understanding the pathophysiology and future therapy for pterygia. Pterygium is a condition of the ocular surface characterized by squamous cell metaplasia and goblet cell hyperplasia. The lesion consists of a wing-shaped mass of fibrovascular conjunctival tissue that invades the normal cornea. Other obvious pathological changes include activation of stromal fibroblasts, a persistent inflammatory component, elastotic degeneration, and destruction of collagenous barriers such as Bowman's layer. Pterygia are particularly prevalent in heavily sun-exposed individuals in whom extensive epidemiological studies link this disease to excessive ultraviolet (UV) radiation.1Coroneo MT Pterygium as an early indicator of ultraviolet insolation: a hypothesis.Br J Ophthalmol. 1993; 77: 734-739Crossref PubMed Scopus (257) Google Scholar, 2Coroneo MT Di Girolamo N Wakefield D The pathogenesis of pterygia.Curr Opin Ophthalmol. 1999; 10: 282-288Crossref PubMed Scopus (229) Google Scholar, 3Hilgers JHC Pterygium: its incidence, heredity and etiology.Am J Ophthalmol. 1960; 50: 635-644Abstract Full Text PDF PubMed Google Scholar, 4Karai I Horiguchi S Pterygium in welders.Br J Ophthalmol. 1984; 68: 347-349Crossref PubMed Scopus (89) Google Scholar, 5Threlfall TJ English DR Sun exposure and pterygium of the eye: a dose-response curve.Am J Ophthalmol. 1999; 128: 280-287Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Despite this evidence, there is still controversy regarding the actual trigger for the development of pterygia and whether or not there is a genetic predisposition to this disease.6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar Recently, we have identified UVB as an environmental agent likely to be responsible for initiating molecular events that lead to the formation of pterygia.7Di Girolamo N Coroneo MT Kumar RK Wakefield D UVB-mediated induction of interleukin-6 and −8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar From our hypothesis,9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar we postulate that UVB radiation activates cells at or near the limbus. This activation may cause 1) phenotypic alterations in a distinct population of epithelial cells, 2) production of proinflammatory and angiogenic cytokines7Di Girolamo N Coroneo MT Kumar RK Wakefield D UVB-mediated induction of interleukin-6 and −8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar and growth factors,8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar and 3) increased invasiveness due to enhanced production of matrix metalloproteinases (MMPs) over and above their natural tissue inhibitors (TIMPs).6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar, 9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar, 10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 11Di Girolamo N Wakefield D Coroneo MT Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head.Invest Ophthalmol Vis Sci. 2000; 41: 4142-4149PubMed Google Scholar, 12Di Girolamo N Coroneo MT Wakefield D Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.Invest Ophthalmol Vis Sci. 2001; 42: 1963-1968PubMed Google Scholar To date, we6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar, 9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar, 10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 11Di Girolamo N Wakefield D Coroneo MT Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head.Invest Ophthalmol Vis Sci. 2000; 41: 4142-4149PubMed Google Scholar, 12Di Girolamo N Coroneo MT Wakefield D Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.Invest Ophthalmol Vis Sci. 2001; 42: 1963-1968PubMed Google Scholar and others13Li D-Q Lee S-B Gunja-Smith Z Liu Y Solomon A Meller D Tseng SCG Overexpression of collagenase (MMP-1) and stromelysin (MMP-3) by cultured pterygium head fibroblasts.Arch Ophthalmol. 2001; 119: 71-80Crossref PubMed Scopus (90) Google Scholar, 14Dushku N John MK Schultz GS Reid TW Pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells.Arch Ophthalmol. 2002; 119: 695-706Crossref Scopus (188) Google Scholar, 15Seifert P Eckert J Spitznas M Topological-histological investigation of the pterygium.Graef's Arch Clin Exp Ophthalmol. 2001; 239: 288-293Crossref PubMed Scopus (17) Google Scholar, 16Wang I-J Hu F-R Chen P-J Lin C-T Mechanism of abnormal elastin gene expression in the pinguecula part of pterygia.Am J Pathol. 2000; 157: 1269-1276Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17Austin P Jakobiec FA Iwamoto T Elastodysplasia and elastodystrophy as the pathologic bases of ocular pterygia and pinguecula.Ophthalmology. 1983; 90: 96-109Abstract Full Text PDF PubMed Scopus (134) Google Scholar have accumulated data from in vitro culture and in vivo tissue-based studies that resemble investigations performed in human UV-exposed skin. These studies have demonstrated increased MMPs in human skin and skin-derived cells in response to UV radiation.18Varani J Warner RL Gharaee-Kermani M Phan SH Kang S Chung J-H Wang Z-Q Datta SC Fisher GJ Voorhees JJ Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.J Invest Dermatol. 2000; 114: 480-486Crossref PubMed Scopus (517) Google Scholar, 19Fisher GJ Choi H-C Bata-Csorgo Z Shao Y Datta S Wang Z-Q Kang S Voorhees JJ Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo.J Invest Dermatol. 2001; 117: 219-226Crossref PubMed Google Scholar, 20Fisher GJ Wang ZQ Datta SC Varani J Kang S Voorhees JJ Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1152) Google Scholar, 21Fisher GJ Datta SC Talwar HS Wang Z-Q Varani J Kang S Voorhees JJ Molecular basis of sun-induced premature skin aging and retinoid antagonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1185) Google Scholar Furthermore, photodamaged skin displays several histological features in common with pterygia, such as regions lacking intact collagen fibrils, the presence of a disrupted matrix, and large deposits of amorphous material composed of cellular debris and elastotic matrix.22Bernstein EF Chen YQ Kopp JB Fisher L Brown DB Hahn PJ Robey FA Lakkakorpi J Uitto J Long-term sun exposure alters the collagen of the papillary dermis: comparison of sun-protected and photoaged skin by Northern analysis, immunohistochemical staining, and confocal laser scanning microscopy.J Am Acad Dermatol. 1996; 34: 209-218Abstract Full Text PDF PubMed Scopus (177) Google Scholar The extensive expression of MMP-1 in cultured pterygium-derived cells and pterygium tissue has been documented by several independent investigators.9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar, 10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 11Di Girolamo N Wakefield D Coroneo MT Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head.Invest Ophthalmol Vis Sci. 2000; 41: 4142-4149PubMed Google Scholar, 13Li D-Q Lee S-B Gunja-Smith Z Liu Y Solomon A Meller D Tseng SCG Overexpression of collagenase (MMP-1) and stromelysin (MMP-3) by cultured pterygium head fibroblasts.Arch Ophthalmol. 2001; 119: 71-80Crossref PubMed Scopus (90) Google Scholar, 14Dushku N John MK Schultz GS Reid TW Pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells.Arch Ophthalmol. 2002; 119: 695-706Crossref Scopus (188) Google Scholar Interestingly, we have also noted diminished expression of MMP-1 in quiescent normal conjunctival tissue specimens.9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar, 10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar The importance of MMP-1 should not be underestimated because it can specifically denature the collagen triple helix at a specific locus, it is required for keratinocyte migration on a type I collagen matrix,23Pilcher BK Dumin J-A Sudbeck BD Krane SM Welgus HG Parks WC The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (491) Google Scholar it can promote endothelial cell migration during angiogenesis,24Partrige CR Hawker Jr, JR Forough R Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration.J Cell Biochem. 2000; 78: 487-499PubMed Google Scholar and its overexpression can result in epidermal hyperplasia, and enhance cell proliferation by activating insulin-like growth factor.25D'armiento J DiColandrea T Dalal SS Okada Y Huang M-T Conney AH Chada K Collagenase expression in transgenic mouse skin causes hyperkeratosis and acanthosis and increases susceptibility to tumorigenesis.Mol Cell Biol. 1995; 15: 5732-5739Crossref PubMed Scopus (127) Google Scholar Thus determining the role and identifying the signals that regulate the expression of MMP-1 in pterygia may be critical for understanding how this disease develops in humans. In a previous investigation we identified the extracellular signal-regulated kinase (ERK1/2) mitogen-activated protein kinase (MAPK) as the intracellular signal transduction pathway activated by UVB that was responsible for induction of MMP-1 in cultured pterygium epithelial cell (PECs). Blocking this pathway with the chemical inhibitor PD98059 resulted in a significant decrease in MMP-1 production, whereas no inhibition was observed with an inhibitor of c-Jun N-terminal kinase (JNK) and p38 (SB202190).9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar In the context of pterygium development, these findings are relevant because activation of the ERK pathway can promote cell survival and proliferation, whereas activation of JNK and p38 signaling pathways can result in apop-tosis.26Xia Z Dickens M Raingeaud J Davis RJ Greenberg ME Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.Science. 1995; 270: 1326-1329Crossref PubMed Scopus (5028) Google Scholar Furthermore, the induction of MMP-1 by UVB,27Fisher GJ Talwar HS Lin J Lin P McPhillips F Wang Z Li X Wan Y Kang S Voorhees JJ Retinoic acid inhibits induction of c-jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.J Clin Invest. 1998; 101: 1432-1440Crossref PubMed Scopus (324) Google Scholar, 28Brenneisen P Wenk J Klotz LO Wlaschek M Briviba K Krieg T Sies H Scharffetter-Kochanek K Central role of ferrous/ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase matrix-degrading metalloproteinase (MMP)-1) and stromelysin (MMP-3) mRNA levels in cultured human dermal fibroblasts.J Biol Chem. 1998; 273: 5279-5287Crossref PubMed Scopus (195) Google Scholar UVA,29Wenk J Brenneisen P Wlaschek M Poswig A Briviba K Oberley TD Scharfetter-Kochanek K Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1 mediated induction of matrix-degrading metalloproteinase-1.J Biol Chem. 1999; 274: 25869-25876Crossref PubMed Scopus (196) Google Scholar and possibly infrared-A radiation30Schieke SM Stege H Kurten V Grether-Beck S Sies H Krutmann J Infrared-A radiation-induced matrix metalloproteinase 1 expression is mediated through extracellular signal-regulated kinase 1/2 activation in human dermal fibroblasts.J Invest Dermatol. 2002; 119: 1323-1329Crossref PubMed Scopus (101) Google Scholar is dependent on AP-1 (c-jun/c-fos) transcriptional activity that is downstream of ERK. An important aim of this study was to trace the intracellular signal cascade and to identify the sensor(s) for the UVB stimulus. One of the earliest events in response to UV radiation involves ligand-independent autophosphorylation of cell surface growth factor receptors such as the keratinocyte growth factor receptor (KGFR)31Marchese C Maresca V Cardinali G Belleudi F Ceccarelli S Bellocci M Frati L Torrisi M-R Picardo M UVB-induced activation and internalization of keratinocyte growth factor receptor.Oncogene. 2003; 22: 2422-2431Crossref PubMed Scopus (59) Google Scholar and the epidermal growth factor receptor (EGFR).32Wan YS Wang ZQ Voorhees J Fisher G EGF receptor crosstalks with cytokine receptors leading to the activation of c-jun kinase in response to UV irradiation in human keratinocytes.Cell Signal. 2001; 13: 139-144Crossref PubMed Scopus (57) Google Scholar It is highly likely that stimulation of such receptors by UV contributes to the activation of MAPK pathways. Furthermore, targeting the EGFR in our model is relevant because this receptor33Liu Z Xei Y Zhang M Overexpression of type 1 growth factor receptors in pterygium.Chin Med J. 2002; 115: 418-421PubMed Google Scholar, 34Maini R Collison DJ Maidment JM Davies PD Wormstone IM Pterygial derived fibroblasts express functionally active histamine and epidermal growth factor receptors.Exp Eye Res. 2002; 74: 237-244Crossref PubMed Scopus (32) Google Scholar and at least one of its ligands heparin-binding epidermal growth factor-like growth factor (HB-EGF)8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 35Nolan TM Di Girolamo N Coroneo MT Wakefield D Proliferative effects of heparin-binding epidermal growth factor-like growth factor on pterygium epithelial cells and fibroblasts.Invest Ophthalmol Vis Sci. 2004; 45: 110-113Crossref PubMed Scopus (35) Google Scholar has been identified in pterygia. Using a pterygium-derived epithelial cell culture model, we demonstrated the partial involvement of the EGFR in transmitting an extracellular UVB signal that resulted in the activation of an intracellular signal transduction pathway. Interestingly, the same molecules and pathways were not activated by UVB in normal conjunctival or limbal8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar epithelial cells and this observation is currently under investigation in our laboratory. Resected pterygia (n = 8) and normal conjunctiva (n = 8) were obtained from the Prince of Wales Hospital, Sydney, Australia. Autologous tissue (n = 3) that remained after grafting was also used in this study and included small segments of conjunctiva. All tissue specimens were formalin-fixed and paraffin-embedded immediately after surgery. Informed consent was obtained from each subject. All research protocols were approved by the University of New South Wales Human Ethics Committee and performed in accordance with the tenets of the World Medical Associations Declaration of Helsinki. Serial sections (4 μm) of pterygia, normal conjunctiva, limbus, and cornea were processed for immunohistochemistry as previously described.6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar, 7Di Girolamo N Coroneo MT Kumar RK Wakefield D UVB-mediated induction of interleukin-6 and −8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar, 10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 11Di Girolamo N Wakefield D Coroneo MT Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head.Invest Ophthalmol Vis Sci. 2000; 41: 4142-4149PubMed Google Scholar, 12Di Girolamo N Coroneo MT Wakefield D Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.Invest Ophthalmol Vis Sci. 2001; 42: 1963-1968PubMed Google Scholar In brief, tissue sections were deparaffinized, hydrated, equilibrated in 0.05 mol/L Tris-buffered saline (TBS), pH 7.6, blocked with the appropriate serum (20% final) diluted in 2% bovine serum albumin/TBS, and incubated at room temperature for 30 minutes. Sections were incubated with primary antibody in 2% bovine serum albumin/TBS at the indicated dilution, temperature, and time (Table 1). A rigorous staining optimization protocol was performed for each antibody. Antibody concentration, time of application (30 minutes to 16 hours), and temperature of incubation (4 to 37°C) was predetermined in preliminary experiments. Antigen retrieval using either microwave treatment or enzymatic digestion was performed but not required for optimal staining. Sections were extensively washed in TBS before the addition of the appropriate biotinylated secondary antibody (Table 1). Sections were rinsed, then incubated with horseradish peroxidase-conjugated streptavidin (DAKO, Carpinteria, CA) for an additional hour at room temperature and the immunoreactivity developed by adding 3-amino-9-ethyl-carbazole (Sigma Aldrich, St. Louis, MO). Some sections were counterstained with hematoxylin. Immunohistochemical controls included either omitting the primary antibody or replacing the primary antibody with a mouse, rabbit, or goat isotype (IgG)-negative control antibody (Table 1). Any difference in the staining intensity between pterygia, conjunctiva, and limbus was comparable because both diseased and normal tissue were analyzed under identical conditions in the same experimental run.Table 1Primary Antibodies Used for Immunohistochemistry and Western BlottingAntibodySourceCatalogue no.ApplicationDilutionTemperature/timePrimary antibodies p-p44/p42 (RaH)CST9101WB, IHC1:1000, (1:30)4°C/16 hours p44/p42 (RaH)CST9102WB, IHC1:1000, (1:30)4°C/16 hours p-p38 (RaH)SCBsc-7975WB1:5004°C/16 hours MMP-1 (MaH)ICN63-178WB1:100022°C/1 hour pEGFR (MaH)CST2236IHC1:4004°C/16 hours c-fos (RaH)SCBsc-52IHC1:20037°C/1 hour c-jun (RaH)SCBsc-1694IHC1:20037°C/1 hour Sp1 (RaH)SCBsc-59IHC1:20037°C/1 hour EGF (RaH)SCBsc-275IHC1:254°C/16 hours HB-EGF (GaH)R&DAF-259-NAIHC1:604°C/16 hours Rabbit IgGSCBsc-2027IHC1:25–1:20037°C/16 hours Mouse IgGDakoX0931IHC1:4004°C/16 hours Goat IgGR&DAB-108-CIHC1:604°C/16 hoursSecondary antibodies SaRHRPDakoP0217WB1:200022°C/1 hour RaMHRPDakoP0260WB1:200022°C/1 hour GaRbDakoE0466IHC1:20022°C/30 minutes GaMbDakoE0433IHC1:20022°C/30 minutes RaGbDakoE0466IHC1:20022°C/30 minutesAbbreviations used: CST, Cell Signaling Technology (GeneSearch, Arundel, Australia); GaH, goat anti-human; GaMb, biotin-conjugated goat anti-mouse; GaRb, biotin-conjugated goat anti-rabbit; ICN, ICN Biomedicals (Sydney, Australia); IHC, immunohistochemistry; MaH, mouse anti-human; R&D, R&D Systems (Minneapolis, MN); RaGb, biotin-conjugated rabbit anti-goat; RaH, rabbit anti-human; RaMHRP, horseradish peroxidase-conjugated rabbit anti-mouse; SC, Santa Cruz Biotechnology (Santa Cruz, CA); WB, (Western blotting); SaRHRP, horseradish peroxidase-conjugated swine anti-rabbit. Open table in a new tab Abbreviations used: CST, Cell Signaling Technology (GeneSearch, Arundel, Australia); GaH, goat anti-human; GaMb, biotin-conjugated goat anti-mouse; GaRb, biotin-conjugated goat anti-rabbit; ICN, ICN Biomedicals (Sydney, Australia); IHC, immunohistochemistry; MaH, mouse anti-human; R&D, R&D Systems (Minneapolis, MN); RaGb, biotin-conjugated rabbit anti-goat; RaH, rabbit anti-human; RaMHRP, horseradish peroxidase-conjugated rabbit anti-mouse; SC, Santa Cruz Biotechnology (Santa Cruz, CA); WB, (Western blotting); SaRHRP, horseradish peroxidase-conjugated swine anti-rabbit. For the EGFR-based studies, PECs were cultured in chamber slides (Nunc, Roskilde, Denmark),10Di Girolamo N McCluskey P Lloyd A Coroneo MT Wakefield D Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar fixed, and stained for this receptor. These conditions ensured no physical disruption of the cell-membrane-bound receptor due to enzymatic digestion. For the EGFR ligand studies, PECs were grown (see below) in 100-mm dishes (Corning, Corning, NY), treated under control conditions or irradiated with 20 mJ/cm2 UVB, trypsin-digested, pelleted, paraffin-embedded, sectioned, and stained for epidermal growth factor (EGF) and HB-EGF (Table 1). PECs or conjunctival epithelial cells (CECs) were seeded at 1 × 106 cells in 100-mm tissue culture dishes (Corning) or seeded at 1.5 × 105 cells per well in 6-well plates (Nunc) and grown in the presence of 10% fetal bovine serum/Eagle's minimum essential medium. Media from semiconfluent cells was aspirated, cells were washed three times with sterile phosphate-buffered saline (PBS), and synchronized in G0 or quiescence by withdrawal of serum growth factors for 16 hours as previously described.6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar, 7Di Girolamo N Coroneo MT Kumar RK Wakefield D UVB-mediated induction of interleukin-6 and −8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 8Nolan T Di Girolamo N Sachdev N Hampartzoumian T Coroneo MT Wakefield D The role of UV irradiation and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the pathogenesis of pterygium.Am J Pathol. 2003; 162: 567-574Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Di Girolamo N Coroneo MT Wakefield D UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (90) Google Scholar This media was replaced with 5 ml of PBS and the monolayer irradiated with 20 mJ/cm2 UVB using TL 20 W/12 RS bulbs (Philips, Sydney, Australia) as previously reported.6Di Girolamo N Chui J Coroneo MT Wakefield D The pathogenesis of pterygia: role of cytokines, growth factors, metalloproteinases, and ultraviolet light.Prog Ret Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (266) Google Scholar, 7Di Girolamo N Coroneo MT Kumar RK Wakefield D UVB-mediated induction of interleukin-6 and −8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 8No
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