Mechanisms of Hypoxic Regulation of Plasminogen Activator Inhibitor-1 Gene Expression in Keloid Fibroblasts
2003; Elsevier BV; Volume: 121; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2003.12564.x
ISSN1523-1747
AutoresQunzhou Zhang, Yidi Wu, David K. Ann, Diana V. Messadi, Tai‐Lan Tuan, Audrey Kelly, Charles N. Bertolami, Anh D. Le,
Tópico(s)Laser Applications in Dentistry and Medicine
ResumoKeloids are an excessive accumulation of extracellular matrix. Although numerous studies have shown elevated plasminogen activator inhibitor-1 (PAI-1) levels in keloid fibroblasts compared with those of normal skin. Their specific mechanisms involved in the differential expression of PAI-1 in these cell types. In this study, the upregulation of PAI-1 expression is demonstrated in keloid tissues and their derived dermal fibroblasts, attesting to the persistence, if any, of fundamental differences between in vivo and in vitro paradigms. We further examined the mechanisms involved in hypoxia-induced regulation of PAI-1 gene in dermal fibroblast derived from keloid lesions and associated clinically normal peripheral skins from the same patient. Primary cultures were exposed to an environmental hypoxia or desferroxamine. We found that the hypoxia-induced elevation of PAI-1 gene appears to be regulated at both transcriptional and post-transcriptional levels in keloid fibroblasts. Furthermore, our results showed a consistent elevation of HIF-1α protein level in keloid tissues compared with their normal peripheral skin controls, implying a potential role as a biomarker for local skin hypoxia. Treatment with antisense oligonucleotides against hypoxia-inducible factor 1α (HIF-1α) led to the downregulation of steady-state levels of PAI-1 mRNA under both normoxic and hypoxic conditions. Conceivably, our results suggest that HIF-1α may be a novel therapeutic target to modulate the scar fibrosis process. Keloids are an excessive accumulation of extracellular matrix. Although numerous studies have shown elevated plasminogen activator inhibitor-1 (PAI-1) levels in keloid fibroblasts compared with those of normal skin. Their specific mechanisms involved in the differential expression of PAI-1 in these cell types. In this study, the upregulation of PAI-1 expression is demonstrated in keloid tissues and their derived dermal fibroblasts, attesting to the persistence, if any, of fundamental differences between in vivo and in vitro paradigms. We further examined the mechanisms involved in hypoxia-induced regulation of PAI-1 gene in dermal fibroblast derived from keloid lesions and associated clinically normal peripheral skins from the same patient. Primary cultures were exposed to an environmental hypoxia or desferroxamine. We found that the hypoxia-induced elevation of PAI-1 gene appears to be regulated at both transcriptional and post-transcriptional levels in keloid fibroblasts. Furthermore, our results showed a consistent elevation of HIF-1α protein level in keloid tissues compared with their normal peripheral skin controls, implying a potential role as a biomarker for local skin hypoxia. Treatment with antisense oligonucleotides against hypoxia-inducible factor 1α (HIF-1α) led to the downregulation of steady-state levels of PAI-1 mRNA under both normoxic and hypoxic conditions. Conceivably, our results suggest that HIF-1α may be a novel therapeutic target to modulate the scar fibrosis process. cycloheximide desferroxamine extracellular matrix hypoxia-inducible factor-1α plasminogen activator inhibitor-1 Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the plasminogen system that, in turn, is central to several essential physiologic and pathologic processes, including fibrinolysis, extracellular matrix turnover, fibrosis, wound healing, and cancer metastasis (Andreasen et al., 1990Andreasen P.A. George B. Lund L.R. Ruccio A. Stacey S.N. Plasminogen activator inhibitors: Hormonally regulated serpins.Mol Cell Endocrinol. 1990; 68: 1-19Crossref PubMed Scopus (382) Google Scholar;Eitzman et al., 1996Eitzman D.T. McCoy R.D. Zheng X. et al.Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or over express the murine plasminogen activator inhibitor-1 gene.J Clin Invest. 1996; 97: 232-237Crossref PubMed Scopus (511) Google Scholar). PAI-1 can be synthesized and secreted by platelets (Erickson et al., 1985Erickson L.A. Heckman C.M. Loskutoff D.J. The primary plaminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.Proc Natl Acad Sci USA. 1985; 82: 8710-8716Crossref PubMed Scopus (103) Google Scholar), vascular endothelial cells (Reilly and McFall, 1991Reilly C.F. McFall R.C. Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator-1 synthesis in vascular smooth muscle cells.J Biol Chem. 1991; 266: 9419-9427Abstract Full Text PDF PubMed Google Scholar), vascular smooth muscle cells (Reilly and McFall, 1991Reilly C.F. McFall R.C. Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator-1 synthesis in vascular smooth muscle cells.J Biol Chem. 1991; 266: 9419-9427Abstract Full Text PDF PubMed Google Scholar), and several nonvascular cell types (Busso et al., 1994Busso N. Niccodeme E. Chesne C. Guillouzo A. Belin D. Hyafil F. Urokinase and type 1 plasminogen activator inhibitor production by normal human hepatocytes: Modulation by inflammatory agents.Hepatology. 1994; 20: 186-190PubMed Google Scholar). PAI-1 gene expression can be regulated by a variety of stimuli, including phorbol ester (Descheemaeker et al., 1992Descheemaeker K.A. Wyns S. Nelles L. Auwerx J. Ny T. Collen D. Interaction of Ap-1, Ap-2 and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myrisate 13-acetate response.J Biol Chem. 1992; 267: 15086-15091Abstract Full Text PDF PubMed Google Scholar), transforming growth factor-β (Sawdey and Loskutoff, 1991Sawdey M.S. Loskutoff D.J. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo: Tissue specificity and induction by lipopolysacchrides, tumor necrosis factor-alpha, and transforming growth factor-beta.J Clin Invest. 1991; 88: 1346-1353Crossref PubMed Scopus (360) Google Scholar;Song et al., 1998Song C.Z. Siok T.E. Gelehrter T.D. Smad4/DPC4 and Smad3 mediate transforming growth factor-α (TGF-α) signaling through direct binding to a novel TGF-α-responsive element in the human plasminogen activator inhibitor-1 promoter.J Biol Chem. 1998; 273: 29287-29290Crossref PubMed Scopus (126) Google Scholar), tumor necrosis factor-α (Sawdey and Loskutoff, 1991Sawdey M.S. Loskutoff D.J. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo: Tissue specificity and induction by lipopolysacchrides, tumor necrosis factor-alpha, and transforming growth factor-beta.J Clin Invest. 1991; 88: 1346-1353Crossref PubMed Scopus (360) Google Scholar), interleukin-1 (Bevilacqua et al., 1986Bevilacqua M.P. Schleef R.R. Gimbrone Jr, M.A. Loskutoff D.J. Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin-1.J Clin Invest. 1986; 78: 587-591Crossref PubMed Scopus (209) Google Scholar), lipopolysaccharide (Crutchley and Conanan, 1986Crutchley D.J. Conanan L.B. Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells.J Biol Chem. 1986; 261: 154-159Abstract Full Text PDF PubMed Google Scholar; Sawdey and Loskutoff, 1991Sawdey M.S. Loskutoff D.J. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo: Tissue specificity and induction by lipopolysacchrides, tumor necrosis factor-alpha, and transforming growth factor-beta.J Clin Invest. 1991; 88: 1346-1353Crossref PubMed Scopus (360) Google Scholar), insulin, and insulin like growth factor-1 (Fattal et al., 1992Fattal P.G. Schneider D.J. Sobel B.E. Billadello J.J. Post-transcriptional regulation of expression of plasminogen activator inhibitor-1 mRNA by insulin and insulin like growth factor-1.J Biol Chem. 1992; 267: 12412-12415Abstract Full Text PDF PubMed Google Scholar;Banfi et al., 2001Banfi C. Eriksson P. Giandomenico G. Mussoni M. Sironi L. Hamsten A. Tremoli E. Transcriptional regulation of plasminogen activator inhibitor type-1 gene by insulin.Diabetes. 2001; 50: 1522-1530Crossref PubMed Scopus (59) Google Scholar), platelet-derived growth factor (Reilly and McFall, 1991Reilly C.F. McFall R.C. Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator-1 synthesis in vascular smooth muscle cells.J Biol Chem. 1991; 266: 9419-9427Abstract Full Text PDF PubMed Google Scholar), and angiotensin II (Feener et al., 1995Feener E.P. Northrup J.M. Aiello L.P. King G.L. Angiotensin II induces plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 expression in vascular endothelial and smooth muscle cells.J Clin Invest. 1995; 95: 1353-1362Crossref PubMed Scopus (254) Google Scholar;Takeda et al., 2001Takeda K. Ichiki T. Tokunou T. et al.Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor-1 gene expression.Arterioscler Thromb Vasc Biol. 2001; 21: 868-873Crossref PubMed Scopus (126) Google Scholar). Recent studies reported an upregulation of PAI-1 gene expression induced by low oxygen tension (hypoxia) in trophoblast cells (Fitzpatrick and Graham, 1998Fitzpatrick T.E. Graham C.H. Stimulation of plasminogen activator inhobitor-1 expression in immortalized human trophoblast cells cultured under low levels of oxygen.Exp Cell Res. 1998; 245: 155-162Crossref PubMed Scopus (75) Google Scholar), hepatocytes (Kietzmann et al., 1999Kietzmann T. Roth U. Jungermann K. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar), endothelial cells (Uchiyama et al., 2000Uchiyama T. Kurabayashi M. Ohyama Y. et al.Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells.Arterioscler Thromb Vasc Biol. 2000; 20: 1155-1161Crossref PubMed Scopus (78) Google Scholar), transformed murine macrophages (Pinsky et al., 1998Pinsky D.J. Liao H. Lawson C.A. Yan S.F. Chen J.X. Carmeliet P. Loskutoff D.J. Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.J Clin Invest. 1998; 102: 919-928Crossref PubMed Scopus (157) Google Scholar), and some cancer cell lines (Fink et al., 2001Fink T. Ebbesen P. Zachar V. Quantitative gene expression profiles of human liver-derived cell lines exposed to moderate hypoxia.Cell Physiol Biochem. 2001; 11: 105-114Crossref PubMed Scopus (24) Google Scholar). Furthermore, studies in cultured rat hepatocytes showed that mild hypoxia (8% O2) induced PAI-1 gene expression directly through a specific promoter sequence (-175/-158) that harbors hypoxia response element-1 (-175/–168) and hypoxia response element-2 (-165/-158), which interact with the hypoxia-inducible factor-1 (HIF-1) (Kietzmann et al., 1999Kietzmann T. Roth U. Jungermann K. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar).Fink et al., 2002Fink T. Kazlauskas A. Poellinger L. Ebbesen P. Zachar V. Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1.Blood. 2002; 99: 2077-2083Crossref PubMed Scopus (118) Google Scholar have reported a tightly regulated hypoxia-response element (-194 to -187) in the promoter of the human PAI-1 gene. Previous studies, using a three-dimensional fibrin gel model system simulating fibroplasias of wound repair, demonstrated that keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of urokinase plasminogen activator (Tuan et al., 1996Tuan T.L. Zhu J.Y. Sun B. Nichter L.S. Nimni M.E. Laug W.E. Increased PAI-1 may account for the altered fibrinolysis by keloid fibroblasts.J Invest Dermatol. 1996; 106: 1007-1011Crossref PubMed Scopus (78) Google Scholar). A marked increase in the PAI-1 transcript and protein was also noted in fibroblasts derived from patients with premature aging disorders (Werner's syndrome), as well as keloid lesions (Higgins et al., 1999Higgins P.J. Slack J.K. Diegelmann R.F. Staiano-Coico L. Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibric phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar). The altered ratio of activator and inhibitor activities is a major contributing factor in both altered fibrin degradation and the subsequent extracellular matrix (ECM) metabolism, leading to the formation of keloids and fibrosis (Niessen et al., 1999Niessen F.B. Spauwen P.H.M. Schalkwijk J. Kon M. On the nature of hypertrophic scars and keloids: A review.Plast Reconstr Surg. 1999; 104: 1435-1458Crossref PubMed Scopus (605) Google Scholar). In light of the growing evidence that the plasminogen system plays an essential role in regulating ECM metabolism in keloid scar formation, we examined the molecular mechanisms underlying the PAI-1 elevation in keloids. Conceivably, elucidating the molecular mechanisms regulating PAI-1 activity is essential for understanding the abnormal ECM metabolism and its pathophysiologic consequences. It has been implied by several histologic studies that a state of local hypoxia exists in hypertrophic scars and keloids secondary to multiple microvascular occlusions (Kischer et al., 1982Kischer C.W. Thies A.C. Chvapil M. Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids.Hum Pathol. 1982; 13: 819-824Abstract Full Text PDF PubMed Scopus (137) Google Scholar;Kischer, 1992Kischer C.W. The microvessels in hypertrophic scars, keloids and related lesions: A review.J Submicrosc Cytol Pathol. 1992; 24: 281-296PubMed Google Scholar). But to date, there is still a lack of direct evidence that hypoxia does exist in the microenvironment of keloids. Numerous studies in cancer cells, in vivo and in vitro, have correlated an increase in HIF-1α level with a local state of tissue hypoxia (Guppy, 2002Guppy M. The hypoxic core: A possible answer to the cancer paradox.Biochem Biophys Res Commun. 2002; 299: 676-680Crossref PubMed Scopus (72) Google Scholar;Zagzag et al., 2002Zagzag D. Zhong H. Scalzitti J.M. Laughner E. Simons J.W. Semenza G.L. Expression of hypoxia-inducible factor 1α in brain tumors.Cancer. 2002; 88: 2606-2618Crossref Scopus (533) Google Scholar). Here, we tested the level of HIF-1α present in fresh keloid tissues as a biomarker for skin hypoxia. Owing to the lack of a well-accepted animal model for the study of fibrotic scar formation, we have adapted an in vitro cell system using primary cultures of dermal fibroblasts derived from both the keloid center and the associated clinically normal peripheral skin from the same patient. We hypothesized that hypoxia-induced upregulation of the PAI-1 gene is, at least in part, responsible for the fibrotic phenotype of aberrant scars, such as keloids, by driving the steady state of ECM metabolism to a state of excessive accumulation. To address the mechanisms underlying the hypoxia-induced regulation of PAI-1 gene expression in an in vitro cell system, we exposed primary cultured dermal fibroblasts either to a hypoxic environment (2% O2) or to desferroxamine (DFO), an iron chelator (Hasegawa et al., 1999Hasegawa T. Sorensen L. Hidemi O. Marshall B.C. Decreased intracellular iron availability suppresses epithelial cell surface plasmin generation: Transcriptional and post-transcriptional effects on u-PA and PAI-1 expression.Am J Respir Cell Mol Biol. 1999; 21: 275-282Crossref PubMed Scopus (12) Google Scholar) that mimics intracellular hypoxia. Using this experimental paradigm, we observed that hypoxia-induced PAI-1 gene expression is regulated at both the transcriptional and the post-transcriptional levels. Our findings also support the fact that HIF-1α plays an essential role in the hypoxia-induced upregulation of PAI-1 gene expression in keloid fibroblasts and suggest that a novel treatment employing antisense oligonucleotides against HIF-1α may be useful in deterring scar fibrosis. The protocol for tissue collection was approved by the institutional review board of the Charles R. Drew University of Medicine and Science. Keloid tissues were taken from patients who underwent excisional biopsies at the King Drew Medical Center via an elliptical incision, which includes a 2- to 4-mm peripheral rim of clinically normal skin (Table I). Clinically raised keloid lesions and associated normal skin borders were histologically confirmed before further studies (Blackburn and Cosman, 1966Blackburn W.R. Cosman C. Histological basis of keloid and hypertrophic scar differentiation.Arch Pathol. 1966; 82: 65-71PubMed Google Scholar;Ehrlich et al., 1994Ehrlich H.P. Desmoulière A. Diegelmann R.F. et al.Morphological and immunochemical differences between keloid and hypertrophic scar.Am J Pathol. 1994; 145: 105-113PubMed Google Scholar). All keloid samples obtained in this study had not been previously treated and were not recurred cases. Using enzymatic digestion (Messadi et al., 1999Messadi D.V. Le A. Berg S. Jewett A. Zhuang W. Kelly P. Bertolami C.N. Expression of apoptosis-associated genes by human dermal scar fibroblasts.Wound Rep Reg. 1999; 7: 511-517Crossref PubMed Scopus (69) Google Scholar), primary dermal fibroblasts were isolated from both keloid lesions and from the associated clinically normal skin peripheral border of the same patient. Fibroblast cells were cultured in Dulbecco's modified Eagle's medium (Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (Gibco). All cultures were maintained at 37°C, 5% CO2, and 20% O2. Cells used for experiments were between passages 1 and 8 and were routinely monitored for cell proliferation, morphology and phenotype.Table ITissue specimens employed in this studyTissuesEthnicitySexAge (years)LocationCauseKeloid lesions and its clinically normal skin border (2- to 4-mm peripheral border)HispanicMale25Posterior earPiercingAfrican AmericanFemale28Lateral neckTraumaAfrican AmericanMale30Mid-chestAcneAfrican AmericanMale42Anterior earPiercingAfrican AmericanFemale23Posterior earPiercing Open table in a new tab Cells growing to about 80% confluence were transferred to a hypoxic chamber with auto purge airlock (Coy Laboratory Products, Inc., Grass Lake, MI). Environmental hypoxic conditions (2%) were achieved in an airtight humidified chamber and continuously flushed with a gas mixture containing 5% CO2 and 95% N2. Maintenance of the desired O2 concentration was constantly monitored during incubation using a microprocessor-based oxygen controller (Coy Laboratory Products, Inc.). Intracellular hypoxic conditions were generated using DFO treatment for 24 h (130 μmol/L). Normoxic conditions were defined as 20% O2, 5% CO2 at 37°C. Total RNA was isolated from cultured cells with guanidinium thiocyanate (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62289) Google Scholar). Total RNA (15 μg) was separated on a 1.4% agarose gel containing formaldehyde and transferred onto nylon membranes (Amersham Pharmacia Biotech UK Limited, Little Chalfont Buckinghamshire, UK). A cDNA fragment (262 bp) complementary to human PAI-1 was amplified using RT-PCR, subcloned into pGEM-T Easy Vector System (Promega, Madison, WI), labeled with [α-32P]dCTP using the Rediprime II Random Prime DNA labeling system (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, UK), and purified through Sephadex G-50 Quick Spin columns (TE; Roche Diagnostics Corp., Indianapolis, IN). After overnight hybridization at 42°C, membranes were washed and exposed to an intensifying screen in cassette (Kodak, Rochester, NY) for 24 h and analyzed with a phosphorimaging scanner (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Images were quantified using ImageQuant analysis software (Molecular Dynamics). After a fresh medium change, fibroblasts were exposed to either hypoxia or DFO for 24 h, followed by treatment with actinomycin D (5 μg/mL, Sigma, St. Louis, MO) to inhibit further mRNA transcription (Kietzmann et al., 1999Kietzmann T. Roth U. Jungermann K. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar) for a fixed time period as indicated. Subsequently, cells were harvested and RNA was prepared for northern blot analysis as described above. Quantitation of autoradiographs was carried out using the ImageQuant System (Molecular Dynamics) and the level of β-actin for loading control. The relative ratios of the 3.2-kb PAI-1 mRNA versus α-actin were graphically depicted and linear regression analyses were performed to assess the half-life of the 3.2-kb PAI-1 mRNA. Cells were pretreated with either cycloheximide (CHX, 10 μg/mL) or actinomycin D (5 μg/mL) for 1 h and then exposed to either hypoxia or DFO for 24 h. Total RNA was isolated for northern blot analyzes. The tissue homogenates were prepared as described previously (Shabsigh et al., 2001Shabsigh A. Ghafar M.A. Alexandre T. Burchardt M. Kaplan S.A. Anastasiadis A.G. Buttyan R. Biomarker analysis demonstrates a hypoxic environment in castrated rat ventral prostate gland.J Cell Biochem. 2001; 81: 437-444Crossref PubMed Scopus (64) Google Scholar). An equal amount (50 μg) of protein sample from each tissue homogenate or aliquots of the conditioned medium from the cultured cells normalized to the cell numbers were subjected to electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gels and electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia). After blocking with Tris-buffered saline/5% skim milk, the membrane was incubated overnight at 4°C with a mouse antihuman PAI-1 antibody (American Diagnostica Inc., Greenwich, CT) or antihuman HIF-1α monoclonal antibody (BD Transduction Laboratory, San Jose, CA). Afterward, the membrane was extensively washed and incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000) (Pierce, Rockford, IL) for 1 h at room temperature, followed by an enhanced chemiluminescent detection (Zeheb et al., 1987Zeheb R. Rafferty U.M. Rodriguez M.A. Andreasen P. Gelehrter T.D. Immunoaffinity purification of HTC rat hepatoma cell plasminogen activator-inhibitor-1.Thromb Haemost. 1987; 58: 1017-1023PubMed Google Scholar). To construct PAI-1 promoter–luciferase reporter genes, the following forward and reverse primers were adopted to perform a PCR using plasmid p800LUC harboring the human PAI-1 5′ flanking promoter (–800 to + 71; gift from D.J. Loskutoff, The Scripps Research Institute, La Jolla, CA) as a template. The forward primer for PCR was 5′-GACAGAGCTCAAGCTTACCATGGTAACCCCT-3′(–800/–779) and the reverse primer was 5′-TCGG AGATCTCAGCTGCTGGAGGGGGGCGT-3′(+ 71/+51), which were encompassed by the SacI (forward primer) and BglII (reverse primer) recognition sites. PCR products were digested, gel-purified, and subcloned into the SacI/BglII sites of the promoterless luciferase reporter gene vector (pGL3-Basic; Promega) to engineer the pGL3PAI-800Luc construct. Transfections of DNA into keloid fibroblasts were performed using SuperFect transfection reagent (Qiagen, Valencia, CA). Cells were cotransfected with 1 μg of pGL3PAI-800Luc and 1 μg of pRL-TK vector as an internal control for normalizing transfection efficiency. Luciferase activities under different conditions were measured using a Dual-Luciferase reporter assay system (Promega) with a Berthold Lumat LB9501 luminometer. Each transfection was repeated at least three times in duplicate. HIF-1α phosphorothioate antisense and sense oligonucleotides were synthesized by GenoMechanix (GenoMechanix, Alachua, FL) according to the sequences described previously (Caniggia et al., 2000Caniggia I. Mostachfi H. Winter J. Gassmann M. Lye S.J. Kuliszewski M. Post M. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGF beta (3).J Clin Invest. 2000; 105: 577-587Crossref PubMed Scopus (507) Google Scholar); antisense sequence, 5′-GCC GGC GCC CTC CAT-3′; sense sequence, 5′-ATG GAG GGC GCC GGC-3′. Cells were washed once with serum and antibiotic-free medium, transfected with the sense and antisense oligonucleotides by using the Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transfected cells were cultured under normal conditions overnight and then exposed to hypoxic conditions for 48 h. Total RNA was isolated from cells using Trizol reagent (Gibco BRL). Analysis of HIF-1α, PAI-1, and β-actin mRNA levels was performed using the one-step RT-PCR kit (Qiagen) with primers specific to HIF-1α (forward primer 5′-TCACCACAGGACAGTACAGGATGC-3′; reverse primer 5′-CCAGCAAAGTTAAAGCATCAGGTTCC-3′), PAI-1 (forward primer 5′-CGCCTCTTCCACAAATCAG-3′, reverse primer 5′-ATGCGGGCTGAGACTATGA-3′) (Caniggia et al., 2000Caniggia I. Mostachfi H. Winter J. Gassmann M. Lye S.J. Kuliszewski M. Post M. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGF beta (3).J Clin Invest. 2000; 105: 577-587Crossref PubMed Scopus (507) Google Scholar), and β-actin (forward primer 5′-TCATGAAGTGTGACGTTGACATCCGT-3′, reverse primer 5′-CCTAGAAGCATTTGCGTGCACGATG-3′). The PCR products were electrophoresed on 1.5% agarose gel containing 0.5 μg per mL ethidium bromide. Although several studies have reported a marked increase in PAI-1 expression in cultured primary keloid fibroblasts compared with that of normal skin (Tuan et al., 1996Tuan T.L. Zhu J.Y. Sun B. Nichter L.S. Nimni M.E. Laug W.E. Increased PAI-1 may account for the altered fibrinolysis by keloid fibroblasts.J Invest Dermatol. 1996; 106: 1007-1011Crossref PubMed Scopus (78) Google Scholar;Higgins et al., 1999Higgins P.J. Slack J.K. Diegelmann R.F. Staiano-Coico L. Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibric phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar), there has been no published report, to our knowledge, indicating that this fundamental difference exists in vivo and then persists through subsequent cultivation. In this study, we collected several sets of elliptically excised keloid lesions with 2- to 4-mm peripheral borders of clinically normal skin from the same patient and carried out tissue homogenate lysates for western blot analyses. Results of three representative sets of keloid lesions and their associated clinically normal skin borders showed a consistent two- to three-fold increase in PAI-1 protein level in keloid tissues compared with their own control normal skins (Figure 1a). Elevated PAI-1 mRNA expression was also demonstrated by northern blot analyses in primary cultured keloid fibroblasts derived directly from the same tissue samples (Figure 1b). The consistent findings of elevated PAI-1 in both tissues and tissue-derived cells confirm that fundamental in vivo biologic differences are recapitulated by an in vitro system. We next examined the expression of HIF-1α protein, a well-characterized biomarker for tissue hypoxia (Shabsigh et al., 2001Shabsigh A. Ghafar M.A. Alexandre T. Burchardt M. Kaplan S.A. Anastasiadis A.G. Buttyan R. Biomarker analysis demonstrates a hypoxic environment in castrated rat ventral prostate gland.J Cell Biochem. 2001; 81: 437-444Crossref PubMed Scopus (64) Google Scholar), using the same sets of tissue homogenate lysates. Western blot analyses (Figure 1a) revealed an intrinsically elevated HIF-1α protein levels (two- to three-fold increase) in keloid tissues compared with their clinically normal bordering skins, suggesting that a local hypoxic state exists in keloid tissues. Next we investigated mechanisms responsible for the elevation of PAI-1 in keloid fibroblasts. Because other studies using nuclear runoff analyses have attributed the overexpression of PAI-1 in keloid fibroblasts to the transcriptional regulation (Higgins et al., 1999Higgins P.J. Slack J.K. Diegelmann R.F. Staiano-Coico L. Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibric phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar), we conducted a study on mRNA stability in keloid and normal fibroblasts after inhibiting transcriptional activities with actinomycin D treatment (Figure 2). Northern blot analyses revealed that under normoxic conditions, the 3.2-kb PAI-1 mRNA was degraded at a much slower rate in keloid fibroblasts (Figure 2a1) than in normal skin fibroblasts (Figure 2b1). Densitometric analyses of 3.2-kb PAI-1 transcripts were graphically depicted (Figure 2c,d), and linear regression analyses revealed a twofold increase in the half-life for the 3.2-kb PAI-1 message in the keloid fibroblasts (t1/2=24.31 h) compared with that in the normal cells (t1/2=10.51 h; p<0.01) (Table II). Conceivably, the increase in PAI-1 mRNA stability may have contributed, at least in part, to the elevated basal PAI-1 expression in keloid fibroblasts. Nevertheless, the mechanisms responsible for the increase in PAI-1 mRNA half-life in keloids remain to be elucidated.Table IIRegression analyses of PAI-1 mRNA half-lives (h)aNumber in parentheses is r value.DFO (-)DFO (+)NormoxiaHypoxiaNormal skin10.51 (-0.96273)52.50 (-0.99720)bp<0.00112.64 (-0.94358)36.17 (-0.97210)cp<0.01.Keloid2
Referência(s)