Fluctuations of Intracellular Iron Modulate Elastin Production
2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês
10.1074/jbc.m409897200
ISSN1083-351X
AutoresSevera Bunda, Nilo Kaviani, Aleksander Hinek,
Tópico(s)Kruppel-like factors research
ResumoProduction of insoluble elastin, the major component of elastic fibers, can be modulated by numerous intrinsic and exogenous factors. Because patients with hemolytic disorders characterized with fluctuations in iron concentration demonstrate defective elastic fibers, we speculated that iron might also modulate elastogenesis. In the present report we demonstrate that treatment of cultured human skin fibroblasts with low concentration of iron 2–20 μm (ferric ammonium citrate) induced a significant increase in the synthesis of tropoelastin and deposition of insoluble elastin. Northern blot and real-time reverse transcription-PCR analysis revealed that treatment with 20 μm iron led to an increase of ∼3-fold in elastin mRNA levels. Because treatment with an intracellular iron chelator, desferrioxamine, caused a significant decrease in elastin mRNA level and consequent inhibition of elastin deposition, we conclude that iron facilitates elastin gene expression. Our experimental evidence also demonstrates the existence of an opposite effect, in which higher, but not cytotoxic concentrations of iron (100–400 μm) induced the production of intracellular reactive oxygen species that coincided with a significant decrease in elastin message stability and the disappearance of iron-dependent stimulatory effect on elastogenesis. This stimulatory elastogenic effect was reversed, however, in cultures simultaneously treated with high iron concentration (200 μm) and the intracellular hydroxyl radical scavenger, dimethylthiourea. Thus, presented data, for the first time, demonstrate the existence of two opposite iron-dependent mechanisms that may affect the steady state of elastin message. We speculate that extreme fluctuations in intracellular iron levels result in impaired elastic fiber production as observed in hemolytic diseases. Production of insoluble elastin, the major component of elastic fibers, can be modulated by numerous intrinsic and exogenous factors. Because patients with hemolytic disorders characterized with fluctuations in iron concentration demonstrate defective elastic fibers, we speculated that iron might also modulate elastogenesis. In the present report we demonstrate that treatment of cultured human skin fibroblasts with low concentration of iron 2–20 μm (ferric ammonium citrate) induced a significant increase in the synthesis of tropoelastin and deposition of insoluble elastin. Northern blot and real-time reverse transcription-PCR analysis revealed that treatment with 20 μm iron led to an increase of ∼3-fold in elastin mRNA levels. Because treatment with an intracellular iron chelator, desferrioxamine, caused a significant decrease in elastin mRNA level and consequent inhibition of elastin deposition, we conclude that iron facilitates elastin gene expression. Our experimental evidence also demonstrates the existence of an opposite effect, in which higher, but not cytotoxic concentrations of iron (100–400 μm) induced the production of intracellular reactive oxygen species that coincided with a significant decrease in elastin message stability and the disappearance of iron-dependent stimulatory effect on elastogenesis. This stimulatory elastogenic effect was reversed, however, in cultures simultaneously treated with high iron concentration (200 μm) and the intracellular hydroxyl radical scavenger, dimethylthiourea. Thus, presented data, for the first time, demonstrate the existence of two opposite iron-dependent mechanisms that may affect the steady state of elastin message. We speculate that extreme fluctuations in intracellular iron levels result in impaired elastic fiber production as observed in hemolytic diseases. Mature elastic fibers and laminae provide extensibility and elastic recoil to vascular walls and ligaments and form a connective tissue framework of lungs, elastic cartilage, and skin (1Uitto J. Hsu-Wong S. Katchman S.D. Bashir M.M. Rosenbloom J. Ciba Found. Symp. 1995; 192: 237-258PubMed Google Scholar, 2Vrhovski B. Weiss A.S. Eur. J. Biochem. 1998; 258: 1-18Crossref PubMed Scopus (386) Google Scholar). They are complex structures made of polymeric (insoluble) elastin and 12-nm microfibrils that consist of several glycoproteins, e.g. fibrillins, fibulins, and microfibril-associated glycoproteins (3Roark E.F. Keene D.R. Haundenschild C.C. Godyna S. Little C.D. Argraves W.S. J. Histochem. Cytochem. 1995; 43: 401-411Crossref PubMed Scopus (161) Google Scholar, 4Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 5Kielty C. Badlock C. 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Iron is a physiologically essential nutritional element for all life forms (35Fraga C.G. Oteiza P.I. Toxicology. 2002; 180: 23-32Crossref PubMed Scopus (205) Google Scholar). It plays critical roles in electron transport and cellular respiration, oxygen transport by hemoglobin, cell proliferation, and differentiation (36Boldt D.H. Am. J. Med. Sci. 1999; 318: 207-212Crossref PubMed Google Scholar). It has been shown that modulating intracellular iron levels may also affect expression of numerous genes that are not directly involved in iron metabolism, such as protein kinase C-β, an important component of intracellular signaling pathways (37Alcantara O. Obeid L. Hannun Y. Ponka P. Boldt D.H. Blood. 1994; 84: 3510-3517Crossref PubMed Google Scholar), or those encoding extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; ROS, reactive oxygen species; DFO, desferrioxamine; DRB, dichlorobenzimidazole riboside; DMTU, dimethylthiourea; FAC, ferric ammonium citrate; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; LIP, labile iron pool; NF-1, nuclear factor-1. 1The abbreviations used are: ECM, extracellular matrix; ROS, reactive oxygen species; DFO, desferrioxamine; DRB, dichlorobenzimidazole riboside; DMTU, dimethylthiourea; FAC, ferric ammonium citrate; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; LIP, labile iron pool; NF-1, nuclear factor-1. components (36Boldt D.H. Am. J. Med. Sci. 1999; 318: 207-212Crossref PubMed Google Scholar, 38Templeton D.M. Liu Y. Biochim. Biophy. Acta. 2003; 1619: 113-124Crossref PubMed Scopus (96) Google Scholar). It has been demonstrated that dietary iron overload in rats resulted in an increase in the steady-state level of pro-α2(I)-collagen in hepatocytes (39Pietrangelo A. Rocchi E. Schiaffonati L. Ventura E. Cairo G. Hepatology. 1990; 11: 798-804Crossref PubMed Scopus (66) Google Scholar) and that 50 μm iron treatment stimulated collagen gene expression in cultured stromal hepatic cells by inducing the synthesis and binding of Sp1 and Sp3 transcription factors to two regulatory elements located in the collagen α1 (I) promoter region (40Ruiz I.G. De Torre LaP. Diaz T. Esteban E. Morrilas J.D. Munoz-Yague T. Solis-Herruzo J.A. DNA Cell Biol. 2000; 19: 167-178Crossref PubMed Scopus (27) Google Scholar). On the other hand, iron loading in cultured cardiac myocytes and fibroblasts decreased the expression of transforming growth factor-β, biglycan, and collagen type I mRNA, whereas it facilitated the expression of decorin mRNA. Interestingly, iron deprivation exerted a similar effect, suggesting that the expression of these genes involved in extracellular matrix production is regulated by certain iron-dependent mechanisms (41Parkes J.G. Liu Y. Sirna J.B. Templeton D.M. J. Mol. Cell. Cardiol. 2000; 32: 233-246Abstract Full Text PDF PubMed Scopus (44) Google Scholar). The molecular basis of iron-dependent mechanism(s) regulating the expression of ECM-encoding genes is not well understood. Because raising levels of iron may overwhelm the iron binding capacity of transferrin, resulting in the appearance of non-transferrin bound iron (42Hershko T.E. Konijn A.M. Link G. Br. J. Hematol. 1998; 101: 399-406Crossref PubMed Scopus (136) Google Scholar), which is capable of catalyzing the formation of the hydroxyl radicals (through the Fenton and Haber-Weiss reactions) (43Pietrangelo A. J. Hepatol. 2000; 32: 862-864Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), it has been suggested that iron-dependent induction of reactive oxygen species (ROS) may modulate the transcription of these genes (36Boldt D.H. Am. J. Med. Sci. 1999; 318: 207-212Crossref PubMed Google Scholar, 44Brenneisen P. Wenk J. Klotz O. Wlaschek M. Briviba K. Krieg T. Scharffetter-Kochanek.Sies H. J. Biol. Chem. 1998; 273: 5279-5287Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The possibility of iron-dependent oxidative damage to elastic fibers has also been suggested, but not proven (30Aessopos A. Farmakis D. Loukopoulos D. Blood. 2002; 99: 30-35Crossref PubMed Scopus (115) Google Scholar). The present study was designed to elucidate the mechanisms by which iron might modulate elastogenesis. We utilized normal human skin fibroblasts that are capable of elastic fiber production in vitro to explore whether changes in concentrations of iron would affect elastin gene expression and the subsequent deposition of elastic fibers. We also tested the influence of different iron concentrations on generation of intracellular ROS, and attempted to identify on which level these iron-induced ROS affect elastogenesis. Materials—All chemical-grade reagents, catalase, desferrioxamine (DFO), dichlorobenzimidazole riboside (DRB), dimethylthiourea (DMTU), ferric ammonium citrate (FAC), superoxide dismutase, and tempol were all obtained from Sigma, and Dulbecco's modified eagle's medium, fetal bovine serum (FBS), 0.2% trypsine-0.02% EDTA, and other cell culture products from Invitrogen. We obtained 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and acetyl ester (CM-H2DCFDA) from Molecular Probes (Eugene, OR). Polyclonal antibody to tropoelastin was purchased from Elastin Products (Owensville, MI). Secondary antibody fluorescein isothiocyanate-conjugated goat anti-rabbit was purchased from Sigma. DNeasy Tissue system for DNA assay and RNeasy Mini Kit for isolation of total RNA were purchased from Qiagen (Mississauga, Ontario, Canada). A OneStep RT-PCR kit was purchased from Qiagen. A SuperScript First-Strand Synthesis System for RT-PCR was purchased from Invitrogen. TaqMan Universal PCR master mix, TaqMan GAPDH control, and Assays-on-Demand Gene Expression probe for elastin were purchased from Applied Biosystems (Foster City, CA). The radiolabeled reagents, [3H]valine and [3H]thymidine, and Rediprime (II) Random Primer labeling system were purchased from Amersham Biosciences. Hybridization solution Miracle Hyb was purchased from Stratagene (Cedar Creek, TX), and the human GAPDH control was purchased from Clontech (Palo Alto, CA). Cultures of Normal Human Skin Fibroblasts—Fibroblasts grown from skin biopsy explants of six normal subjects, aged from 2 months to 10 years, were obtained from the cell repository at The Hospital for Sick Children in Toronto with the permission of the Institutional Ethics Committee. Fibroblasts were routinely passaged by trypsinization and maintained in Dulbecco's modified eagle's medium supplemented with 1% antibiotics/antimycotics, and 10% FBS. In all described experiments passages 2–6 were used. In experiments aimed at assessing ECM production, fibroblasts were initially plated (100,000 cells/dish) and maintained in normal medium until confluency at which point they produce abundant ECM. Confluent cultures were then treated for 72 h with or without FAC producing iron concentrations from 2 to 200 μm. The low iron concentration (2 and 20 μm) of iron utilized in the present study remained in the range that did not induce any disturbances in cellular metabolism when tested by other investigators (45Breuer W. Epsztein S. Cabantchik I. FEBS Lett. 1997; 382: 304-308Crossref Scopus (95) Google Scholar). The high iron concentration (200 μm) was comparable to concentrations used in studies of iron overload (41Parkes J.G. Liu Y. Sirna J.B. Templeton D.M. J. Mol. Cell. Cardiol. 2000; 32: 233-246Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 46Traore H.N. Meyer D. Met. Cell Sci. 2002; 23: 175-184Crossref Scopus (12) Google Scholar). In some experiments the membrane permeable ferric iron chelator, DFO, was added 30 min prior to FAC treatment. For the experiments conducted in the presence of various antioxidants, the antioxidants were applied 1 h prior to FAC treatment. For experiments conducted in serum-free conditions, 7-day-old confluent fibroblast cultures were starved for 12 h in serum-free medium and incubated with various concentrations of iron (as FAC) for additional 72 h in serum-free medium. Immunostaining—At the end of the incubation period confluent cultures were fixed in cold 100% methanol at -20 °C for 30 min and blocked with 1% normal goat serum for 1 h at room temperature. Cultures were then incubated for 1 h with 10 μg/ml polyclonal antibody to tropoelastin followed by a 1-h incubation with fluorescein-conjugated goat anti-rabbit. Nuclei were counterstained with propidium iodide. Secondary antibody alone was used as a control. All of the cultures were then mounted in ELVANOL, and examined with an Nikon Eclipse E1000 microscope attached to a cooled charge-coupled device camera (QImaging, Retiga EX) and a computer-generated video analysis system (Image-Pro Plus software, Media Cybernetics, Silver Spring, MD). Quantitative Assays of Tropoelastin and Insoluble Elastin—Normal human skin fibroblasts were grown to confluency in 35-mm culture dishes (100,000 cells/dish) in quadruplicates. Then, 2 μCi of [3H]valine/ml of fresh media was added to each dish, along with or without 2, 20, 100, and 200 μm FAC. Cultures were incubated for 72 h, and the soluble and insoluble elastin was assessed separately in each dish. The cells were extensively washed with PBS, and the soluble proteins present in the intracellular compartments were extracted overnight at 4 °C with 0.1 m acetic acid in the presence of proteinase inhibitors. After centrifugation, the supernatants were pre-cleaned by 30-min incubation with 50 μl of 4% protein A-beaded agarose, then 500 μl of the supernatant was incubated with 5 μg of polyclonal antibody to tropoelastin for 2 h and subsequently with 50 μl of 4% protein A-beaded agarose for 3 h at 4 °C. The protein A-containing beads were sedimented by centrifugation, washed with immunoprecipitation buffer, mixed with scintillation fluid, and counted (21Hinek A. Rabinovitch M. J. Cell Biol. 1994; 126: 563-574Crossref PubMed Scopus (147) Google Scholar). The remaining cultures containing cell remnants and deposited insoluble extracellular matrix were scraped and boiled in 500 μl of 0.1 n NaOH for 45 min to solubilize all matrix components except elastin. The resulting pellets containing the insoluble elastin were then solubilized by boiling in 200 μl of 5.7 n HCl for 1 h, and the aliquots were mixed in scintillation fluid and counted (21Hinek A. Rabinovitch M. J. Cell Biol. 1994; 126: 563-574Crossref PubMed Scopus (147) Google Scholar). Aliquots taken from each culture were also used for DNA determination according to Ref. 47Rodems S.M. Clark C.L. Spector D.H. J. Virol. 1998; 72: 2697-2704Crossref PubMed Google Scholar, using the DNeasy Tissue System from Qiagen. Final results reflecting amounts of metabolically labeled insoluble elastin in individual cultures were normalized per their DNA content and expressed as cpm/μg of DNA. In separate experiments, the specified treatment described in the figure legends was added along with 2 μCi of [3H]valine/ml media to confluent cultures of normal human skin fibroblasts in 35-mm culture dishes (100,000 cells/dish) in quadruplicates for 72 h. The conditioned media was then removed, the cell layers were washed, and incorporation of [3H]valine into the insoluble elastin was assessed as described above. Assessment of Cell Proliferation—Normal human skin fibroblasts were suspended in Dulbecco's modified eagle's medium containing 10% FBS and plated in 35-mm culture dishes (100,000 cells/dish) in quadruplicates. Twenty-four hours later, the cells were transferred to the serum-free medium for synchronization of their cell cycle and then maintained in the presence or absence of FAC (2–200 μm) and 2 μCi of [3H]thymidine/ml in media with 10% FBS for 72 h. These cultures were then washed in PBS and treated with cold 5% trichloroacetic acid twice for 10 min at 4 °C. One-half milliliter of 0.3 n NaOH was added to all dishes, incubated at room temperature for 30 min, and 200-μl aliquots of each culture mixed with scintillation fluid were counted. Assays of Intracellular ROS Levels—The ROS-sensitive fluorescent probe, CM-H2DCFDA has been used to detect oxidative activity in cultured fibroblasts (48Royall J.A. Ischiropoulos H. Arch. Biochem. Biophys. 1993; 302: 348-355Crossref PubMed Scopus (1042) Google Scholar). This probe passively diffuses into the cell interior, and only upon oxidation is a fluorescent product released that can be visualized under fluorescent microscope or captured by flow cytophotometry, when it is excited at 480 nm. To measure intracellular ROS production, normal human skin fibroblasts were plated on glass coverslips in 35-mm dishes (50,000 cells/dish) and grown to confluency. The cells were then washed with PBS and incubated with or without 10 μm CM-H2DCFDA for 30 min in fresh media. The cells were then washed again in PBS and incubated with new media in the presence or absence of FAC (2–400 μm) for three additional hours. The cells were then washed twice with PBS before being mounted to the glass slides, and the images were captured using a fluorescence microscope under identical parameters of contrast and brightness. In addition, the quantification of this reaction was performed by flow cytometry (λ excitation, 480 nm; λ emission, 520 nm). Quadruplicate cultures of fibroblasts were preincubated with CM-H2DCFDA and maintained in the presence or absence of FAC as described above. To reduce stress-induced oxidant activation, the cells were cooled and harvested by trypsinization at 4 °C. They were then collected by centrifugation (4 °C, 1000 rpm for 3 min), washed in cold PBS, and fixed with 4% formaldehyde for 10 min in the dark and analyzed by flow cytometry (FACSCalibur, BD Biosciences). Northern Blots—Normal human skin fibroblasts were grown to confluency in 100-mm culture dishes. Fresh media was added along with or without 2, 20, and 200 μm of FAC for 24 h. Total RNA was isolated using RNeasy Mini Kit according to manufacturer's instructions, and 10 μg were resolved by electrophoresis on formaldehyde-1% agarose gels. Recovery of 18 S and 28 S rRNA was analyzed using ethidium-bromide staining and image analysis on a Gel Doc 1000 optical-system (Bio-Rad). RNA was transferred onto Hybond-N membrane (Amersham Pharmacia Biotech) by capillary transfer in 10× SSC and immobilized by UV cross-linking. Human elastin cDNA recombinant probe H-11 (49Olson T.M. Michels V.V. Urban Z. Csiszars K. Christiano A.M. Driscoll D.J. Feldt R.H. Boyd C.D. Thibodeau S.N. Hum. Mol. Genet. 1995; 4: 1677-1679Crossref PubMed Scopus (83) Google Scholar, 50Urban Z. Riazi S. Seidl T.L. Katahira J. Smoot L.B. Chitayat D. Boyd C.D. Hinek A. Am. J. Hum. Genet. 2002; 71: 30-44Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) was radiolabeled with 32P random primer method and incubated overnight at 42 °C with the membrane in Miracle Hyb solution at a concentration of 2.5–5 × 106 cpm/ml. The membrane was washed to high stringency, and the bound radioactivity was visualized by autoradiography and quantified by scanning densitometry (Gel Doc 1000). RNA loading and transfer were evaluated by probing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to which relative elastin mRNA values were normalized. Quantitative TaqMan RT-PCR—To confirm the expression level of elastin mRNA in the presence of 2, 20, and 200 μm FAC obtained by Northern blot analysis, we also conducted quantitative RT-PCR. To assess the effect of iron on elastin mRNA stability, parallel quadruplicate cultures were grown to confluency in 100-mm dishes. Media were then changed, supplemented with 60 μm transcription blocker, DRB (51Zhang M-C. Giro M.G. Quaglino D. Davidson J.M. J. Clin. Invest. 1995; 95: 986-994Crossref PubMed Scopus (33) Google Scholar), and cultures were maintained in the presence or absence of 20 and 200 μm FAC for 0, 6, 12, and 24 h. Total RNA was extracted using the RNeasy Mini kit, according to manufacturer's instructions, at indicated time points. The reverse transcriptase reaction was performed using 1.5 μg of total RNA, oligo(dT)s, and the SuperScript First-Strand synthesis system (Invitrogen) according to the manufacturer's instructions. Elastin mRNA levels were measured by real-time quantitative PCR method performed on the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). For each treatment two distinct amplifications were carried out in parallel to amplify elastin cDNA and GAPDH cDNA. The amplification reactions were performed in 25-μl volumes containing 30 ng of cDNA per treatment in triplicate, 12.5 μlof 2× TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μl of 20x Assays-on-Demand Gene Expression probe for elastin (Applied Biosystems) or TaqMan GAPDH probe (Applied Biosystems). Elastin mRNA levels from each treatment were normalized to the corresponding amount of GAPDH mRNA levels. Water controls and samples without PCR mixtures were set up to eliminate the possibility of significant DNA contamination. Final results were expressed as the mean of two independent experiments. One-step RT-PCR—To further confirm the effect of iron on elastin mRNA levels, confluent normal human skin fibroblast cultures were treated with or without intracellular ferric iron chelator, 20 μm DFO in the presence or absence of 20 μm FAC for 24 h. Total RNA was extracted using the RNeasy Mini kit, according to the manufacturer's instructions, and 1 μg of total RNA was added to each one-step RT-PCR (Qiagen OneStep RT-PCR kit), and reactions were set up according to the manufacturer's instructions in a total volume of 25 μl. The reverse transcription step was performed for elastin and β-actin reactions at 50 °C for 30 min followed by 15 min at 95 °C. The elastin PCR reaction (sense primer: 5′-GGTGCGGTGGTTCCTCAGCCTGG-3′, antisense primer: 5′-GGGCCTTGAGATACCCCAGTG-3′; designed to produce a 255-bp product) was performed under the following conditions: 25 cycles at 94 °C denaturation for 20 s, 63 °C annealing for 20 s, and 72 °C extension for 1 min, then 1 cycle at 72 °C with a final extension for 10 min. The β-actin PCR reaction (sense primer: 5′-GGTCAGAAGGATTCCCTATGTG-3′; antisense primer: 5′-ATTGCCCAATGGTGATGACCTG-3′; designed to produce a 615-bp product) was performed under the following conditions: 25 cycles at 94 °C denaturation for 60 s, 60 °C annealing for 60 s, and 72 °C extension for 120 s, the 1 cycle at 72 °C with a final extension for
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