From www.bloodjournal.org by guest on December 22, 2014. For personal use only. Effects of Cyclosporin A on Erythropoietin Production by the Human Hep3B Hepatoma Cell Line By Alessandro M. Vannucchi, Alberto Grossi, Alberto Bosi, Daniela Rafanelli, Marinella Statello, Stefan0 Guidi, Riccardo Saccardi, and Pierluigi Rossi-Ferrini There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsAunrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 pmol/L) not affecting total protein synthetic rate nor the constitutive secretion of a-fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M,of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo t o remain unsecreted within the cells; almost all (97%of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears t o be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway. 0 1993 by The American Society of Hematology. C planted with a T-cell-depleted BM graft, exhibited higher serum Epo levels than those treated with cyclosporin-A (CsA) for the prevention of graft-versus-host disease (GVHD). Moreover, in a recent study” the Epo response to anemia was found to be impaired in autologous transplant patients, most of which had been treated with CsA for the induction of autologous GVHD, although the influence of CsA on Epo production could not be definitely clarified in comparison with patients not receiving CsA. Finally, we have recently shown that mice treated with therapeutical doses of CsA have an impaired capacity to produce adequate amounts of Epo in response to anemia.13Because this effect occurred in the animal model in the absence of any biochemical or histologic evidence of kidney toxicity, we hypothesized that the inhibitory effects of CsA might be somehow specific, and different from the classical CsA-induced nephrot~xicity.’~ Further support to this idea derived from the observation that both captopril14 and pentoxyfillineI5 failed to prevent the inhibition on Epo production in mice treated with CsA (June 199 1, personal observations). In this paper, we provide experimental evidence supporting the hypothesis that the inadequate Epo production characteristic of allogeneic BMT patients might be ascribed, at least in part, to an inhibitory effect of CsA on the mechanisms regulating Epo production. To this end, we took advantage of the human hepatoma Hep3B cell line, which is a well-characterized in vitro system for studies of the mechanisms regulating Epo production.16 In fact, these cells release high amounts of Epo in the culture medium in response to hypoxia or to certain transition metals,” and this regulation has been shown to occur at the Epo mRNA level.’’ IRCULATING levels of erythropoietin (Epo), the main regulator of red blood cell (RBC) homeostasis in humans,’ are finely adjusted according to the oxygen tension of the blood. In fact, Epo levels are maintained constant within a relatively narrow range ( I O to 25 mU/mL) under steady-state conditions, but they increase several fold when the RBC mass is reduced, as in acute or chronic anemias. An inverse, exponential correlation exists between serum Epo concentrations and hematocrit levels in normal subjects and in the majority of anemic patients.’ However, this correlation may be lost in some types of nonrenal anemias, such as in rheumatoid a r t h r i t i ~~, ~a n c e rand , ~ the acquired immunodeficiency syndrome (AIDS);’ therefore, these patients are considered to have an “inappropriate” Epo production. In studies of patients subjected to bone marrow transplantation (BMT), an inappropriate Epo production has generally been observed in patients receiving an allogeneic graft,”” while there is some disagreement about the behavior of autologous rescue patient^.'-^^''^'^ In this regard, Abedi et aI6 interestingly observed that allogeneic BMT patients not receiving immunosuppressive therapy, being trans- From the Bone Marrow Transplant Unit, the Division ofHematology, Careggi Hospital and University of Florence, Italy. Submitted October 26, 1992; accepted March 25, 1993. Supported by grants,from Boehringer Mannheim (Milano), Associazione ltaliana per le Leucemie (Firenze), Associazione Italiana per la Ricerca SUI Cancro, and MURST 40% -60%. Address reprint requests to Alessandro M . Vannucchi, MD, Bone Marrow Transplant Unit, Division of Hematology, Ospedale di Careggi, 50134 Florence, Italy. The publication costs of this article were defayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this ,fact. 0 1993 by The American Society of Hematology. 0006-49 71/93/8Z03-0035$3.00/0 978 MATERIALS AND METHODS Cell cultures. Hep3B cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD), and cultured in Iscove’s modified minimal essential medium (IMEM; GIBCO, Grand Island, NY), supplemented with penicillin (100 U/mL), Blood, Vol 82, No 3 (August 1). 1993: pp 978-984 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 979 CYCLOSPORIN A AND ERYTHROPOIETIN PRODUCTION streptomycin (100 pLg/mL), and 10% heat-inactivated fetal calf serum (FCS; Boehringer Mannheim GmbH, Germany). Cells were maintained in a fully humidified, 5%COzatmosphere at 37"C, and the culture medium was changed every other day during the logarithmic growth. Stock solutions of CsA and MeAla-6 (a synthetic analog of CsA, modified at the 6 position) at 1 mg/mL were prepared by dissolvingthe drugs in 10%vol/vol ethanol, 0.2% vol/vol Tween 80 (Boehringer Mannheim) in IMEM. FK-506 was dissolved at 1 mg/mL in 10% vol/vol ethanol in IMEM. From these stock solutions, maintained frozen in aliquots at -7O"C, working dilutions in IMEM were freshly prepared. In preliminary experiments, we observed that the addition of the buffers used for dissolving the drugs to mock cultures had no effects on the levels of Epo, when compared with control cultures. Starting from the day before experiment, nearly confluent cultures were fed with a serum-free medium, consisting of IMEM, 1% Nutridoma-SP (Boehringer Mannheim), L-glutamine (0.29 mg/mL), plus penicillin-streptomycin. Epo production by Hep3B cells was routinely induced by incubating cultures with 50 pmol/L cobalt chloride (CoCl,) for 24 hours; in some experiments, cells were also stimulated with 300 pmol/L nickel chloride (NiCl,) or with a low (1%)oxygen tension atmosphere. At the end of the incubation period, supernatants were harvested, clarified by centrifugation, and stored frozen at -20°C until assayed. Epo levels in supernatants and cell lysates were determined with an enzyme-linked immunoassay kit (Clinigen; Amgen Diagn, Thousand Oaks, CA); each sample was assayed in duplicated using at least two dilutions. Epo levels were normalized to the amount of total cellular protein in cell pellets (Bradford's dye test''), and expressed as mU/mg cell protein. a-fetoprotein (a-FP) levels were measured with a radioimmunoassay kit (Cis Diagnostici, Vercelli, Italy), and expressed as ng/mg cell protein. Characterization of Hep3B cytosolic CsA-binding protein($. To prepare cytosolic proteins from Hep3B cells, about 5 X 10' unstimulated cells were collected by mild trypsinization, washed three times with calcium- and magnesium-free phosphate buffered saline (PBS), and either used immediately or stored frozen as a cell pellet at -70°C. Cytosolic protein extraction was performed by homogenizing cells with a ground-glasshomogenizer in ice-cold 10 mmol/L Tris-HCI, pH 7.2, containing 150 mmol/L KCl, 0. I % sodium azide, and l mmol/L phenylmethylsulphonyl fluoride (TrisKCl-PMSF buffer), at a 1:4 cell/buffer ratio. 2-mercaptoethanol (2-ME) was then added to give a final concentration of 5 mmol/L, and supernatants from crude homogenates spun at lO0,OOOg for 1 hour (Beckman TL- 100 Ultracentrifuge; Beckman Instruments, Palo Alto, CA) were saved. Protein concentration in the supernatant was then determined, and the samples either used immediately for binding assay or stored at -70°C in aliquots. The binding of ['HI-CsA ([mebmt-B-3H]cyclosporin A, specific activity 10.0 Ci/mmol; Amersham Int plc, Buckinghamshire, England) to cytosolicproteins was performed as described." The binding buffer consisted of 20 mmol/L Tris-HCI, pH 7.2, containing 5 mmol/L 2-ME, 7.5% FCS, and 0.1% sodium azide. One hundred pL-volumes of cytosolicproteins, of different protein content, were mixed with 50 pL of ['HI-CsA to give 0.5 nmol/L of labeled ligand, in the presence (nonspecificbinding) or absence (total binding) of a 200-fold molar excess of unlabeled CsA. The mixture was incubated for 30 minutes at room temperature with occasional agitation, then kept in ice. Free-labeled ligand was separated from the one bound to receptofls) by loading the sample on minicolumns (total volume, 3 mL) of LH-20 resin (Pharmacia P-L Biochem Inc, Milwaukee, WI); in these experimental conditions, free ['HI-CsA is significantly retarded in the column, and separated from the protein-containing void volume. The fraction corresponding to the void volume was collected and radioactivity counted in a liquid scintillator. Specific binding was calculated by the difference between total and nonspecific binding. Size-exclusion high performance liquid chromatography (HPLC) was performed with an LKB apparatus (Pharmacia LKB Biotechnology, Uppsala, Sweden) equipped with a TSK-G 3000 SW column. About 1 mL of cytosolic protein extract was concentrated to 0.2 mL with a Centricon 10 microconcentrator (Amicon-Grace, Beverly, MA), 0.45 pm filtered, and injected; the column was developed with 20 mmol/L KH2P0,, pH 7.2, at 1.0 mL/min. Fractions of 0.25 mL were collected, and immediately additioned with 2-ME to give 5 mmol/L final concentration; each fraction was then processed for CsA binding with the LH-20 assay, The identification of CsA-binding protein(s) by ligand blotting was performed by running a concentrated cytosolic protein sample on a 12%sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE); the sample was simply diluted with unreducing SDS-sample buffer, but not boiled. Separatedproteins were electrophoretically transferred to nitrocellulose filters (Bio-Rad, Richmond, CA), and the blot incubated in a blocking buffer, consisting of 25 mmol/L Tris-HC1, pH 7.4, 140 mmol/L NaC1, 4% bovine serum albumin (BSA), and 1% nonfat dried milk. Thereafter, the nitrocellulose sheet was incubated overnight at room temperature with 5 nmol/L ['HI-CsA, in the presence or absence of a 500-fold excess of unlabeled CsA, and then extensively washed. Fluorography-treated gels were exposed at -70°C for 7 to 10 days. Northern blot analysis. The probe for human Epo was a 40-mer oligonucleotide, whose sequence is derived from the exon 2 of the Epo gene (Oncogene Science,Manhasset, NY). A 7-mer oligonucleotide probe for human 0-actin was purchased from Clontech Lab (Palo Alto, CA). Both oligonucleotides were 5'4abeled with [-y3'P] adenosine triphosphate (ATP) (specific activity 6,000 Ci/mmol; NEN-DuPont, GmbH, Dreieich,Germany) and T4-polynucleotide kinase to a specific activity of 3.5 to 5.0 X lo6 cpm/pmol. Total RNA was prepared by cultured cells as described." The RNA was denatured by formaldehyde-formamide,and subjected to 3w t 0.4 0.8 1.2 1.6 DRUG CONCENTRATION (pM) Fig 1 . Dose-dependent reduction of Epo levels in the medium of Hep3B cells incubated with CsA ( 0 )or MeAla-6 (A).The effects of FK-506 (m) are also shown. Cells were fed with serum-free media containing 5 0 rmol/L CoCI,, in the absence or in the presence of the drugs. Epo levels were determined after a 24-hour incubation, and normalized to the amount of cell proteins. Data are derived from five experiments for CsA and MeAla-6, and two experiments for FK-506. *P < .05; **P < .01, Student's t-test. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. VANNUCCHI ET AL 980 Table 1. Effects of CsA on Total Cellular Protein Synthetic Rate Experimental Condition Control CsA (pmol/L) 1.6 0.8 0.1 0.01 [3H]-Aminoacids Incorporated (cpm x 77 f 16 75 f 19 81 2 2 5 87 f 27 75 f 23 Hep3B cells were incubated for 16 hours in media containing 50 pmol/L cobalt chloride, in the presence or absence of CsA, and then pulsed for 2 hours with 15 pCi of a mixture of [3H]-aminoacids.Cellular proteins were precipitated with TCA and counted in a beta counter. Results are expressed as the mean ? SD from two experiments. electrophoresis in I % agarose gels in 2.2 mol/L formaldehyde gel running buffer. The transfer of denatured RNA to Hybond-N filters (Amersham) was performed by standard procedures in 1OX SSC (saline-sodium citrate buffer).22Prehybridization was performed at 42°C for 6 hr with prehybridization buffer (6X SSPE [saline-sodium phosphate-EDTA], 50%formamide, 0. I % SDS, 10% Denhart solution, 100 pg/mL denatured salmon sperm DNA). Hybridization was performed at 42°C for 16 hours with hybridization buffer (6X SSPE, 50% formamide, 0.1% SDS). The final washing was in 1X SSC at 60°C. Autoradiography was performed with two intensifying screens at -70°C with Hypefilm-MP (Amersham) for 1 to 3 days. Intracellular Epo total content and distribution. For the determination of total intracellular Epo content, trypsinized cells were washed twice with PBS, and then dissolved in Tris-KC1-PMSF buffer containing0.2%Triton X-100 (BDH Chem, Poole, England) for 30 minutes on ice. The sample was centrifuged at 1.OOOg for 10 minutes at 4°C to precipitate insoluble material, and the clear supernatant was used for Epo determination. In experiments where the intracellular distribution of Epo was determined, trypsinized cells were washed twice, and finally resuspended in Tris-KCI-PMSF buffer containing 0.25 mol/L sucrose. Cell lysis was obtained by forcing the cells through a 27-gauge needle for six times, on ice; the loss of cellular integrity was confirmed by phase-contrast microscopy. Crude nuclei were prepared at 1,OOOg for 10 minutes, the Table 2. Effects of CsA and MeAla-6 on the Level of a-FP in the Culture Medium of Hep3B Cells Experimental Condition Control CsA (pmol/L) 1.6 0.8 0.1 0.01 MeAla-6 (pmol/L) 1.6 0.8 0.1 0.01 a-FP (ng/mg protein) 445 f 56 425 437 427 455 f39 f 45 f 27 f 33 445 f 35 438 f 18 4 1 6 ? 69 445 f 3 0 Hep3B cells were incubated in media containing5 0 pmol/L cobalt chloride, in the presence or absence of CsA and MeAla-6, and the amount of a-FP was measured 2 4 hours later. Results are the mean f SD from two experiments. :I ,-45.0 ; p 30.0 B 3 , 0Q: 15.0 D;o ,;x I 0 0 10 20 30 40 M CYTOSOLIC PROTEINS, pg Fig 2. Identification of CsA-binding protein(s) in Hep3B cells. The specific binding of r3H]-CsA by increasing amounts of cellular cytosolic proteins, and the displacement after incubation with an excess of unlabeled CsA, are shown in the figure. The line represents specific binding, which was calculated by subtracting from total binding the unspecific binding measured in the presence of a 200-fold molar excess of unlabeled CsA. supernatant collected and spun down at 100,OOOg for 6 0 minutes at 4°C. The final supernatant (soluble) was saved, while the membrane pellet was dissolved on ice in Tris-KC1-PMSF buffer containing 0.2% Triton X- 100 to yield the membrane-associated Epo fraction. RESULTS In nearly confluent, unstimulated Hep3B cells maintained for 16 to 24 hours under serum-free conditions, Epo was barely detected in the medium (4.0 ? 3.5 mU/mg protein). On the other hand, cultures stimulated with CoCI, (50 pmol/L) released high amounts of Epo in the medium, up to 255 t- 40 mU/mg protein (Fig 1). Comparable results were obtained when cells were incubated in an atmosphere containing 1% 0, (305 f 95 mU/mg), while stimulation with 300 pmol/L NiCI, proved to be less efficient ( 1 90 f 75 mU/ mg). Therefore, owing to its simplicity and reproducibility, we have routinely used CoCI, for inducing Epo production by Hep3B cells in all subsequent experiments. However, in preliminary experiments not reported in detail, similar effects of CsA on Epo production were observed when Hep3B cells were stimulated with either hypoxia or NiCI,. Cultures incubated with CsA (in the range of 0.0 1 to I .6 pmol/L) showed a dose-dependent reduction in the amount of Epo released into the culture medium after CoC1, stimulation (Fig 1). At CsA concentrations of 0.4 pmol/L, about half of the controls’ Epo was found in the medium, and decreased further up to about 30% of controls (78 k 15 mU/mg) at 1.6 pmol/L. The inhibitory effect was not caused by a reduction in the total rate of protein synthesis by Hep3B cells; in fact, the amount of tritiated amino acids incorporated in trichloroacetic acid (TCA) precipitates was virtually comparable in control and CsA-treated cultures (Table 1). Moreover, concentrations of CsA up to 2.0 pmol/ L were found not to be toxic to the cells, because no significant modification of total cell number per plate, nor of the From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CYCLOSPORIN A AND ERYTHROPOIETIN PRODUCTION 981 Fig 3. Size-exclusion HPLC characterization of CsA receptor(s) in cytosolic extracts from Hep3B cells. The fractions containing functional CsA receptor(s) were identified by the LH-20 assay. Insert, ligand blotting identification of CsA-binding protein. Unseparated cytosolic proteins were resolved on a 12% SDS-PAGE, transferred to nitrocellulose filters, and incubated with [3H]-CsA in the presence (lane A) or absence (lane B) of an excess of unlabeled CsA. percentage of Trypan-blue’ cells, was observed (data not shown in detail). Finally, to evaluate the specificity of the inhibitory effects of CsA on Epo, the amount of a-FP, a constitutively secreted protein by Hep3B cells, was also measured in culture media. As shown in Table 2, the levels of this protein were not significantly modified by CsA. We next tested whether the nonimmunosuppressive, stable CsA analog MeAla-6 had similar effects as the parental molecule. As reported in Fig 1, dosedependently reduced Epo levels were found in cultures incubated with MeAla-6 (0.01 to 1.6 pmol/L), although on a molar basis the analog proved to be less efficient than CsA, especially at the lower concentrations. No inhibitory effect on a-FPlevels was observed with MeAla-6 (Table 2). On the other hand, the CsA-unrelated,immunosuppressiveagent FK506 failed to reduce Epo levels in the medium, in comparison with control cultures (Fig 1). Because it is known that the effects of CsA on target cells are mediated by specific cytoplasmic receptors, we searched for the presence of (a) [email protected])in Hep3B cells. As shown in Fig 2, cytosolic proteins prepared from Hep3B cultures were found to bind [’HI-CsA in a dose-related fashion, and the bound labeled molecule was efficiently displaced by the cold substrate. By hypothesizinga 1:1 stoichio- metric interaction between [3H]-CsA and the receptor($, the calculated concentration of the CsA receptor(s)was 0.12 f 0.04 pglmg cytosolic proteins. To further characterizethe CsA receptor(s),a molecular size-exclusion HPLC analysis of cytosolic extracts was performed, which allowed for the identification of a major peak of [3H]-CsAbinding protein correspondingto a calculated M, of about 18.0 Kd (Fig 3). The CsA-receptorprotein was also identified by ligand-blotting experiments,in which gels were deliberatelyoverloaded to allow evidentiation of even less represented CsA-binding species. However, only a single band was observed in these experimental conditions, with a M, of 17.5 Kd (Fig 3, insert). To verify the hypothesis that the reduced levels of Epo found in the medium of cultures incubated with CsA were caused by an effect on the expression of the Epo gene, total RNA was prepared from CoCI,-stimulated Hep3B cultures that had been incubated for 16 hours in the presence or absence of CsA. However, in four separateexperiments,one of which is reported in Fig 4, we were unable to document any apparent reduction in the amount of Epo mRNA in cultures incubated with up to 1.6 pmol/L CsA. Then, we reasoned that alterations of posttranslational processes might be involved in causing the reduced Epo levels found From www.bloodjournal.org by guest on December 22, 2014. For personal use only. VANNUCCHI ET AL 982 18s- + 275 Fig 4. Effects of CsA on the expression of Epo mRNA in Hep3B cells stimulated with CoCI,. Total RNA (20 pg) was resolved on a 1%fonnaldehydeagarose gel, and hybridized with a [3ZP]-labeledoligonucleotide probe for human Epo. Filters were then stripped, and incubated with a probe for human ,!?-actin,to provide a control of the amount of RNA loaded in the gel. Epo levels in the medium, normalizedto the amount of proteins in cell pellets, are also shown. 93 Actin: in the medium ofcultures incubated with CsA. To this end, the amount of Epo remaining within the cells at different times after stimulation with CoCI, was measured. While no effect was noticed within the first 4 hours of incubation, a progressive increase in the intracellular Epo content was observed in the presence of CsA (1.2 pmol/L) at 8 to 24 hours after CoCI, stimulation (Fig 5): at 24 hours, cells incubated with CsA contained about IO-fold the amount ofcontrols’ Epo. The bulk (97% oftotal) ofthe intracellular Epo in W 5 a - 1 cl w/o CsA 0 with CsA] 60 cells incubated with CsA was found in the fraction precipitated at lO0,OOOg and dissolved with Triton X- 100, suggesting that it was membrane-associated (Table 3). DISCUSSION Results presented in this paper indicate that in Hep3B cultures incubated with CsA at nanomolar concentrations, the amount of Epo released in the medium is significantly reduced when compared with control cultures. The following considerations support the idea that the inhibitory effect of CsA was specific: (1 ) at the doses used, CsA did not cause a general reduction in total cellular protein synthetic rate, nor did it affect the secretion of a-FP, a constitutively secreted protein by Hep3B cells; (2) the nonimmunosuppressive CsA analog MeAla-6 proved to be inhibitory, although .** Table 3. Intracellular Distribution of Epo in Hep3B Cells dl 24 hr INCUBATION TIME Fig 5. Total intracellular Epo content as a function of incubation time. Hep3B cells were stimulated with CoCI,, in the presence or absence of CsA (1.2 pmol/L), and the amount of Epo in detergentlysed cells was measured at different times. Epo levels were normalized to the amount of proteins in cell lysates. Data from two experiments. **P < .01, Student’s t-test. €PO (mU/mg protein) Subcellular Fraction Control CsA Crude nuclei Membrane-associated Soluble NA 5.5 2 3.0 0.2 f 0.2 NA 57.4? 10.0 1.7 f 0.5 Hep3E cells were incubatedin media containing 50 pmol/L cobalt chloride, in the presence or absence of CsA (1.2 pmol/L), and cells were collected 16 hours later. The subcellular fractionation was performed as described in Materials and Methods. Results are expressed as the mean 2 SD from two experiments. Abbreviation: NA, not measureable. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CYCLOSPORIN A AND ERYTHROPOIETIN PRODUCTION about 40% less efficiently on a molar basis than the parental drug. This correlates well with the previous demonstration that MeAla-6 binds to the CsA receptor, the protein cyclophilin, with an efficiency of about 50% in comparison with CSA;’~and (3)the CsA-unrelated immunosuppressive agent FK506 had no effect on the release of Epo by Hep3B cells. We have also found that Hep3B cells contain a protein capable of binding t3H]-CsA, with a molecular weight comparable with the main cyclophilin species of T-lymphocytes ( 18.0 Kd).24Finally, evidence has been provided that the reduced levels of Epo found in the culture medium of Hep3B cells are not caused by an effect of CsA on the expression of the Epo mRNA, at least at the level of sensitivity of Northern analysis; rather, they appeared to be dependent on (an) alteration(s) in the mechanisms leading to hormone secretion in the culture medium. In fact, the amount of Epo remaining inside the cells was significantly higher in cultures incubated with CsA than in controls, and subcellular fractionation studies indicated that the most part of the unsecreted, intracellular Epo was associated with the plasmamembrane fraction. The immunosuppressive activity of CsA results from the inhibition of some receptor-mediated signal transduction pathways in T lymphocyte^.^^^^^ CsA becomes active after the binding to a specific cytoplasmicreceptor, named cyclophilin, which is part of a family of ubiquitous immunosuppressant receptor proteins, the imm~nophilins.~’ These are all rotamases (peptidyl-prolylcis-trans isomerases; PPIase), enzymes that catalize the cis-trans isomerization of peptidyl-prolyl amide bonds of peptide and protein substrates, and which may act by accelerating the correct folding of proteins during protein synthesis. Although the rotamase activity is strongly inhibited by C S A , ~ *there ~ * ~ is evidence that the immunosuppressive activity of CsA derives from the interaction of the active drug-receptor complex with the protein phosphatase c a l c i n e ~ r i nmore , ~ ~ than from the inhibition of PPIase activity. It has also been suggested that the physiologic role of the ubiquitous PPIases in the cells may be that of interacting with proline-containing proteins, perhaps stabilizing their structure and/or allowing their translocation within different cellular compartments.” The results of our experiments seem to suggest that the formation of an active complex between CsA and the cyclophilin-like protein found in Hep3B cells may elicit some alterations in the posttranslational processing of Epo, with the consequent accumulation of the molecule within the endocellular membranes and a net reduction of the amount of hormone secreted. Theoretically, this might be caused by (1) a defect in the correct folding of the Epo molecule, because of the inhibition of PPIase activity, and/or ( 2 )a defect in the intracellular translocation processes of the Epo molecules required for the secretion in the medium. About the first mechanism, it may be worthy to recall that Lodish and Kong’’ observed that CsA inhibited the secretion of transferrin from another human hepatoma cell line (HepG2) at concentrations not affecting the release of other proteins, and suggested this effect might be caused by a lag in the folding of transferrin molecules and to a retarded maturation from the endoplasmic reticulum (ER). However, ex- 983 periments performed up to now failed to document significant structural alterations of the Epo molecule produced by Hep3B cells incubated with CsA (unpublished data, September 1992), possibly indicating that the first mechanism is less likely. Therefore, although the unequivocal intracellular localization of Epo in CsA-treated cultures will require the use of specific antibodies and further subcellular fractionation studies, at the moment we favor the second hypothesis, which has analogies with studies of a cyclophilinlike protein encoded by the n i n d gene of Drosophila m e l a n ~ g a s t e rThe . ~ ~ n i n d gene product, highly homologous to the B isoform of mammalian cyclophilin, is found only in the retina of Drosophila, and it has been shown to be essential for the posttranslational processing of rhodopsin Rhl molecules, which require n i n d product for the exit from the ER and their traveling through the cytoplasm to the rhabdomeres. In fact, n i n d mutant flies showed a dramatic accumulation of Rh 1 opsin molecules in the cisternae of ER.33A similar mechanism of action has also been proposed by Hultsch et who observed that both CsA and FK-506 can inhibit receptor-mediated exocytosis of secretory granules from a rat basophilic leukemia cell line. They suggested that the two drugs might act by inhibiting the intracellular translocation of the secretory granules, in the same way as the translocation of nuclear factors required for the activation of the early genes involved in the immune response is affected in T lymphocytes. Our proposed model is that the immunophilin found in Hep3B cells is physiologically involved in some steps of the intracellular processing and/or secretion of the Epo molecules, and that these processes are hindered in the presence of CsA. In this regard, it has been shown that a cyclophilin identified from a murine cDNA library (cyp C) binds specifically to a 77-Kd protein in the absence of the drug, while in the presence of CsA it binds to a 55-Kd protein.35The authors suggested that the formation of the cyp C-p77 complex may reflect the physiologic function of this immunophilin in the cells, while the cyp C-p55 complex is involved in signal transduction events elicited by CsA binding. Furthermore, an immunophilin specific for FK-506 has been recently shown to associate, in the absence of the drug, with two heat-shock proteins and with the glucocorticoid receptor to form the inactive glucocorticoid receptor complex,36 thus suggesting a physiologic role for the F’K506-binding protein different from that involved in immunosuppression. In conclusion, we have observed that CsA is able to significantly affect the release of Epo in vitro by the Hep3B hepatoma cell line, thus supporting previous suggestions that the reduced Epo levels characteristic of allogeneic BMT patients might be ascribed, at least in part, to the drug. Although it might be questioned whether Hep3B cells are a physiologic model of Epo production, the previous demonstration that CsA impairs Epo production in vivo’3seems to indicate that results obtained with the hepatoma cell model are possibly applicable also to kidney cells, which represent the major source of the hormone in the intact animal. Finally, our data add some further insights into the roles of immunophilins in the regulation of protein metabolism in From www.bloodjournal.org by guest on December 22, 2014. For personal use only. VANNUCCHI ET AL 984 cells other than T lymphocytes, and suggest the existence of a complex network of target molecules for CsA, whose knowledge might also help in understanding the molecular mechanisms of the toxicity associated with the drug. ACKNOWLEDGMENT Authors are indebted to Prof F. Paoletti for helpful suggestions. Dr G. Corbetta (Sandoz, Milano, Italy) kindly provided CsA and MeAla-6; FK-506 was a gift of Dr M.P. Piccinni (University of Florence). REFERENCES 1. Krantz SB: Erythropoietin. Blood 77:419, 199 I 2. Erslev AJ: Erythropoietin titers in health and disease. Semin Hematol 28:2, 1991 3. Hochberg MC, Arnold CM, Hogans BB, Spivak JL: Serum immunoreactive erythropoietin in rheumatoid arthritis: Impaired response to anemia. Arthritis Rheum 31:1318, 1988 4. Miller CB, Jones RJ, Piantadosi S, Abeloff MD, Spivak JL: Decreased erythropoietin response in patients with the anemia of cancer. New Engl J Med 322:1689, 1990 5. 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Cell 67:255, 1991 34. Hultsch T, Albers MW, Schreiber SL, Hohman RJ: Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proc Natl Acad Sci USA 88:6229, 1991 35. Friedman J, Weissman I: Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: One in the presence and one in the absence of CsA. Cell 66:799, 1991 36. Tai P-KK, Albers MW, Chang H, Faber LE, Schreiber SL: Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex. Science 256: 1315, 1992 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 1993 82: 978-984 Effects of cyclosporin A on erythropoietin production by the human Hep3B hepatoma cell line AM Vannucchi, A Grossi, A Bosi, D Rafanelli, M Statello, S Guidi, R Saccardi and P Rossi-Ferrini Updated information and services can be found at: http://www.bloodjournal.org/content/82/3/978.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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