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Conjoint Endocrine Laboratory (B.M., H.L., K.R., R.M.), Clinical Research Centre, Royal Brisbane and Women’s Hospital and Queensland Health Pathology Services, Herston, Queensland 4029, Australia; and the Department of Obstetrics and Gynaecology (R.M.), The University of Queensland, St. Lucia, Queensland 4072, Australia
Address all correspondence and requests for reprints to: Dr. Robin Mortimer, Department of Endocrinology, Royal Brisbane and Women’s Hospital, Base Hospitals PO, Herston, Queensland 4029, Australia. E-mail: robin_mortimer{at}health.qld.gov.au.
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Abstract |
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Objective: The objective of this study was to investigate trophoblast thyroid hormone binding proteins that may modulate interactions between D3 and T4.
Design: Placentas were obtained by informed consent from women delivering normal infants by repeat cesarean section at 38–40 wk gestation. T4 and T3 binding was examined in human placenta. Serum thyroid hormone binding proteins were identified by Western blotting, and their mRNA was examined by RT-PCR. Presence of these proteins in trophoblast was determined by immunocytochemistry and immunofluorescence. Cytosol was progressively purified to reveal additional thyroid hormone binding proteins that were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Effects of mefenamic acid on placental deiodination were examined by HPLC.
Results: We detected high-affinity T4 and T3 binding in human placental cytosol. All three major serum-binding proteins, T4 binding globulin (TBG), transthyretin (TTR), and albumin, were present in cytosol. TTR mRNA and albumin mRNA were detected in human placenta, and TTR and albumin were identified histochemically in syncytiotrophoblasts. Neither TBG mRNA nor TBG was detected, suggesting that plasma TBG had contaminated the cytosol preparation. Low-affinity thyroid hormone binding proteins 
-1-antitrypsin and -1-acid glycoprotein were also identified. Addition of mefenamic acid, a potent inhibitor of thyroid hormone binding, to placental cytosol significantly enhanced deiodination of T4 by D3.
Conclusions: Placenta produces a series of thyroid hormone binding proteins that may modify thyroid hormone deiodination and materno-fetal thyroid hormone transport.
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Introduction |
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Along with the thyroid hormone binding proteins produced by liver and secreted into blood, several cell types produce nonsecreted cytosolic thyroid hormone binding proteins (7, 8, 9). Although production of thyroid hormone binding proteins has not been described in trophoblast, we postulated that such a protein or proteins could modulate T4 delivery to the deiodinase and regulate materno-fetal thyroid hormone transfer. The purpose of this study was therefore to examine human placental cytosol for the presence of specific high-affinity thyroid hormone binding proteins and, if present, identify any synthesized by trophoblast. We found three thyroid hormone binding proteins in cytosol: transthyretin (TTR), albumin (ALB), and T4 binding globulin (TBG). TBG appears to be a contaminant from placental blood, but TTR, a high-affinity thyroid hormone binding protein, is produced by trophoblast and can be localized to the subapical region of the cell. ALB also appears to be synthesized by the cell and can be found on the apical membrane of trophoblasts. Examination of cytosol failed to detect any novel high-affinity intracellular binding proteins.
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Materials and Methods |
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Thyroid hormones (T4, T3, and rT3), mefenamic acid, antisheep secondary antibody, mouse antihuman ALB antibody, antimouse peroxidase-conjugated antibody, and all standard chemicals were obtained from Sigma (New South Wales, Australia). Antimouse fluorescein-conjugated antibody, rabbit antihuman TTR antibody, labeled streptavidin-biotinylated antibody immunohistochemistry kit, serum-free blocking solution, and 4',6-diamidino-2-phenylindole (DAPI) staining mounting media were supplied by Dako (Glostrup Denmark) and TBG antibody by Abcam (Cambridge, UK). The 125I-T3, 125I-T4, column chromatography media (Mono Q Sepharose, Phenyl Sepharose 6 fast flow, and Sephacryl S-200 HP), and the enhanced chemiluminescence Western blotting analysis system were obtained from Amersham Biosciences (Sydney, Australia). TRIzol and superscript III reverse transcriptase was supplied by Invitrogen (Grand Island, NY). The Affi-Gel Blue, Bio-Rad protein assay and Bio-Rad precision plus electrophoresis molecular markers, and Gel filtration molecular weight markers were supplied by Bio-Rad (New South Wales, Australia).
Thyroid hormone binding in placental cytosol
Relevant institutional research ethics committees approved the study, and patient consent was obtained before collection of placentas after uncomplicated cesarean section at 38–40 wk gestation. Villus tissue was separated from amnion, and chorion and cytosol were prepared from approximately 200 g of tissue by standard methods. Sample was stored at –70 C before additional analysis.
Cytosolic T4 and T3 binding affinity was estimated by incubating 1 pmol of radiolabeled T4 in incubation buffer (0.05 M Tris HCl, 2.4 mM CaCl2, 0.78 mM MgCl2, 4.1 mM KCl, and 104 mM NaCl) with cytosol (protein concentration 10 ng/ml) overnight at 4 C. Bound and free thyroid hormones were separated by addition of 15 µl of 2% dextran-coated charcoal and centrifuged for 30 sec. The supernatant was kept and counted in a Packard Cobra II series 
counter (Hewlett Packard, Palo Alto, CA) to determine total bound. Nonspecific binding was determined by addition of an excess of unlabeled T4 (1 µM). The dissociation constant for T4 and inhibition constant for T3 binding were determined by adding increasing amounts of unlabeled T4 or T3, respectively, to the incubation buffer. Each experiment was performed in triplicate and repeated three times. A negative control experiment was performed with no protein in the sample. Results were fitted to a one-site competitive binding curve (Prism; GraphPad Software, San Diego, CA). Fractions of cytosol obtained after each purification step were assayed for T3 binding as described previously (10).
Western blotting
To separate proteins in cytosol, 30 µg protein was denatured by heating to 95 C for 5 min in Laemmli buffer. Samples were run on a 4–15% SDS-PAGE using reducing conditions in a buffer containing [25 mM Tris, 192 mM glycine, 0.1% SDS, and dithiothreitol (DTT)] as described previously (11)
Proteins were transferred onto a nitrocellulose membrane at 350 mV for 1 h in Tris/glycine buffer (25 mM Tris and 192 mM glycine). Membranes were blocked for 1 h with blocking buffer [5% skim milk, 0.1% (v/v) Tween 20, and 0.01 M PBS]. After brief washing with washing buffer [0.01 M PBS and 0.1% (vol/vol) Tween 20], membranes were incubated with primary antibody (1:1500 for TTR; 1:1500 for TBG, and 1:5000 for ALB) in blocking buffer overnight at 4 C. Membranes were again washed briefly and incubated with secondary antibody conjugated with horseradish peroxidase at 1:1500 in blocking buffer for 1 h at room temperature. Detection was performed with an enhanced chemiluminescence Western blotting analysis system, and chemiluminescence was captured by charge-coupled device camera (Fuji image LAS-3000; Fujifilm, Tokyo, Japan).
RT-PCR
Total RNA was extracted from fresh placenta by homogenization in TRIzol reagent (10 ml/g tissue) according to the instructions of the manufacturer. Chloroform (0.2 ml/ml TRIzol) was added and incubated for 3 min. The sample was centrifuged at 12,000 x g for 15 min at 4 C, and supernatant, containing total RNA, was precipitated with isopropanol.
Five hundred nanograms of total RNA from fresh placenta and a human hepatocellular carcinoma cell line (Hep G2) were reverse transcribed with the Superscript III Reverse Transcription kit using oligo-dT primers in a reaction volume of 20 µl. Four microliters of the cDNA product was used in PCR with 2.5 mM MgCl2, 0.2 mM dNTP, 0.15 µM specific primers, and 0.5 U Platinum Taq DNA Polymerase in a total volume of 20 µl. The primers used to amplify specific mRNA were as follows: ALB, 5'-CCTGCTGACTTGCCTTCAT-3' (5' 19-mer) and 5'- CGAGCTCAACAAGTGCAGT-3' (3' 19-mer), which generated a specific 703 bp ALB product; TBG, 5'- CCACTGTGCATCACCTGAA-3' (5' 19-mer) and 5'-GCAGCTTCCACTGACTCCA-3' (3' 19-mer), which generated a specific 755 bp TBG product; and TTR, 5'-GTCCACTCATTCTTGGC-3' (5' 17-mer) and 5'-CATTCCTTGGGATTGGTG-3' (3' 18-mer), which generated a specific 469 bp TTR product. The PCR conditions were as follows: after an initial denaturing step of 5 min at 95 C, 35 cycles of 30 sec at 95 C, 30 sec at 60 C, and 30 sec extension step at 72 C. The final extension step was allowed to proceed for 10 min. The resultant product was run on a 1.5% agarose/Tris-borate EDTA gel, stained with ethidium bromide, and viewed under UV light against a DNA size marker. Nucleotide sequencing of the products was performed on an Applied Biosystems (Foster City, CA) Prism dye primer sequencing kit and Applied Biosystems automatic sequencer.
Immunohistochemistry
Human placental tissue sections were provided by Queensland Health Pathology Services, Royal Brisbane and Women’s Hospital. All immunostaining procedures were performed at room temperature unless otherwise stated. Paraffin sections were dewaxed, rehydrated, and incubated with 3% (vol/vol) H2O2 in water for 15 min to eliminate endogenous peroxidase activity. Sections were washed in PBS twice for 3 min each and incubated with serum-free blocking solution for 30 min. Sections were then incubated with primary antibodies for TTR (1:4000 dilution) for approximately 18 h at 4 C. After washing, sections were incubated with biotinylated swine antirabbit IgG, streptavidin-peroxidase conjugated for 15 min each, and treated with diaminobenzidine for 3 min. Sections were washed twice with 0.1% Triton X-100-PBS and once in PBS after each step. The sections were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
The negative control for TTR antibodies comprised absorption of the primary antibody with antigen overnight before being applied to control sections. The negative control also comprised replacement of the primary antibody with nonimmune rabbit serum or omission of the primary antibody. The specificity of the TTR antibodies was confirmed by Western blotting in this study.
Immunofluorescence
For immunofluorescence staining, the placental sections were incubated with serum-free blocking solution for 30 min after dewaxing and rehydration. The TTR antibody (1:2000 dilution) and ALB antibody (1:1000 dilution) were applied to the sections overnight at 4 C. Sheep antirabbit IgG-conjugated with Texas Red (1:400) for TTR and goat antimouse fluorescein-conjugated IgG (1:200) were applied to sections for 20 min. The sections were washed in 0.1% Triton X-100 in PBS twice and PBS once after incubated with each antibody. Sections were mounted with DAPI staining mounting media. The staining was examined by Leica (Nussloch, Germany) TCS SP2 confocal laser scanning microscopy or Leica IM 1000 fluorescence microscopy.
Purification of thyroid hormone binding proteins
The first step in the purification process was removal of the high levels of ALB from the sample. Disposable plastic columns were filled with 20 ml Affi-Gel blue matrix and equilibrated with buffer A [10 mM Tris-HCl, 2 mM EDTA, and 0.5 mM DTT (pH 8.0)]. Sample was applied to column in 4 ml aliquots and eluted with 40 ml buffer A. Flow-through material was kept for subsequent purification steps.
A Mono Q Sepharose high-performance anion exchange column (3 x 5 cm) was preequilibrated with 60 ml buffer B [1.0 M NaCl, 10 mM Tris-HCl, 2 mM EDTA, and 0.5 mM DTT (pH 8.0)]. The sample was applied to the anion exchange column at a flow rate of 0.7 ml/min and washed with buffer A overnight at the same flow rate. Proteins were eluted with a continuous gradient of NaCl from 0.01–0.3 M, and 2 ml fractions were collected. Protein content of each fraction was determined with the Bio-Rad Protein Assay kit (Bradford-Lowry) using the instructions of the manufacturer. Fractions containing protein were tested for specific 125I-T3 binding with binding assay described above. Fractions with binding activity were pooled.
A phenyl Sepharose 6 fast-flow (low sub) column (30 x 1 cm) was equilibrated with 200 ml buffer C [1.5 M ammonium sulfate, 10 mM Tris HCl, 2 mM EDTA, and 0.5 mM DTT (pH 8.0)]. The pooled fraction with 125I-T3 binding activity from the ion exchange column was diluted 100 times with buffer C and concentrated with a Millipore (Bedford, MA) stirring cell according to the instructions of the manufacturer to equilibrate the sample in start buffer. The sample was loaded onto column at 0.5 ml/min, protein was eluted at the same flow rate with a continuous gradient of ammonium sulfate at 1.5–0 M. Fractions (2 ml) were collected, and protein content and 125I-T3 binding activity were assessed as above.
A Sephacryl S-200 HP gel filtration column (3 x 30 cm) was preequilibrated with buffer D [0.15 M NaCl, 10 mM Tris-HCl, 2 mM EDTA, and 0.5 mM DTT (pH 8.0)]. Samples with binding activity were pooled from the phenyl Sepharose column and concentrated with a Millipore stirring cell. The concentrated sample (5 ml) was loaded onto the column at a flow rate of 0.3 ml/min. Proteins were eluted with buffer A, and 2 ml fractions were collected. Molecular weight of binding protein was estimated by comparison with the elution pattern of five compounds of known molecular weight: thyroglobulin, globulin, ovalalbumin, myoglobin, and vitamin B12 (Bio-Rad protein standards), applied to the column under identical conditions.
The sample obtained after purification was run on an SDS-PAGE gel as described above. Bands visible after Coomassie blue R250 staining were excised from SDS-PAGE and sent to the Australian Proteome Analysis Facility (Sydney, New South Wales, Australia), where identification was performed by in-gel trypsin digest, followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).
Deiodination study
Homogenates were prepared from human placental tissue collected as described previously. All steps were performed at 4 C unless otherwise specified. Tissue was washed extensively in EDTA buffer [0.1 M KH2PO4 and 0.01 M EDTA (pH 7.4)], cut into 2-cm cubes, and homogenized in EDTA/DTT buffer (EDTA buffer supplemented with 100 mM DTT, 3 ml buffer/1 g tissue). Tissue homogenate was centrifuged at 700 x g for 10 min, and supernatant was kept.
The effect of mefenamic acid on placental metabolism of T4 was examined. Placental homogenate (equivalent to 1 mg protein) was incubated with 125I-T4 (0.2 µCi) in the presence and absence of 100 µM mefenamic acid in a total volume of 400 µl for 120 min at 37 C. At the end of the incubation, 800 µl of 95% ethanol (vol/vol) was added to each tube to precipitate protein. Samples were centrifuged at 3000 x g for 10 min, and supernatants were analyzed by HPLC (Brownlee RP18 column, mobile phase: 50% methanol and 50% phosphate buffer [0.05% KH2PO4 and 0.05% hyaluronan synthase: 6.65 g KH2PO4 plus 1.01 g heptane sulfonic acid per liter (pH 3), adjusted with orthophosphoric acid), flow rate, 0.9 ml/min with UV at 254 nm at 37 C to examine the degree of metabolism of T4]. Peak identity was confirmed by parallel analysis of pure standard solutions of T4, T3, and rT3.
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Results |
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We demonstrated specific high-affinity T4 and T3 binding in placental cytosol (Fig. 1
) with association constant for T4 of 9.5 x 1010 M (95% confidence intervals 7.6 x 1010 M to 1.2 x 1011 M). Unlabeled T3 displaced 125I-T4 with an inhibition constant of 3.7 x 109 M (95% confidence intervals, 3.1 x 109 to 4.5 x 109).
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Western blots performed on placental cytosol revealed bands of appropriate size for all three serum thyroid hormone binding proteins (Fig. 2). TBG both in serum and cytosol appeared as two bands at approximately the 50–55 kDa mark that were more prominent in serum. The additional band probably represents differently glycosylated forms of the protein. (Fig. 2, lanes 2 and 3). TTR appeared as equally strong bands in serum and cytosol at approximately 15 and 60 kDa, as well as a less intense band at 30 kDa. The three bands represent the TTR monomer, dimer, and tetramer. The majority of TTR runs as a monomer under reducing conditions, but some will remain in the dimeric and tetrameric forms (Fig. 2, lanes 4 and 5). Strong bands for ALB were also identified in serum and cytosol at approximately the 60 kDa mark, consistent with its size (Fig. 2, lanes 6 and 7).
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The primers designed to specifically amplify TBG yielded a product of 438 bp from Hep G2 cells but not placenta. The TTR and ALB primers also amplified product in Hep G2 cells. These latter products (703 and 469 bp) were, however, also amplified from placenta (Fig. 3). Nucleotide sequencing of the amplified products positively established the identity of the bands as TTR and ALB message.
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Placental sections convincingly demonstrated TTR immunoreactivity as a dark brown subapical stain within villous trophoblasts (Fig. 4A). No staining was visible in capillary endothelial cells, and no staining could be observed in the negative control (Fig. 4B). Magnification (Fig. 4C) and additional immunofluorescence studies (Fig. 4D) confirmed the subapical localization of the protein in these cells. ALB immunofluorescence was observed as a light haze at the apical surface of villous trophoblasts (Fig. 4E). Confocal microscopy confirmed the immunoreactivity at the trophoblast apical membrane (Fig. 4F).
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Approximately 240 ml cytosol containing 500 mg protein was isolated from 200 g of placental tissue as described above. SDS-PAGE analysis revealed thick bands at approximately 60 kDa, which we assumed to be ALB and which can interfere with the thyroid hormone binding assay. The majority of the ALB was removed by passage through an Affi-Gel Blue column. A number of distinct protein peaks were visible in the elution profile of the Mono Q Sepharose ion exchange column. Elution of the 125I-T3 binding activity began at approximately 244 ml, at approximately 0.2 M NaCl (Fig. 5A). Significant 125I-T3 binding activity eluted from the phenyl Sepharose 6 fast-flow column with a concentration of approximately 0.7 M ammonium sulfate (Fig. 5B). Elution of the 125I-T3 binding activity from the Sephacryl 200 HP gel filtration column occurred at a volume of 140 ml (Fig. 5C). This corresponded to a molecular weight of approximately 56 kDa compared with Bio-Rad molecular weight standards.
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-1-B glycoprotein, -1-antitrypsin, and -1-acid glycoprotein. Studies of T4 metabolism
Chromatography of pure standards indicated that the first peak obtained from the HPLC column was free iodine, the second major peak was rT3, and the third was T4. HPLC analysis of placental homogenates incubated with 125I-T4 in the absence of drug showed that T4 was partially metabolized to rT3 and iodine (Fig. 6A). When 100 µM mefenamic acid was added, thyroid hormone was predominantly in the form of rT3 with barely detectable levels of T4 (Fig. 6B).
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Discussion |
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TTR was found to be abundant in the trophoblast by immunohistochemistry and by immunofluorescence. These techniques show TTR to be primarily located in the apical compartment of the cell. ALB was also demonstrated as a thin rim on the apical trophoblast membrane. Similar apical localization of ALB has been reported previously (10, 11), but it is unclear whether it is of maternal plasma or of trophoblast origin.
Because there might have been additional cytosolic high-affinity thyroid hormone binding proteins, we examined thyroid hormone binding by cytosol further. We used T3 binding rather than T4 binding assays because T3 is less hydrophobic and is technically easier to work with. We removed much of the ALB (exogenous or endogenous) with Affi-Gel Blue and used a series of chromatographic techniques to separate proteins with T3 binding capacity. The remaining sample, with high-affinity T3 binding preserved, eventually yielded a restricted number of protein bands visualized by electrophoresis. Western blotting of the partially purified sample showed that both TTR and TBG were still present. To examine whether any other proteins could be responsible for this high-affinity binding, we determined the identity of remaining proteins in the cytosol by MALDI-TOF MS sequencing. We identified three proteins, -1-antitrypsin, -1-acid glycoprotein, and -1-B glycoprotein. Both -1-antitrypsin and -1-acid glycoprotein are secreted proteins, are produced in the human (13) and rat (14) placenta, respectively, and have been shown previously to bind thyroid hormone at low affinity (15). Little is known of the biological function of the final protein identified, -1-B glycoprotein. It is a member of the Ig superfamily and is present in high levels in serum (16). Although Ig are minor thyroid hormone binders, neither placental production of nor thyroid hormone binding by -1-B glycoprotein has been reported, and this protein may well be a serum contaminant.
Placenta thus synthesizes at least four proteins with thyroid binding properties, namely TTR, ALB, -1-antitrypsin, and -1-acid glycoprotein. There are many potential roles for thyroid hormone binding proteins in modulating materno-fetal thyroid hormone transport, including effects on thyroid hormone uptake and efflux and modifying deiodination. TTR is a homotetrameric protein that carries retinol binding protein as well as thyroid hormones. TTR is produced predominantly in liver and secreted into plasma. TTR has also been identified in other tissues, including choroid plexus and the retinal pigment epithelium (17), both of which secrete the protein (18). TTR is produced in relatively large amounts in choroid plexus in which it plays an important role in the transport of T4 from blood into cerebrospinal fluid (19). Choroid plexus TTR appears to have evolved 200 million years earlier than hepatic TTR (20). Type 2 deiodinase has been reported in chicken choroid plexus in which it generates T3 from T4. TTR then, it is postulated, carries T3 into cerebrospinal fluid (21). TTR has also been found in low levels in heart, stomach, skeletal muscle, and spleen of the rat (22). TTR has not been reported previously in trophoblast but has been found in rat embryonic yolk sac (23), the precursor of liver.
TTR, predominantly a secreted protein, is taken up by some cells. In renal tubules, free, T4 bound, and retinol binding protein bound TTR is taken up by a megalin-mediated process (24). Megalin has been reported in rat placenta (25).
There are therefore several possible roles for TTR in the materno-fetal thyroid hormone transport process. Secreted trophoblastic TTR could bind maternal T4 and then be taken up by a megalin-mediated process into trophoblast, perhaps then providing T4 to the fetal circulation. Secreted TTR could also facilitate thyroid hormone efflux (26), which in choriocarcinoma cells appears to occur by passive diffusion (27).
The nonsteroidal antiinflammatory drugs competitively inhibit binding of T4 to a number of thyroid hormone binding proteins, both serum and cytosolic (28). The nonsteroidal antiinflammatory drug mefenamic acid is a potent inhibitor of T4 binding to TTR (29). We showed that incubation of placental homogenates with mefenamic acid increases deiodination of T4. This suggests that T4 displaced from TTR, ALB, and possibly other thyroid hormone binding proteins would be more available for deiodination. D3 in other tissues is a membrane protein (30). Although this has not been confirmed in trophoblast, competition at the cell membrane between secreted TTR and deiodinase for T4 could modulate T4 deiodination. At this stage, however, the roles of TTR, ALB, and other thyroid hormone binding proteins in placental thyroid hormone transfer are uncertain, and the limited materno-fetal transfer of T4 remains unexplained.
ALB, a low-affinity but high-capacity thyroid hormone binding protein, also appears to be synthesized by trophoblast and adheres to the trophoblast glycocalyx. ALB is internalized by megalin in opossum kidney (31), and this might provide a thyroid hormone uptake pathway, although cell binding of ALB to trophoblast appears weak (32). ALB does enhance thyroid hormone efflux from fibroblasts and Hep G2 cells (26).
We also confirmed the presence of two other minor carrier proteins for thyroid hormone, -1-antitrypsin and -1 acid glycoprotein in placental cytosol. These are low-affinity thyroid hormone binding proteins (15) but could play qualitatively similar roles to TTR and ALB.
Last, plasma TTR binds retinol-binding protein and is an important vitamin A carrier (33). Trophoblast contains cellular retinol-binding protein (34), and placenta actively transports maternal vitamin A to the fetus. TTR of trophoblastic origin may play an important part in this transport.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALB, Albumin; D3, type 3 iodothyronine deiodinase; DTT, dithiothreitol; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; TBG, T4 binding globulin; TTR, transthyretin.
Received March 30, 2005.
Accepted September 1, 2005.
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References |
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-alanine. Biochem Biophys Res Commun 167:587–592[Medline]
1-antichymotrypsin and 1-antitrypsin by human trophoblast. Pediatr Res 34:312–317[Medline]
(1)-acid glycoprotein mRNA during rat development. High levels of expression in the decidua. J Biol Chem 264:5784–5790 1B-glycoprotein: homology to the immunoglobulin supergene family. Proc Natl Acad Sci USA 83:2363–23672-macroglobulin and albumin by human placental syncytiotrophoblast in vitro. J Cell Biochem 68:427–435[CrossRef][Medline]
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) or T3 (
). The association constant for T4 and inhibition constant for T3 were calculated by fitting the data to a one-site competition curve using nonlinear regression.
