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Original Studies |
Children’s Hospital of Philadelphia (S.A.W.), University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Pharmacology (T.B.G.), University of Texas, Southwestern Medical Center, Dallas, Texas 75235; Department of Pediatrics (P.F.C.-S.), Duke University, Durham, North Carolina 27710; Diagnostic Systems Laboratories, Inc. (A.K.), Webster, Texas 77598; and Mattel Children’s Hospital at UCLA (B.L., P.C.), University of California at Los Angeles, Los Angeles, California 90095-1752
Address all correspondence and requests for reprints to: Pinchas Cohen, M.D., Professor and Director of Research and Training, Division of Endocrinology, Department of Pediatrics, Mattel Children’s Hospital at UCLA, 10833 Le Conte Avenue, MDCC 22-315, Los Angeles, California 90095-1752. E-mail: hassy{at}mednet.ucla.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of IGFBP-3 to bind other molecules in addition to the IGFs, acid labile subunit, and IGFBP-3 proteases has been previously demonstrated. IGFBP-3 is noted to have a heparin-binding domain (heparin-BD) in its midregion and is known to interact with heparin-containing molecules in the extracellular matrix (5). IGFBP-3 has been observed in the nucleus of certain cells and contains a nuclear localization sequence that may allow it to interact with nuclear transcription factors (6, 7). Several groups, including our own, have demonstrated specific binding of IGFBP-3 to other proteins in cell lysates (8, 9) and cell membrane preparations (10, 11, 12, 13), and it has been proposed that IGFBP-3 may share a common receptor with transforming growth factor-ß (14).
We have previously demonstrated serum proteins of 70, 100, and 150 kDa that specifically bind IGFBP-3 and that may function as IGFBP-3 carriers in human serum (15). We report here the identification of the 70-kDa protein as transferrin (Tf), confirm the validity of Tf/IGFBP-3 binding through multiple independent in vitro methods, and demonstrate physiologically significant consequences of Tf/IGFBP-3 binding on cell proliferation and apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum samples were collected from healthy young adult volunteers (with informed consent). Samples were collected in serum separator tubes, centrifuged, and frozen at -70 C.
Recombinant human IGF-I was provided by Pharmacia (Stockholm, Sweden). Recombinant human IGFBP-3 and IGFBP-3 mutants were provided by Protigen Inc. (Mountain View, CA). IGFBP-1 was the gift of Genentech, Inc. (San Francisco, CA). IGFBP-2 was the gift of Sandoz Pharmaceuticals Corp. (Basel, Switzerland). IGFBP-4, -5, and -6 was purchased from Austral (San Francisco, CA). Recombinant human 125I-IGFBP-3 and affinity-purified antihuman IGFBP-3 antibody were purchased from Diagnostic Systems Laboratories, Inc.. Human Tf was purchased from Life Technologies, Inc. (Gaithersburg, MD). Holo-Tf, partially-saturated Tf, dimethylsulfoxide, and Igepal CA-630 were purchased from Sigma (St. Louis, MO). Tris (crystallized free base) was purchased from Fisher (Fair Lawn, NJ). Antihuman Tf antibody was purchased from Chemicon (Temecula, CA). SDS-PAGE reagents, Tween, and fat-free milk were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). Yeast strain Saccharomyces cerevisiae HF7c and yeast two-hybrid screening kit, including liver complementary DNA (cDNA) library, were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA).
Reverse ligand blot
Reverse Western ligand blots (WLB) were used to assess the presence of serum IGFBP-3 binding proteins/association proteins. Two microliters of normal serum were electrophoresed on SDS-PAGE (8%), at constant voltage, overnight, then transferred to nitrocellulose for 4 h at 170 mA. The nitrocellulose was buffered in Tris-buffered saline (TBS)/3% Igepal CA-630 for 30 min. The membranes were blocked for 3 h with TBS/1% BSA, and then incubated overnight with 125I-IGFBP-3 (106 cpm) in TBS/0.1% Tween/1% BSA. The nitrocellulose was washed four times with TBS/0.1% Tween and TBS. The resulting bands were visualized by autoradiography or phosphorimaging.
Reverse ligand dot blot
Increasing concentrations of Tf were carefully dot-blotted directly onto nitrocellulose (2 µL at a time) and allowed to dry completely. The nitrocellulose was then treated as described above for a reverse ligand blot.
Western immunoblot
Two microliters of serum were separated by SDS-PAGE (8%) at constant voltage overnight, then transferred to nitrocellulose for 4 h at 170 mA. The nitrocellulose was immersed in blocking solution (5% nonfat milk/TBS) for 45 min, washed with TBS/0.1% Tween, and incubated with primary antihuman Tf or antihuman IGFBP-3 antibody (1:4,000) for 2 h. After washing off any unbound antibodies, the nitrocellulose was incubated with a general secondary antibody (1:10,000) for 1 h. The membrane was washed four times with TBS/0.1% Tween and TBS. Bands were visualized using the peroxidase-linked enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Uppsala, Sweden).
Western immunodot blot
Five-microgram samples of IGFBP-1 through -6 were carefully dot-blotted directly onto nitrocellulose (2 µL at a time) and allowed to dry completely. The membranes were then incubated overnight with holo-Tf (4 µg/mL) followed with a series of washes in TBS. The nitrocellulose was then treated, as described above, for a Western immunoblot, using an antihuman Tf antibody (1:4,000) as the primary antibody.
Affinity chromatography
One milliliter of human plasma was diluted (1:3) with 0.05 mol/L sodium phosphate buffer, pH 7.4, and then filtered through a 0.4-µm filter and loaded onto an IGFBP-3 antigen affinity column. Bound proteins were first eluted with 0.05 mol/L sodium phosphate buffer, pH 7.4 containing 0.6 mol/L NaCl, followed by 0.1 mol/L glycine (pH 2.75) containing 0.15 mol/L NaCl (no. 1) or 0.1 mol/L citrate buffer, pH 4.5 and pH 2.5 (no. 2). Five milliliter fractions were collected. Fractions with optical density more than 0.03 were pooled and concentrated. Two fractions were collected for elution condition no. 1, and three fractions were collected for elution condition no. 2. The fractions were combined and dialyzed overnight at 4 C in dH2O to remove excess salt and to neutralize the buffer. Two microliters were loaded onto SDS-PAGE (8%) and then processed for amido black staining, immunoblotted, or reverse ligand blotted as described above.
Amino acid sequencing
Amino acid sequencing was provided by the Protein Chemistry Laboratory of the University of Pennsylvania Medical School, supported by core grants of the Diabetes and Cancer Centers (DK-19525 and CA-16520). Twenty microliters of samples eluted of the IGFBP-3 column were separated by 10% SDS-PAGE under nonreducing conditions, then transferred to a polyvinylidene difluoride membrane for 1.5 h. Bands were stained with 2% amido black. Bands were excised and subjected to Edmon degradation for sequencing in x machine. The first 10 amino acids of each indicated band were determined.
Sensor chip immobilization
The BIAcore 2000, Sensorchip CM5 (certified grade), amine
coupling reagents
(N'-ethyl-N'-(dimethylaminopropyl)-carbodiimide,
N-hydroxysuccinimide, and ethanolamine hydrochloride)
were purchased from Pharmacia Biosensor (Sweden).
Biosensor chips were made by immobilizing wild-type IGFBP-3
(IGFBP-3WT) and NLS-IGFBP3 mutant via primary
amine groups.
N'-ethyl-N'-(dimethylaminopropyl)-carbodiimide
coupling chemistry was used to activate the carboxymethylated dextran
surface for protein immobilization. Approximately 20 µg/mL of each
IGFBP-3 solution (10 mmol/L sodium acetate, pH 5.5) were injected over
the chip surface, at 5 µL/min at 25 C, to a level of
approximately1000 response units (
1 ng/mm2).
Ethanolamine solution (1 mol/L) was added to inactivate unbound
carboxyl groups. The surface was then exposed to 5 mmol/L HCl solution
for washing excess bound protein after each injection. Association and
dissociation phases were 180 sec.
BIAcore kinetic assays
The sensor chip was used to screen human serum Tf binding to IGFBP-3. All experiments were conducted at 25 C, with a constant flow rate of 30 µL/min PBS/0.005% Tween buffer. Holo-Tf or IGF-I were automatically injected over the chip surface using the kinject command in the BIAlogue control software. The association phase lasted 3 min. PBS buffer alone was then flowed over the chip to commence the dissociation phase for an additional 3 min. The sensor chip surface was regenerated, after each cycle, by injecting PBS buffer, at 10 µL/min twice, followed by 5 mM HCl at 100 µL/min for 30 sec three times, and a final wash of PBS buffer at 100 µL/min.
Biosensor interaction analysis (BIA)
BIAevaluation 3.0 software was used to perform global analysis of the resulting sensorgrams. The bulk refractive index effect of PBS buffer alone on the sensor chip was subtracted from each kinetic curve. The association and dissociation curves of each interaction between Tf and IGFBP-3 were fitted using standard kinetic equations as previously described in detail (16, 17). The program calculated the kinetic rates and the binding affinities for these reactions.
Yeast two-hybrid screening
The yeast strain Saccharomyces cerevisiae HF7c [MATa, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3, 112, gal4–542, gal80–538, LYS2::GAL1—HIS3, URA3::(GAL4 17 mers)3—CYC1—lacZ] was purchased from CLONTECH Laboratories, Inc. The fusion gene IGFBP-3/BD was constructed by splicing cDNA encoding the human IGFBP-3 gene into the plasmid pGBT9 directly 5' to and in phase with the gene encoding the BD of the yeast transcriptional activator GAL4 (CLONTECH Laboratories, Inc.). A human liver cDNA library with the activation domain (AD) of the GAL4 gene was purchased (CLONTECH Laboratories, Inc.) and screened by cotransforming yeast with both plasmids. Yeast colonies were made competent for transformations according to the manufacturer’s instructions (CLONTECH Laboratories, Inc. Matchmaker protocol handbook). Positive cotransformants were selected by growth on histidine-deficient agar media and assayed for ß-galactosidase activity according to the manufacturer’s instructions (CLONTECH Laboratories, Inc. Matchmaker protocol handbook). Genes encoding IGFBP-3-binding proteins identified through this method were isolated by plasmid recovery, amplified using PCR, sequenced, and compared with known sequences in GenBank using the MacVector software program (Oxford Molecular Ltd., Williamstown, MA).
Cell proliferation assay
Cell proliferation assays were performed using the CellTiter 96 aqueous nonradioactive proliferation assay kits (Promega Corp., Madison, WI). Sheep bladder smooth muscle (sBLSM) cells in primary culture were obtained from Dr. Ed Macarak at the University of Pennsylvania School of Dentistry. An equal number of cells were plated in 96-well plates in serum-free medium treated with various concentrations of IGFBP-3, Tf, and a combination thereof, and grown for 72 h. The quantity of formazan product was measured spectrophotometrically by the amount of absorbance at 490 nM (Bio-Rad Laboratories, Inc.). Sample size for each condition in the experiment was eight. Samples were pipetted with a multistep multidispenser using fine pipettes to eliminate any operator errors. Each experiment was repeated three times for reproducibility.
Apoptosis enzyme-linked immunosorbent assay (ELISA) assay
Photometric cell death detection ELISA (Roche Molecular Biochemicals, Indianapolis, IN) was performed to quantitate the apoptotic index by detecting the histone-associated DNA fragments (mono- and oligonucleosomes) generated by the apoptotic cells. The assay is based on the quantitative sandwich-ELISA principle using mouse monoclonal antibodies directed against DNA and histones, respectively, for the specific determination of these nucleosomes in the cytoplasmic fraction of cell lysates. In brief, an equal number of prostate cancer PC-3 cells (from ATCC, Manassas, VA) were plated in 24-well culture plates (1 x 104/cm2) in serum supplemented FK-12 medium, and grown to confluency for 72 h. At the time of sample collection, the confluent cells were washed with PBS and treated with various concentrations of IGFBP-3, Tf, and a combination thereof. The cells were dissociated gently (PBS with 0.1 M EDTA) and pelleted along with the floating cells (mostly apoptotic cells) collected from the conditioned media. The cell pellets were used to prepare the cytosol fractions, which contained the smaller fragments of DNA. Equal volumes of these cytosolic fractions were incubated in antihistone antibody-coated wells (96-well plates), and the histones of the DNA fragments were allowed to bind to the antihistone antibodies. The peroxidase-labeled mouse monoclonal DNA antibodies were used to localize and detect the bound fragmented DNA using photometric detection with 2,2'-azino-di-[3-ethylbenzathiazoline sulfonate] as the substrate. Calcium ionophore treated conditions were used as positive controls (see 20). Serum-free-medium-treated conditions were used as negative controls. Each experimental condition was performed with at least three samples and was repeated at least three times. The reaction products in each 96-well plate were read using a Bio-Rad Laboratories, Inc. microplate reader (Model 3550-UV). Averages of the values ± SEM from double absorbance measurements of the samples were plotted.
Immunofluorescence confocal microscopy
PC-3 cells (1 x 104) were plated on coverglasses in F12K medium containing 10% FBS for 2 days. The cells were then incubated in serum-free media, with or without IGFBP-3 (500 ng/mL) or Tf (1000 ng/mL), for 3 h before staining for immunofluorescence. After three washes in PBS, fixation and permeabilization of the cells were performed with 1% paraformaldehyde in PBS for 15 min at room temperature and 0.2% Triton X-100 in PBS for 15 min on ice, and cells were washed twice with PBS. IGFBP-3 protein localization was detected using the DSL hIGFBP-3 goat polyclonal antibody (which was previously purified in an IGFBP-3 column), diluted 1:200, followed by Fluorescein anti-Goat antibody from Vector Laboratories, Inc. (Burlingame, CA). Tf protein localization was detected using a human Tf chicken polyclonal antibody, from Chemicon, diluted 1:150, followed by fluorescein isothiocyanate antichicken IgG from Sigma. Specimens were incubated with primary antibodies in PBS for 1 h at room temperature, with secondary antibodies in PBS for 40 min at room temperature, and then incubated with HOECHST from Electron Microscopy Sciences (Ft. Washington, PA) for 2 min. Samples were analyzed using the Inverted Confocal Microscope (Leica Corp., Heidelberg, Germany), and operated by TCS-nt software (Leica Corp.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using rWLBs with 125I-IGFBP-3, we
demonstrated that IGFBP-3 binds to two specific proteins in normal
serum, which are separated by gel electrophoresis (Fig. 1A
and Ref. 15). Similarly, normal serum
that has been passed over an IGFBP-3 affinity column also demonstrates
these same specific proteins that bind to
125I-IGFBP-3 in an rWLB (data not shown) and were
uniquely separated from other serum proteins as shown in Fig. 1B, demonstrating amido black protein staining. These proteins, at 70 and
100 kDa, were subjected to N-terminal amino acid sequencing, and the
70-kDa protein was identified through the Swiss Protein database,
searching as human serum Tf (N-terminal sequence: PDKTVRWCA). Once this
initial identification was performed, we further confirmed the identity
of this protein as Tf by performing a Western immunoblot on the IGFBP-3
affinity-purified serum fraction. Antibodies to human Tf specifically
bound to the 70-kDa band separated by the IGFBP-3 column (Fig. 1C).
Using specific antihuman IGFBP-3 and antihuman Tf, we
coimmunoprecipitated IGFBP-3 and Tf from human serum (data not
shown).
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We tested the degree of specificity of Tf binding to IGFBP-3 by
comparing binding to all six human IGFBPs. A nitrocellulose membrane
was dot-blotted with 5 µg each of IGFBP-1 (0.2 nmol), IGFBP-2 (0.16
nmol), IGFBP-3 (0.11 nmol), IGFBP-4 (0.21 nmol), IGFBP-5 (0.17
nmol), and IGFBP-6 (0.15 nmol), incubated with a Tf solution, washed,
and probed with anti-Tf antibodies in manner similar to that of a
Western immunoblot (Fig. 2). The results
indicate that Tf preferentially binds to IGFBP-3 at several orders of
magnitude greater than the other IGFBPs, indicating a strong
specificity of Tf for IGFBP-3.
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We then investigated the effect of iron saturation on Tf-IGFBP-3
binding. Increasing amounts [10 µg (0.14 nmol) to 80 µg (1.14
nmol)] of unsaturated and iron-saturated (holo-) Tf were carefully
dot-blotted onto a nitrocellulose membrane, allowed to dry, probed with
125I-IGFBP-3, and visualized by autoradiography
(Fig. 3). The results demonstrate that
holo-Tf binds to IGFBP-3 with approximately twice the affinity as
unsaturated Tf.
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We also compared the affinity of IGFBP-3 for Tf vs.
IGF-I with a reverse ligand dot blot. IGF-I and Tf, in equimolar
amounts ranging from 25–1000 nmol, were dot-blotted to a
nitrocellulose membrane, allowed to dry, probed with
125I-IGFBP-3, and visualized by autoradiography.
Tf-IGFBP-3 binding was dose-responsive and of a magnitude similar to
that of IGF-I (Fig. 4). Insulin and
albumin, at the same molar quantities, had no appreciable binding to
IGFBP-3.
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We employed BIAcore technology (Pharmacia) to directly measure protein-protein interactions between IGFBP-3 and Tf. Using surface plasmon resonance, the system detects and displays a signal from resultant evanescent waves that is proportional to the change in mass as one protein molecule binds to the immobilized ligand on the sensorchip. In this manner, we observed changes in the association and dissociation phases of Tf/IGFBP-3 interactions and compared them with those of IGF-I/IGFBP-3 interactions. We performed kinetic analyses using both IGFBP-3WT and a IGFBP-3 protein mutated in the nuclear localization sequence (IGFBP-3NLS(-) generously provided by Protigen Inc.)
Qualitative inspection of the IGF-I/IGFBP-3 curve (Fig. 5A) reveals a rapid initial association
followed by a gradual dissociation, corresponding to a specific,
high-affinity binding event. IGF-I bound to
IGFBP-3NLS(-) with a
similar affinity as IGFBP-3WT, suggesting that
the NLS domain is not involved in IGF-I/IGFBP-3 binding. Inspection of
the Tf–IGFBP-3WT curve (Fig. 5B, black
line) reveals a rapid association phase, equal in magnitude to
that of the IGF-I -IGFBP-3 association, followed by a relatively rapid
dissociation phase. The
Tf–IGFBP-3NLS(-) curve
(Fig. 5B, gray line) is characterized by a significantly
slower (by two orders of magnitude) association phase and rapid
dissociation phase. Taken together, these data suggest that Tf/IGFBP-3
binding occurs rapidly at high affinity but with rapid dissociation,
such that functional interactions are probably short-lived.
Furthermore, comparison of the Tf–IGFBP-3WT and
Tf–IGFBP-3NLS(-) curves
suggests that the Tf binding site on IGFBP-3 involves the NLS
region.
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The ability of Tf to bind IGFBP-3 in an in vivo situation was tested using a yeast two-hybrid system. We used the yeast strain Hf7c (which is unable to grow in histidine-, leucine-, or tryptophan-deficient media) with a histidine-selection marker and ß-galactosidase marker under control by the GAL1 promoter. The transcriptional activator GAL4 consists of two separable domains: a DNA-BD that binds to an upstream activating sequence, and a transcriptional AD necessary for RNA synthesis. We performed cotransformations with: 1) pGBT9 plasmid containing the fusion gene IGFBP-3/GAL4 BD and a histidine-selection marker; plus 2) pGAD424 plasmid containing a liver cDNA library/GAL4 AD fusion gene and a tryptophan-selection marker. Positive cotransformants were isolated by growth on tryptophan-, leucine-, and histidine-deficient media; and colonies with ß-galactosidase activity were harvested to recover library plasmid. Library fragments were amplified using PCR and were sequenced. We isolated a 830-base pair cDNA fragment encoding 160 amino acids of the C-terminal portion of the human Tf gene followed by 350 base pairs of the 3' untranslated region of the human Tf cDNA (GenBank sequence 339452; M12530.1).
In vivo effects of Tf-IGFBP-3 interactions
Tf, IGF-I, and IGFBP-3 were added to sBLSM and a human prostate
cancer cell line (PC-3) to determine the physiological consequences of
Tf-IGFBP-3 interactions. We have previously demonstrated the
stimulatory role of IGFBP-3 on growth in sBLSM (9). As
demonstrated in Fig 6, incubation of
sBLSM with IGF-I [100 ng/mL (13.3 pmol/L)], IGFBP-3 [0.5 µg/mL
(11.4 pmol/L)] or Tf [4 µg/mL (57.4 pmol/L)] alone increased cell
proliferation by 35 ± 3%, 38 ± 4%, and 51 ± 2%,
respectively. The coincubation of sBLSM with both Tf and IGFBP-3,
however, resulted in a 33 ± 11% inhibition of growth, as
compared with baseline conditions.
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. The addition of Tf alone [4 µg/mL
(57.4 pmol/L)] to PC-3 cells inhibited apoptosis, consistent with the
well-known viability-enhancing effects of Tf in cell culture systems.
IGFBP-3 [0.5 µg/mL (11.4 pmol/L)] alone induced apoptosis in
PC-3 cells, to 200% of baseline, but this effect was blocked by the
coincubation of IGFBP-3-treated PC-3 cells with Tf [4 µg/mL (57.4
pmol/L)].
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PC-3 cells were plated on coverglasses and treated with either
IGFBP-3 [0.5 µg/mL (11.4 pmol/L)] or Tf [1 µg/mL (14.3 pmol/L)]
for 3 h. As Fig. 8 shows, both
IGFBP-3 and Tf are localized intracellularly and demonstrate some
nuclear and perinuclear localization. However, the addition of either
molecule results in the enhancement of the intracellular distribution
of the other. In Fig. 8A, IGFBP-3 protein was localized to the
cytoplasm and to the nucleus under serum-free conditions; the addition
of Tf increased both. In Fig. 8B, Tf protein was localized to the inner
plasma membrane, the cytoplasm, and to the perinuclear areas under
serum-free conditions; the addition of IGFBP-3 resulted in cytoplasmic
enhancement of Tf and to some nuclear staining.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The discovery of IGF-independent modulation of growth by IGFBP-3 provides indirect evidence for the presence of specific IGFBP-3 binding proteins, which may be cell-surface associated, cytosolic, or nuclear in location. Oh et al. have demonstrated specific binding of IGFBP-3 to cell surface proteins of 20, 26, and 50 kDa in the estrogen receptor negative breast cancer cell line Hs578T, by affinity cross-linking and immunoprecipitation (10, 11, 12). We have similarly identified proteins with IGFBP-3 binding ability in prostate (8) and BLSM whole-cell lysates (9). Evidence for specific IGFBP-3 receptors also comes from experiments characterizing IGFBP-3 as a growth-inhibitory factor in murine knockout cells lacking the IGF-R (20) and as the mediator of apoptosis induced by retinoic acid and TGF-ß (12, 13).
In this report, we describe, for the first time, the identification of Tf as another IGFBP-3 binding protein. We have demonstrated specific high-affinity Tf/IGFBP-3 binding through several in vitro methods, including column chromatography, coimmunoprecipitation, Western ligand blot, and Western immunoblot techniques. These studies demonstrate not only that Tf binds IGFBP-3 in human serum but that binding is dose-responsive, specific for IGFBP-3, and dependent on the degree of iron saturation. Biosensor BIA kinetic data suggests that the binding of Tf to IGFBP-3 occurs with short duration, perhaps involving the IGFBP-3 nuclear localization sequence. We have also independently cloned Tf as an IGFBP-3 binding protein, through the screening of a liver cDNA library with a yeast two-hybrid system. This method has been demonstrated to be a powerful tool in characterizing protein-protein interactions, even those that involved cell membrane-associated receptors (21, 22).
We have further demonstrated physiologically significant ramifications of Tf/IGFBP-3 interactions on the modulation of cell proliferation and apoptosis in several mammalian cellular systems. Tf and IGFBP-3 are both growth-potentiating in BLSM, but the coincubation of these proteins in BLSM-conditioned media resulted in a dramatic reduction in growth. This phenomenon is consistent with Tf/IGFBP-3 binding with such affinity as to prevent either ligand from interacting with its own natural cell surface receptor, or alternately, affecting access of IGFBP-3 to transcriptional modulators in the nucleus.
Tf is a 70-kDa glycoprotein that functions as the primary iron-carrying protein in serum (23). It is synthesized predominantly in the liver, but smaller amounts are produced in other organs, such as brain and testis (24). Tf normally binds two iron molecules after absorption of iron from the gastric and duodenal mucosa; Tf-iron binding at physiological conditions is nearly complete, so that essentially no free unbound iron exists in the blood (25). The Tf-iron complex circulates in serum and binds to cell surface Tf receptors (Tf-R), undergoes receptor-mediated endocytosis, and is recycled after dissociation of iron and exocytosis (26). Elevated intracellular iron levels are associated with destabilization of Tf-R messenger RNA (mRNA), whereas intracellular iron depletion increases Tf-R mRNA half- life (27).
The growth-enhancing effects of Tf at the cellular level are well-recognized. Tf is required for cell viability or proliferation in many cell culture systems, including cortical neuron (28), lymphocyte (29, 30), renal cortex (31), liver (32, 33), and bladder (34). Tf also seems to be required for differentiation of fetal metanephric mesenchyme during embryogenesis (35). Tf deprivation from cells is associated with elevated indices of apoptosis (36). Growth and aggressiveness of malignancies are also Tf-dependent; Tf-R number is positively correlated with tumor growth rate (37) and metastasis (38). In prostate cancer cells, Tf stimulates both proliferation and invasiveness (39, 40); and suramin, a compound employed in metastatic prostate cancer therapy, has recently been demonstrated to exert its effects by antagonizing the binding of Tf to the Tf-R (41).
The relationship between Tf and the IGF-IGFBP-3 axis is poorly understood. Serum concentrations of Tf are significantly correlated with IGF-I in pubertal girls (42) and boys (43) and with IGFBP-3 in pubertal girls (44). Tf levels are low in growth-hormone deficient children (45, 46) and increase after chronic GH administration (47, 48). At the molecular level, IGF-I seems to increase the expression of cell-surface Tf-Rs by redistributing Tf-Rs from an intracellular compartment to the cell surface (49, 50, 51). Tf has been shown to be localized intracellularly and has specifically been shown to be perinuclear (52). Our observations that IGFBP-3 nuclear localization is enhanced by Tf and that Tf is localized perinuclearly in an IGFBP-3-dependent manner suggest that IGFBP-3-Tf interactions may involve cotransport into cellular compartments and possibly additional molecule(s), which facilitate these events.
The nature of the IGFBP-3 to Tf interaction seems to involve an association, which is short-lived, and indeed suggests that a third molecule may be involved, or that this interaction may facilitate presentation of one or the other molecule to a receptor or a transporter. Physically, the interaction seems to occur between the NLS domain region of the IGFBP-3 molecule (as the IGFBP-3 mutant studies suggest) and the C-terminal portion of the Tf molecule (as evident from the two-hybrid screen). The observation that both IGFBP-3 and Tf stimulate smooth-muscle cell growth, but that their combination inhibits growth, suggests that the IGFBP-3-Tf complex may still be able to inhibit endogenously produced IGF-II, which we have shown to be made in these cells (7).
It is exciting to speculate that some of the previously recognized survival effects of Tf, which where, until now, completely unexplained, may be mediated through the inactivation of IGFBP-3 effects, as we have shown in PC-3 cells. There is substantial production of IGFBP-3 by epithelial cells, which may is responsible for the baseline apoptosis observed in such cells, as apoptosis is reduced in the presence of an IGFBP-3 neutralizing antibody (8).
The discovery of Tf as an IGFBP-3 binding protein adds a further level of complexity to the modulation of growth at the cellular level. In addition to the modulation of IGF activity at the cell membrane and direct interactions with its own specific receptors, IGFBP-3 may also affect cell growth through several Tf- and Tf-R-dependent mechanisms. Sequestration or presentation of IGF-I to the cell surface by IGFBP-3 may indirectly influence growth by regulating Tf-R density at the cell surface, and Tf/IGFBP-3 binding may directly interfere with each ligand from binding its own natural receptor, depending on the iron status of the tissue or organism. Whether these Tf/IGFBP-3 interactions occur predominantly at the cell surface or in the cytosolic or nuclear compartments is unknown, although it is intriguing to speculate that Tf may also modulate IGFBP-3 activity by binding IGFBP-3 and preventing its nuclear entry. These findings suggest that IGFBP-3 may serve an important role in coordinating growth to the nutritional state of the tissue or organism and that further IGFBP-3 binding proteins remain to be discovered.
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Footnotes |
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Received March 4, 2000.
Revised August 14, 2000.
Revised January 3, 2001.
Accepted January 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Oesterreicher, W. F. Blum, B. Schmidt, T. Braulke, and B. Kubler Interaction of Insulin-like Growth Factor II (IGF-II) with Multiple Plasma Proteins: HIGH AFFINITY BINDING OF PLASMINOGEN TO IGF-II AND IGF-BINDING PROTEIN-3 J. Biol. Chem., March 18, 2005; 280(11): 9994 - 10000. [Abstract] [Full Text] [PDF]
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 N Wilczak, G S. Ramsaransing, J Mostert, D Chesik, and J De Keyser Serum levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in relapsing and primary progressive multiple sclerosis Multiple Sclerosis, February 1, 2005; 11(1): 13 - 15. [Abstract] [PDF]
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E. M. Ongeri, Q. Zhu, M. F. Verderame, and J. M. Hammond Insulin-Like Growth Factor-Binding Protein-3 in Porcine Ovarian Granulosa Cells: Gene Cloning, Promoter Mapping, and Follicle-Stimulating Hormone Regulation Endocrinology, April 1, 2004; 145(4): 1776 - 1785. [Abstract] [Full Text] [PDF]
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K.-W. Lee, B. Liu, L. Ma, H. Li, P. Bang, H. P. Koeffler, and P. Cohen Cellular Internalization of Insulin-like Growth Factor Binding Protein-3: DISTINCT ENDOCYTIC PATHWAYS FACILITATE RE-UPTAKE AND NUCLEAR LOCALIZATION J. Biol. Chem., January 2, 2004; 279(1): 469 - 476. [Abstract] [Full Text] [PDF]
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B. Singh, D. Charkowicz, and D. Mascarenhas Insulin-like Growth Factor-independent Effects Mediated by a C-terminal Metal-binding Domain of Insulin-like Growth Factor Binding Protein-3 J. Biol. Chem., January 2, 2004; 279(1): 477 - 487. [Abstract] [Full Text] [PDF]
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L. D. Payet, X.-H. Wang, R. C. Baxter, and S. M. Firth Amino- and Carboxyl-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-3 Cooperate to Bind IGFs with High Affinity and Inhibit IGF Receptor Interactions Endocrinology, July 1, 2003; 144(7): 2797 - 2806. [Abstract] [Full Text] [PDF]
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 S. L. Franklin, R. J. Ferry Jr., and P. Cohen Rapid Insulin-Like Growth Factor (IGF)-Independent Effects of IGF Binding Protein-3 on Endothelial Cell Survival J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 900 - 907. [Abstract] [Full Text] [PDF]
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