| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Medicine (J.C., K.E.C., D.N., T.B., U.B., M.W., J.-M.L., P.C.T., Q.C., V.A.C., H.E.R., A.B.G., I.R.R.) and School of Biological Sciences (H.M.B., E.N.B.), University of Auckland, Auckland 1001, New Zealand; Fonterra Research Centre (K.P.P., N.W.H.), Palmerston North 5315, New Zealand; and Institute of Medical and Veterinary Science (N.F.), Adelaide 5000, Australia
Address all correspondence and requests for reprints to: Dr. J. Cornish, Department of Medicine, University of Auckland, Private Bag 92019, Auckland 1001, New Zealand. E-mail: j.cornish{at}auckland.ac.nz.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B ligand. In contrast, lactoferrin had no effect on bone resorption by isolated mature osteoclasts. Lactoferrin was administered over calvariae of adult mice for 5 d. New bone formation, assessed using fluorochrome labels, was increased 4-fold by a 4-mg dose of lactoferrin. Thus, lactoferrin has powerful anabolic, differentiating, and antiapoptotic effects on osteoblasts and inhibits osteoclastogenesis. Lactoferrin is a potential therapeutic target in bone disorders such as osteoporosis and is possibly an important physiological regulator of bone growth. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lactoferrin is an 80-kDa iron-binding glycoprotein that belongs to the transferrin family of proteins (1). It is produced by many exocrine glands and, consequently, is widely distributed in body fluids including tears, saliva, bile, pancreatic fluid, vaginal secretions, semen, and milk (2). Lactoferrin is also a major constituent of the secondary granules of neutrophilic leukocytes, from which it is released during acute inflammation (3). Serum levels of lactoferrin in healthy subjects range from 2 to 7 µg/ml and are predominantly neutrophil derived, but during inflammation and sepsis its local concentrations may be much higher (4). It acts as an iron chelator, which may contribute to its antimicrobial activity (5), but it also has effects on cell growth and differentiation (6), embryonic development (7), myelopoiesis (8), endothelial cell adhesion (9), cytokine (10, 11) and chemokine (12) production, regulation of the immune system (13), and modulation of the inflammatory response (14). Its effects on bone have received little attention.
The present studies address the skeletal effects of lactoferrin in vitro (using assays of osteoblast growth, differentiation and survival, osteoclast development and activity, and bone organ culture) and in vivo. These studies establish lactoferrin as a potent novel anabolic factor in osteoblasts, which also reduces bone resorption and increases bone mass when administered in vivo. These findings pose important questions regarding the role of lactoferrin in bone physiology, both during growth and in adulthood, and provide a potential target for drug development in the therapeutics of osteoporosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cultures of primary human osteoblasts were prepared using normal human trabecular bone obtained from 50- to 70-yr-old patients undergoing knee or hip arthroplasty. Osteoblasts were grown from enzyme-treated bone chips, using a modified method of Robey and Termine (16).
Cell lines were grown to 80% confluency in 5% fetal calf serum-supplemented media and then seeded into 24-well plates for proliferation assays, as described for primary rat osteoblasts. Human osteoblast-like cell line (SaOS-2) cells were seeded at a density of 7.0 x 103 cells/cm2 and cultured in MEM. Murine bone marrow stromal cell line (ST2) cells were seeded at 1.4 x 104 cells/cm2 in DMEM.
Apoptosis assay
For the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, primary rat osteoblasts were seeded into 8-well chamber slides (Lab-Tek, Nalge Nunc International, Naperville, IL) in 5% fetal calf serum/MEM (3 x 104 cells/chamber) and incubated for 24 h. Media were changed to 0.1% BSA/MEM and the cells incubated overnight. Fresh media and treatments were added and cells incubated a further 24 h. Cells were fixed in 2% paraformaldehyde for 15 min and washed thoroughly in PBS before being permeabilized with 1% Triton X-100 (BDH Chemicals Ltd., Poole, UK)/PBS for 5 min. Cells were rinsed thoroughly and then a modified TUNEL assay performed using the Dead End Colorometric TUNEL system (Promega Corp., Madison, WI) according to manufacturer’s instructions. Results are expressed as the number of apoptotic bodies per microscopic field. There were eight wells for each group, and each experiment was repeated two to three times.
Bone nodule assay
Osteoblasts were isolated from 20-d fetal rat calvariae as described above. Cells were grown for 48 h in 10% FBS/DMEM in T75 flasks. Cells were then trypsinized (GIBCO, Invitrogen) and plated into 35-mm tissue culture dishes at a density of 3.5 x 104 cells/dish in 15% FBS/
MEM supplemented with 50 µg/ml L-ascorbic acid-2-phosphate (Sigma Chemical Co., St. Louis, MO). When cells were confluent (approximately 5 d), media were changed to 15%FBS/MEM supplemented with L-ascorbic acid-2-phosphate and 10 mM ß-glycerophosphate (Sigma-Aldrich Co., St. Louis, MO), and test substances were added. These supplemented media were changed twice weekly and test substances were replaced. After 21 d the cells were fixed in neutral buffered formalin for 15 min, rinsed thoroughly with distilled water, and the cultures stained for mineral using Von Kossa stain. The number and area of mineralized bone nodules greater than 1 mm in diameter were quantified using image analysis.
Chondrocyte culture
Chondrocytes were isolated by removing cartilage (full-depth slices) from the tibial and femoral condyles of adult sheep under aseptic conditions. Slices were placed in 5% FBS/DMEM and chopped finely with a scalpel blade. Tissue was weighed and then incubated at 37 C in pronase solution (0.8% wt/vol in 5% FBS/DMEM) for 90 min followed by collagenase (0.1% wt/vol in 5% FBS/DMEM) for 18 h to complete the digestion. The cells were isolated from the digest by centrifugation (5 min at 1300 rpm), resuspended in 5% FBS/DMEM, passed through a nylon mesh screen of 90 µm pore size to remove any undigested fragments, and recentrifuged. The cells were washed twice and seeded into a 75-cm2 flask containing 10% FBS/DMEM and ascorbic acid (50 µg/ml). The cells were incubated under 5% CO2/95% air at 37 C. Confluence was reached within 7 d, at which time the cells were seeded into 24-well plates at a density of 1.4 x 104 cells/cm2 in 5% FBS/DMEM + ascorbic acid. Proliferation assays were performed as for primary rat osteoblasts.
Bone marrow culture
Bone marrow was obtained from the long bones of normal Swiss male mice aged 4–6 wk, as previously described (15). Briefly, marrow cells were cultured for 2 h in 90-mm petri dishes. Nonadherent cells were then collected and grown in 48-well plates; 1,25 dihydroxyvitamin D3 (10–8 M) was added (d 0) to all wells except to negative controls. On d 2 and 4, cultures were fed by removing 0.5 ml medium from each well and replacing with 0.5 ml fresh medium containing test substances and 1,25 dihydroxyvitamin D3. After culture for 7 d, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (containing three or more nuclei) were counted in all wells. Each experiment had three wells in which cells were grown on bone slices and checked for resorptive pits, indicating that the TRAP-positive multinucleated cells in these cultures were capable of resorbing bone. There were at least eight wells for each group, and each experiment was repeated three or four times.
Mature isolated osteoclast culture
Rat osteoclasts were isolated from the long bones of 1-d-old neonatal rats, as previously described (15). Briefly, an osteoclast-rich suspension prepared by homogenizing the bone tissue was placed onto bovine bone slices (9 mm2) in 96-well plates and incubated for 35 min to allow the mature osteoclasts to settle. The bone slices were then placed in 12-well plates (four slices per well) containing acidified media and incubated with test substances or vehicle for 20 h. After incubation, the bone slices were fixed and stained for TRAP. The number of TRAP-positive multinucleated cells (containing more than three nuclei) on each bone slice were quantified, the cells were removed by gentle scrubbing, and then the bone slices were stained for 30 sec with toluidine blue. After several washes in water, the bone slices were dried and assessed for the pits excavated by the osteoclasts, using reflected light microscopy with metallurgic lenses. The results for each bone slice were expressed as the ratio of the number of pits to the number of osteoclasts. There were 6–12 bone slices in each group and each experiment was repeated two or three times.
Real-time PCR
RNA for real-time PCR was prepared from human primary osteoblasts and mouse bone marrow cultures. The culture conditions were as previously described for the proliferation assays and osteoclastogenesis assays, but for RNA preparation the cells were plated in 6-well plates at densities of 1.5 x 105 primary osteoblasts/well and 4 x 106 bone marrow cells/well. Two wells were used for every experimental point. Bovine lactoferrin (100 µg/ml) or vehicle was added, and cells were harvested at the indicated time points after the beginning of treatment. Total cellular RNA was purified from the cultures using RNeasy minikit (Qiagen, Valencia, CA), and genomic DNA was removed using RNase-free DNase set (Qiagen). Reverse transcription was carried out following the previously published protocol (17), and cDNA was used for real-time PCR. The primer-probe sets were purchased as Assay-on-Demand from Applied Biosystems (Foster City, CA). Multiplex PCR was performed with FAM-labeled specific probes [osteoprotegerin (OPG) and receptor activator of nuclear factor-B ligand (RANKL) from human and OPG, RANKL, interferon-ß and -
, IL-18, and TNF from mouse)] and VIC-labeled 18S rRNA probes according to the company’s instructions, using ABI PRISM 7700 sequence detection system (Applied Biosystems). The experiments were performed in triplicates and repeated three times with similar results.
Bone organ culture
Mice were injected sc with 5 µCi 45Ca at 2 d of age, and hemicalvariae were dissected out 4 d later. Hemicalvariae were preincubated for 24 h in medium 199 with 0.1% BSA and then changed to fresh medium containing test substances or vehicle. Incubation was continued for a further 48 h. To assess DNA synthesis, [3H]thymidine (0.6 µCi/ml) was added in the last 4 h of the incubation. The experiment was terminated, and both calcium release and thymidine incorporation were assessed. There were five to seven hemicalvariae in each group, and each experiment was repeated three or four times.
Culture media for all studies described above contained penicillin (100 U/ml) and streptomycin (100 µg/ml).
In vivo study
Five groups of sexually mature male mice were given daily sc injections over the right hemicalvaria for a consecutive 5 d, as previously described (18, 19). Three groups (n = 15) received one of three doses of bovine lactoferrin (0.04, 0.4, or 4 mg), and a further two groups (n = 9) received vehicle or BSA, 4 mg. The animals were killed 10 d after the last injection. Fluorochrome labels were injected sc at the base of the tail on d 1 (calcein), 5 (alizarin red), and 14 (calcein). Calvariae were excised, fixed in 10% neutral-buffered formalin, dehydrated, and embedded in methylmethacrylate resin. Sections were cut, mounted on gelatin-coated slides, and histomorphometric indices measured using image analysis.
All animal procedures were approved by the Animal Ethics Committee of our institution.
Lactoferrin preparations
Bovine lactoferrin was isolated from fresh skim milk by cation exchange chromatography and gel filtration. Briefly, the milk at native pH was passed through S Sepharose fast flow at 4 C and the bound proteins eluted in steps with 0.1, 0.35, and 1 M NaCl, respectively. The 1 M NaCl fraction containing lactoferrin was dialyzed and freeze dried. The resulting material was then dissolved in 25 mM sodium phosphate buffer (pH 6.5) and reapplied to the cation exchanger, which had been equilibrated in the above buffer. Lactoferrin was eluted by application of a salt gradient to 1 M NaCl in phosphate buffer and the recovered material dialyzed and freeze-dried. Final purification of lactoferrin was achieved by gel filtration through Sephacryl S300 in phosphate buffer and the protein recovered as a dialyzed freeze-dried powder. Purity of the final product was greater than 98% as assessed by resource reversed-phase HPLC and mono-S HPLC (20).
Native human lactoferrin was prepared from breast milk as previously described (21) and fully saturated with iron by the addition of ferric nitrilotriacetate. Full-length recombinant human lactoferrin was expressed in baby hamster kidney cells, as described by Stowell et al. (22), and was again used in its iron-saturated form. The degree of iron saturation was determined from the spectral ratios A280/A466 and A412/A466 (23), which have values of 20–22 and 0.70–0.74 for the fully iron-saturated protein. The protein solutions were dialyzed exhaustively against 0.05 M Tris-HCl (pH 8.0), 0.2 M NaCl, to remove any excess, unbound, ferric iron before use.
Statistics
Data were analyzed using ANOVA with post hoc Dunnett’s tests. A 5% significance level is used throughout. Data are presented as means ± SE, unless indicated otherwise.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). This effect was present at concentrations of lactoferrin that occur in vivo (1–100 µg/ml). The same range of concentrations of lactoferrin stimulated proliferation in the human osteoblast-like cell line, SaOS-2 (Fig. 1B) (although the size of the effect was less in these transformed cells) and the stromal cell line, ST2 (Fig. 1C). Comparable effects were found in primary cultures of human osteoblasts treated with bovine lactoferrin (Fig. 1D), and recombinant human lactoferrin was possibly more potent at low concentrations in these cells, increasing thymidine incorporation 2-fold at 10 µg/ml (data not shown). With this exception, the species of origin and method of preparation of the lactoferrin did not substantially impact the extent of the proliferative effect.
|
). These effects were significant only at lactoferrin concentrations of 100 µg/ml and greater, suggesting that the concentrations needed to stimulate osteoblast differentiation are 10-fold greater than those required to stimulate proliferation in these models.
|
). This effect was dose dependent and evident at similar concentrations to those causing osteoblast proliferation. In this assay, lactoferrin reduced osteoblast apoptosis by up to 50–70%, indicating that lactoferrin not only stimulates osteoblast activity but also acts as a potent osteoblast survival factor.
|
). At concentrations of 10 and 20 µg/ml, lactoferrin increased thymidine incorporation in these cells. These effects were comparable in magnitude with those seen in osteoblasts.
|
). At 100 µg/ml, osteoclastogenesis was completely arrested. To determine at which stages of osteoclast development lactoferrin acts, it was added to bone marrow cultures from the outset or from d 2 (Fig. 5B). Both interventions reduced osteoclastogenesis, with more frequent additions, resulting in greater effects. This implies that lactoferrin acts on both preosteoclasts and more mature cells of this lineage. However, lactoferrin had no effect on bone resorption by isolated mature osteoclasts (Fig. 5C).
|
). Exposure of osteoblasts to lactoferrin resulted in reduced OPG transcription and a transient increase in expression of RANKL. When these experiments were repeated in bone marrow cultures, OPG mRNA levels decreased at 96 h but not at earlier time points. However, RANKL mRNA levels were diminished at both 72 and 96 h. Similar studies of expression of IL-18, TNF, interferon-ß, and interferon- in bone marrow cultures showed no changes in response to treatment with lactoferrin (data not shown).
|
), which previous studies in this model suggest probably reflects increased proliferation of cells of the osteoblast lineage (24), although effects on other cells in the calvariae might also have contributed. However, bone resorption, measured as 45Ca release, was unaffected (Fig. 7B). Because there is virtually no bone marrow in the calvariae, bone resorption in these explants predominantly reflects mature osteoclast function. Thus, this finding is consistent with that in isolated mature osteoclasts, shown in Fig. 5C
.
|
. The dramatic increases in bone area in the calvariae from lactoferrin-treated animals, compared with control, can be appreciated in these photomicrographs. New bone formation (assessed by measuring the distance between the first fluorochrome label and bone surface) was dose-dependently increased by lactoferrin, such that the 4-mg dose induced changes 4-fold greater than those observed in control animals (Fig. 9A). Local injections of lactoferrin also increased the mineral apposition rate (Fig. 9B) and the bone formation rate (Fig. 9C).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The actions of lactoferrin on osteoclasts are strikingly different from those we observed in osteoblasts, in that it produces an almost total arrest of osteoclastogenesis in mouse bone marrow cultures. Even though lactoferrin does not influence the activity of mature osteoclasts, the inhibition of osteoclastogenesis is still likely to result in a profound reduction in bone resorption. There is one previous report of the effects of lactoferrin on bone resorption, in which Lorget et al. (27) demonstrated that bovine lactoferrin reduces bone-resorbing activity in a rabbit mixed bone cell culture. This effect appeared to be mediated by an inhibition of the development of mature osteoclasts and operated by a mechanism independent of the RANK/RANKL/OPG system. The finding in the present study, that lactoferrin reduces RANKL expression in bone marrow cultures, could in part explain the inhibition of osteoclastogenesis, although this will tend to be counterbalanced by the effects of lactoferrin to also inhibit expression of OPG. We did not find changes in the levels of expression of other known regulators of osteoclastogenesis. It should be noted that lactoferrin has previously been demonstrated to inhibit the survival of progenitor cells in the bone marrow (28), implying that it might also act earlier in osteoclast development.
As a result of its effects on osteoblast growth, lactoferrin produces substantial increases in local bone formation in vivo, even with the very short-term exposure studied here. The bone growth resulting from local lactoferrin injection is considerably greater than we have found previously in response to factors such as insulin, amylin, adrenomedullin, C-terminal PTH-related peptide, calcitonin, or calcitonin gene-related peptide in the same model (29, 30, 31, 32). It is qualitatively different from the effects of PTH in this model, which produces a powerful stimulation of bone resorption in addition to its effect on formation (33). The magnitude of the lactoferrin effect approaches those reported following local injection of statins (34) or TGFß (35, 36). This potency is further attested to by increases in new bone formation seen at sites remote from the injection site: there is evidence of increased formation on the intracranial aspect of the calvariae (see Fig. 8B) and the contralateral, uninjected, hemicalvariae (data not shown), something we have not seen with most other agents studied in this model. This anabolic potency suggests that lactoferrin or its analogs should be explored as therapies for osteoporosis that can restore skeletal strength because most current interventions merely arrest further structural decline.
The present findings pose the question as to whether lactoferrin has a physiological role in skeletal development or homeostasis. Lactoferrin is expressed biphasically in embryogenesis (7). It appears first in the two- to four-cell embryo. In the postblastocyst stage, its expression declines but increases again dramatically in the latter half of gestation. Therefore, it could play a significant role in the development and function of chondrocytes and osteoblasts in the fetal skeleton. Lactoferrin is present in high concentrations in milk, particularly in colostrum, and it is likely that large proteins such as lactoferrin can cross the neonatal gut and enter the systemic circulation (37). Therefore, it is possible that the anabolic actions of lactoferrin might continue into the neonatal period. There is some evidence of biological effects of orally administered lactoferrin in adults (38, 39, 40), although, as yet, none that it impacts on bone. Indeed, milk supplementation in human adults produces similar effects on bone density to calcium supplementation alone (41, 42), so it is unlikely that dietary lactoferrin has an important effect on human skeletal health later in life. Lactoferrin plays an important immunomodulatory function (14), decreasing the secretion of a number of osteolytic cytokines such as TNF and IL-1ß (11, 43, 44) and stabilizing mast cells (45). Thus, its direct effects on the activity and development of bone cells may be complemented by these cytokine-mediated effects. In adult life, lactoferrin production is believed to be principally influenced by stimuli causing inflammation because it is present in the secretory granules of neutrophils. Therefore, in inflammatory states, lactoferrin may play a role in counterbalancing the catabolic effects on the skeleton of some of the mediators of the inflammatory response, although bone loss still predominates in most cases.
Clearly, identification of the mechanism(s) by which lactoferrin acts on bone cells is important because of the potency of the effects demonstrated, and this is being actively investigated by our group (45A ). A putative lactoferrin receptor in the intestine has been identified by Suzuki et al. (46), but we have not been able to detect this receptor in primary rat osteoblasts. However, we have presented preliminary evidence that lactoferrin acts through low-density lipoprotein receptor-related protein-1, a member of the low-density lipoprotein-related receptor family (47). These receptors, which are expressed in primary cultures of osteoblasts as well as in osteoblastic cell lines, have a large number of potential ligands (48), so they represent a novel pathway by which many factors might impact on bone cell function.
Taken together, these data demonstrate that the naturally occurring glycoprotein, lactoferrin, is anabolic to bone in vivo, an effect that is consequent upon its potent proliferative, differentiating, and antiapoptotic actions in osteoblasts and its ability to inhibit osteoclastogenesis. Lactoferrin may therefore have a physiological role in bone growth. In addition, it is a potential therapeutic target in bone disorders such as osteoporosis and might have utility as a local agent to promote bone repair.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: FBS, Fetal bovine serum; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-B ligand; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
Received September 30, 2003.
Accepted for publication May 18, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) chain expression in peripheral blood T lymphocytes from cervical cancer patients. Anticancer Res 22:1897–1901[Medline]
levels in mice subjected to surgery. Arch Immunol Ther Exp (Warsz) 46:97–104[Medline]
This article has been cited by other articles:
 |
|
 |
|
  G. W. Krissansen Emerging Health Properties of Whey Proteins and Their Clinical Implications J. Am. Coll. Nutr., December 1, 2007; 26(6): 713S - 723S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cornish, K. E. Callon, U. Bava, M. Watson, X. Xu, J. M. Lin, V. A. Chan, A. B. Grey, D. Naot, C. M. Buchanan, et al. Preptin, another peptide product of the pancreatic beta-cell, is osteogenic in vitro and in vivo Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E117 - E122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R A Stein and D P McDonnell Estrogen-related receptor {alpha} as a therapeutic target in cancer Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S25 - S32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Naot, A. Grey, I. R Reid, and J. Cornish Lactoferrin - A Novel Bone Growth Factor Clin. Med. Res., May 1, 2005; 3(2): 93 - 101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Bonnelye and J. E. Aubin Estrogen Receptor-Related Receptor {alpha}: A Mediator of Estrogen Response in Bone J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3115 - 3121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Grey, T. Banovic, Q. Zhu, M. Watson, K. Callon, K. Palmano, J. Ross, D. Naot, I. R. Reid, and J. Cornish The Low-Density Lipoprotein Receptor-Related Protein 1 Is a Mitogenic Receptor for Lactoferrin in Osteoblastic Cells Mol. Endocrinol., September 1, 2004; 18(9): 2268 - 2278. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) or with vehicle (
) for the duration of the experiment. The expression level of RANKL (A and C) and OPG (B and D) were determined in triplicates using multiplex real-time PCR. The relative expression levels were normalized to the level of 18S rRNA. Data are mean ± SEM. *, Significantly different from control (P < 0.05). For some data points, error bars are not visible because they are smaller than the size of the symbol.
