![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 152-157
2-Macroglobulin Receptor in Human Mammary Epithelial Cells1,2
Department of Nutrition, University of California, Davis, CA 95616, U.S. Fax: (530) 752-8966 Tel: (530) 752-8347 E-mail: bllonnerdal{at}ucdavis.edu
 |
ABSTRACT |
|---|
Abstract
Introduction
Results
Discussion
References
|
|---|
Several cases of zinc (Zn) deficiency in human infants caused by abnormally low concentrations of Zn in breast milk were recently reported, the underlying mechanism of which is not known. Alpha2-macroglobulin (
2-M), a major Zn-binding ligand in serum, presents a potential vehicle for mammary Zn uptake. This study was conducted to determine if an 2-M receptor is present in human mammary epithelial cells, where it may be involved in the endocytosis of 2-M into the mammary gland. Normal human mammary epithelial cells were grown to confluency in serum-free medium. For all binding and uptake studies, 2-M, preactivated with methylamine and labeled with 125I, was added to cells for varied lengths of time to determine saturation over time and at varied concentrations to determine saturation over increasing concentration of ligand. Nonspecific and competitive binding were measured by addition of a 100-fold molar excess of unlabeled 2-M and serum albumin or lactoferrin, respectively. Binding at 4°C was specific for 2-M and approached saturation kinetics at 56 nmol/L. Scatchard plot analysis of the binding data demonstrated more than one binding site: a high affinity, saturable binding site and a low affinity, nonsaturable binding site. Uptake of 2-M at 37°C was rapid and continuous over increasing concentrations of 2-M, and internalized 2-M was rapidly degraded. Results from this study present evidence for receptor-mediated uptake of 2-M in human mammary epithelial cells, which in turn, provides a potential mechanism for Zn acquisition by the cell.
2-macroglobulin ·
2-macroglobulin receptor ·
mammary gland ·
humans
The importance of zinc (Zn) nutriture for normal growth and development of human infants and of human milk as a source of bioavailable Zn to the neonate was clearly shown by clinical studies of acrodermatitis enteropathica (AE)4 (Moynahan 1974 In humans, ~98% of plasma Zn is associated with albumin and Materials.
Normal human mammary epithelial cells and serum-free growth medium were obtained from Clonetics, (San Diego, CA). Cell culture supplies were obtained from Falcon, Becton Dickinson Labware (Franklin Lakes, NJ). Purified human plasma Culture of mammary epithelial cells.
Normal human mammary epithelial cells, obtained from mammoplasty, were grown to confluency in a 95% air-5% CO2 incubator at 37°C. Cells were purchased from a commercial supplier at passage number 7 and were maintained for 6-8 additional passages. Cells were cultured in 24-well plates in serum-free growth medium supplemented with epidermal growth factor (10 µg/L), hydrocortisone (0.5 mg/L), insulin (5.0 mg/L) and bovine pituitary extract (protein content 13 g/L). Cell viability was assessed by Trypan blue exclusion and was routinely determined to be greater than 95%. Confluency was determined by microscopic examination. Cell homogeneity was evaluated by cytokeratin determination.
Iodination of Measurement of binding and uptake of Cell permeabilization.
In order to differentiate between surface binding sites and intracellular binding sites for Fate of Binding of
Uptake and fate of
INTRODUCTION
Abstract
Introduction
Results
Discussion
References
, Nelder and Hambidge 1975). AE is a genetic disorder of Zn metabolism characterized by chronic dermatitis, infection, diarrhea, anorexia and failure to thrive. The efficacy of human milk in preventing the development of symptoms in AE infants and in ameliorating symptoms when they occur postweaning has led to the notion that breast-fed infants are protected from the development of Zn deficiency. However, a growing number of case studies now suggests that human milk may not always provide protection. Acquired Zn deficiency was recently reported in exclusively breast-fed infants due to low levels of milk Zn in otherwise healthy mothers (Ando et al. 1993, Atkinson et al. 1989, Bilinski et al. 1993, Buehnig and Goltz 1993, Connors et al. 1983, Heinen et al. 1995, Khoshoo et al. 1992, Kuramoto et al. 1986, Lee et al. 1990, Weymouth et al. 1982, Zimmerman et al. 1982). While infants responded rapidly to oral Zn therapy, attempts to increase milk Zn concentrations through maternal Zn supplementation failed to alter milk Zn in these women (Atkinson et al. 1989, Kuramoto et al. 1986, Moore et al. 1984, Zimmerman et al. 1982) and in clinical trials of dietary Zn modification and/or supplementation in lactating women (Kirksey et al. 1979, Krebs et al. 1995, Moore et al. 1984, Moser-Veillon and Reynolds 1990). The failure of nutritional modulation to affect milk Zn concentration suggests the regulation of mammary Zn. However, the mechanism involved in regulating Zn uptake into the mammary gland and Zn secretion into milk is not known. A possible defect in the underlying regulatory mechanism could be responsible for the low-milk Zn concentrations reported in cases of acquired Zn deficiency.
2-macroglobulin (2-M). Less than 2% of plasma Zn is associated with amino acids, of which histidine and cysteine predominate (Prasad and Oberleas 1970). Serum albumin binds Zn with relatively low affinity and in no particular stoichiometric ratio, while each molecule of 2-M binds four atoms of Zn with high affinity (Giroux 1975, Prasad and Oberleas 1970). The importance of these complexes as sources of cellular Zn is poorly understood. Although prior investigations attempted to clarify the role of albumin in cellular Zn uptake, these studies failed to define its involvement. Several in vitro studies suggest that albumin may actually have a negative effect on Zn uptake in certain cell types (Page et al. 1992, Patterson et al. 1991, Taylor and Simons 1994). Furthermore, no specific cellular binding sites were determined for albumin. Although Zn may be taken up by the mammary cell in association with amino acid uptake, it is unlikely that this process would be regulated. In addition, the low proportion of Zn associated with amino acids in plasma makes this an unlikely regulatory mechanism for mammary Zn uptake. Because of its high affinity for Zn and its abundance in plasma, 2-M presents a potential vehicle for regulation of Zn uptake into the mammary gland.
2-M is a 720 kDa, multifunctional, plasma protein that is involved in lipoprotein metabolism and in the inhibition of a wide range of serum proteases as well as in Zn transport (Borth 1992). A receptor for 2-M (2-MR) was isolated, purified and characterized in hepatocytes and in trophoblasts (Jensen et al. 1989, Moestrup and Gliemann 1989). Although the 2-MR is reportedly expressed in a wide spectrum of noncarcinogenic cell types (Herz et al. 1988, Moestrup et al. 1992) as well as in several carcinogenic cell types (Li et al. 1997, Li et al. 1998), whether the receptor is expressed in the mammary gland is unknown. The objective of this study was to determine if an 2-MR is present in normal human mammary epithelial cells.
EXPERIMENTAL PROCEDURES
2-M was purchased from Biodesign International (Kennebunk, ME). Human serum albumin, lactoferrin and methylamine were obtained from Sigma Chemical Company (St. Louis, MO). Carrier-free Na-125I was purchased from Amersham Life Sciences Inc. (Arlington Heights, IL). Iodo-Gen was purchased from Pierce Chemical Co. (Rockford, IL).
2-M.
Prior to iodination, purified human 2-M was activated from its native, nonreceptor recognized form to its protease/methylamine modified, receptor recognized form using the method described by Imber and Pizzo (1981). Hereafter, 2-M refers to the methylamine modified form. 2-M (100 µg) was labeled with 125I (250 µCi) in phosphate buffered saline (PBS) to a final volume of 1 mL in a glass vial coated with 100 µg Iodo-Gen (1,3,4,6-tetrachloro-3-6-diphenylglycouril). The iodination proceeded for 10 min at room temperature. After 10 min, the reaction solution was removed from the vial and run through a Bio-Gel P-6 column that had been equilibrated with PBS (pH 7.0). The specific activity for the 125I-labeled 2-M was ~1000 cpm/ng.
2-M.
When confluency was attained, cells were incubated with 125I-labeled 2-M for varied lengths of time (ranging from 30 min to 4 h) for determination of saturation over time and at varied concentrations (ranging from 1.75 to 56 nmol/L) for determination of saturation over increasing concentrations of ligand. All variables within a study were done in triplicate and each study was repeated at least three times. Cells were incubated at 4°C for all binding studies and at 37°C for all uptake studies. Nonspecific binding was assessed in the presence of a 100-fold molar excess of unlabeled 2-M. Competitive binding assays were performed in the presence of a 100-fold molar excess of unlabeled serum albumin or unlabeled lactoferrin.
2-M. Separate wells of nonradiolabeled mammary cells were counted by hemocytometry for estimation of cell number. Analysis of the binding data was done by Scatchard plot (Scatchard 1949).
2-M, confluent cells were incubated at 4°C. The culture medium was removed and replaced with 0.5% saponin in fresh medium (0.5 mL) for 25 min. Following the treatment period, the detergent was removed and cells were incubated with new medium containing the radiolabeled ligand. Binding was then measured as described above.
2-M.
To determine the fate of cell-associated 125I-labeled 2-M in human mammary epithelial cells, internalization and degradation studies were conducted. Uptake of 2-M at 37°C was allowed to proceed for 4 h as previously described. Cells were washed three times with PBS, collected with a sterile cell scraper and frozen in 300 µL of PBS at 
20°C. The cell extract was lysed by thawing in a 37°C water bath and refreezing in an ethanol-dry ice bath. After repeating the freeze-thaw procedure five times, the sample was centrifuged to remove insoluble matter. The supernatant was collected, concentrated using a microconcentrator and subjected to gel electrophoresis. The gel was stained, destained and dried between minicellophane paper. Intracellular processing of 2-M was detected by autoradiography.
RESULTS
Abstract
Introduction
Results
Discussion
References
2-M at 4°C.
Studies were conducted to characterize the binding kinetics of 125I-labeled 2-M to normal human mammary epithelial cells. As binding kinetic studies are based on the premise that unbound ligand is essentially removed during the washing procedures while surface-bound ligand remains cell-associated, preliminary studies were done to ensure that these assumptions held true under our experimental conditions. Results (data not shown) verified the following critical assumptions: >98% of unbound radioactivity was removed by the initial PBS wash; the remaining cell-associated counts did not change significantly after the third wash; and a final wash with 1% SDS was effective in removing the cell-associated ligand for analysis of radioactivity and determination of ligand binding.
2-M to human mammary epithelial cells reached equilibrium after 3 h (Fig. 1). All follow-up binding studies were carried out at 4 h to ensure that equilibrium had been attained. The concentration-dependent binding studies are shown in Fig. 2. The specific binding curve indicates that as increasing concentrations of ligand were added, saturation kinetics were attained. Specific binding was defined as total binding (the amount of 125I-labeled 2-M bound) minus nonspecific binding (the amount bound in the presence of a 100-fold molar excess of unlabeled 2-M). Scatchard analysis of the binding data (Fig. 3) yielded a nonlinear plot suggesting the presence of both a high-affinity, saturable binding site (Fig. 4A) and a low-affinity, nonsaturable binding site (Fig. 4B). The apparent dissociation constant (Kd) of the high-affinity binding site was 5.4 pmol/L, whereas the Kd of the low-affinity binding site was 4.1 nmol/L.
View larger version (10K):
[in a new window]
Fig 1.
Binding of 125I-labeled 2-macroglobulin (2-M) to normal human mammary epithelial cells at 4°C over increasing time. Each point represents the mean ± SD, n = 9.
View larger version (19K):
[in a new window]
Fig 2.
Specific binding of 125I-labeled 2-macroglobulin (2-M) to normal human mammary epithelial cells at 4°C. Each point represents the mean ± SD, n = 9.
View larger version (7K):
[in a new window]
Fig 3.
Scatchard plot analysis of binding of 125I-labeled 2-macroglobulin (2-M) to normal human mammary epithelial cells at 4°C. Each point represents the mean of nine values derived from three studies repeated in triplicate.
View larger version (9K):
[in a new window]
Fig 4.
Nonlinear Scatchard plot analysis of 2-macroglobulin (2-M) binding to normal human mammary epithelial cells. A) High affinity binding; B) low affinity binding. Each point represents the mean ± SD, n = 9.
2-M to human mammary epithelial cells, nonspecific binding studies were conducted using a 100-fold molar excess of unlabeled 2-M while competitive binding studies were conducted using either a 100-fold molar excess of unlabeled human serum albumin or unlabeled lactoferrin. As shown in Table 1, binding of 125I-labeled 2-M to human mammary epithelial cells was reduced by ~81% in the presence of excess unlabeled 2-M. In contrast, the 100-fold molar excess of unlabeled human serum albumin and lactoferrin led to only small reductions in the cell-associated 2-M.
View this table:
Table 1.
Effect of various unlabeled proteins in excess on 125I-labeled 2-macroglobulin binding to normal human mammary epithelial cells1
2-M. No differences were noted in the binding of 2-M to permeabilized and nonpermeabilized cells, hence indicating the absence of intracellular binding sites.
2-M at 37°C.
Using the same methods as described for binding studies, uptake of 2-M by mammary epithelial cells was analyzed at 37°C (Fig. 5A and B). Results of time-dependent studies (Fig. 5A) demonstrated that uptake of 2-M occurred most rapidly within the first hour and plateaued after 3 h. In addition, data from concentration-dependent studies (Fig. 5B) indicated that there was a continuous uptake of radiolabeled 2-M by the epithelial cells with increasing concentrations of added ligand. Autoradiography from internalization studies demonstrated intracellular processing of 125I-labeled 2-M following uptake and endocytosis into the mammary cell.
View larger version (11K):
[in a new window]
Fig 5.
Uptake of 125I-labeled 2-macroglobulin (2-M) to normal human mammary epithelial cells at 37°C. A) effect of time B) effect of ligand concentration. Each point represents the mean ± SD, n = 9.
DISCUSSION
Abstract
Introduction
Results
Discussion
References
2-M is a major plasma Zn-binding ligand in humans. Yet, despite the high affinity of 2-M for Zn, little is known about the role of 2-M in cellular Zn uptake, particularly with regard to mammary gland Zn metabolism. The failure of maternal Zn supplementation to alter milk Zn concentration in cases of acquired Zn deficiency in exclusively breast-fed infants and in recent clinical supplementation trials suggests that regulation of mammary Zn uptake occurs. However, the underlying mechanism involved in the regulation of Zn uptake into the mammary gland has not previously been determined. Receptor-mediated uptake of 2-M by the mammary gland, as shown by the results of this study, provides a potential mechanism for Zn acquisition by the cell and for regulation of Zn uptake into the mammary gland.
2-M by normal human mammary epithelial cells. Binding of 125I-labeled 2-M at 4°C was both saturable and specific, consistent with ligand binding to cell surface receptors. Specific binding reached equilibrium after 3 h of incubation which is similar to what has been shown for binding of 2-M to other cell types (Moestrup 1994, Petersen 1993). Binding approached saturation at a concentration of ~56 nmol/L and was not significantly affected by addition of either excess unlabeled albumin or lactoferrin. Although the 2-MR is known to bind lactoferrin, recent findings of lactoferrin binding to the 2-MR showed binding to occur at a site distinct from that of 2-M, thus not favoring competition between the two ligands (Hüettinger et al. 1992).
2-M reported linear Scatchard plots, most kinetic analyses of 2-M binding yielded curvilinear plots supporting the possibility of both high- and low-affinity receptor binding sites (Dickson et al. 1981). However, interpretation of 2-M binding kinetics is somewhat controversial and is complicated by the possibility of receptor aggregation and by the multivalent nature of 2-M (Petersen 1993). Results from the current study suggest the existence of both high- and low-affinity binding sites in mammary epithelial cells. The apparent Kd of 5.4 pmol/L for the high-affinity binding site and of 4.1 nmol/L for the low-affinity binding site is of similar magnitude to the Kd reported for other cell types (Moestrup 1994, Petersen 1993). The estimated cell-associated receptor number (22 × 10
5 binding sites/cell) is also within the range reported for other types of cells. Saponin-permeabilized cell studies of 2-M binding did not indicate the presence of intracellular binding sites as binding did not differ significantly between nonpermeabilized and saponin-permeabilized cells.
2-M were well characterized in a variety of cell types. Prior studies demonstrated that uptake of 2-M follows the same pathway in all receptor-bearing cells investigated (Petersen 1993). The continuous uptake of 2-M at 37°C over increasing time and concentration of added ligand, as well as the intracellular processing of 2-M noted in the studies described herein, suggest a similar mechanism of receptor-mediated internalization and degradation of 2-M by mammary epithelial cells. Although Christensen et al. (1996) did not find 2-M in their cells from breast carcinomas, which is in contrast to our findings, several authors have documented the presence of such receptors in breast cancer cells (Li et al. 1997, Li et al. 1998). Receptor expression seemed to vary with both cell type and stage. When comparing carcinoma and noncarcinoma cells, evidently 2-M receptor expression is lower or absent in carcinoma cells (Gonias et al. 1994, Van Leuven et al. 1979), making such cell lines inappropriate for our studies.
2-M in human mammary epithelial cells. However, due to methodological problems, we were unable to execute these studies to completion. While we were successful in stripping the native Zn from 2-M, we were unable to reincorporate 65Zn into the 2-M molecule. Possibly incorporation of Zn only occurs during biosynthesis and apo-2-M has a conformation which does not allow binding of Zn. To our knowledge, no reported studies exist of 65Zn-labeled 2-M in the literature, which may be explained by the difficulty in incorporating 65Zn into 2-M. However, as 2-M always binds four atoms of Zn, apparently these atoms of Zn will be internalized into the cell with 2-M and will be released when 2-M is degraded intracellularly.
2-M has ~20 binding sites for Zn, with four binding sites having considerably higher affinity for Zn (Giroux 1975). Zn bound to the high-affinity sites of 2-M is inevitably transported via the plasma to different tissues bearing 2-MR for potentially varied functions. Although the function of 2-MR in the mammary gland is not known, results from the current study suggest that 2-MR-mediated uptake of Zn by the mammary gland is a likely mechanism to provide Zn to the mammary gland for milk synthesis. To strengthen this hypothesis, we calculated the potential physiological "drain" on the pool of 2-M within the body that would be required to maintain milk Zn concentrations. Assuming an average maternal Zn requirement of 1 mg/d solely for the purpose of milk synthesis as determined by Krebs et al. (1994), 2.77 mg/d of 2-M would be needed to supply the necessary Zn. Since the human body contains ~4-5 g of 2-M/L of plasma (Petersen 1993), 2-MR-mediated uptake of Zn would not represent a physiologically "expensive" means of providing Zn to the mammary gland. While 2-M is known to be a multifunctional protein, the array of its metabolic functions is seemingly not yet fully understood. However, it is likely that its critical role in protease inhibition and in combating infection takes precedence over other metabolic functions of 2-M. Thus, during systemic inflammation or infection, 2-M may be directed toward tissues other than the mammary gland. The presence of maternal, subclinical infection could therefore result in low concentrations of milk Zn as reported in cases of acquired Zn deficiency in exclusively breast-fed infants. In our previous study on marginal Zn deficiency in rats during pregnancy and lactation, we found that Zn was redirected toward the liver of deficient dams rather than to the mammary gland and milk (Beshgetoor & Lönnerdal 1997). The existence of a prior marginal Zn deficiency in the mothers with low milk Zn can not be ruled out, and it is possible that Zn supplementation could not overcome this effect. Evidently, further studies are needed to clearly establish the role of 2-M and the 2-MR in Zn metabolism and in the regulation of Zn uptake by the mammary gland.
| |
FOOTNOTES |
|---|
2-M, 2-macroglobulin; 2-MR, 2-M receptor; PBS, phosphate buffered saline; Zn, zinc; SDS, sodium dodecyl sulfate.
Manuscript received 14 January 1998. Initial reviews completed 27 March 1998. Revision accepted 15 October 1998.
| |
ACKNOWLEDGMENT |
|---|
The authors acknowledge the helpful suggestions by Suhasini Iyer.
| |
LITERATURE CITED |
|---|
|
Abstract
Introduction
Results
Discussion
References
|
|---|
2-macroglobulin, a multifunctional binding protein with targeting characteristics.
FASEB J.
1992;
6:3345-3353[Abstract]2-macroglobulin receptor in human breast carcinomas.
Int. J. Cancer
1996;
66:441-452[Medline]2-macroglobulin by cultured fibroblasts.
J. Biol. Chem.
1981;
256:3454-34592-Macroglobulin and the 2-macroglobulin receptor/LRP: a growth regulatory axis.
Ann. N.Y. Acad. Sci.
1994;
737:273-290[Medline]2-macroglobulin.
J. Biol. Chem.
1992;
267:18551-185572-macroglobulin receptor expression in breast cancer cell T-47D.
Anticancer Res.
1998;
18:1197-1202[Medline]2-macroglobulin receptor/low density lipoprotein receptor-related protein is cell culture density-dependent in human breast cancer cell line BT-20.
Biochem. Biophys. Res. Commun.
1997;
240:122-127[Medline]2-macroglobulin receptor and epithelial glycoprotein-330: two giant receptors mediating endocytosis of multiple ligands.
Biochim. Biophys. Acta
1994;
1197:197-213[Medline]2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues.
Cell Tissue Res.
1992;
269:375-382[Medline]2-macroglobulin and pregnancy zone protein.
Dan. Med. Bulletin
1993;
40:409-4462-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell-lines.
J. Biol. Chem.
1979;
254:5155-5160This article has been cited by other articles:
|
|
 |
|
  M. Moriya, Y.-H. Ho, A. Grana, L. Nguyen, A. Alvarez, R. Jamil, M. L. Ackland, A. Michalczyk, P. Hamer, D. Ramos, et al. Copper is taken up efficiently from albumin and {alpha}2-macroglobulin by cultured human cells by more than one mechanism Am J Physiol Cell Physiol, September 1, 2008; 295(3): C708 - C721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hollborn, G. Birkenmeier, A. Saalbach, I. Iandiev, A. Reichenbach, P. Wiedemann, and L. Kohen Expression of LRP1 in Retinal Pigment Epithelial Cells and Its Regulation by Growth Factors Invest. Ophthalmol. Vis. Sci., June 1, 2004; 45(6): 2033 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Kelleher and B. Lonnerdal Zinc Transporters in the Rat Mammary Gland Respond to Marginal Zinc and Vitamin A Intakes during Lactation J. Nutr., November 1, 2002; 132(11): 3280 - 3285. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
