Fetuin:

its enigmatic

property

of growth promotion

ZETAN NIE Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115 Nie, Zetan. Fetuin: its enigmatic property of growth promotion. Am. J. Physiol. 263 (Cell Physiol. 32): C551C562, 1992.-A variety of cell types in culture respondto fetuin, a glycoprotein from fetal bovine serum,which is often an important supplementto many serum-freemedia. Bovine fetuin preparation hasbeenshownto inhibit trypsin activity and promote cellular attachment, growth, and differentiation in many different culture systems. In addition, fetuin associateswith various growth factors or growth-promoting substances.However, whether the growth-promoting activity of fetuin preparation is due to fetuin per seor to its minor contaminant(s) has beena long-standingpuzzle. The present review surveys the literature concerning this enigmatic property of fetuin and summarizesthree possibilities: 1) fetuin itself is active, although the majority of studiesdo not support this; 2) various contaminants of fetuin preparations, including potentially unidentified ones, are responsiblefor the activity, a possibility supported by numerousreports; and 3) one of the fetuin subspecies,one of its contaminants, or a combination of both of these is responsiblefor growth of a specific cell type. In addition, the basic physicochemicalproperties and other biological functions of fetuin have alsobeenpresented. cell culture; contaminant; glycoprotein; growth factor; growth and differentiation; serum; serum-freemedia

was first identified and termed by Pedersen in fetal calf serum almost fifty years ago. Fetuin is a major component of fetal serum and comprises ~45% of the total serum proteins (27, 46). It is an acidic glycoprotein and is heterogeneous in molecular weight and isoelectric point (PI) (10, 51, 85, 87, 102, 103). Fetuin is found in the earliest fetuses examined. During development, the concentration of fetuin increases to -10-22 mg/ml at 3- to 9-mo gestations (13) and then rapidly decreases at birth to a level of -0.5 mg/ml in the adults (3,7, 13,76). Fetuin also exists in both allantoic and amniotic fluids (76, 93) as well as in cerebrospinal fluid (32, 68). Fetuin (or fetuin-like proteins) has also been found in at least five mammalian orders (Artiodactyla, Primates, Rodentia, Carnivora, and Perissodactyla), including lamb (3,86), pig (32,93), rat, human, and other species (30). Fetuin is, in fact, the bovine homologue of human QHS glycoprotein and may be related to the cystatin superfamily (21, 31). Fetuin, like many other secretory proteins, is synthesized in liver with an 18-amino acid signal peptide on membrane-bound polyribosomes (69)) and it is degraded through the lysosomal pathway (75, 132). Bovine fetuin can be prepared by three major methods from fetal calf serum. The first isolation of fetuin was described by Pedersen (104, 105), in which the serum was first diluted with an equal volume of 0.2 M sodium chloride and then precipitated by ammonium sulfate. The precipitate between 37 and 45% saturated ammonium sulfate was washed twice with 50% saturated amFETUIN

(104) as an a-globulin

0363-6143/92

$2.00

Copyright

monium sulfate and centrifuged down. This fetuin preparation (referred to hereafter as Pedersen fetuin) was a rather crude fraction and contained several minor components that can be readily detected by gel electrophoresis (72, 84, 107). Deutsch (27) found that fetuin was soluble in 5% trichloroacetate buffer (pH 3.5) and used a combination of 5% sodium trichloroacetate precipitation at pH 3.5, 40% saturated ammonium sulfate precipitation at pH 7, and fractionation between 15 and 50% (vol/vol) cold ethanol to prepare a purer fetuin. However, this preparation still contained a small amount of a high-molecular-weight contaminant but was otherwise homogeneous on electrophoresis at neutral pH (27). A few years later, Spiro (125) employed a cold ethanol precipitation method to obtain a fetuin preparation that behaved as a single component in the ultracentrifuge and on electrophoresis over a pH range of 1- 11. This method involved sequential precipitations with ethanol and heavy metals of zinc and barium at -5 to -1OOC for various time intervals. Fetui n was p recipitated out between 25 and 40% ethanol at pH 6.7 and dialyzed against distilled water for several days at 4°C to remove the zinc and barium ions. Pedersen fetuin has long been known to possess growth-promoting activity. Since Fisher et al. (37) first demonstrated that fetuin promoted attachment of HeLa cells, numerous reports in the literature have illustrated its ability to stimulate the attachment and proliferation of a wide variety of cell types in culture (described below). However, highly purified fetuin prepared by

0 1992 the American

Physiological

Society

c551

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

C552

INVITED

Spiro’s or Deutsch’s method is biologically inactive in most growth assays (36, 50, 84, 109, 117). Thus the existing question is whether the growth-promoting activity of fetuin preparation resides in fetuin itself or in some minor contaminant(s). The present review is a survey of studies concerning this enigmatic growth-promoting property of fetuin. For a better understanding, the basic physicochemical properties of fetuin are discussed first. Finally, other biological functions of fetuin are also enumerated for the purpose of reference. BASIC CHEMICAL AND OF BOVINE FETUIN

PHYSICAL

PROPERTIES

The basic physicochemical properties of bovine fetuin are summarized in Table 1, and its chemical composition is listed in Table 2. Fetuin is a relatively small acidic glycoprotein compared with other serum proteins. The small variations in the parameters listed in Table 1 may result from different preparations and different analytic methods employed. However, the variation in molecular weight and p1 primarily reflects the microheterogeneity of fetuin and might result from differences in glycosylation as well as in the protein structure of fetuin (10, 14, 51, 68, 85, 87, 102, 103). The acidic nature of bovine fetuin is mainly due to the sialic acids because removal of -95% of sialic acids from fetuin raises the p1 up to 5.23 (10). The coefficients of sedimentation and diffusion of bovine fetuin vary markedly as the concentration changes (27, 105, 125). The sedimentation coefficient is independent of pH above the p1, but, at or below the p1 value, the measured sedimentation coefficient increases (27, 125, 134) and reaches a maximum value at pH -2.5 (47). Fetuin is soluble in water, 35% saturated ammonium sulfate, 7% trichloroacetic acid, 1.2 M perchloric acid, 25% cold ethanol, and 25% polyethylene glycol (PEG) but can be precipitated out by 5% phosphotungstic acid in 2 M HCl (15, 27, 83, 125). Deutsch (27) first reported the presence of carbohydrate in bovine fetuin and reported values of 8% glucosamine, 9.5% mannose, and a positive reaction for sialic acid with diphenylamine. Subsequently, it was shown that the bovine fetuin molecule contains ~78% protein (36,124), with carbohydrate contributing ~22% with a total of 52 sugar residues in the form of sialic acid, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine (125, 127). Bovine fetuin has three N-linked and also three O-linked carbohydrate side chains. It is well establ shed that the N-glycosylation of glycoproteins , inc luding fetuin, occurs in rough endoTable 1. Basic phys ‘icochemicalproperties of bovzne fetuin Value

Molecular mass, Da Isoelectric point, pH

43,360-54,300 3.3-4.32 4.78-6.23

Electrophoretic mobility,

5.16 (at pH 6.85) 5.6 (at pH 8.6)

Ref. No.

10, 27, 47, 105, 125, 134, 10, 27, 102, 105, 125, 134,

(asialo-fetuin) 10-s cm2. V-1.

47 125

s-1

so , w (Svedberg) 2.70-3.55 27, 47, 102, 105, 125, 134, @, , w, low7 cm2/s 4.97-5.73 27, 105, 125 S& and DgO,coefficients of sedimentation and diffusion, respectively. w, water .

REVIEW

Table 2. Chemical composition of bovine fetuin Average pmol/lOO Ref.

Amount, mg protein

124

Aspartic acid 68.1 Threonine 51.4 Serine 54.1 Glutamic acid 70.0 Proline 69.6 Glycine 50.0 Alanine 69.1 Valine 83.2 Isoleucine 30.8 Leucine 55.4 Tyrosine 14.0 Phenylalanine 22.4 Lysine 34.0 Histidine 21.1 Arginine 24.3 Half-cystine 25.0 Tryptophan 4.6 Amide N 49.2 Sialic acids 8.20* Hexosamines 7.20* Galactose 4.15* Mannose 2.70* * Expressed as g/100 g protein.

Residues/Molecule

Ref. 36

Ref. 124

Ref. 36

60 36 46 69 78 41 62 73 27 62 17 21 35 24 35 40 4.2

33.0 24.9 26.2 33.9 33.7 24.2 33.4 40.3 14.9 26.8 6.8 10.8 16.5 10.2 11.8 12.1 2.2 23.8 13.6 17.1 12.4 8.1

26.8 16.0 20.6 30.8 34.9 18.3 27.7 32.6 12.1 27.7 7.6 9.4 15.6 10.7 15.6 17.9 1.9

30.1

13.5

plasmic reticulum with the cotranslational transfer of a core oligosaccharide unit from a lipid carrier to asparagine residues (53). 0-glycosylation of fetuin was recently found to be a posttranslational event taking place in the Golgi apparatus (69). A biochemical study revealed that as many as 35 fractions of asparagine-linked oligosaccharides of fetuin (after hydrazinolysis/re-N-acetylation/ mild acid treatment of pronase-digested bovine fetuin) could be detected by two different high-performance liquid chromatography (HPLC) systems (11). These fractions contained di-, tri-, tetra-, and pentasialylated oligosaccharides. Three triantennary structures having sialic acids linked only to galactose residues have been elucidated (11). The protein portion of bovine fetuin was identified as a single peptide chain of 361 amino acid residues with six intrachain disulfide bonds (124). Methionine was not detected by Spiro and Spiro (124) but was identified by Fisher et al. (36) at a low concentration (1.2 hmol/lOO mg protein). It was recently confirmed by direct sequencing that native bovine fetuin does not contain methionine (31). This discrepancy might have been caused by some prefetuin contamination in the fetuin preparation used by Fisher et al. (36), because methionine is the only NH2terminal residue in prefetuin (3 1,68). Isoleucine occupies both NH2- and COOH-terminals of fetuin (31, 126). Bovine fetuin and its complete cDNA have been sequenced, and the deduced amino acid sequence was identical with that obtained from direct amino acid sequencing, with the number of amino acid residues in protein part of bovine fetuin confirmed to be 341 (21,31). The Southern analysis with the bovine fetuin cDNA probe on the bovine genomic DNA digested by both BgZ II and EcoR I restriction enzymes showed that there was only one hybridization band, suggesting that bovine fetuin is probably encoded by a single gene (31) .

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

INVITED GROWTH-PROMOTING FETUIN PREPARATIONS

PROPERTY

Fetuin Preparations Promote Growth, and Differentiation

OF

BOVINE

Cell Attachment,

Many types of normal cells and nontransformed cell lines, as well as transformed cells, respond to fetuin preparation in culture. These cell types are listed in Table 3. Generally speaking, bovine fetuin preparations promote cell attachment, growth, and differentiation in many different culture systems. It should be noted that the term “growth-promoting activity of fetuin preparations” is sometimes used in a broad sense, implying all three aspects of the activity. Attachment. The fetuin activity was first demonstrated by Fisher et al. (37) on the S3 strain of HeLa cells for promoting cell attachment and stretching (flattening) on the glass. The HeLa cell line was derived from a human cervical carcinoma, and during passage in culture they exhibited an increase in epithelium-like properties (44). Table 3. Cell types stimulated

Normal cells Blastocysts Brain cells Cardiac cells

Mouse Rat Rat

Epithelia

Rat cells

Lymphocytes

Mouse Mouse Rat Monkey Human

Phagocytes Smooth muscle cells Spermatogonia

Human Rat Mouse cow Mouse

Spleen cells

Mouse

Myoblasts

Cell lines CHO (ovary) HeLa (epithelia)

Hamster Human Human

HMT-3909Sl HMT-3909S8

A year later, this attachment activity of fetuin was reported again in a serum-free medium containing defined micromolecular constituents plus serum albumin (38). In this medium, HeLa S3 cells could not attach in the absence of fetuin and albumin or in the presence of either one alone. However, the plating efficiency was dramatically increased to almost 100% when both fetuin and albumin were added. Subsequently, other investigators also reported the attachment and growth-promoting effect of bovine fetuin preparation for other cell types. For example, Matsuya (90) demonstrated this fetuin activity for L cells (fibroblasts). Anchorage is one of the critical requirements of normal cells and nontransformed cell lines to be able to grow in culture. Thus promoting cell attachment is often an important function of the fetuin activity. Growth. As shown in Table 3, the growth of a variety of mammalian cells from different species is stimulated by fetuin prepa ratio ns. This includes most cell types grown

by bovine fetuin preparation Species

Hematopoietic Kidney cells

c553

REVIEW

Fetuin

Type

P Unknown D P Unknown P P P P P P S wm P D P P P P s/p

Concentration

Ref. No.

500 pg/ml

110

0.5-5 pg/ml 2.5 mg/ml 250 pg/ml

88 23 96 71 117 116 65 29 116 135 61 62 50 4 140 81 52

1 mg/ml 1.0 mg/ml >l mg/ml* 1 mg/ml In vivo dose? 1 mg/ml* 1 mg/ml 4-8 mg/ml 4 mg/ml 500 pg/ml 500 pg/ml l-4 mg/ml l-10 mg/ml 5 mg/ml 5-10 mg/ml 0.2-0.8 mg/ml

P P P P P

50- 100 pg/ml 0.4-2 mg/ml 30-960 pg/ml 50- 100 pg/ml 20 dml

P P m/s

50- 100

100 18 107 37,38 84

107 106

(breast carcinoma)

L (fibroblasts) L6 (myoblasts) MRC -5 (fibroblasts) Ob 17 (adipogenic) Opossum kidney cells (epithelia) 1246 (adipogenic) OTT-6050 (embryonal carcinoma)

Mouse Rat Human Mouse Opossum Mouse Mouse

Unknown P Unknown P P P Rat PA-III (prostate adenocarcinoma) Unknown P PCC.4 aza-1 and Fg (embryonal carcinoma) Mouse Hamster Syrian hamster fibroblast P P 3T3-Ll (adipogenic) Mouse Monkey P Vero (kidney) Unknown P, Pedersen fetuin; D, Deutsch fetuin; S, Spiro fetuin. * Approximate equivalence to unfractionated was given by injection before analysis with hematopoietic cells.

1 mg/ml pg/ml

1 mg/ml 1 mg/ml 500 pg/ml l-2 mg/ml 500 pg/ml 1 mg/ml 1 mg/ml* 150 pg/ml 500 pg/ml 2.5 mg/ml 300 pg/ml l-10 mg/ml 1 mglml fetuin preparation. t Fetuin (20

90 107 40, 41 22 42,98 80 141 115 116 20

109 91 122 33 22

mg/mouse)

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

c554

INVITED

in culture: adipogenic cells, epithelia, fibroblasts, hematopoietic cells, lymphocytes, myoblasts, spermatogenic cells, and so on. Fetuin is often required for optimum growth for cells that can be grown under serum-free conditions. The optimum concentration of fetuin for maximum growth is usually from 100 pg to a few milligrams per milliliter among different cell types. The majority require -0.5-l mg fetuin/ml for optimum growth (Table 3) . The growth-promoting effect of fetuin does not necessarily result from its enhancement of cell attachment, although this could be true for some of the cell types or culture systems listed in Table 3. The attachment requirement of normal cells and nontransformed cell lines can be satisfied in vitro by special treatment of the surface of the culture dishes, by coating dishes with collagen or other extracellular matrices, or by adding these substances directly into the medium. For example, fibronectin, an extracellular matrix protein (138), can promote attachment of cardiac and skeletal muscle and other cells, but fetuin is still required for optimum growth of these cells (4, 23, 28, 99, 115). We found that the myogenic cell line L6 could grow well in medium MCDB 120 plus serum-free supplement SF [dexamethasone, epidermal growth factor (EGF), fetuin, insulin, and serum albumin], which was specifically developed for clonal growth of normal human skeletal muscle satellite cells (50), but they failed to attach in MCDB 120 plus SF minus fetuin. Coating dishes with calf collagen or adding fibronectin into the medium could make the L6 cells attach initially in the absence of Pedersen fetuin; however, they stopped growing and detached again in another 24 h (Ref. 99; Z. Nie and R. G. Ham, unpublished observations). Thus the growth-promoting activity of fetuin is clearly separable from its attachment function. Differentiation. In addition to attachment and growthpromoting effects, fetuin aiso demonstrates an ability to stimulate differentiation of some cell types. Rizzino and Sherman (110) reported that fetuin stimulated development and differentiation of mouse blastocysts in a serumfree medium. In the presence of fetuin, hatching, attachment, and outgrowth of the blastocysts could all occur to give rise to cells resembling parietal endoderm cells, but this could not happen with transferrin or insulin alone. Both fetuin and bovine serum albumin together were required for these events to occur. Haneji et al. (52) observed that Pedersen type III fetuin with folliclestimulating hormone plus insulin and transferrin synergistically stimulated in vitro differentiation of type A spermatogonia. However, Deutsch fetuin, Spiro fetuin, and type IV Pedersen fetuin were found to be inactive. Nishimune et al. (100) also reported this stimulatory effect of fetuin preparation during spermatogenesis. Fetuin preparation also stimulates growth and adipose conversion of Ob 17 adipogenic cell line in a serum-free medium, and this effect of fetuin cannot be replaced by other factors, such as EGF, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) (42). Some cell types respond mitogenically to several growth factors, including EGF, by producing collagen. A fraction of Pedersen fetuin has been shown to stimulate collagen pro-

REVIEW

duction of normal rat kidney cells and rat mammary epithelium cells (116). Another report indicated that fetuin and other serum proteins could enhance the aggregate formation of fetal rat brain cells leading to elongated neurites on a collagen gel matrix (88). There is also a report demonstrating in vivo effect of fetuin. Fetuin (20 mg) was injected into a mouse, and the colony-forming units of hematopoietic precursors from irradiated mice were increased compared with controls (29) It’should be noted that the requirement for fetuin in some tissue culture systems turned out not to be specifitally for the growth-promoting activity of fetuin. However, when the actu .a1 requirement of the cells was satisfied by adding the specific substances, fetuin was still needed for optimum growth by many other cells. For example, in most early cell culture experiments, including the original cell culture study on fetuin (37), the trypsin used to disperse cells was not completely removed. This had little effect in serum-containing media, but for serum-free growth, fetuin, which is a potent trypsin inhibitor (37,43,79), or other trypsin inhibitors must be used. Thus neutralization of trypsin action appeared to be one of the major roles of fetuin under such conditions (56, 135). However, subsequently, in more controlled studies, when either the cells were washed and diluted carefully with the medium or a trypsin inhibitor such as soybean trypsin inhibitor was used, fetuin was still required for the cell growth (33, 50, 135). Ham (48, 49) also reported that the fetuin requirement for serum-free clonal growth of Chinese hamster ovary cells could be substituted by addition of putrescine and related amines, but these polyamines cannot replace fetuin for mouse L cells (90) or for human muscle satellite cells (50, 99). Type of fetuin preparation. Pedersen fetuin is a rather crude fetal bovine serum (FBS) fraction, Spiro fetuin is the purest preparation commercially available so far, and Deutsch fetuin is something in between. Thus the purity of the preparation has contributed to the mystery of the growth-promoting property of bovine fetuin, and it is worthwhile to examine what preparation of fetuin was used in those culture systems. As seen in Table 3, Pedersen fetuin preparation was the most widely used type as a cell attachment and growth-promoting substance. In these systems, neither Spiro fetuin nor Deutsch fetuin was active, and even the Pedersen fetuin type IV was not active compared with the Pedersen fetuin type III in some of these systems (52). It should be pointed out that a fetuin preparation used in some culture systems in Table 3 was prepared from fetal calf serum by a slightly modified Pedersen method that gave a higher yield of material with no sacrifice in the fetuin activity (37, 38). Thus this preparation has been classified as Pedersen type. On the other hand, Spiro and Deutsch fetuin preparations have been demonstrated to be active for cardiac cells (23), lymphocytes (61,62), and rat L6 myogenic cells (41), but there are not many such reports compared with those showing the fetuin activity from Pedersen type. Possible mechanism of fetuin activity. The mechanism by which fetuin promotes cell attachment, growth, and

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

INVITED

differentiation is unknown, although a number of suggestions have been made. It has been suggested that the binding of fetuin to the cell membrane might be the first step of its growth-promoting function in hepatoma cells (73) and probably is followed by internalization as shown in Sertoli cells and spermatocytes (1). It has also been suggested that the cell attachment and growth-promoting activity of fetuin might be mediated via inhibition of the residual trypsin activity in culture media and inhibition of other cellular proteases (37, 56, 135), because fetuin is a potent inhibitor of trypsin (37,43, 79) and other serine proteases (111). It has also been reported that fetuin might behave as a source of contaminant growth factors and mitogenic substances (discussed in detail below). Fetuin may promote cellular attachment, growth, and differentiation by involving various mechanisms for different cell types. However, the principal mechanism will probably not be revealed before the mystery of whether the growth-promoting activity of fetuin preparation resides in fetuin itself or in some minor contaminant(s) has been finally solved. Does Fetuin

Itself Possess

Growth-Promoting

Activity?

Because the relatively impure Pedersen fetuin is the type of preparation frequently showing the growth-promoting activity, whether the fetuin itself possesses the activity has been a long-standing question. Basically, there are two opposite opinions in the literature. However, the hypothesis of contaminants seems to be dominant. Arguments for contaminants. Pedersen fetuin that has growth-promoting property for many cell types is a rather crude ammonium sulfate fraction of FBS (104, 105) and contains many minor components (72, 74, 84, 130). In addition, the amount of fetuin in culture media is generally high enough (Table 3) for small contaminants to become responsible for or contributory to the cell growth. For example, if fetuin is used at a concentration of 100 pg/ml, a contaminant of l/l,000 would still be fully active if it has a specific activity in the nanogram-per-milliliter range. Also, fetuin has been used sometimes at a concentration up to a few milligrams per milliliter (Table 3). Furthermore, the highly purified Spiro or Deutsch fetuin is biologically inactive in most growth assays (36, 50, 52, 84, 109, 117). Even in some cases where Spiro fetuin or Deutsch fetuin was found to be active, the investigators could still not rule out the possibility of contaminants due to the high concentration of fetuin used and the lack of homogeneity of Spiro fetuin (4,61,62). For example, Hsu and Floyd (61) reported that anti-Spiro fetuin antibody revealed three heat-labile minor components in Spiro fetuin that was stimulatory to lymphocytes, and one of the minor components appeared to be a fetal protein distinct from fetuin. Therefore there have been extensive speculations and many reports that the growth-promoting activity of the fetuin preparation is carried by trace contaminants rather than by the fetuin itself. These arguments appear to be reasonable and valid. Arguments for a direct effect of fetuin. Although the highly purified Spiro or Deutsch fetuin is biologically inactive in most growth assays, it could not be ruled out that the use of organic solvents and heavy metal ions in

REVIEW

c555

the preparation of Spiro or Deutsch fetuin might have produced subtle denaturation of fetuin or introduction of toxins, leading to subsequent loss of the fetuin activity (27, 41, 47, 111, 125). For example, Florini and Roberts (41) reported that all three types of fetuin preparations were active in promoting rat L6 cell growth in a serumfree medium MM-l, but there was a huge variability in activity among different Spiro fetuin preparations from total inactivity to stimulation equaling that of Pedersen fetuin. However, a consistent activity of Spiro fetuin could be restored by removal of toxic levels of zinc and barium introduced during the purification process through exhaustive dialysis against EDTA. In addition, Florini (39) stated in a recent review that the use of various chromatographic methods, including reversephrase HPLC, had failed to separate any active contaminant from the bulk of fetuin protein. Puck et al. (107) also reported that the fetuin activity could be specifically neutralized by anti-fetuin y-globulin and that several purification methods completely destroyed the biological activity of fetuin preparation. Taken together, these reports argued that the mitogenic activity of fetuin preparation might reside in fetuin itself, at least for some cell types, despite the fact that fetuin is used at high concentrations. Fetuin Contaminants Shown to Be Either or Partially Responsible for Its Activity

Completely

Within a short time after the first demonstration (37) that Pedersen fetuin promoted cellular attachment and stretching (flattening), Lieberman et al. (82) reported that fetuin purified by DEAE-cellulose chromatography was inactive in promoting attachment and a separate fraction from the column contained the cell flattening activity. However, because so little activity was recovered and DEAE cellulose had been reported to be able to completely inactivate fetuin (60), critics at that time suggested that the procedure used by Lieberman et al. (82) was denaturing most of the fetuin activity and leaving only a small residual fraction with the biological activity (36, 107). However, their work represented the first attempt in the course of identifying the active component from Pedersen fetuin. In the subsequent years, several substances have been identified as fetuin contaminants that could either completely replace fetuin or contribute to its activity for those cell types studied. These reported contaminants are summarized in Table 4 and discussed individually below. Thyroid hormones. Fetuin has been shown to bind thyroid hormones as demonstrated by lslI-labeled thyroxine (T4) or 3,5,3’-triiodothyronine (T3) association with the fetuin band on reverse-flow paper electrophoresis (35). Purified bovine fetuin also binds T4 in vitro with a relatively high specificity, and fetuin appears to contain one primary T4-binding site per molecule with an estimated association constant of 1.2 x 10’ l/M (35). Thyroid hormones are capable of promoting cell growth in culture. For example, T4 is stimulatory for rat cardiac muscle cells (96), both T3 and T4 stimulate growth of GH1 cells (a rat pituitary tumor cell line), and thyroid hormone (T3) synergistically enhances the glucocorticoid-stimulated secretion of growth hormone by these cells in culture (119, 120) .

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

C556

Table 4. Growth-promoting

INVITED

substances

associated

with fetuin preparation Ref. No.

Thyroid hormones Fibronectin a2-Macroglobulin GH’ NGF* PDGF* TGF-P, and -&* IL-lo* IL-6* bFGF* TNF-CY* PDGF Lipids Adipogenic factors

35 28, 115, 34, 116, 118, 2 112 63 24, 92, 101, 17 89 26 136 81 74 141

GH, growth hormone; NGF, nerve growth factor; PDGF, plateletderived growth factor; TGF, transforming growth factor; IL, interleukin; bFGF, basic fibroblast growth factor; TNF, tumor necrosis factor. * Associated with cu2-macroglobulin.

Fibronectin. Fetuin preparation has also been suggested to contain fibronectin (28, 115), an extracellular matrix protein that promotes cellular attachment (138). Fibronectin has been observed to substitute for the attachment function of fetuin activity (Ref. 99; Nie and Ham, unpublished observations) as discussed in the previous section. Thus it is possible that the contaminant fibronectin of fetuin could be responsible in some culture systems if these systems require the attachment factor only. cu2-Macroglobulin and its bound growth factors. It was reported that a growth-promoting glycoprotein fraction from fetal calf serum contained two major components, fetuin and cu-macroglobulin, and both proteins had the growth activity (84). Salomon et al. (116, 118) isolated a fetuin contaminant called embryonin from Pedersen fetuin. The embryonin could completely replace fetuin for the growth of a mouse embryonal carcinoma cell line (OTT-6050) and rat mammary epithelial cells. However, embryonin resembled human az-macroglobulin in many physicochemical and immunologic properties. In addition, human a2-macroglobulin produced a comparable stimulation of the embryonal carcinoma cell growth in the absence of serum (116). Subsequently, it was determined that embryonin was in fact bovine az-macroglobulin (34). az-Macroglobulin itself is a rather interesting protein in plasma, and cu-macroglobulins, including cy2macroglobulin from different species, have been reported to have growth-promoting activity either alone or in combination with other proteins associated with the a-globulin fraction (9, 57, 59, 116, 133). Human cu2-macroglobulin is a 720-kDa homotetramer composed of four 180-kDa subunits (8,55), and a 420-kDa glycoprotein has been identified as a cell surface receptor for the activated forms of a2-macroglobulin (6). cu2-Macroglobulin is a strong proteinase inhibitor with a broad specificity (8). More importantly, ar,-macroglobulin has been shown to bind and interact with many cytokines and growth factors (recently reviewed in Refs. 67, 78). It has been reported that ar2-macroglobulin is a major serum-binding protein for transforming growth factor (TGF)-& and -p2

REVIEW

and with a lo-fold greater affinity for TGF-P2 than for TGF-& (24, 101). The biological activity of TGFs can be regulated by.association and dissociation with Cu2-macroglobulin (24, 77, 92). cu2-Macroglobulin is also a binding protein for basic FGF (26) and for PDGF (63, 108). In addition, it binds interleukin (IL)-lfl (16), IL-6 (89), nerve growth factor (112), tumor necrosis factor-a (135), and human growth hormone (2). All these growth factors are well characterized and very potent, and some have a broad activity. Therefore it is reasonable to suggest that when a,-macroglobulin is a contaminant of the fetuin preparation, these a2-macroglobulin-bound growth factors could be responsible for the fetuin activity for those cell types that respond to the particular growth factor in question. PDGF. PDGF is a well-characterized mitogen (reviewed in Refs. 58, 113). It has been shown that PDGF may also be a direct contaminant of fetuin (81). In fact, PDGF may occur in Pedersen fetuin both in a free form and in a bound form with a2-macroglobulin, and this contaminant PDGF could completely replace the Pedersen fetuin preparation in promoting arterial smooth muscle cell growth (81). Thus the contribution of these possible free and/or a2-macroglobulin-bound PDGF should not be ignored in the evaluation of the growth promoting effect of fetuin. It is possible that this contaminant PDGF may explain the requirement of certain cell types for fetuin. Lipids. Fetuin was reported to be associated with a variety of lipids, predominantly cholesterol, cholesteryl ester with small amounts of phospholipids, triglycerides, and free fatty acids (74). A lipoprotein-like particle similar to high-density lipoprotein was also found to be contained in Pedersen fetuin but not in Spiro fetuin (74). Lipids are capable of stimulating serum-free growth of cultured cells. For example, phospholipids containing polyunsaturated fatty acyl groups are growth-promoting for normal mouse mammary epithelial cells in serum-free culture (64), cholesterol is required for serum-free growth of NS-1 mouse myeloma cells (121), and human lowdensity lipoprotein is also essential for the serum-free growth of these NS-1 cells (70). Thus the contribution of these contaminant lipids to the growth-promoting activity of fetuin for certain cell types should be taken into account. Adipogenic factors. Zaitsu and Serrero (141) reported that Pedersen fetuin contained three adipogenic factors and that these factors were all bigger than fetuin with distinct p1 values. Their study showed that the adipogenie factors contained in fetuin could completely replace fetuin for both growth and differentiation of mouse adipogenic cell line 1246 and that these factors were also distinct from ar2-macroglobulin. A fetuin-replacing activity for human muscle cells from FBS. Ham et al. (50) developed a nutrient medium

MCDB 120 and serum-free supplement SF (dexamethasone, EGF, fetuin, insulin, and serum albumin) for the clonal growth of normal human skeletal muscle satellite cells (HMSC). Pedersen fetuin at 0.5 mg/ml was strictly required for the serum-free growth but not for the growth

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

INVITED

in serum-containing MCDB 120. In addition, Spiro fetuin was biologically inactive in promoting growth of HMSC (50). In the absence of fetuin in MCDB 120 SF, HMSC could attach to Petri dishes but could not grow. Fibronectin could not replace fetuin, suggesting that the requirement was not for the attachment but for the growth-promoting activity from fetuin (Refs. 50, 99; Fig. 1). In the course of further defining the role of fetuin for HMSC, we worked under a hypothesis that a contaminant(s) from FBS might be involved (99). In view of the problems that others have experienced in attempts to fractionate Pedersen fetuin, we undertook direct fractionation of FBS to verify that the fetuin-replacing activity for HMSC could be separated from fetuin. We first precipitated FBS with 50% saturated ammonium sulfate; the precipitate could be regarded as “Pedersen fetuin plus” (Pedersen fetuin is prepared with 45% saturated ammonium sulfate precipitation). This precipitate was redissolved in the buffer and reprecipitated with 25% PEG. PEG at this concentration would precipitate every protein except fetuin in the fraction (83). The precipitate from the PEG step was redissolved and precipitated again with 50% saturated ammonium sulfate, resulting in a fraction designated as 50 PEG 2X that was essentially free of fetuin. The subsequent mitogenic assays revealed, however, that 50 PEG 2X completely replaced Pedersen fetuin for the clonal growth of HMSC and the further purification with fast-protein liquid chromatography (FPLC) anion exchange chromatography achieved 99fold enrichment of the activity with 30% overall recovery (99). The active fraction from FPLC anion exchange of 50 PEG 2X not only replaced Pedersen fetuin but also pushed the clonal growth well above the level in MCDB 120 SF and approaching that in MCDB 120 SF plus 15% fetal bovine serum (Fig. 1). Elimination of fetuin in the fetuin-replacing activity was verified by size determina-

REVIEW

c557

tion with Sephadex G-150 gel filtration and sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) analyses. The most mitogenic activity resided in two peaks of -290 and 130 kDa, and all the activity was above 70 kDa (99). The SDS-PAGE also showed that several bands with molecular mass ~80 kDa were intensified in the most active fraction, whereas bands in the expected weight range for fetuin were further reduced (99) This fetuin-replacing activity seems to be different from all other fetuin-replacing substances discussed above. Replacements tested so far, including commercially prepared az-macroglobulin, failed to replace Pedersen fetuin for HMSC in our tests (Refs. 50, 99; Nie and Ham, unpublished data). This activity was heat labile, and heating at 100°C for 3 min totally destroyed it. It was also pH sensitive, and most of its growth-promoting activity was lost upon treatment at pH 12 or 2 overnight (99). In addition, this fetuin-replacing activity for HMSC had an affinity for heparin and required -1 M sodium chloride for elution from a heparin-Sepharose column (Nie and Ham, unpublished observation). The heat and pH sensitivity is consistent with previous reports of the growth-promoting activity of fetuin. For example, Gaillard et al. (42) observed that the activity of Pedersen fetuin for adipogenic Ob 17 cells was lost with heating at 95°C for 15 min or pH 1 treatment for 60 min. Florini and Roberts (41) also reported that the activity of Pedersen fetuin for rat L6 cells was eliminated upon heating at 60-70°C for 20 min. We speculated that the fetuin-replacing activity for HMSC from FBS might be one of macroglobulin species or related proteins based upon the observations that all the activity was above 70 kDa and that large proteins became more prominent as the activity was concentrated (99). However, this has to be tested when a homogeneous preparation of the activity is isolated. Is There Any Possible Biological Significance for the Association of Growth Factors With Fetuin?

The fetuin-replacing

FPLC fractions

@g/ml)

Fig. 1. Effects of fetuin and fetuin-replacing activity from fetal bovine serum (FBS) on clonal growth of human skeletal muscle satellite cells. A total of 300 cells were inoculated in triplicate to 60-mm tissue culture Petri dishes containing 5.0 ml MCDB 120 SF-fetuin with the fetuinreplacing FPLC fractions added as indicated. After 14 days of incubation (37”C, 5% CO, in air, with saturated humidity) without medium change, the colonies were fixed with methanol and stained with 0.1% crystal violet, and total colony area per dish was determined with an Artek 880 colony counter [data from Nie et al. (99)]. SF contains epidermal growth factor, dexamethasone, fetuin (0.5 mg/ml), bovine serum albumin, and insulin [Ham et al. (50)].

It is known that cu2-macroglobulin plays a regulatory role in the action of many growth factors in vitro (reviewed in Refs. 67, 78). For example, the biological activity of TGFs is regulated by association and dissociation with c+-macroglobulin (24, 77, 92). Another example is that cuz-macroglobulin can synergistically enhance the PDGF-stimulated fibroblast proliferation probably by increasing the local concentration of PDGF at the cell surface (16). Furthermore, many binding proteins for growth factors are glycoproteins that also regulate the activity of those growth factors (reviewed in Ref. 114). Two such recent reports indicate that betaglycan and decorin bind TGF-fl and regulate its activity (5, 139). As discussed previously, fetuin is also a glycoprotein that either binds or associates with a number of growth factors and other growth-promoting substances. Could fetuin also modulate the activity of growth factors through interaction with them? If so, is this modulation by fetuin more prevalent during fetal development, because the concentration of fetuin is very high in fetuses and decreases at birth to a low level in adults? Although the only data available are the specific binding of thyroid hormones with fetuin,

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

C558

these questions directions. Three Possibilities

INVITED

are important About the Fetuin

for

future

research

Mystery

After careful evaluation of the literature, three possibilities for the fetuin enigma appear most tenable. The first one is that the growth-promoting activity of fetuin resides in various contaminants, including potentially unidentified ones, rather than in fetuin itself. This is supported by many reports and a large body of experimental data in the literature, as discussed above, and seems to be reasonable and valid, at least for the majority of cell types in culture. Such a possibility helps to explain why the highly purified Deutsch or Spiro fetuin preparations are biologically inactive in promoting cell growth and why fetuin is needed at such high concentrations (Table 3). In fact, the growth-promoting activity of fetuin has been replaced by various growth factors or mitogenic substances in many studies. Of course, not every cell type that responds to fetuin has been tested for contaminant effects. The second possibility is that fetuin itself has the growth-promoting activity. However, this notion receives little support from the majority of experimental studies. It is highly unlikely that fetuin per se possesses the growth-promoting activity, at least for the majority of cell types that respond to fetuin in culture, because large amounts of Pedersen fetuin are required in the culture media, and highly purified Spiro fetuin, Deutsch fetuin, or even Pedersen type IV fetuin is inactive. On the other hand, it is difficult to rule out the possibility of subtle denaturation of the active component of fetuin or introduction of toxins during preparation of Spiro or Deutsch fetuin. Thus it is impossible at present to exclude the possibility that fetuin itself has the growth-promoting activity, at least for some cell types, until more conclusive experimental data are available. Because biological mechanisms are not so simple in mammalian cells, the third possibility of the fetuin mystery might be somewhat tricky. One of the fetuin subspecies, one of its contaminants (e.g., growth factors), or both of them in combination (e.g., possible modulation of growth factor activity by fetuin) is responsible for growth of one cell type. Alternatively, another combination of fetuin subspecies and the contaminants is growth promoting for another cell type or for the same type of cells but from another species. As mentioned in previous sections, fetuin represents a population of a-globulin in fetal serum, which differ in molecular weight, p1, glycosylation, and immunoreactivity (10, 14, 51, 85, 87, 94, 102, 103, 105). The microheterogeneity of fetuin and various contaminants has undoubtedly complicated the elucidation of the growth-promoting property of fetuin. It is possible that one of the fetuin subspecies might have the fetuin activity for a particular cell type and another fetuin subspecies for another cell type. This idea is supported by reports showing the growth-promoting activity from the highly purified Deutsch or Spiro fetuin. Moreover, the possibility of active subspecies of fetuin for certain cells might be further complicated by possible synergistic enhancement by some of the fetuin contaminants (or, more likely, fetuin acts in synergy with contaminant growth

REVIEW

factors). For instance, Burger (18) reported that the mitogenic activity of different ammonium sulfate-precipitated fetuin fractions for mouse spleen cells was correlated with their content of fetuin and that Spiro fetuin was fully active; in addition, another factor from serum could greatly enhance the fetuin activity. Thus this third possibility is a difficult challenge. To be able to test this, highly purified subspecies of fetuin free of contaminants are required, or a recombinant bovine fetuin preparation should be used because the bovine fetuin gene has been cloned (31). The growth assays could then be performed with these highly purified populations of fetuin on different cell types both individually and with different combinations of growth factors. Unfortunately, however, to prepare a fetuin preparation pure enough to be free of some very potent growth-promoting contaminants is a difficult technical challenge, not to mention that subspecies of fetuin have not yet been well studied and characterized. However, if this third possibility turns out to be true, the term “fetuin” would probably no longer be precise or appropriate under such situations. Thus one probably would use “fetuins” when the whole population of fetuin is referred to, and “fetuin-1,” “fetuin-2,” and so on to precisely identify each member of the fetuin family. It is obvious that more studies are needed to finally resolve the puzzle as whether the growth-promoting property of fetuin (or “fetuins”) is due to fetuin per se or to its contaminants or whether fetuin exhibits its growth-promoting activity by modulating the mitogenic effect of other growth factors. OTHER

BIOLOGICAL

FUNCTIONS

OF BOVINE

FETUIN

Throughout the years since it was first isolated from fetal calf serum, fetuin has been reported to inhibit trypsin and other proteases as already indicated (37, 43, 79, 111)) inhibit hemagglutination by heat-inactivated influenza virus (45), stimulate cellular uridine uptake on mouse embryo cells (54), enhance transport of hexose into mouse 3T3 fibroblasts (131), inhibit phytomitogeninduced lymphocyte transformation probably resulting from perturbation or “blindfolding” of the cell membrane similar to other immunosuppressive serum a-globulins (137), bind specifically to lectin or lectin-like molecules (25), suppress bovine T- and B-lymphocyte responses (128)) bind specifically to the transformation-sensitive heat shock protein (hsp 47) from chick embryo fibroblasts in vitro (97), protect protein synthesis during thermotolerance in vitro (95), inhibit sperm motility (12), inhibit zona pellucida hardening (resistant to chymotrypsin digestion) during spontaneous maturation of mouse oocyte in vitro (123), accelerate exogenous fatty acid incorporation into cellular triglycerides on rabbit aortic smooth muscle cells (19), and induce alkaline phosphatase in epiphyseal growth plate chondrocytes (66). In addition, it has been proposed that fetuin is involved in fatty acid transport and delivery to cells in fetal life (129). The author thanks Dr. Richard G. Ham for giving him the opportunity to work on the fetuin question, Dr. Richard Odessey for reading the manuscript, and the editor and unknown reviewers for helpful suggestions. This work was supported by National Institutes of Health Grant AR-39860 to Dr. R. G. Ham; Z. Nie was also initially supported by this

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

INVITED grant and then by a research fellowship from the Muscular Dystrophy Association of America during preparation of this article. Address for reprint requests: 2. Nie, Dept. of Cellular and Molecular Physiology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115.

20. 21.

22.

REFERENCES 1. Abdullah, M., J. A. Crowell, L. L. Tres, and A. L. Kierszenbaum. Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture. J. Cell. Physiol. 127: 463-432, 1986. 2. Adham, N. F., Z. H. Chakmakjian, J. W. Wehl, and J. E. Bethune. Human growth hormone and a*-macroglobulin: a study of binding. Arch. Biochem. Biophys. 132: 175-178, 1969. 3. Aliau, S., J. Marti, and J. Moretti. Levels of fetoproteins in fetal serum and extra embryonic fluids during ontogenesis in the ovine species. Int. J. Biochem. 11: 49-54, 1980. 4. Allen, R. E., M. V. Dodson, L. S. Luiten, and L. K. Boxhorn. A serum-free medium that supports the growth of cultured skeletal muscle satellite cells. In Vitro Cell. Dev. Biol. 21: 636-

23. 24.

25.

26.

27.

640, 1985. 5.

6.

7.

8.

9.

10.

11.

Andres, J. L., K. Stanley, S. Cheifetz, and J. Massague. Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-p. J. Cell Biol. 109: 3137-3145, 1989. Ashcom, J. D., S. E. Tiller, K. Dickerson, J. L. Cravens, W. S. Argraves, and D. K. Strickland. The human cu2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of a2-macroglobulin. J. Cell Biol. 110: 1041-1048, 1990. Barboriak, J. J., G. de Bella, I. Setnikar, and W. A. Krehl. Age-related changes in plasma proteins of the fetal goat. Am. J. Physiol. 193: 89-91, 1958. Barrett, A. J., and P. M. Starkey. The interaction of a2macroglobulin with proteinases: characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133: 709-724, 1973. Baudysova, M., and J. Michl. Complete replacement of serum in primary cultures of chick embryo brain cells by growth-promoting alpha-globulin. Experientia Base1 37: 826-828, 1981. Begbie, R. Studies on fetuin from foetal bovine serum. The composition and amino acid sequences of glycopeptides from fetuin. Biochim. Biophys. Acta 371: 549-567, 1974. Bendiak, B., M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming. Separation of the complex asparagine-linked oligosaccharides of the glycoprotein fetuin and elucidation of three triantennary structures having sialic acids linked only to glactose residues. Biochemistry 28: 6491-6499,

28.

1981.

Dons, R. W. H., F. E. Trobaugh, Jr., and W. Fried. Effect of fetuin and of spleen extracts on hematopoietic precursors and on differentiated hematopoietic cells in irradiated mice. CeLZTissue Kinet. 7: 371-379, 1974. 30. Dziegielewska, K. M., E. Bock, M. E. P. Cornelis, K. Mgllgard, H. New, and N. R. Saunders. Identification of fetuin in human and rat fetuses and in other species. Comp. Biochem. Physiol. A Comp. Physiol. 76: 241-245, 1983. 31. Dziegielewska, K. M., W. M. Brown, S.-J. Casey, D. L. Christie, R. C. Foreman, R. M. Hill, and N. R. Saunders. The complete cDNA and amino acid sequence of bovine fetuin: its homology with cv2HS glycoprotein and relation to other members of the cystatin superfamily. J. Biol. Chem. 265: 4354-4357, 1990. 32.

33.

34.

35. 36. 37.

1977.

19. Cayatte, A. J., L. Kumbla, and M. T. R. Subbiah. Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin. J. Biol. Chem. 265: 5883-5888, 1990.

Dziegielewska, K. M., G. Kocsis, and N. R. Saunders. Identification of fetuin and other proteins in cerebrospinal fluid and plasma of fetal pigs during development. Comp. Biochem. Physiol. B Comp. Biochem. 66: 535-541, 1980. Engelhardt, D. L., and J.-H. Mao. A serum factor requirement for the passage of cultured Vero cells through G2. J. Cell. Physiol. 90: 307-320, 1977. Feldman, S. R., S. L. Gonias, K. A. Ney, C. W. Pratt, and S. V. Pizzo. Identification of “embryonin” as bovine ar2-macroglobulin. J. Biol. Chem. 259: 4458-4462, 1984. Fisher, D. A., and R. W. Lam. Thyroid hormone binding by bovine and ovine fetuin. Endocrinology 94: 49-54, 1974. Fisher, H. W., D. O’Brien, and T. T. Puck. The hydrolytic products of fetuin. Arch. Biochem. Biophys. 99: 241-248, 1962. Fisher, H. W., T. T. Puck, and G. Sato. Molecular growth requirements of single mammalian cells: the action of fetuin in promoting cell attachment to glass. Proc. Natl. Acad. Sci. USA 44: 4-10,

38.

39. 40.

1989.

18. Burger, M. The stimulation of the in vitro immune response by mouse spleen cells using fetuin. Immunochemistry 14: 11-14,

Chan, S. Y. A chemically defined medium for the propagation of rat prostate adenocarcinoma cells. Prostate 2: 291-298, 1981. Christie, D. L., K. M. Dziegielewska, R. M. Hill, and N. R. Saunders. Fetuin: the bovine homologue of human cv2HS glycoprotein. FEBS Lett. 214: 45-49, 1987. Clark, J. Reducing the requirement for serum supplements in high yield microcarrier cell culture. In: Hormonally Defined Media, edited by G. Fisher and R. J. Wieser. Berlin: Springer-Verlag, 1983, p. 6-15. Claycomb, W. C. Culture of cardiac muscle cells in serum-free media. Exp. Cell Res. 131: 231-236, 1981. Danielpour, D., and M. B. Sporn. Differential inhibition of transforming growth factor & and p2 activity by a2-macroglobulin. J. Biol. Chem. 265: 6973-6977, 1990. Decker, J. M. Lectin-like molecules on murine thymocytes and splenocytes. I. Molecules specific for the oligosaccharide moiety of the fetal a-globulin fetuin. Mol. Immunol. 17: 803-808, 1980. Dennis, P. A., 0. Saksela, P. Harpel, and D. B. Rifkin. cu2-Macroglobulin is a binding protein for basic fibroblast growth factor. J. BioZ. Chem. 264: 7210-7216, 1989. Deutsch, H. F. Fetuin: the mucoprotein of fetal calf serum. J. Biol. Chem. 208: 669-678, 1954. Dollenmeier, P., D. C. Turner, and H. M. Eppenberger. Proliferation and differentiation of chick skeletal muscle cells cultured in chemically defined medium. Exp. Cell Res. 135: 47-61,

29.

1989.

12. Berger, T., and J. W. Beierle. Inhibition of sperm motility by bovine serum components. BioZ. Reprod. 42: 545-551, 1990. 13. Bergmann, F. H., L. Levine, and R. G. Spiro. Fetuin: immunochemistry and quantitative estimation in serum. Biochim. Biophys. Acta 58: 41-51, 1962. 14. Berman, E., and P. Bendel. One- and two-dimensional 90.5MHz lsC-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin. FEBS Lett. 204: 257-260, 1986. 15. Bodman, J. Development of foetal protein. In: Protides of the Biological Fluids, edited by H. Peeters. Amsterdam: Elsevier, 1959, p. 173-179. 16. Bonner, J. C., A. Badgett, A. R. Osornio-Vargas, M. Hoffman, and A. R. Brody. PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized cy2macroglobulin. J. Cell. Physiol. 145: l-8, 1990. 17. Borth, W., and T. A. Luger. Identification of ar2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-l@ to “F” cu2-macroglobulin. J. BioZ. Chem. 264: 5818-5825,

c559

REVIEW

41. 42.

1958.

Fisher, H. W., T. T. Puck, and G. Sato. Molecular growth requirements of single mammalian cells. III. Quantitative colonial growth of single S3 cells in a medium containing synthetic small molecular constituents and two purified protein fractions. J. Exp. Med. 109: 649-660, 1959. Florini, J. R. Hormonal control of muscle growth. Muscle Nerve 10: 577-598, 1987. Florini, J. R., and D. Z. Ewton. Insulin acts as a somatomedin analog in stimulating myoblast growth in serum-free medium. In Vitro 17: 763-768, 1981. Florini, J. R., and S. B. Roberts. A serum-free medium for the growth of muscle cells in culture. In Vitro 15: 983-992, 1979. Gaillard, D., G. Ailhaud, and R. Negrel. Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone supplemented medium. Biochim. Biophys. Acta 846: 185-191,

1985.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

C560

INVITED

F., and J. R. Cann. Fetuin as a trypsin 43. Galembeck, Arch. Biochem. Biophys. 164: 326-331, 1974. 44. Gey, G. O., W. D. Coffman, and M. T. Kubicek.

inhibitor.

Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Res. 12: 264-265, 1952. E. R. B. Some aspects of the structure of the carbo45. Graham, hydrate moiety of fetuin. Aust. J. Sci. 24: 140-141, 1961. 46. Graham, E. R. B. Fetuin. In: Glycoproteins: Their Composition, Structure and Function, edited by A. Gottschalk. Amsterdam: Elsevier, 1966, p. 353-361. 47. Green, W. A., and C. M. Kay. The influence of buffer composition on the hydrodynamic behavior of fetuin. Arch. Biochem. Biophys. 102: 359-366, 1963. 48. Ham, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29: 515-526, 1963. 49. Ham, R. G. Putrescine and related amines as growth factors for a mammalian cell line. Biochem. Biophys. Res. Commun. 14: 34-38, 1964. 50. Ham, R. G., J. A. St. Clair, C. Webser, and H. M. Blau.

Improved media for normal human muscle satellite cells: serumfree clonal growth and enhanced growth with low serum. In Vitro Cell. Dev. 51. Hamann,

Biol. A.

24: 833-844,

1988.

Microheterogeneity of serum glycoproteins as revealed by flat-bed gel isoelectric focusing. In: Electrofocusing Isotachophoresis. Electrofocusing

Proceedings Isotachophoresis,

of the International

Symposium

of

edited by B. J. Radola and D. Graesslin. Berlin: de Gruyter, 1977, p. 329-336. 52. Haneji, T., M. Maekawa, and Y. Nishimune. In vitro differentiation of type A spermatogonia from mouse cryptorchid testes in serum-free media. Biol. Reprod. 28: 1217-1223, 1983. 53. Hanover, J. A., and W. J. Lennarz. Transmembrane assembly of membrane and secretory glycoproteins. Arch. Biochem. Biophys. 211: 1-19, 1981. 54. Hare, J. D. Mechanism

of serum stimulation

in serumless mouse cells. Biochim.

Biophys.

of uridine uptake Acta

255: 905-916,

1972. 55. Harpel,

P. C. Studies on human plasma a,-macroglobulin-enzyme interactions: evidence for proteolytic modification of the subunit chain structure. J. Exp. Med. 138: 508-521, 1973. 56. Havemann, K., S. Buerger, and H. M. Dosch. Growth-promoting action of antiproteases on lymphocytes in vitro. Acta Haematol. 46: 282-293, 1971. 57. Healy, G. M., and R. C. Parker.

Cultivation of mammalian cells in defined media with protein and nonprotein supplements.

J. Cell Biol. 30: 539-553, 1966. 58. Heldin, C.-H., and B. Westermark.

Platelet-derived growth factor: mechanism of action and possible in vivo function. CeZL

Regul. 1: 555-566, 1990. R. Preparation 59. Holmes,

from human serum of an alpha-one protein which induces the immediate growth of unadapted cells in vitro. J. Cell Biol. 32: 297-308, 1967. 60. Hood, S. In: Animal Cell Information Service Newsletter 1: 4, 1959. 61. Hsu,

C. C. S.,

immunochemical

M. Floyd. Lymphocyte stimulating and properties of fetuin preparation. Protides Biol.

and

Fluids 24: 295-302, 1976. 62. Hsu, C. C. S., W. I. Waithe, P. Hathaway, and K. Hirschhorn. Effects of foetuin on lymphocytes: lymphocyte-stimulating property. Clin. Exp. Immunol. 15: 427-434, 1973. J. S., S. S. Huang, and T. F. Deuel. Specific cova63. Huang,

lent binding of platelet-derived growth factor to human plasma cu2-macroglobulin. Proc. Natl. Acad. Sci. USA 81: 342-346, 1984. 64. Imagawa, Nandi.

W., 0. K. Dandyopadhyay,

D. Wallace,

and

C.

Phospholipids containing polyunsaturated fatty acyl groups are mitogenic for normal mouse epithelial cells in serum free primary cell culture. Proc. Natl. Acad. Sci. USA 86: 4122-

4126, 1989. 65. Inaba, T., W. G. Wiest,

R. C. Strickler, and J. Mori. Augmentation of the response of mouse uterine epithelial cells to estradiol by uterine stroma. Endocrinology 123: 1253-1258, 1988.

66. Ishikawa,

Y., L. N. Wu,

W. B. Valhmu,

and

R. E. Wuthier.

Fetuin and (r*HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes. J. Cell. Physiol. 149: 222234, 1991.

REVIEW 67. James, K. Interactions between cytokines and lin. Immunol. Today 11: 163-166, 1990. 68. Johnson, W. V., and E. C. Heath. Structural

a2-macroglobu-

features of bovine fetuin revealed from analysis of the primary translation product: anomalous behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is due largely to peptide and not solely to carbohydrate. Arch. B&hem. Biophys. 251: 732-737,

1986. 69. Johnson,

W. V., and

tional 0-glycosylation 1986.

E. C. Heath. Evidence for posttranslaof fetuin. Biochemistry 25: 5518-5525,

70. Kawamoto, T., J. D. Sato, A. Le, D. B. McClure, and G. H. Sato. Development of a serum-free medium for growth of NS-1

mouse myeloma cells and its application to the isolation of NS-1 hybridomas. Anal. Biochem. 130: 445-453, 1983. 71. Kessler-Icekson, Wasserman.

G.,

0.

Sperling,

C.

Rotem,

and

L.

Cardiomyocytes cultured in serum-free medium. Growth and creatine kinase activity. Exp. Cell Res. 155: 113-120,

1984. 72. Knox,

P., S. Griffiths,

and

Foetal calf se-

J. G. Whateley.

rum and the adhesion of cells in culture. B&hem.

Sot. Trans. 7: 1011-1012, 1979. 73. Knox, P., and C. A. Pasternak. The binding of serum components to cultured cells. In: Radioimmunoassay in Clinical Biochemistry, edited by C. A. Pasternak. London: Heyden, 1974, p.

289-295. 74. Kumbla,

L.,

A. J. Cayatte,

ation of a lipoprotein-like

and M. T. R. Subbiah. Associparticle with bovine fetuin. FASEB J.

3: 2075-2080, 1989. 75. LaBadie, J. H., K. P. Chapman,

and

N. N. Aronson,

Jr.

Glycoprotein catabolism in rat liver: lysosomal digestion of iodinated asialo-fetuin. Biochem. J. 152: 271-279, 1975. P. 76. Lai, Church,

C. W., L. L. Huang, and F. L. Lorscheider.

D.

E.

Panrucker,

R.

B.

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development. J. Reprod. Fertil. 63: 53-60, 1981.

77. LaMarre, Hayes.

J.,

78. LaMarre, Hayes.

J.,

G.

K.

Wollenberg,

J.

Gauldie,

and

M.

A.

cuz-Macroglobulin and serum preferentially counteract the mitoinhibitory effect of transforming growth factor+32 in rat hepatocytes. Lab. Invest. 62: 545-551, 1990. G. K. Wollenberg,

S. L. Gonias,

and

M.

A.

Cytokine binding and clearance properties of proteinaseactivated cuz-macroglobulins. Lab. Invest. 65: 3-14, 1991. 79. Lampreave, F., and A. Pineiro. Characterization of a’new alpha-glycoprotein as the major serum component in later fetal and newborn pigs. Comp. Biochem. Physiol. B Comp. Biochem. 72: 215-219, 80. Leiderman,

1982. L. J.,

and V. W. Dennis. Mycoplasma provides for the spreading of opossum kidney cells in a serum-free, defined medium. In Vitro Cell. Dev. Biol. 25: 655-658, 1989.

81. Libby,

P.,

E. W.

Raines,

P. M.

Cullinane,

and

R. Ross.

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor. J. Cell. Physiol. 125: 357-366, 1985. 82. Lieberman, I., F. Lamy, and P. Ove. Nonidentity of fetuin and protein growth (flattening) factor. Science Wash. DC 129: 43-44, 1959. 83. Loskutoff, D. J. Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture. J. Cell. Physid. 96: 361-369, 1978. A. G. M., J. A. Owen, 84. Marr,

the growth-promoting Biochim. 85. Marti,

and G. S. Wilson. Studies on glycoprotein fraction of foetal calf serum.

Biophys. Acta 63: 276-285, 1962. J., S. Aliau, C. Bonfils, C. Vigne,

and

J. Moretti.

Preparation, con&antes physiques et constitution chimique de la fetuin D’agneau. Biochim. Biophys. Acta 303: 348-359, 1973. 86. Marti, J., S. Aliau, and J. Moretti. Characterization of lamb fetuin. C. R. Acad. Sci. Ser. D 273: 1054-1056, 1971. 87. Marti, J., C. Bonfils, and J. Moretti. Microheterogeneity of lamb fetuin. Biochimie 56: 873-880, 1974. 88. Matsuda, M. Serum proteins enhance aggregate formation of dissociated fetal rat brain cells in an aggregating culture. In Vitro Cell. Dev. 89. Matsuda,

Biol. 24: 1031- 1036, 1988. T., T. Hirano, S. Nagasawa,

Identification

of aZ-macroglobulin

and

T. Kishimoto.

as a carrier protein for IL-6. J.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

INVITED Immunol. 142: 148-152, 1989. 90 Matsuya, Y. The replacement of the active compounds in a serum-free medium and the serial transfer of L line cells in it. Tohoku J. Exp. Med. 86: 84-92, 1965. 91 Matsuya, Y., and I. Yamane. Culture of Syrian hamster fibroblasts in albumin-fortified medium. I. Action of animal albumin preparations on the growth of lung cells of newborn hamster. Tohoku J. Exp. Med. 101: 379-385, 1970. 92 McCaffrey, T. A., D. J. Falcone, C. F. Brayton, L. A. Agarwal, F. G. P. Welt, and B. B. Weksler. Transforming growth factor-p activity is potentiated by heparin via dissociation of the transforming growth factor-@/a2-macroglobulin inactive complex. J. CeZZBiol. 109: 441-448, 1989. 93. McKenna, P. Proteins in the amniotic and allantoic fluids of fetal pigs. Comp. Biochem. Physiol. B Comp. Biochem. 78: 663-

Ross, R., E. W. Raines, and D. F. Bowen-Pope. The biology of platelet-derived growth factor. Cell 46: 155-169, 1986. 114. Ruoslahti, E. Proteoglycans in cell regulation. J. Biol. Chem. 113.

264: 13369-13372, 115.

116.

117.

667, 1984.

94. Meyers, W. M., and H. F. Deutsch. Immunological studies of fetuin. Arch. Biochem. Biophys. 54: 38-44, 1955. 95. Mivechi, N. F., and P. D. Ogilvie. Effects of heat shock proteins (M, 70,000) on protein and DNA synthesis at elevated temperatures in vitro. Cancer Res. 49: 1492-1496, 1989. 96. Mohamed, S. N. W., R. Holmes, and C. R. Hartzell. A serum-free, chemically defined medium for function and growth of primary neonatal rat heart cell cultures. In Vitro 19: 471-478, 1983. 97. Nakai, A., K. Hirayoshi, S. Saga, K. M. Yamada, and K. Nagata. The transformation-sensitive heat shock protein (hsp 47) binds specifically to fetuin. Biochem. Biophys. Res. Commun. 164: 259-264,

399-405,

Prostacyclin as a Biochem. J. 257:

1989.

99. Nie, 2. T., D. Jellinek, and R. G. Ham. Separation of growth-promoting activity for human muscle cells from fetuin. Biochem. Biophys. Res. Commun. 178: 959-966, 1991. 100. Nishimune, Y., T. Haneij, M. Maekawa, K. Sakamaki, and Y. Kitamura. Effects of steel mutation of spermatogenesis stimulated by fetuin. Arch. Androl. 15: 117-120, 1985. M. D., and L. M. Wakefield. Latent 101 O’Connor-McCourt, transforming growth factor 0 in serum: a specific complex with cup-macroglobulin. J. Biol. Chem. 262: 14090-14099, 1987. 102 Oshiro, Y., and E. H. Eylar. Physical and chemical studies on glycoproteins. III. The microheterogeneity of fetuin, a fetal calf serum glycoprotein. Arch. Biochem. Biophys. 127: 476-489, 1968. 103. Page, M. Demonstration of the microheterogeneity of fetuin by electrofocusing. Biochim. Biophys. Acta 236: 571-577, 1971. 104. Pedersen, K. 0. Fetuin, a new globulin isolated from serum. Nature Land. 154: 575, 1944. 105. Pedersen, K. 0. Ultracentrifugal and electrophoretic studies on fetuin. J. Phys. Colloid Chem. 51: 164-171, 1947. 106. Petersen, 0. W., B. van Deurs, K. V. Nielsen, M. W. Madsen, I. Laursen, I. Balslev, and P. Briand. Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909Sl and HMT-3909S8, established in serum-free medium. Cancer Res. 50: 1257-1270, 1990. 107. Puck, T. T., C. A. Waldren, and C. Jone. Mammalian cell growth proteins. I. Growth stimulation by fetuin. Proc. Nutl. Acad. Sci. USA 59: 192-199, 1968. 108. Raines, E. W., D. F. Bowen-Pope, and R. Ross. Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors. Proc. Nutl. Acud. Sci. USA 81: 3424-3428,

Rizzino, A., and G. Sato. Growth of embryonal carcinoma cells in serum-free medium. Proc. Natl. Acad. Sci. USA 75: 1844-

110.

Rizzino, A., and M. I. Sherman. Development and differentiation of mouse blastocysts in serum-free medium. Exp. Cell Res.

120.

122

123

124 125.

126.

1979.

Rohrlich, S. T., and D. B. Rifkin. Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin. J. Cell. Physiol. 109: l-15, 1981. 112. Ronne, H., H. Anundi, L. Rask, and P. A. Pederson. Nerve growth factor binds to serum alpha-2-macroglobulin. Biochem. Biophys. Res. Commun. 87: 330-335, 1979.

1989.

Salomon, D. S. Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium. Exp. Cell Res. 128: 311-321, 1980. Salomon, D. S., M. Bano, K. B. Smith, and W. R. Kidwell. Isolation and characterization of a growth factor (embryonin) from bovine fetuin which resembles crz-macroglobulin. J. Biol. Chem. 257: 14093-14101, 1982. Salomon, D. S., L. A. Liotta, and W. R. Kidwell. Differential response to growth factor by rat mammary epithelium plated on different collagen substrata in serum-free medium. Proc. Natl. Acad. Sci. USA 78: 382-386, 1981. Salomon, D. S., K. B. Smith, I. Losonczy, M. Bano, W. R. Kidwell, G. Alessandri, and P. M. Gullino. az-Macroglobulin, a contaminant of commercially prepared Pedersen fetuin: isolation, characterization, and biological activity. In: Cell CuLture Methods for Molecular and Cell Biology, edited by D. W. Barnes, D. A. Sirbasku, and G. H. Sato. New York: Liss, 1984, vol. 3, p. 125-153. Samuels, H. H., 2. D. Horwitz, F. Stanley, J. Casanova, and L. E. Shapiro. Thyroid hormone controls glucocorticoid action in cultured GH cells. Nature Lond. 268: 254-257, 1977. Samuels, H. H., J. S. Tsai, and R. Cintron. Thyroid hormone action: a cell culture system responsive to physiological concentrations of thyroid hormones. Science Wash. DC 181: 1253-1256,

121

1973.

Sato, J. D., T. Kawamoto, D. B. McClure, and G. H. Sato. Cholesterol requirement of NS-1 mouse myeloma cells for growth in serum-free medium. Mol. Biol. Med. 2: 121-134, 1984. Schmidt, W., G. Poll-Jordan, and G. Loffler. Adipose conversion of 3T3-Ll cells in a serum-free culture system depends on epidermal growth factor, insulin-like growth factor I, corticosterone, and cyclic AMP. J. BioL. Chem. 265: 15489-15495, 1990. Schroeder, A. C., R. M. Schultz, G. S. Kopf, F. R. Taylor, R. B. Becker, and J. J. Eppig. Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse occyte maturation in vitro in the absence of serum. Biol. Reprod. 43: 891-897, 1990. Spiro, M. J., and R. G. Spiro. Composition of the peptide portion of fetuin. J. Biol. Chem. 237: 1507-1510, 1962. Spiro, R. G. Studies on fetuin, a glycoprotein of fetal serum. I. Isolation, chemical composition, and physicochemical properties. J. Biol. Chem. 235: 2860-2869, 1960. Spiro, R. G. Demonstration of a single peptide chain in the glycoprotein fetuin: terminal amino acid analyses and studies of the oxidized and reduced alkylated preparations. J. Biol. Chem. 238: 644-649,

1963.

127.

Spiro,

128.

Splitter, G. A., and K. M. Everlith. Suppression of bovine Tand B-lymphocyte responses by fetuin, a bovine glycoprotein. Cell Immunol. 70: 205-218, 1982. Subbiah, M. T. Newly recognized lipid carrier proteins in fetal life. Proc. Sot. Exp. Biol. Med. 198: 495-499, 1991. Temin, H. M., R. W. Pierson, and N. C. Dulak. The role of serum in the control of multiplication of avian and mammalian cells in culture. In: Growth, Nutrition and Metabolism of Cells in Culture, edited by G. H. Rothblat and V. J. Cristofalo. New York: Academic, 1972, vol. 1, p. 50-81. Thrash, C. R., and D. D. Cunningham. Dissociation of increased hexose transport from initiation of fibroblast proliferation. Nature Lond. 252: 45-47, 1974. Tolleshaug, H., and T. Berg. Uptake and degradation of asialo-fetuin by isolated rat hepatocytes. Acta Biol. Med. Ger. 36:

163-167,

129. 130.

131.

1978.

121: 221-233,

119.

1984.

109.

1848,

118.

1989.

98. Negrel, R., D. Gaillard, and G. Ailhaud. potent effector of adipose cell differentiation.

C561

REVIEW

132.

111.

1753-1762,

R. G. Isolation of fetuin. Methods Carbohydr. Chem. 7: 1976.

1977.

Tozer, B. T., and S. J. Pirt. Suspension culture of mammalian cells and macromolecular growth-promoting fractions of calf serum. Nature Land. 201: 375-378, 1964. 134. Turner, K. J. Some physical studies on the two a-glycoproteins of foetal calf serum. Biochim. Biophys. Acta 69: 518-523, 1963. 133.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

C562 135.

INVITED Wallis, C., B. Ver, and J. L. Melnick. Role of serum and fetuin in the growth of monkey kidney cells in culture. Exp. Cell Res. 58: 271-282, 1969.

136. Wollenberg, G. K., J. LaMarre, S. Rosendal, S. L. Gonias, and M. A. Hayes. Binding of tumor necrosis factor alpha to activated forms of human plasma alpha2 macroglobulin. Am. J. Pathol. 138: 265-272, 1991. 137. Yachnin, S. Fetuin, an inhibitor of lymphocyte transformation. The interaction of fetuin with phytomitogens and possible role for fetuin in fetal development. J. Exp. Med. 141: 242-256, 1975.

REVIEW 138. Yamada, K. M., and K. Olden. Fibronectins-adhesive glycoproteins of cell surface and blood. Nature Lo&. 275: 179-184, 1978. 139. Yamaguchi, Y., D. M. Mann, and E. Ruoslahti. Negative regulation of transforming growth factor-p by the proteoglycan decorin. Nature Lond. 346: 281-284, 1990. 140. Yen, S. E., and C. C. Stewart. Effects of serum fractions on the growth of mononuclear phagocytes. J. Cell. Physiol. 112: 107-114, 1982. 141. Zaitsu, H., and G. Serrero. Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics. J. Cell. Physiol. 144: 485-491, 1990.

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 9, 2018. Copyright © 1992 American Physiological Society. All rights reserved.