Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Fetuin and alpha-2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes.

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.     

Growth of normal human ovarian surface epithelial cells in reduced-serum and serum-free media

Summary The human ovarian surface epithelium (OSE) is believed responsible for over 85% of ovarian cancers, yet little is known about the normal biology of these cells. To date, culture of OSE has only been reported in media with high serum supplements. We have developed two media, one with less than 1% of serum (OSEM-1) and the other comprised of highly purified and defined materials (OSEM-2), which allow us to study OSE under relatively defined conditions. By substituting 0.05% of Pedersen’s fetuin for 15% fetal bovine serum (FBS) with Medium 199/MCDB105 basal medium, the cell numbers reached 50 to 60% of those in the presence of 15% FBS over 7 days. However, over several weeks, the total number of population doublings achieved were comparable to those in 15% FBS. Addition of insulin, transferrin, ethanolamine, lipoic acid, and phosphatidylcholine to the medium with Pedersen’s fetuin (OSEM-1) enhanced growth up to 20% more than in their absence. Supplementation of M199/105 with highly purified (>99%) fetuin, alpha₂-macroglobulin, and hydrocortisone resulted in a defined medium (OSEM-2) that permitted 1 to 2 doublings/7 days. In addition, cells maintained a more normal, epithelial-like morphology in culture for a longer period in the presence of Pedersen’s or purified fetuin than in M199/105/15% FBS, thus increasing the number of morphologically normal cells available for experimentation. Addition of 0.05% Pedersen’s fetuin to M199/105 in the presence of 6 to 8% FBS resulted in levels of growth equivalent to those in M199/105/15% FBS alone. We are now able to study the effects of various compounds on the growth and differentiation of OSE under defined conditions, and have reduced the requirement for FBS to produce large numbers of OSE cells.     

Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics.

Crude Pedersen fetuin, derived from fetal bovine serum, contains adipogenic activity. Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S-300, the majority of adipogenic activity eluted in two distinct peaks, FI (molecular weight greater than 669 kDa) and FII (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, FIII (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. FI has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. FII has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein alpha 2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.     

The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones

Epidermal growth factor (EGF) added in a single dose (between 10^–16 and 1.7 ± 10^–9M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (³H)thymidine labeling and autoradiography and by a 1-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10^–11M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10^–14M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10^–14M) mixture of the two pancreatic hormones or EGF by itself at 10^–14M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.     

Assay and partial purification of factors from serum that control multiplication of human diploid fibroblasts

The nature of the set of poorly characterized macromolecular factors from fetal bovine serum needed for sustained multiplication of isolated human diploid fibroblasts $\text{in vitro}$ depends on the qualitative and quantitative composition of the set of defined small molecules used in the assay. The apparent multiplication-promoting activity of a subset of serum factors is interchangeable with multiple precisely defined modifications of the assay system. Use of optimized assay conditions that eliminate dependence on “replaceable” activities from serum reduces the number of crude fractions that affect multiplication. This facilitates quantification and purification of the remaining “nonreplaceable” multiplication-promoting activities, which can now be separated from fetuin and serum albumin and purified approximately 15-fold with 46 per cent recovery.     

Effect of Fetal Bovine Serum Glycoproteins on the In Vitro Proliferation of the Oyster Parasite Perkinsus marinus: Development of a Fully Defined Medium

ABSTRACT. The oyster parasite Perkinsus marinus replicates in our medium consisting of Dulbecco modified Eagle's medium: Ham's F12 nutrient mixture (1:1) supplemented with 1–5% fetal bovine serum, with a doubling time of 24 hours during the exponential phase of the culture. Fetal bovine serum concentrations above 5% dramatically reduced parasite proliferation in a dose-dependent manner. We tested the individual effects of the three major protein components of fetal bovine serum (fetuin, transferrin and albumin) on the replication of the parasite in a serum-free medium. At the concentrations tested, fetuin enhanced parasite growth, whereas albumin had a modest positive effect and transferrin was inhibitory. Proteolytic digestion of fetuin, strongly diminished its growth-enhancing properties, indicating that the overall glycoprotein architecture may be required for activity. On the contrary, desialylation of fetuin slightly enhanced its growth-promoting activity. The addition of fetuin at 1.7 mg/ml to the serum-free DME: Ham's F12 medium yielded growth rates that are comparable to those obtained with our standard culture methodology. This has resulted in a fully defined culture medium that will allow for a rigorous characterization of excretory/secretory products involved in modulating or blocking the host's humoral and cellular defense mechanisms.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

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

Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.     

Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.     

Algal phycocyanins promote growth of human cells in culture

Summary The growth-promoting substances in a non-dialyzable extract of Synechococcus elongatus var. on RPMI 8226 cells (a human myeloma cell line) were separated by gel filtration and ion exchange chromatography. By gel filtration with Sepharose 4B, the dialyzate was separated into two fractions. One fraction was green-colored (P-1) and the other was blue-colored (P-2). The P-2 fraction had a higher growth-promoting activity than P-1. By ion exchange chromatography, the P-2 fraction was separated into two blue-colored fractions of phycocyanin and allophycocyanin. Both biliproteins promoted the growth of RPMI 8226 cells; however, allophycocyanin was more active than phycocyanin. Editor's statement This reporrt that phycobiliprotein from algae is capable of stimulating animal cell growth is unique, and raises the possiblity that related compounds such as biliverdin might also be similarly active. Some puzzling aspects, such as the lack of increase in specific activity during purification, as well as the possibility that the activity might be due to a contaminant, remain to be resolved.     

Inhibition of sperm motility by bovine serum components.

Various sources and components of mammalian sera were evaluated for their ability to maintain or inhibit sperm motility. Human, rabbit, hamster, and porcine sera were equal in ability to maintain motility of human sperm. Four sources of fetal calf serum and one source of neonatal calf serum were unable to maintain motility of human sperm or sperm-fertilizing potential. In the presence of human serum, fetal calf serum actually inhibited human sperm motility. Fetuin, a component of fetal calf serum, contained the inhibitory activity. An inhibitory effect of fetuin on porcine and caprine sperm motility was also observed. The inhibitory activity resided in the second peak when fetuin was separated by isoelectric focusing. The sperm head membranes remained impermeable to dye, and mitochondrial membrane potential was maintained after motility had been reduced to almost zero by incubation with fetuin and fetuin fractions. Fetuin or the active portion of the molecule may be a useful component of a vaginal contraceptive and in research where inhibition of motility is desirable.     

Macrophage-mediated fungistasis: requirement for a macromolecular component in serum

Peritoneal macrophages from Mycobacterium bovis- or Toxoplasma gondii-infected mice cultured in vitro in Dulbecco's medium containing 10% fetal bovine serum (FBS) and endotoxin stopped replication of Cryptococcus neoformans for 30 hr, whereas yeast cells cultured alone reproduced with a 3.0-hr doubling time. Without at least 5% FBS, macrophage fungistasis was poor. FBS without macrophages enhanced the growth rate of cryptococci. Macrophages preincubated in vitro for 24 hr without serum became fungistatic when challenged with cryptococci in medium with FBS but were not fungistatic without FBS. Macrophages preincubated in medium with FBS were never subsequently fungistatic. Dialyzed, heated (56 degrees C, 30 min), or delipidated FBS supported macrophage fungistasis, whereas FBS heated at 70 degrees C for 30 min did not. FBS contained no measurable opsonic activity for C. neoformans. Inclusion of endotoxin and/or murine IFN-gamma over wide concentration ranges did not substitute for FBS. Ultrafiltration estimation of FBS activity localized to 50 to 150 Kd. By gel filtration chromatography, FBS activity ran in the 25 to 100 Kd range. Dye-ligand affinity chromatography on Cibacron blue agarose gel dissociated the FBS activity from the albumin and lipoprotein fractions. Anion-exchange chromatography on DEAE-Sephacel revealed activity in the first fraction eluting at low ionic strength, pointing to a protein(s) with an isoelectric point toward neutral. Activated macrophages can prevent microbial replication within host tissues; the local environment is critical for fulfillment of this important physiologic function. These results point to a macromolecular factor(s) present in serum that is essential for full fungistatic capability of activated macrophages.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

Glycoprotein biosynthesis: Folic acid effects on glycoprotein:glycosyl transferase activities of rat kidney and liver

Folic acid at 14 μM to 1.4 mM increased the activity of the collagen:glc and fetuin:gal and decreased the activity of the fetuin:NANA glycoprotein:glycosyl transferases of rat liver and kidney in vitro; highest effects were found with 1.4 mM folic acid. 1.4 mM folic acid increased kidney fetuin:gal activity 5-fold and decreased fetuin:NANA activity 3-fold. At 1.4 mM, folinic acid and p-methylaminobenzoic acid were totally inactive toward the transferases, methasquin was moderately active, and homofolic, tetrahydrohomofolic and methotrexate were very active toward the transferases. In all instances, however, the fetuin:gal and collagen:glc transferases were activated while the fetuin: NANA transferase was inhibited. From the data presented, folic acid is viewed as a possible control molecule in the synthesis of glycoprotein.     

Effect of purine supplementation on the growth of salmonid cell lines in different mammalian sera

The Chinook salmon embryo cell line, CHSE-214, grew well in fetal bovine serum (FBS) but poorly in dialyzed (d) FBS. Purines restored most but not all growth-promoting activity to dFBS, which suggests that purines account for a large portion of the dialyzable fraction's growth-promoting activity. CHSE-214 died in newborn calf serum (NCS) but grew slightly in dNCS, which suggests that the dialyzable fraction of NCS contains a toxic component(s). Little or no proliferation occurred in calf serum (CS); some took place in horse serum (HS). Porcine serum (PS) was very toxic. In all these sera except PS and HS, the purine nucleoside, inosine, significantly enhanced growth, whereas the pyrimidine nucleoside, uridine, was without effect. The other purines, hypoxanthine, adenine, adenosine and guanosine also stimulated proliferation but not as well as inosine. Inosine also enhanced the growth of the rainbow trout gonadal cell line, RTG-2. Although their morphology underwent minor alterations in medium with inosine, CHSE-214 cells could be grown indefinitely in CS and inosine as effectively as in the more expensive FBS.     

Biochemical characterization of the serum fetuin-mineral complex

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9-24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.     

Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone-supplemented medium

A serum-free hormone-supplemented medium able to support the growth of rodent adipose precursor cells has been used to characterize additional components from serum required for the differentiation of preadipose Ob17 cells into adipose-like cells. Fetuin is shown to behave as a growth-promoting agent for these cells. In addition to growth hormone, triiodothyronine and a low-molecular weight component(s) also purified from serum, fetuin is required for the full expression of the differentiation program. Other serum proteins as well as other mitogenic factors are unable to substitute for fetuin. A possible role of fetuin in the development of adipose tissue is discussed.