Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors.

Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.     

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.     

Isolation of heparin-binding growth factors from dogfish (Mustela canis) brain and retina

We have purified two growth factors from dogfish brain and retina by using their binding affinity for heparin-Sepharose. These growth factors were eluted at 1 and 2 M NaCl similarly to those purified from bovine brain or retina. Their mitogenic activity was assayed in vitro on the same mammalian cells: bovine lens epithelial cells or human fibroblasts. All these data seem to indicate that these growth factors belong to the families of other well defined mammalian growth factors: EDGF I, brain basic FGF, AGF II, on the one hand and EDGF II, brain acidic FGF, AGF I, ECGF, on the other. Thus, these growth factors have been widely distributed during evolution and retain at least a conservative sequence to stimulate cell proliferation, in mammalian cells.     

Acidic fibroblast growth factor (FGF) from bovine brain: amino-terminal sequence and comparison with basic FGF.

Acidic fibroblast growth factor (FGF) from bovine brain has been isolated by a combination of salt precipitation, ion-exchange chromatography, heparin-Sepharose affinity chromatography and reverse phase h.p.l.c. The amino acid composition of the mitogen is indistinguishable from that of acidic FGF previously purified. The amino-terminal sequence of acidic FGF was established as Phe-Asn-Leu- Pro-Gly-Asn-Tyr-Lys-Pro-Lys-Leu-X-Tyr-X-Ser-Asn-Gly-X-Tyr-Phe-Leu-Arg-Il e-Leu-Pro-Asp-Gly. Acidic FGF is structurally different from basic FGF as judged by mol. wt., amino acid composition and sequence. In vitro biological comparison of the two growth factors indicates that acidic and basic FGFs possess the same intrinsic activities to stimulate the proliferation of aorta, vein or capillary endothelial cells and adrenal cortex cells, but acidic FGF is 30-100 times less potent, depending on the cell type.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Isolation and partial characterization of mitogenic factors from cementum.

Cementum is the mineralized structure through which soft connective tissues are attached to the teeth. It is a unique calcified tissue characterized by a low metabolic turnover, lack of blood supply, and presence of very few cells. However, it contains substances that influence the biological activities of fibroblasts of adjacent soft tissues. We have partially characterized cementum proteins that have mitogenic activity toward fibroblasts. Cementum was harvested from bovine teeth, and mitogenic factors were extracted in 0.5 M CH3COOH. Heparin-Sepharose chromatography separated the mitogenic activity into a major and a minor fraction eluted by 0.5 and 2.0 M NaCl, respectively. The distribution of cementum mitogens in heparin-Sepharose fractions was different from that of alveolar bone and other bones. The cementum mitogenic factor eluting with 2.0 M NaCl from a heparin-Sepharose column was shown to be basic fibroblast growth factor (bFGF) on the basis of inhibition by anti-bFGF antibody and Western blots. The 0.5 M NaCl fraction was purified by HPLC with use of a combination of a DEAE-3W column followed by TSK-250 and C18 columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified fraction contained two protein bands with Mr 22,000 and 19,000, and mitogenic activity was associated with the Mr 22,000 species. The activity of this mitogen, designated as CGF, was potentiated by small quantities of plasma-derived serum or epidermal growth factor. It was heat resistant, but was destroyed by reduction. Assays of CGF preparations revealed that they contained no detectable platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response

Salt extracts of the extracellular matrix (ECM) that is produced by vascular and capillary endothelial cells contain mitogens that are indistinguishable from basic and acidic fibroblast growth factors (FGFs). The biological activity found in these extracts is retained by heparin-Sepharose affinity columns and elutes with salt concentrations similar to those required to elute FGFs (i.e. 1.1 - 2M NaCl). Antisera raised against synthetic fragments of basic and acidic FGF crossreact with the ECM-derived mitogens. Radioiodinated basic FGF binds to the ECM formed by both vascular and capillary endothelial cells, a result that is consistent with the observation that FGF-like mitogens are found on the ECM. The binding of FGF to the ECM is negligible when the ECM has been pretreated with heparinase or heparitinase suggesting that the mitogen is interacting with a heparin-like glycosaminoglycan in the ECM. The digestion of the ECM with several grades of hyaluronidase, chondroitinase or chondro-4-sulfatase or chondro-6-sulfatase has little or no effect on 125I-FGF binding to the ECM. In view of the fact that many, if not all cells, produce heparan sulfates and that these glycosaminoglycans are associated with the external surface of the cell and the ECM, a model is proposed suggesting that the neovascular response induced by tumours and some normal tissues may be mediated at least in part, by the initial release of heparinase-like enzymes rather than angiogenic factors (FGFs) per se. The release of these enzymes would effectively mobilize a secondary local release of FGF from the ECM which then induces a proliferative response.     

Endothelial cell growth factors in embryonic and adult chick brain are related to human acidic fibroblast growth factor.

We have investigated the nature of endothelial cell growth factors in 14-day embryonic and adult chick brain extracts. Mitogenic activity was isolated by a combination of cation-exchange, heparin-Sepharose affinity, and reverse-phase HPLC. Two major mitogenic fractions eluted from heparin-Sepharose at 0.8-1.3 M and 1.5-2 M. Biologically active proteins eluting at 0.8-1.3 M NaCl, after purification to homogeneity from embryonic and adult brain, were found to possess the same amino-terminal sequence as human acidic fibroblast growth factor (aFGF). The notion that the isolated mitogens represent chick aFGF is further supported by the findings that their affinity for heparin and their retention behavior in highly resolutive HPLC are indistinguishable from those of genuine aFGF. Mitogenic activities eluting at 1.5-2 M NaCl were also present in embryonic and adult brain, but in quantities insufficient for preliminary characterization. The high specific mitogenic activity for endothelial cells, high affinity for heparin and cross-reactivity with antibodies against bovine basic FGF (bFGF) suggest a relationship of those materials with basic FGF. Our data also suggest that the sequence of aFGF is highly conserved among vertebrates. While angiogenesis occurs predominantly in the embryonic brain, the absence of notable differences in the contents of the potent angiogenic factors aFGF and bFGF in embryonic versus adult chick brain is interesting.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

An androgen-dependent subclone derived from a mouse mammary tumor, Shionogi carcinoma 115, secretes a heparin-binding growth factor having an apparent molecular weight of 31,000 in response to androgen.

An androgen-dependent cell line denoted SC2G is a clone of an androgen-dependent mouse mammary tumor, Shionogi Carcinoma 115. Fibroblast growth factors (FGFs), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are stimulatory for the growth of SC2G cells in the absence of androgen. This clone was found to secrete an androgen-induced growth factor mostly eluting at 1.8 M NaCl on a heparin-Sepharose column. This factor was partially purified by chromatography on two consecutive heparin-Sepharose columns followed by cation-exchanging chromatography on an S-Sepharose column from the chemically defined serum-free medium conditioned by SC2G cells in the presence of androgen. The factor was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, most of the growth-promoting activity of this factor was found at approx. 31 kDa under non-reduced conditions. Neither neutralizing antibody against basic-FGF nor that against EGF inhibited the growth-promoting activity of this factor in cell culture, suggesting the factor was distinct from basic FGF or EGF. However, the possibility that the factor was another FGF- or EGF-like growth factor was not excluded.     

Endothelial cell mitogens derived from retina and hypothalamus: biochemical and biological similarities

Bovine retina and hypothalamus contain anionic endothelial cell mitogens that display unusual affinities for the negatively charged glycosaminoglycan heparin. Both growth factor activities are acidic polypeptides (pl's of 5.0) as determined by isoelectric focusing and DEAE-affinity chromatography. In spite of their anionic nature, the factors bound to heparin-Sepharose columns with high affinity and could be eluted only at high salt concentrations (0.9-1.1 M NaCl). The affinity of the retina-derived growth factor (RDGF) for heparin permitted a 15,000-fold purification of the mitogen in two steps: heparin-affinity chromatography and size exclusion high-performance liquid chromatography. RDGF and the anionic hypothalamus-derived factor (aHDGF) exhibit three major biochemical similarities including isoelectric point, (pl's of 5.0), heparin affinity (elution at 0.9-1.1 M NaCl) and molecular weight (18,000). Additionally, the two factors display similar biological activities, stimulating the proliferation of capillary and human umbilical vein endothelial and 3T3 cells but not vascular smooth muscle cells. We suggest that RDGF and aHDGF are related if not identical growth factor molecules.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.     

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.     

Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells

A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF.     

Modulation of growth of human carcinoma SW-13 cells by heparin and growth factors

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.     

Monoclonal antibodies to avian lipoprotein lipase. Purification of the enzyme by immunoaffinity chromatography

Three monoclonal antibodies to avian lipoprotein lipase have been isolated by fusing spleen cells from immunized BALB/c mice with myeloma P3X-63 Ag 8. The antibodies were detected by their ability to bind immobilized lipoprotein lipase in enzyme-linked immunosorbent assay (ELISA) and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibodies. Two of these antibodies, CAL1-7 and CAL1-11, inhibited catalytic activity, whereas with CAL1-2 interaction with lipoprotein lipase could be demonstrated only in ELISA and in Western blot assays following denaturation of the enzyme with sodium dodecyl sulfate. An immunoadsorbent column was prepared by coupling immunopurified CAL1-11 to Sepharose-4B. When acetone powder extracts of adipose tissue were applied on the column, 70% of the catalytic activity bound to the matrix. Effective elution was achieved with 1.8 M NaCl, 40% glycerol, 5% acetone, 20 mM Chaps (3[(3-cholamidopropyl)dimethylammonio]propanesulfonate), 0.5 mM EDTA, 1 mM phosphate (pH 6.5). After concentration of the active fractions on a heparin-Sepharose 4B column, the purified enzyme was obtained with an overall recovery of 25%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrates that the preparation is homogeneous with a major band at Mr 60900. Thus, avian adipose lipoprotein lipase has been purified by a one-step immunoaffinity followed by a concentrating step on heparin-Sepharose 4B.     

Purification of cartilage-derived growth factor by heparin affinity chromatography

Cartilage-derived growth factor (CDGF) was found to bind tightly to columns of immobilized heparin and could be eluted with concentrations of salt in the order of 1.6-1.8 M NaCl. The molecular weight of CDGF was estimated to be 18,000-20,000 by high performance liquid-size exclusion chromatography. The affinity of CDGF for heparin greatly facilitated its purification. Highly purified CDGF active at about 1-2 ng/ml was obtained when crude cartilage extract was applied to heparin-Sepharose and the growth factor activity was recycled over heparin-Sepharose two more times. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain visualization of highly purified CDGF showed one major polypeptide band with a molecular weight of about 19,000 containing over 95% of the protein and one minor polypeptide band containing the rest of the protein. Only the Mr 19,000 polypeptide was active after elution from the polyacrylamide gel. Although CDGF bound tightly to immobilized heparin, it did not bind to immobilized chondroitin sulfate or hyaluronic acid. In addition, CDGF bound to heparin much more tightly than did platelet-derived growth factor even though these two growth factors had similar isoelectric points of about 10. These results suggest that the binding of CDGF to heparin was due to a specific affinity of the 2 molecules for each other.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.