Ceruloplasmin releases pH-induced inhibition of cell proliferation stimulated by growth factors

Summary

Swiss 3T3 fibroblasts can be weakly stimulated to grow by bombesin, epidermal growth factor or ceruloplasmin when cells are maintained in Dulbecco's Modified Essential Medium (DMEM), the pH of which is 7.75. Addition of insulin synergizes with the other mitogens. However, only ceruloplasmin promotes DNA synthesis in Minimum Essential Medium (MEM). The pH in this medium is 7.0. All the other growth factors synergize with the ceruloplasmin effects, but such synergism is not evident with insulin. If the pH in MEM is increased to 7.25 or 7.75 by supplementation with HEPES or NaHCO₃, respectively, the results are similar to those found in DMEM. Since the oxidation of iron is increased at alkaline pH, the reoxidation of iron at the cell surface may facilitate growth at alkaline pH. We propose that iron reoxidation is limiting for cell growth and that part of the ceruloplasmin effect is mediated by its action as a terminal oxidase for ferrous iron on the cell surface. Observations consistent with this explanation include: 1) combinations of insulin with bombesin or epidermal growth factors do not promote cell proliferation at pH 7.0; 2) fetal calf serum, which has ferroxidase activity, and ceruloplasmin plus or minus other growth factors stimulate cell proliferation at pH 7.0; and 3) alkaline pH also restores the mitogenic effect of growth factors.     

Control of normal and transformed cell proliferation by growth factor-nutrient interactions

The proliferation of normal mammalian cells, similar to that of single bacterial and lower eukaryotic cells, is restricted by space and nutrients. Cultured human lung fibroblasts have been used as a model to show that, in the absence of spatial restrictions, the requirement for specific nutrients limits normal cell proliferation. Serum-derived hormonelike growth factors transiently reduce the requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for normal cell proliferation. Oncogenic transformation by virus causes a constitutive reduction in the requirement for multiple nutrients for proliferation. A constitutive reduction in the proliferative requirement for Ca2+, K+, and Mg2+ allows transformed cells to escape the restrictions imposed on normal cell growth by suboptimal external concentrations of Ca2+, K+, Mg2+, and hormonelike growth factors. An understanding of the processes that determine the nutrient requirements of different normal cell types and their alteration by hormonelike growth factors and oncogenic agents is needed to understand and suppress the growth advantage possessed by malignant cells.     

Enhancement of survival and proliferation of clonogenic cells by monocyte-derived growth factors

Human peripheral blood monocytes release a factor that enhances the clonal growth of human epithelial tumor cells in soft agar. The monocyte-derived growth factor was needed for both cellular proliferation and survival. Survival of SW-13 colony-forming cells decreased linearly in the absence of monocyte-conditioned media (MO-CM). Cells failed to respond to MO-CM after four days in culture. Although MO-CM enhanced growth when cells were plated at low density, growth was also enhanced when cell density was not a limiting factor. MO-CM increased DNA synthesis of SW-13 cells growing in monolayer culture as measured by tritiated-thymidine incorporation. These findings support evidence indicating inflammatory products may play a role in maintenance of the transformed phenotype.     

Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium

Pure neuronal cultures prepared from 6-day-old embryonic chick brains incorporated [3H]-thymidine in serum-free medium up to the 4th day in culture. The addition of insulin any time within this culture period caused an increase in thymidine incorporation. This increase in [3H]-thymidine was correlated with an increase in cell number and percentage of labeling index. Triiodothyronine and endothelial cell growth factor were also active in stimulating [3H]-thymidine incorporation into chick neuroblasts. The effect of these trophic agents is unique since a variety of known mitogens tested were negative.     

Transforming growth factors and the regulation of cell proliferation

The number of different growth regulatory molecules which have been isolated and characterized is continuing to increase. As more information is obtained, it has become apparent that the cooperative actions of many factors with distinct activities is necessary for appropriate proliferative responses. An interplay of both growth stimulatory and growth inhibitory factors is essential for normal growth. Of crucial importance, therefore, is the appropriate regulation of growth factors. Unregulated expression, synthesis, posttranslational processing or activation of either positive or negative growth signals may contribute to neoplastic transformation (Fig. 3). Altered responses to normally positive or negative signals by transformed cells have been demonstrated by several investigators [64, 79, 84]. While altered growth factor responses in transformed cells are well documented, the mechanisms responsible for the loss of growth control are poorly understood and are likely to be both complex and numerous. Continued efforts to dissect and comprehend fully growth factor action on normal cells will be necessary before an understanding of neoplastic transformation can be achieved.     

Control of CFUc proliferation by selective endogenous inhibitors.

The susceptibility of mouse bone marrow colony forming cells (CFUc) to three different types of proliferation inhibitors in capillary semisolid agar gel was studied. GI-3, a target specific peptide containing granulocyte fraction, T4-1, an oligospecific thymic factor of proteid nature, and the alkylating cytostatics dianhydrogalactitol (DAD) inhibit myeloid colony formation as a function of concentration. The respective MED values amount to 8, 10, and 0.002 microgram/ml. When compared with this same parameter 3H-TdR incorporation into DNA of liquid bone marrow cultures showed a single fold charge for the endogenous inhibitors (GI-3, T4-1) for the cytostatic (DAD) a 3 to 4 fold lower difference. It was demonstrated, that in competitive antagonism of GI-3 and colony stimulating factor the inhibitor prevails over CSF.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.     

Therapeutic implications of serum factors inhibiting proliferation and inducing differentiation of myeloid leukemic cells

Murine post-endotoxin sera contain high levels of myeloid colony-stimulating factor(s) (GM-CSF) and factors capable of inducing terminal granulocyte and macrophage differentiation of the murine myelomonocytic leukemic cell line WEHI-3. The combination of C. parvum and endotoxin induced a serum activity capable of inducing tumor necrosis and inhibiting leukemic colony formation in vitro. This factor (TNF) could be separated from the differentiation-inducing factor (GM-DF) and from CSF. In conjunction with a Phase I trial of highly purified endotoxin in patients with advanced malignancy, we monitored human post-endotoxin sera for CSF and GM-DF. Induction of GM-DF occurred maximally at 2-6 h and was associated with increased serum levels of CSF active against the patient's own bone marrow. Following repeated injections of escalating doses of endotoxin, persistent levels of GM-DF were detected both pre-endotoxin and 24 h post-endotoxin treatment. The ability to induce repeatedly a serum protein with potent capacity to promote terminal differentiation of myelomonocytic leukemic cells suggests a possible therapeutic role in human myeloid leukemias.     

Erythropoietin and myeloid colony stimulating factors.

The production of blood cells in the body is controlled by at least 20 polypeptide growth factors. Most of these factors have been cloned and many expressed in bacterial and eukaryotic systems to give biologically active proteins. Currently, these recombinant human proteins are undergoing intensive evaluation for their use in treating primary haemopoietic diseases, or stimulating normal haemopoiesis following drug-, radiation- or virus-induced trauma of the bone marrow. Erythropoietin (EPO) and the myeloid colony stimulating factors (IL-3, G-CSF, GM-CSF and M-CSF) were among the first to be cloned and expressed.     

Regulation of fracture repair by growth factors.

Fractured bones heal by a cascade of cellular events in which mesenchymal cells respond to unknown regulators by proliferating, differentiating, and synthesizing extracellular matrix. Current concepts suggest that growth factors may regulate different steps in this cascade (10). Recent studies suggest regulatory roles for PDGF, aFGF, bFGF, and TGF-beta in the initiation and the development of the fracture callus. Fracture healing begins immediately following injury, when growth factors, including TGF-beta 1 and PDGF, are released into the fracture hematoma by platelets and inflammatory cells. TGF-beta 1 and FGF are synthesized by osteoblasts and chondrocytes throughout the healing process. TGF-beta 1 and PDGF appear to have an influence on the initiation of fracture repair and the formation of cartilage and intramembranous bone in the initiation of callus formation. Acidic FGF is synthesized by chondrocytes, chondrocyte precursors, and macrophages. It appears to stimulate the proliferation of immature chondrocytes or precursors, and indirectly regulates chondrocyte maturation and the expression of the cartilage matrix. Presumably, growth factors in the callus at later times regulate additional steps in repair of the bone after fracture. These studies suggest that growth factors are central regulators of cellular proliferation, differentiation, and extracellular matrix synthesis during fracture repair. Abnormal growth factor expression has been implicated as causing impaired or abnormal healing in other tissues, suggesting that altered growth factor expression also may be responsible for abnormal or delayed fracture repair. As a complete understanding of fracture-healing regulation evolves, we expect new insights into the etiology of abnormal or delayed fracture healing, and possibly new therapies for these difficult clinical problems.     

Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner

Adiponectin, an adipocyte-specific secretory protein, is present in serum as three oligomeric complexes. Apart from its roles as an anti-diabetic and anti-atherogenic hormone, adiponectin has been implicated as an important regulator of cell growth and tissue remodeling. Here we show that some of these functions might be mediated by the specific interactions of adiponectin with several important growth factors. Among six different growth factors examined, adiponectin was found to bind with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF) with distinct affinities. The bindings of adiponectin with these growth factors are oligomerization-dependent. PDGF-BB bound to the high molecular weight (HMW) and middle molecular weight (MMW) complexes, but not to the low molecular weight (LMW) complex of adiponectin. Basic FGF preferentially interacted with the HMW form, whereas HB EGF bound to all three forms with comparable affinities. These three growth factors did not compete with each other for their bindings to adiponectin, suggesting the involvement of distinct binding sites. The interactions of adiponectin with PDGF-BB, basic FGF, and HB EGF precluded the bindings to their respective membrane receptors and attenuated the DNA synthesis and cell proliferation induced by these growth factors. Small interfering RNA-mediated down-regulation of adiponectin receptors did not affect the suppressive effects of adiponectin on cell proliferation stimulated by these growth factors. These data collectively suggest that the oligomeric complexes of adiponectin can modulate the biological actions of several growth factors by controlling their bioavailability at a pre-receptor level and that this effect might partly account for the anti-atherogenic, anti-angiogenic, and anti-proliferative functions of adiponectin.     

Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors

TGF beta is a potent (ED50 approximately 10(-11) M) inhibitor of the proliferative activities of both acidic and basic FGF on vascular and capillary endothelial cells in vitro. The inhibition of cell growth is dose-dependent and characteristic of a non-competitive interaction. The results demonstrate that TGF beta and FGF can interact at the cellular level to modulate growth and suggest that many of the biological activities of FGF observed in vitro and in vivo (ie angiogenesis, cell growth, cell differentiation) may be regulated by the presence of TGF beta and related proteins (ie inhibin) in the local cellular milieu. The possible identity of TGF beta with the inhibitors of endothelial cell growth detected in in vitro assays of crude extracts is discussed.     

Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-I and follicle-stimulating hormone in vitro.

Granulosa cells of antral follicles both proliferate and undergo differentiation. The aim of the present work was to study the mechanisms controlling the balance between proliferation and differentiation in granulosa cells during the development of antral follicles in the ewe. For this purpose, the responses of both activities to insulin-like growth factor-I (IGF-I) and to FSH in vitro were studied comparatively in granulosa cells from small antral follicles (1-3 mm in diameter) and large antral follicles (5-7 mm in diameter). In granulosa cells from large follicles, IGF-I enhanced both basal and FSH-induced progesterone secretion after a 24-h delay period; this effect was lower and further delayed in cells from small follicles. Reciprocally, FSH increased IGF-I-stimulated progesterone secretion in cells from large follicles. IGF-I increased the thymidine labeling index of granulosa cells from small follicles within 24 h and enhanced cell multiplication. In cells from large follicles, this effect was lower and delayed, but IGF-I also enhanced cell survival. Culture at high density of plating inhibited the proliferative response of both types of cells to IGF-I. FSH was without effect on granulosa cell multiplication. These results suggest that the cytodifferentiative and the growth-promoting effects of IGF-I are clearly distinct. We propose that they would be exerted on two distinct granulosa cell subpopulations, nonproliferating and proliferating cells, respectively. Differences in the responsiveness of cells from small and large follicles could be related to differences in the proportion of these two cellular subtypes.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Secretion of stromelysin by cultured dermal papilla cells: differential regulation by growth factors and functional role in mitogen-induced cell proliferation.

To understand better the molecular nature of the epithelial-mesenchymal interactions that govern folliculogenesis and hair growth, we have studied the behavior of cultured rat dermal papilla cells (rDP), the mesenchymal component of the hair follicle. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) both potentiated the growth of rDP in culture, and transforming growth factor-beta (TGF-beta) inhibited rDP proliferation. Biosynthetic labeling studies demonstrated that both PDGF and bFGF induced synthesis of a major secreted protein(s) with Mr = 55-60 kD. It was noted that PDGF and bFGF differentially regulated synthesis of this major secreted protein; PDGF-mediated induction was found to be transient, while bFGF allowed prolonged synthesis of the protein. Sodium dodecyl sulfate (SDS)-substrate gel analysis of rDP-conditioned media revealed that this protein is a metalloproteinase with casienolytic activity and Mr approximately 51 kD (unreduced). We have identified the growth factor-regulated rDP protein as the matrix metalloproteinase stromelysin by immunoprecipitation. Northern analysis established that increased secretion of stromelysin was accompanied by an increased expression of stromelysin-specific mRNA. Remarkably, stromelysin antisera interfere with stimulation of dermal papilla cell growth, demonstrating that stromelysin production serves a functional role in mitogen-induced proliferation in these cells. These findings provide insight into the mechanism by which the connective tissue remodeling required for formation of hair embryonically and the postembryonic hair cycle may be regulated.