Expression of TGF-beta stimulated clone-22 (TSC-22) in mouse development and TGF-beta signalling.

TSC-22 is a highly conserved member of a novel family of transcription factors, that is a direct target of transforming growth factor-beta (TGF-beta) in osteoblastic cells. We have investigated the expression of TSC-22 in detail during mouse development using in situ hybridization. We detected strong expression of TSC-22 in the embryo proper first at embryonic day 8.5 (E8.5), in the primitive heart, intermediate mesoderm and the neural tube. The dynamics of the TSC-22 distribution in the neural tube was particularly striking, with ubiquitous expression rostrally and restriction to neural tissue nearer the floor plate more caudally; between E8.5 and E9.5 the zone of restricted expression extended rostrally. At later stages of development, TSC-22 was detected in the mesenchymal compartment of many tissues and organs, including the lung, trachea, kidney, stomach, intestine, tooth buds, and in precartilage condensations. Furthermore, TSC-22 was highly expressed in the floor plate itself and notochord, and the endothelium lining the blood vessels, in particular the major arteries. Many of these sites have been proposed previously as possible TGF-beta target tissues; the results imply that TSC-22 may also be a direct TGF-beta target gene during mouse embryogenesis. Experiments on TSC-22 expression in embryoid bodies of embryonic stem (ES) cells expressing dominant negative TGF-beta binding receptors initially supported this hypothesis. However, examination of somatic chimeras derived from these same mutant ES cells at nominal E9.5 showed that TSC-22 expression in the heart and neural tube was still detectable despite obvious phenotypic abnormalities. We therefore propose that although TSC-22 may be a direct target of TGF-beta in late development, other factors are likely to be major regulators of expression at earlier stages.

Phenotypic transformation of normal rat kidney cells by transforming growth factor beta is not paralleled by enhanced production of a platelet-derived growth factor.

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum-deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density-inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor beta (TGF-beta) and retinoic acid is delayed when compared to induction by platelet-derived growth factor (PDGF), indicating that both TGF beta and retinoic acid may exert their growth-stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGF beta 1, PDGF A-chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGF beta or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF-like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti-PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGF beta and retinoic acid is not the result of enhanced production of a PDGF-like growth factor.

Identification and characterization of polypeptide growth factors secreted by murine embryonal carcinoma cells.

Undifferentiated P19 and PC13 murine embryonal carcinoma (EC) cells have been analyzed for their ability to secrete polypeptide growth factors. This has been carried out by a combination of specific bioassays and the use of biochemical and immunological detection methods. Both P19 and PC13 EC cells secrete a platelet-derived growth factor (PDGF)-like growth factor, a type beta transforming growth factor, and insulin-like growth factors. In addition, PC13 EC cells secrete a heparin-binding growth factor functionally related to fibroblast growth factor, while P19 EC cells secrete transforming growth factor-alpha. This is the first demonstration for secretion of transforming growth factor-alpha by an equivalent of early embryonic cells. The possible paracrine growth stimulating effects of these growth factors have been tested on differentiated derivatives of P19 EC cells, corresponding to all three germ layers. The differences in growth factor production by various embryonal carcinoma cells are discussed in relation to the developmental origin of these cell lines.

Production of insulin-like growth factors, platelet-derived growth factor, and transforming growth factors and their role in the density-dependent growth regulation of a differentiated embryonal carcinoma cell line.

P19 EPI-7, a differentiated murine embryonal carcinoma cell line with an epithelioid morphology, does not require external growth factors for proliferation under clonal and subconfluent conditions. At saturation density, however, cells become quiescent in the G1/G0 phase of the cell cycle from which they can be restimulated, particularly upon addition of epidermal growth factor. Medium conditioned by confluent P19 EPI-7 cultures is able to enhance clonal outgrowth of this cell line, suggesting that autocrine growth factor loops may be acting in these cells. Analysis of conditioned serum-free medium shows that this cell line produces a platelet-derived growth factor-like growth factor, next to a type beta transforming growth factor and large amounts of insulin-like growth factor II (IGF-II) and an IGF-binding protein with high specificity for IGF-II. This latter observation has been confirmed by the use of a specific bioassay for IGFs, based on their ability to specifically stimulate proliferation of MCF-7 human breast cancer cells. The amount of IGF-II produced (0.5 mg/liter conditioned medium) makes P19 EPI-7 one of the best producing cell lines for this factor described so far. Receptor cross-linking analysis shows that this cell line contains IGF-I receptors, but no specific receptors for IGF-II. Depending on the conditions tested, transforming growth factor-beta 1 either act as a growth-stimulating factor or as a strong growth inhibitory factor. These data demonstrate that upon cellular differentiation, embryonal carcinoma cells can be formed which produce polypeptide growth factors and are also able to respond to such factors. These observations are discussed in the light of the role of autocrine and paracrine growth stimulation processes during early murine development.

Purification of a growth factor related to platelet-derived growth factor and a type beta transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors.

A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets.

The role of polypeptide growth factors in phenotypic transformation of normal rat kidney cells.

A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of [3H]thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested (epidermal growth factor, platelet-derived growth factor, transforming growth factor-beta, and retinoic acid) is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.

Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents.

The chromatographic behavior of a number of globular proteins was studied on a Bio-Sil TSK CM-2-SW weak cation exchange HPLC column under acidic conditions. A linear gradient of 0-1 M NH4Ac in 1 M HOAc, inducing a convex pH gradient from 2.4-4.8, resulted in an excellent separation of highly basic proteins. For these proteins a linear relationship between isoelectric point and retention time was determined experimentally. The effect of pH and the ion composition of the eluting buffer system on this linear correlation was studied. Although the exact basis for protein separation on the CM-2-SW column at low pH is not clear yet, both the pH-dependent net positive charge per unit surface area and most likely the relative percentage of arginine in the total number of basic residues contribute to this separation. Because of the high resolving power and the high protein recovery obtained in a system using only acidic volatile buffer solutions, the cation exchanger is particularly suitable for the purification of nanogram amounts of acid-stable basic growth factors. The present sterile conditions (1 M HOAc/NH4Ac system, pH less than 4) and the easy removal of salt by lyophilization facilitate the detection of these proteins by biological assays.

PC13 embryonal carcinoma cells produce a heparin-binding growth factor.

A polypeptide growth factor has been isolated from serum-free medium conditioned by mouse PC13 embryonal carcinoma cells, which is strongly mitogenic for Swiss 3T3 fibroblasts. On a CM-2-SW high-performance liquid chromatography cation-exchange column at low pH, this growth factor elutes at a salt concentration very close to that of basic fibroblast growth factor (FGF). The growth factor is mitogenic for a mesodermal derivative of embryonal carcinoma (EC) cells, but not for differentiated derivatives with endodermal or ectodermal characteristics, again similar to FGF. The PC13-derived growth factor binds to heparin-Sepharose, and elutes from this column at similar salt concentrations as FGF. These data demonstrate that PC13 embryonal carcinoma cells produce a basic heparin-binding growth factor (HBGF). Since the initial purification steps are similar to those used by Heath & Isacke (EMBO j 3 (1984) 2957 [7]) for isolation of a PC13 embryonal carcinoma-derived growth factor (ECDGF), which is cationic with a molecular weight (MW) close to that of FGF, the present heparin-binding growth factor (HBGF) is most likely identical with ECDGF.

Production of transforming growth factors by simian sarcoma virus-transformed cells.

Cellular transformation of normal rat kidney (NRK) cells by simian sarcoma virus (SSV) results in a complete loss of the cellular requirement of externally added polypeptide growth factors for proliferation. Moreover, SSV-transformed NRK cells have a strongly reduced ability to bind both external platelet-derived growth factor and epidermal growth factor, when compared to nontransformed NRK cells. Analysis of serum-free medium conditioned by SSV-transformed NRK cells shows that this cell line secretes both types alpha and beta transforming growth factor (TGF). The level of TGF alpha production (300 ng/liter conditioned medium) by SSV-transformed NRK is among the highest described to date. Since addition of TGF alpha and beta in combination is sufficient to induce phenotypic transformation of NRK cells, it is concluded that although expression of the sis oncogene is essential for transformation, expression of additional genes may be required for the phenotypic alterations accompanying complete cellular transformation.

PDGF-like growth factor induces EGF-potentiated phenotypic transformation of normal rat kidney cells in the absence of TGF beta.

Using a growth factor defined assay for anchorage-independent growth (van Zoelen, E.J.J., van Oostwaard, Th.M.J., van der Saag, P.T. and de Laat, S.W. (1985) J. Cell. Physiol. 123, 151- 160, we have studied the ability of polypeptide growth factors produced by Neuro-2A neuroblastoma cells to induce anchorage-independent growth of normal rat kidney cells. Neuro-2A cells produce and secrete a PDGF-like growth factor in addition to TGF beta, which can be fully separated from each other by means of reverse-phase HPLC. Using a new, very sensitive technique for detection of TGF beta in growth factor samples based on its additional ability to act as a growth inhibitory factor, it is shown that the PDGF-like growth factor does not contain any detectable TGF beta. Still this neuroblastoma derived PDGF-like growth factor is able to induce anchorage-independent growth of NRK cells, particularly in the additional presence of EGF. It is concluded that under growth factor defined assay conditions TGF beta is not essential for phenotypic transformation of NRK cells.

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor.

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.

Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.

Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor.

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.

Neuroblastoma cells produce transforming growth factors during exponential growth in a defined hormone-free medium.

Mouse neuroblastoma Neuro-2A cells have been cultured in a chemically defined serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 30 nM selenite and 10 micrograms of transferrin per ml. In this medium, which does not contain any externally added polypeptide growth factor, cells proliferate rapidly with a doubling time of approximately equal to 10 hr. During exponential growth in this serum-free medium, Neuro-2A cells secrete a 15- to 20-kDa transforming growth factor with strong mitogenic action and the ability to induce anchorage-independent growth on nontransformed cells. This neuroblastoma-derived transforming growth factor (ND-TGF) is acid and heat stable but is sensitive to treatment with trypsin or dithiothreitol. However, it does not compete with epidermal growth factor (EGF) for receptor binding and does not require EGF receptors for its mitogenic activity. Experiments on the effects of EGF on ND-TGF-induced soft agar growth of normal rat kidney cells indicate that Neuro-2A cells secrete an EGF-potentiated TGF in addition to ND-TGF. It is suggested that Neuro-2A cells can proliferate in the absence of externally added growth factors as a result of autocrine production of polypeptide growth factors.