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.