A.J.M. van den Eijnden-van Raaij

Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A

Aggregation of P19 embryonal carcinoma cells in the presence of a factor, secreted by the visceral endoderm-like cell line END-2, induces differentiation to cell types including visceral endoderm, mesoderm-derived muscle tissue and neurons. This factor is different from activin A, type beta transforming growth factors (TGF beta) and fibroblast growth factors (FGF) although its acid- and heat-lability and its stability in the presence of reducing agents resemble the properties of the FGFs. The END-2 factor is completely inhibited in its action by activin A. This inhibitory effect of activin A is not specific for the END-2 factor as retinoic acid (RA)-induced differentiation of aggregated P19 EC cells into neurons (10(-8) M RA) or mesoderm-derived muscle tissue (10(-9) M RA) is also completely inhibited by activin A. The results of this study suggest that the END-2 activity and activin A are intimately involved in the induction and regulation, respectively, of early differentiation processes in vertebrate embryogenesis.

Differential localization of TGF-β2 in mouse preimplantation and early postimplantation development

The localization of transforming growth factor type beta 2 (TGF-beta 2) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-beta 2. The staining pattern showed that TGF-beta 2 is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed.

TGF-beta 1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CCl64 cells.

Type beta transforming growth factors represent a family of polypeptides that modulate growth and differentiation. TGF‐beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are largely unresolved as yet. In this study we report that TGF‐beta 1 induces a rapid phosphorylation of the cyclic AMP responsive element binding protein (CREB) in mink lung CCl64 cells. Phosphorylation induced by TGF‐beta 1 is not mediated by the cAMP‐dependent protein kinase. Parallel to the increase in phosphorylation of CREB, an increase in binding to the collagenase TPA responsive element was observed. CREB participates in the binding to this element, probably as a heterodimer with another as yet unknown protein. The modification imposed on CREB and its involvement in an enhanced TRE‐binding could be a mechanism by which TGF‐beta 1 induces the TRE‐mediated transcriptional activation.

Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development

Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.

Charge and molecular weight heterogeneity of EDTA-extractable proteins from calf lens membranes

The EDTA-extractable proteins (EEP) of calf lens fiber cell membranes have been further characterized. Fiber EEP has been purified by gel filtration and resolved into eight bands with molecular weights of 30-38 K dalton by SDS-polyacrylamide gel electrophoresis. For epithelial EEP the same range has been obtained. In agreement with these findings a value of 33 K has been determined for fiber EEP by Sephadex G200 thin-layer gel filtration, while 34 K dalton was found by high-performance gel permeation chromatography in combination with low-angle laser light scattering (HPGPC-LALLS). The isoelectric focusing patterns of fiber and epithelial EEP show considerable charge heterogeneity. By two-dimensional electrophoresis the relation molecular weight-isoelectric point has been established for most EEP components. Peptide maps of the individual protein bands of fiber EEP differ from each other and from those of the beta Bp-, beta B1a- and beta B1b-crystallin bands, which have about the same molecular weight. From our results we conclude that EEP is not an oligomeric nor a multisubunit protein, but a collection of different extrinsic membrane proteins, biochemically unrelated to lens crystallins. The fact that removal of the cytoskeleton by urea-treatment of the membranes is a prerequisite for its isolation by EDTA or EGTA suggests that EEP is bound to the inner surface of the plasma membranes, probably via calcium.

Ultrastructural localization of platelet-derived growth factor and related factors in normal and transformed cells

Visualization of platelet-derived growth factor (PDGF) and PDGF-like growth factors in cultured cells has been achieved by cryo-ultramicrotomy in combination with immunogold labeling. Immunogold staining of cryosections requires a mild chemical fixation in order to ensure preservation of antigenicity and ultrastructural details. Therefore the effect of several chemical fixatives on the antigenic properties of PDGF and PDGF-like growth factors was studied by indirect immunofluorescence using a polyclonal anti-PDGF antiserum. These studies demonstrated that formaldehyde has no effect on antigenicity, in contrast to glutaraldehyde or acrolein. For this reason formaldehyde was used as the only fixative for the visualization of PDGF in cryosections. PDGF was visualized in cryosections of normal human fibroblasts, preincubated with PDGF under various conditions. Preincubation at 4 degrees C with PDGF resulted in partial internalization of the growth factor. During subsequent warming of the cells to 37 degrees C PDGF was translocated to the nucleus. PDGF was also detected in the cytoplasm of tumor cells producing endogenous PDGF-like growth factors (neuroblastoma and simian sarcoma virus-transformed cells) but in these cases no significant amounts of these growth factors were present in the nucleus or at the extracellular surface of these cells. These results will be discussed in view of the intracellular routing of PDGF in normal responsive cells and of PDGF-like growth factors in factor-producing cells.

Immunological relationship between the EDTA-extractable proteins from calf lens fiber membranes.

An antiserum has been prepared against the EDTA-extractable proteins (EEP) from calf lens fiber membranes. It was shown to be highly specific for EEP. Using this anti-EEP antiserum in (crossed-line) immunoelectrophoresis and (crossed) immunoelectrofocusing experiments, evidence was obtained that all of the EDTA-extractable proteins are immunologically related. They have at least one of six different antigenic determinants in common, while some of the proteins with pI values above 4.8 probably have a second common determinant. The acidic proteins of EEP comprise specific determinants with a low immunogenicity. Furthermore, it was demonstrated that no crystallin-like determinant was present on the EEP molecules.

Calcium-dependent binding of EDTA-extractable proteins to calf lens fiber membrane structures.

Calf lens fiber membranes and fractions enriched in junction-like structures have been isolated in the absence and presence of EDTA. Their biochemical features have been studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting experiments have provided evidence that a distinct group of EDTA-extractable proteins, being one of the main protein components of calf lens fiber membranes and very likely also of junction-like structures, is bound to these membranes via calcium ions. In addition to these proteins, four polypeptides with apparent molecular weights between 14000 and 17000 are characteristic for detergent-insoluble lens fiber structures prepared in calcium-rich medium. The absence of EDTA-extractable proteins in the urea-soluble calcium-containing fraction implies that they are not components of the cytoskeleton and that the calcium-dependent binding of these proteins to the membrane is urea-resistant. The use of EDTA throughout the whole membrane isolation procedure results in their complete removal from the membranes which already starts during buffer washing. This indicates that EDTA-extractable proteins exclusively consist of extrinsic membrane proteins which probably are not involved in cytoskeleton binding.

Characterization of polyclonal anti-peptide antibodies specific for transforming growth factor β2

An antiserum was prepared against a synthetic peptide corresponding to the first 29 N-terminal amino acid residues of transforming growth factor beta type 2 (TGF beta 2) from porcine platelets. The anti-TGF beta 2 peptide antiserum appeared to be completely specific for TGF beta 2 in several immunological assays, including enzyme-linked immunosorbent assays, immunoblotting and immunofluorescence experiments. Furthermore, this antiserum completely neutralized the growth inhibitory effect of TGF beta 2 on mink lung carcinoma (ML-CC164) cells and the transforming capacity of this factor on quiescent monolayers of NRK cells in the presence of epidermal growth factor. These data indicate that the N-terminal region of TGF beta 2 may be involved in the biological activity of this growth factor. TGF beta 1 was not recognized by the anti-TGF beta 2 peptide antiserum. The specificity of the anti-TGF beta 2 peptide antiserum for TGF beta 2 appeared to be useful in identifying TGF beta 2 produced by different cell systems and will be helpful in determining possible functional differences between TGF beta 1 and TGF beta 2.

Complex formation of EDTA-extractable proteins from calf lens fiber membranes with calcium and acidic phospholipids

The nature of the membrane components, which are involved in the calcium-dependent binding of EDTA-extractable proteins (EEP) to calf lens fiber membranes, has been investigated. Association experiments of radioiodinated EEP with several lens membrane preparations by means of the gel-overlay technique have shown that only phospholipids have the ability to bind EEP in the presence of calcium. The water-soluble crystallins, cytoskeletal proteins and membrane proteins of lens cells lack EEP-binding properties in this technique. Binding studies of EEP with sonicated vesicles of separate phospholipids revealed that acidic calcium-binding phospholipids, i.e. phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) are effective in binding provided that calcium is present. Phosphatidylcholine (PC) and sphingomyelin (SM) do not bind EEP in the presence of calcium. The results of association and binding studies indicate that the EEP-lipid binding is purely electrostatic and is accomplished very likely by coupling of the anionic groups of EEP and phospholipids via calcium ions. The interaction of EEP with (isolated) lens fiber membranes is resistant to the non-ionic detergent Triton X-100 in the presence of calcium. This result confirms the earlier idea that the EEP-membrane binding is predominantly or even exclusively electrostatic. It is suggested that calcium-binding phospholipids, rather than proteins, function as binding sites for EEP in this hydrophilic calcium-dependent binding of EEP to lens fiber membranes.