Expression patterns of follistatin and two follistatin-related proteins during mouse development.

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.

The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

ActRIA (or ALK2), one of the type I receptors of the transforming growth factor-beta (TGF-beta) superfamily, can bind both activin and bone morphogenetic proteins (BMPs) in conjunction with the activin and BMP type II receptors, respectively. In mice, ActRIA is expressed primarily in the extraembryonic visceral endoderm before gastrulation and later in both embryonic and extraembryonic cells during gastrulation. To elucidate its function in mouse development, we disrupted the transmembrane domain of ActRIA by gene targeting. We showed that embryos homozygous for the mutation were arrested at the early gastrulation stage, displaying abnormal visceral endoderm morphology and severe disruption of mesoderm formation. To determine in which germ layer ActRIA functions during gastrulation, we performed reciprocal chimera analyses. (1) Homozygous mutant ES cells injected into wild-type blastocysts were able to contribute to all three definitive germ layers in chimeric embryos. However, a high contribution of mutant ES cells in chimeras disrupted normal development at the early somite stage. (2) Consistent with ActRIA expression in the extraembryonic cells, wild-type ES cells failed to rescue the gastrulation defect in chimeras in which the extraembryonic ectoderm and visceral endoderm were derived from homozygous mutant blastocysts. Furthermore, expression of HNF4, a key visceral endoderm-specific transcription regulatory factor, was significantly reduced in the mutant embryos. Together, our results indicate that ActRIA in extraembryonic cells plays a major role in early gastrulation, whereas ActRIA function is also required in embryonic tissues during later development in mice.

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse.

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB-/- embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB-/- ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB-/- cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB-/- blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation.

Truncated activin type II receptors inhibit bioactivity by the formation of heteromeric complexes with activin type I. receptors.

Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP-lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors.

Expression of type I and type IB receptors for activin in midgestation mouse embryos suggests distinct functions in organogenesis.

Activins exert their effects by inducing heteromeric complexes of either of two different type I receptors, ActR-I or ActR-IB, and either of two type II receptors, ActR-II or ActR-IIB. We describe the cDNA cloning of the mouse homologue of human ActR-IB and analyze binding of radio-iodinated activin on type I/type II combinations of mouse receptors expressed from cDNA. We studied the distribution of ActR-I and ActR-IB mRNAs in postimplantation mouse embryos by in situ hybridization. In the 12.5-day postcoitum embryo, both mRNAs are found in the brain, spinal cord, some ganglia, vibrissae, lungs, body wall, stomach, gonads, ribs, limbs and shoulders. ActR-I mRNA, but not ActR-IB, is expressed in blood vessels, the heart, tongue, intervertebral discs and diaphragm. Conversely, only ActR-IB mRNA is detected in the olfactory region, eye, tooth primordium, esophagus, mesonephros, dorsal root ganglia and is strongly expressed in the spinal cord. Our results demonstrate similarities, but also differences and complementarities (mesenchymal versus epithelial expression) between the expression patterns of these type I receptors. Moreover, their expression patterns overlap with at least one of the type II activin receptors and/or one of activin subunits in some regions of the embryo, such as the brain, spinal cord, pituitary, whisker follicles, and the inner nuclear neuroblastic layer of the eye.

Regulation of Oct-4 gene expression during differentiation of EC cells.

The stem cell-specific factor Oct-4 is expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down regulated upon RA-induced differentiation. Irrespective of the direction of differentiation, Oct-4 repression in P19 EC cells requires treatment with high doses of either all-trans or 9-cis RA. Unlike in P19 cells, no RA-induced down regulation of Oct-4 expression is observed in the P19-derived RA-resistant RAC65 cells. However, in these cells Oct-4 promoter repression can be rescued in a RA-dependent manner by cotransfection of RAR alpha 2 or RAR beta 2 but not RARr gamma 1, matching previously reported transactivation properties of these receptor types. In the vicinity of the transcription initiation site of the Oct-4 gene, three Hormone Response Element HRE half sites are present which are arranged as direct repeats with different spacing. In vitro translated RAR and RXR proteins bind to this HRE as heterodimers with low affinity, in such a way that all three HRE half sites contribute to complex formation. Although P19 EC cells contain weak binding activity interacting with the Oct-4 promoter HRE, strong binding activity is observed in nuclear extracts from RA-treated P19 cells. This binding activity was shown to correspond to COUP-TFs but not nuclear RA receptors. Moreover, the presence of these binding factors in nuclear extracts corresponds to silencing of Oct-4 expression. These results implicate RA and the action of its nuclear receptors in silencing Oct-4 expression upon differentiation of EC cells. The observed silencing is most likely not exerted by direct binding of RARs to the Oct-4 proximal promoter HRE. Our results support models in which different nuclear receptor complexes sequentially occupy different sites in the Oct-4 promoter HRE to silence Oct-4 expression during RA-induced differentiation.

Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins.

Using in situ hybridization we have studied the localization of the messenger RNAs encoding the inhibin/activin subunits (alpha, beta A, beta B), the activin-binding protein follistatin and activin receptors (IIA, IIB) in mouse embryos during postimplantation development. From 6.5- to 9.5-days post coitum (p.c.) activin beta A and beta B subunit expression was restricted to the decidua, while activin receptor type IIB messages were exclusively detected in the embryo. Expression of activin receptor type IIA was apparent in the embryo as early as 9.5 days p.c. In contrast, follistatin transcripts were present in both the decidua and the embryo at the early postimplantation stages. In particular, the primitive streak region, specific rhombomeres in the developing hindbrain, somites, paraxial mesoderm and parietal endoderm cells attached to the Reichert's membrane showed strong expression of follistatin. In 10.5- and 12.5-day embryos expression of the beta A subunit message was abundant in mesenchymal tissue, in particular in the developing face, the body wall, the heart, precartilage condensations in the limb and in the mesenchyme of structures that show both epithelial and mesenchymal components, including tissues of the embryonic digestive, respiratory and genital tracts. The distribution of beta B transcripts was quite different from that observed for beta A. beta B is strongly expressed in selected regions of the brain, in particular the fore- and hindbrain, and in the spinal cord. Specific hybridization signals were also present in the epithelium of the stomach and oesophagus. Common sites of beta A and beta B expression are blood vessels, intervertebral disc anlagen, mesenchymal condensations in the flank region and the gonad primordium. The latter organ is the only site in the embryo where the alpha subunit is expressed, and thus where inhibit activity may be present. During the period of organogenesis the sites of expression of activin receptors type IIA and IIB messenger RNA (mRNA) generally coincide with or are adjacent to the sites of beta subunit expression. Differences in the expression patterns of the receptor RNAs are the whisker follicles, where type IIA is expressed, and the metanephros and the forebrain where type IIB transcripts are present. Taken together, the present data suggest that follistatin, but not one of the known activin forms (A,B,AB) is involved in early postimplantation development.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of activin expression by type beta transforming growth factors.

Activins are potentially important regulators of early developmental processes in vertebrates. Although the different forms of activin appear to be differentially expressed during early amphibian, avian, and murine development, little is known about the factors that regulate their expression. In this study we report the qualitative effects of several growth and differentiation factors on the expression of inhibin subunits in three differentiated cell lines derived from P19 embryonal carcinoma cells. These cell lines include mesodermal (MES-1), neuroepithelial (EPI-7), and visceral endoderm-like (END-2) cell types, expressing both inhibin beta A and beta B subunit mRNAs. We have shown for the first time that this expression is modulated by transforming growth factor (TGF)beta 1 and TGF beta 2 but not significantly by other growth factors such as leukemia inhibitory factor or members of the fibroblast growth factor family (aFGF, bFGF, or kFGF). beta A mRNA expression is increased while beta B expression is simultaneously decreased by TGF beta. Furthermore, TGF beta increased the amount of bioactive activin secreted by MES-1 and END-2 cells. Inhibin alpha subunit mRNA expression is not affected by TGF beta. These results point to a possible role of type beta transforming growth factors as regulators of activin expression in embryonal cells.

Secretion of transforming growth factor-beta isoforms by embryonic stem cells: isoform and latency are dependent on direction of differentiation.

Murine embryonic stem (ES) cells are maintained in an undifferentiated state when cultured in medium conditioned by Buffalo rat liver (BRL) cells. BRL conditioned medium (CM) contains a differentiation inhibitory activity (DIA) that is synonymous with leukemia inhibitory factor (LIF). ES cells in monolayer culture can be induced to differentiate by addition of all-trans retinoic acid (RA) to the BRL CM, when they mainly form cells resembling parietal endoderm, or by culture in medium not conditioned by BRL cells. ES cells thus deprived of LIF/DIA differentiate spontaneously to a cell type that expresses Brachyury (T), a marker of early mesoderm. Northern blot analyses have shown previously that transcripts for transforming growth factor beta 1 (TGF-beta 1) are detected in undifferentiated cells while transcripts for TGF-beta 2 and TGF-beta 3 only become detectable after differentiation. We have now determined levels of TGF-beta protein in CM and in the extracellular matrix (ECM) and have used neutralizing antibodies specific for TGF-beta 1 and TGF-beta 2 that do not react with recombinant human TGF-beta 3 to determine the isoform secreted. Using the growth inhibition of mink lung CCL64 cells as a bioassay for TGF-beta activity, we demonstrate that undifferentiated ES cells secrete latent TGF-beta 1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF-beta 2 in both active and latent forms in their CM. Likewise their ECM contains TGF-beta 2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF-beta 1 and TGF-beta 2 isoforms in their CM but TGF-beta-like activity remains after addition of neutralizing antibodies for TGF-beta 1 and TGF-beta 2. This active TGF beta is the major component of the TGF-beta activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF-beta 1 and beta 2 isoforms. The remaining activity in CM correlates with high expression of TGF-beta 3 by Northern blot analysis in these cells. We speculate that TGF-beta 3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF-beta 1 and TGF-beta 2, since it is present in CM only in its active form.

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.

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.

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RAR alpha.

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.

Survey of neuropeptide gene expression in tumor cell lines.

The presence of 3 different neuropeptide mRNAs with a strict cell-specific expression in vivo was investigated in 13 tumor cell lines from neuroendocrine and in 23 tumor cell lines from non-neuroendocrine origin. Northern blots showed no expression of mRNA for vasopressin (VP) in the 36 tested cell lines. Very low oxytocin (OT) mRNA hybridization signals were detected in the rat pituitary tumor cell line GH4C2 and the rat pancreas tumor cell line RIN5. Both the rat pituitary tumor cell line AtT-20 and the human myeloid leukemia cell line K562, contained proopiomelanocortin (POMC) mRNA. The low incidence of VP, OT and POMC gene expression in the tested tumor cell lines was not influenced by treatments inducing differentiation. In contrast, the cholecystokinin (CCK) gene which is widely present in nervous and endocrine systems was abundantly expressed in the human primitive neuroepithelioma cell line SK-N-MC and its clonal derivative SK-N-MC-IX-C. The results indicate that the expression of neuropeptide genes is very rare in tumor cell lines. The lack of expression in undifferentiated cells agrees with the appearance of expression after day 13 of the embryogenesis when maturation of neurons begins.

Expression of growth factors during the differentiation of embryonic stem cells in monolayer.

Embryonic stem (ES) cells, derived in culture directly from the inner cells mass (ICM) of blastocysts, more closely resemble their embryonic counterparts than the more commonly used embryonal carcinoma (EC) cells derived from teratocarcinomas. In view of the potential role of growth factors in early development, we have now followed changes in the expression of transforming growth factor beta (in particular TGF beta 1, beta 3, beta 4), platelet-derived growth factor (PDGF-A, PDGF-B) and insulin-like growth factor (IGF II) during the differentiation of ES cells in monolayer. When maintained in medium conditioned by Buffalo rat liver cells (BRL-CM) to inhibit differentiation, ES cells expressed 2.5 and 1.8 kb transcripts for TGF beta 1, as well as transcripts for TGF beta 4, PDGF-A, and low levels of PDGF-B, but not TGF beta 3 or IGF II. After formation of parietal endoderm-like cells by addition of retinoic acid (RA) to BRL-CM, the 1.8-kb transcript of TGF beta 1 and PDGF-A expression were reduced, IGF II mRNA and a single TGF beta 3 transcript of 3.8 kb were induced while PDGF-B and TGF beta 4 remained virtually unchanged. By contrast, in ES cells induced to differentiate by the absence of BRL-CM, unusual transcripts for TGF beta 3 of 3.0 and 6.0 kb became detectable and PDGF-B expression increased. The changes in growth factor expression in ES cells are compared with those in F9 and P19 EC cells induced to differentiate in monolayer by RA.

Expression of transforming growth factor beta 2 during the differentiation of murine embryonal carcinoma and embryonic stem cells.

Transforming growth factor beta 2 (TGF beta 2) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF beta 2 transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, P10, and ES. Following induction of differentiation, however, TGF beta 2 became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF beta 2 transcripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF beta 2 did not stain P19 EC cells by immunofluorescence but 2-3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF beta 2, while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF beta 2 after plating on a substrate. Neither TGF beta 1 nor TGF beta 2 affected the growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF beta 2 being marginally more effective than TGF beta 1 at the same concentration.

Transforming growth factor-beta and its receptor are differentially regulated in human embryonal carcinoma cells.

The human embryonal carcinoma cell lines Tera-2 clone 13 and NTera-2 clone D1 can be induced by retinoic acid to differentiate in vitro into neuroectodermal derivatives. The undifferentiated cells are rapidly proliferating and tumorigenic, whereas retinoic-acid-treated cells possess a decreased growth rate, lose their transformed phenotype and show a finite lifespan. Differentiation is accompanied by a marked increase in the levels of mRNA for TGF-beta 1 and TGF-beta 2 and the production of TGF-beta activity. Just like murine embryonal carcinoma cells the growth of Tera-2 clone 13 cells is not affected by the addition of either TGF-beta 1 or TGF-beta 2 to the culture medium. In contrast to published data on murine embryonal carcinoma cells, Tera-2 clone 13 and NTera-2 clone D1 cells bind TGF-beta 1 with high affinity, which is due to the presence of type-III TGF-beta receptors. Furthermore, and again in contrast to murine embryonal carcinoma cells, treatment of the human embryonal carcinoma cells with retinoic acid causes a nearly complete loss of TGF-beta 1 binding sites. These results are discussed in the light of similarities and differences in the regulation of growth and differentiation of human and murine embryonal carcinoma 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.

The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C.

The effects of hydrophobic interaction on the activation of Ca2+-stimulated phospholipid-dependent protein kinase (protein kinase C), isolated from mouse brain, by phosphatidylserine (PS) and diacylglycerol (DAG) or phorbol 12-myristate 13-acetate were studied. To maintain bilayer structure during assay conditions, phosphatidylcholine was added to the PS vesicles. The vesicular structure of all types of PS was confirmed by freeze-fracture electron microscopy. The PS-dependent activation of purified protein kinase C from mouse brain is affected by the fatty acid composition of PS: an inverse relationship between the unsaturation index of PS (isolated from bovine heart, bovine spinal cord or bovine brain) and the ability to activate protein kinase C was demonstrated. In highly saturated PS lipid dispersions, only slight additional activation of protein kinase C by DAG was found, in contrast with highly unsaturated PS lipid dispersion, where DAG increased protein kinase C activity by 2-3-fold at optimal PS concentrations. We quantified the formation of the protein kinase C-Ca2+-PS-phorbol ester complex by using [3H]phorbol 12,13-dibutyrate [( 3H]PDBu). The efficiency of complex-formation, determined as the amount of [3H]PDBu bound, is not affected by variations in the hydrophobic part of PS. These results indicate a role of the hydrophobic part of the activating phospholipid in the activation mechanism of protein kinase C and in the action of cofactors.

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line.

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.

EDTA-extractable proteins from calf lens fiber membranes are phosphorylated by Ca2+-phospholipid-dependent protein kinase.

A distinct group of EDTA-extractable proteins (EEP), being a major protein component of calf lens fiber membranes, is bound to these membranes in a calcium-dependent way. Both purified and membrane-bound EEP can be phosphorylated in vitro by a Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). In general, this protein kinase preferentially phosphorylates serine and threonine residues of protein substrates. Phosphoamino-acid analysis of the two major bands of EEP phosphorylated by protein kinase C, representing the 33,000 + 34,000 EEP proteins and the 30,700-31,800 proteins, respectively, revealed differences in the phosphoamino-acid patterns. For the 33,000 + 34,000 EEP proteins, only phosphothreonine was detected whereas for the 30,700-31,800 proteins, the label was incorporated in both threonine and serine residues. No label was found on tyrosine residues. These results implicate differences in the primary structure of the individual EEP proteins. Regarding previous observations that EEP is a main protein component of lens fiber junctions and of the many covering epithelial and endothelial cells, and considering the fact that protein kinase C is involved in cell-cell communication, growth and differentiation processes we suggest that a correlation exists between phosphorylation-dephosphorylation of EEP and the regulation of a number of cellular processes.