Variants of the alpha 6 beta 1 laminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin alpha 6 subunit.

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the alpha 6 subunit of murine alpha 6 beta 1 and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The alpha 6 subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than alpha 6 and has a more restricted distribution until the late blastocyst stage. alpha 6 beta 1 is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin alpha 6 subunit from a mammary gland lambda gt11 library showed, as in man, an open reading frame encoding two variants of alpha 6, alpha 6A and alpha 6B. The identity of the alpha 6 amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine alpha 6 was mapped to chromosome 2. While undifferentiated ES and EC cells express only alpha 6B, alpha 6A is co-expressed in ES cells after differentiation is induced by retinoic acid. alpha 6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both alpha 6A and alpha 6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.

Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines.

Embryonic stem (ES) cells isolated from late blastocysts can now be maintained in culture in an undifferentiated state provided they are grown in the presence of a specific differentiation inhibitor, known variously as leukaemia inhibiting factor (LIF) or differentiation inhibiting activity (DIA), found at high concentrations in medium conditioned by Buffalo rat liver (BRL) cells. ES cells acquired a differentiated phenotype in monolayer, either when in the absence of LIF/DIA or in the presence of retinoic acid (RA). We have now characterized this bipotential differentiation of ES cells in terms of a series of extracellular matrix and cell surface proteins as well as cytokeratin expression, and compared it with the changes observed during the differentiation of two embryonal carcinoma (EC) cell lines, P19 and F9. ES cells exposed to RA in the presence of LIF/DIA largely resembled F9 EC + RA after 5 days, while ES cells deprived of LIF/DIA formed a culture with mixed phenotype resembling P19 EC + RA. This study therefore establishes the predominantly parietal endoderm-like phenotype of cells derived from ES by RA induction, and suggests that a mixed population of endoderm- and ill-defined mesoderm-like cells are formed after removal of specific inhibitor(s) of differentiation.

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.

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.

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP.

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.