Molecular Mechanisms in Cellular Growth and Differentiation

354 Pages·1991·10.624 MB·English

by  A.R. Bellve (Eds.)

MOLECULAR MECHANISMS IN
CELLULAR GROWTH AND
DIFFERENTIATION
Edited by
Anthony R. Bellve
Henry J. Vogel
College of Physicians and Surgeons
Columbia University
New York, New York
ACADEMIC PRESS, INC.
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Library of Congress Cataloging-in-Publication Data
Molecular mechanisms in cellular growth and differentiation /
[edited
by] Anthony R. Bellve, HenryJ. Vogel.
p. cm.
Includes index.
ISBN 0-12-085360-4 (alk. paper)
1. Cells-Growth-Regulation. 2. Growth factors. 3. Paracrine
mechanisms. 4. Autocrine mechanisms. I. Bellve, Anthony R.
II. Vogel, Henry J. (Henry James), Date.
QH604.M65 1991
574.87'61-dc20 90-1034
CIP
Printed in the United States of America
91 92 93 94 9 8 7 6 5 4 3 2 1
Preface
If to genetics, embryology, and molecular biology, we add biochemistry,
physiology, and pathology, and if we focus on the region in which all six of
these fields overlap, we are looking at the general area underlying this
volume. Within this interdisciplinary area, we find three processes of broad
import for all the life sciences: mitogenesis, oncogenesis, and differentiation.
These processes are interrelated, partly because all three characteristically
can involve a sequence of three major types of mechanisms, namely, cell-
cell communication, signal transduction, and regulation of gene expression.
Overall, this sequence provides a means by which information from a cell's
chemical environment can modify the expression of the cell's genetic endow-
ment. Mitogenesis, oncogenesis, and differentiation are also interrelated
because all three can additionally involve another mode of regulation of gene
expression, which depends on intracellular factors, such as sequence-specific
DNA-binding proteins, rather than on cell-cell communication.
A significant type of cell-cell communication involves the interaction of a
polypeptide growth factor, secreted by one cell, with a corresponding specif-
ic membrane receptor on the surface of a neighboring cell. Polypeptide
growth factors are involved not only in cell growth, as their name suggests,
but also in the control of multiple cell functions during embryogenesis,
development, and adulthood. Such factors thus can cause discrete popula-
tions of cells to proliferate or to differentiate, or both. Proliferation and
differentiation can occur in spatially and temporally regulated patterns to
bring about coordinated interactions and development of the cell popula-
tions forming a tissue. In cell proliferation as well as in differentiation,
growth factors can act singly or in concert or in opposition to coordinate the
expression of multiple functions.
Molecules such as polypeptide growth factors can be regarded as signals
that, through interaction with membrane receptors, are capable of triggering
a series of signal transduction events culminating in the regulation of gene
expression. Signal transduction tends to be specific, complex, and versatile,
and may be pleiotypic, i.e., productive of more than one specific effect.
Characteristically, signal transduction involves phosphorylation mechanisms
in which membrane-associated kinases and guanine nucleotide-binding pro-
teins may participate. Between the triggering of transduction and gene ex-
pression, various translocation-type processes must occur through which the
xv
XVI PREFACE
information supplied by the signal is delivered to appropriate cytoplasmic or
nuclear sites.
Signals may be (a) autocrine, i.e., produced by a cell and active on the
same cell, or on a neighboring sister cell; (b) paracrine, i.e., secreted by a
cell and active on a neighboring cell of different lineage (as in some types of
cell-cell communication); or (c) endocrine, i.e., produced by a source cell
and active on a target cell, at some distance. In this volume, autocrine and
paracrine signals are emphasized. Because of their role in cell-cell commu-
nication, paracrine signals are discussed first.
An important alternative to the role of paracrine signals, such as growth
factors, in the regulation of gene expression is the functioning of autocrine
regulatory elements, e.g., sequence-specific DNA-binding proteins. Such
regulatory elements include proteins that are capable of interacting, in re-
ceptor-ligand fashion, with small effector molecules, e.g., steroid hor-
mones.
Gene expression may be regulated (a) directly, by the positive or negative
control of transcription, through mechanisms including induction, repres-
sion, and derepression, or (b) indirectly, at any of several posttranscriptional
levels up to and including translation. Induction (positive control) and re-
pression (negative control) represent an increase or decrease in the rate of
transcription of a structural gene and hence in the rate of synthesis of the
protein encoded by that gene, respectively. Both of these mechanisms typ-
ically involve interactions between sequence-specific DNA-binding proteins
and part of the promoter region for the structural gene. In induction, the
transcription-accelerating effect is brought about, in the promoter region, by
the DNA-binding protein in conjunction with a small-molecule inducer,
such as an estrogen. The DNA-binding protein thus can function as an
intracellular receptor for the small-molecule inducer. In repression, the
transcription-decelerating effect is produced, in the promoter region, by the
DNA-binding repressor protein, in conjunction with a small repressive mol-
ecule, e.g., in some cases, a glucocorticoid. In the absence of the small
repressive molecule, derepression occurs. However, the regulation of gene
expression is not unrestricted. For instance, in cells that have undergone
commitment during embryogenesis, regulation is limited to the expression
of those genes that are within the regulable repertoire of the committed
cells. Thus, a particular signal may elicit different responses from different
cell types.
Among the polypeptide growth factors are the transforming growth factors,
which are so designated because, for appropriate cell types, they can bring
about not only proliferation, but also morphological transformation, in mono-
layer culture, and anchorage-independence. Their special characteristics
relate the transforming growth factors to neoplastic transformation, oncogen-
PREFACE XV11
esis, and oncogenes, although such growth factors can occur in normal cells.
Oncogenes, which are capable of inducing neoplastic transformation, bear
a remarkable relationship to the growth factor-signal transduction-gene
expression sequence method above. Thus, oncogene-encoded proteins in-
clude polypeptide growth factors, growth factor receptors, membrane-asso-
ciated kinases, and guanine nucleotide-binding proteins, and other proteins
that can function in various steps of signal transduction pathways, with con-
sequent autocrine production of cellular abnormalities, including stimula-
tion of the growth of cultured cells and of solid tumors. Oncogenes are
activated derivatives of protooncogenes, which are oncogene homologs pre-
sent in normal cells. Mechanisms of activation include (a) changes in regula-
tion of gene expression; (b) point mutations; and (c) formation of recombinant
fusion proteins. There are indications that some protooncogenes encode
growth factors and receptors, which can function in the control of normal cell
proliferation.
Protooncogenes, with or without involvement of proliferative functions,
can play key roles in normal processes of differentiation. A number of such
roles have been identified for specific protooncogenes. Thus, the src pro-
tooncogene, which is the cellular progenitor of the tyrosine kinase oncogene
of Rous sarcoma virus, can be involved in neuronal differentiation in the
absence of cell proliferation.
In line with the various connections between protooncogenes and on-
cogenes, on the one hand, and peptide growth factors, on the other (Parts I
and II of this volume), these growth factors importantly participate in differ-
entiation phenomena. In the present treatment of differentiation and devel-
opment, emphasis is placed on pattern formation, i.e., the genesis of spatial
relationships among the parts of an organism, embryonic or adult. Verte-
brate (mammalian and avian) and invertebrate (dipteran) organisms are dis-
cussed. Parts III to V of this volume are arranged according to molecular
mechanisms that are exhibited in illustrative organ systems: paracrine mech-
anisms in gonadal and vascular systems and one case of a segmentation
system (Part III); autocrine mechanisms in other cases of segmentation and
segmentation-like systems (Part IV); and autocrine-type mechanisms with
responsiveness to an exogenous small-molecule morphogen or effector in
limb and intestinal cell systems (Part V).
Part I deals with major steps leading from growth factor-receptor interac-
tions, via transduction and modulation mechanisms, to the proliferative re-
sponse. Insulin and insulin-like growth factors and their receptors, which are
of broad relevance to differentiation, are considered from the points of view
of ligand-receptor binding (Van Wyk and Casella) and membrane protein
recycling (Czech et ah). Insulin and other growth factors, including epider-
mal growth factor, can trigger, among others, a transduction mechanism with
xviii PREFACE
widespread consequences, namely, the multiple phosphorylation of the 40 S
ribosomal protein S6, which can lead to a considerable increase in the rate of
general protein synthesis, as a significant step in the transition of animal cells
from the quiescent to the proliferative state of growth (Jenö, Ballou, and
Thomas). The complexity of phosphorylation events at the cytosolic face of
cell membranes is exemplified by the functioning of pp60csrc, a protein
product of the c-src gene, which is thought capable of participating in the
phosphorylation of other proteins. In pp60csrc, there is a multiplicity of
phosphorylation sites, and there is a multiplicity of protein kinases that
can phosphorylate these sites, with the possibility of interactive effects,
either synergistic or counteractive, allowing pp60c_src to act as a node in a
regulatory circuit, which receives inputs through the different protein
kinases able to phosphorylate it, and which provides an integrated output by
phosphorylating its substrates (Hunter et al.). In signal transduction, a large
number of receptors can interact with GTP-binding proteins whose proper-
ties resemble those of certain cytoskeletal proteins. GTP-binding proteins
appear to have higher order polymeric structures which are coiled and
springlike, and which are adapted to rapid transmission of information pro-
vided by subtle changes in receptor structure (Rodbell). Transduction can be
accompanied by the appearance of modulators capable of regulating the
activity of enzymes relevant to the transduction signal. Thus, in response to
insulin, plasma membranes can release modulators identified as inositol
phosphate glycans (Saltiel).
In addition to soluble signals, such as polypeptide growth factors, certain
insoluble signals, such as extracellular matrix components, can function as
regulators of gene expression. One such component, a heparan sulfate pro-
teoglycan, bound to the surface of hepatocytes by inositol phosphate, can be
internalized, and the heparan sulfate chains can be translocated to the cell
nucleus. Since heparins are capable of binding growth factors, the possibility
is considered that heparins or related molecules can serve as vehicles that
would permit the growth factors to reach the nucleus (Reid). One of the
components of the extracellular matrix is the basement membrane, and a
component of this membrane is laminin, which is a large, complex, and
versatile glycoprotein. Laminin can interact with tumor cells through a cell
membrane receptor. This interaction plays a significant role in the attach-
ment of tumor cells to the basement membrane. Once attached, the tumor
cell can secrete, or induce host cells to secrete, enzymes that can locally
degrade the basement membrane. Such degradation is an essential step in
tumor cell invasiveness and in metastasis (Sobel and Liotta).
A polypeptide growth factor, transforming growth factor-ß, can counteract
the degradation of the basement membrane not only by decreasing protease
production and stimulating protease inhibitor, but also by increasing the
PREFACE XIX
synthesis of extracellular matrix components. This growth factor is mitogenic
for fibroblastic and certain other mesenchymal cells, but is growth-inhibitory
for many cell types. Transforming growth factor-α, however, is a potent
mitogen for most cell types. Cell proliferation and its positive or negative
control, under the influence of these two growth factors, are considered
(Moses et al.).
Part II is concerned with the relation of growth factors and their receptors
to oncogenes and to protooncogenes. Transforming growth factor-α shows
partial sequence and structural homology to epidermal growth factor, and
appears to function through interaction with epidermal growth factor recep-
tor. The major structural elements of the mature receptor are an extra-
cellular epidermal growth factor-binding domain, a transmembrane region of
hydrophobic amino acids, and a cytoplasmic domain. The binding of the
growth factor to its receptor activates the receptor tyrosine kinase. A trun-
cated form of the epidermal growth factor receptor is encoded by the v-erbB
oncogene of the avian erythroblastosis virus, and the v-erbB protein appears
to transform by functioning as an activated growth factor receptor
(Schlessinger). The w-erbB oncogene can transform erythroblasts and fibro-
blasts in vitro. A second oncogene, v-erbA, having an enhancing function, is
carried by the same virus, which can induce erythroblastosis and sarcomas in
vivo. The cellular protooncogene c-erbA, corresponding to this viral on-
cogene, encodes a high-affinity nuclear receptor for thyroid hormones, pre-
sumably acting by direct regulation of transcription. The v-erbA protein,
however, is ligand-independent. Nucleotide sequences, effects of mutations,
and the mechanism of transformation are discussed (Vennström et al.).
Transformation of NIH 3T3 cells with the ras oncogene induces the syn-
thesis of basic fibroblast growth factor. Tumors produced by ras-transformed
cells express this growth factor. In vitro, the expression of the growth factor
in ras-transformed cells is increased by a factor of 25, in comparison with
that in the parental cells (Klagsbrun et al.). The finding that NIH 3T3 cells,
cultured in a defined medium, die in the absence of fibroblast growth factor
or platelet-derived growth factor forms the basis of a new assay for the
detection of oncogenes, which has led to the identification of an oncogene
encoding a novel fibroblast growth factor (Goldfarb). In connection with the
normal cellular functioning of several protooncogenes and with their rela-
tionships to peptide growth factors or growth factor receptors, the ex-
pression of the mos protooncogene in murine male germ cells and oocytes is
described (Cooper).
Part III pertains to roles of growth factors and receptors in differentiation
and development, particularly, in pattern formation. In male gonadal devel-
opment and functions, a number of polypeptide growth factors and their
receptors are involved. The growth factors include seminiferous growth fac-
XX PREFACE
tor, which appears to be related to acidic fibroblast growth factor; basic
fibroblast growth factor; insulin-like growth factor-I; transforming growth
factors a and ß; ß-nerve growth factor; and, probably originating from extra-
gonadal sites, epidermal growth factor and insulin-like growth factor-II.
Growth factors of local origin may act primarily in an avascular environment,
whereas those present in serum may act within the interstitial or peritubular
regions where appropriate cell membrane receptors are accessible. In the
developing murine seminiferous epithelium, the expression of seminiferous
growth factor is consistent with levels of mRNA encoded by the c-myc
protooncogene. Seminiferous growth factor and the acidic and basic fibro-
blast growth factors are related, and have significant effects, which may or
may not be concerted, on the regulation of the growth and function of Ley dig
and Sertoli cells. All three of these growth factors can bind heparin, can
induce the proliferation of endothelial cells (among others), and are thought
to be involved in neovascularization of the developing gonads (Bellve and
Zheng). Endothelial cells, which form the inner surface of all functional
blood vessels, are polarized: the luminal surface has nonthrombogenic prop-
erties, whereas abluminally the cells, early in development, produce an
extracellular matrix, which is subsequently remodeled into a basement
membrane. Laminin expression appears to be an early marker for vascular
maturation. Two heparin-binding endothelial growth factors, which are also
angiogenic in vivo, are structurally similar to the fibroblast growth factors.
Endothelial cells cultured on nitrocellulose membranes secrete a platelet-
derived growth factor-like chemotactic factor into the abluminal compart-
ment. Such polarized secretion would be expected for a factor involved in
the development of the vascular wall. These growth factor-dependent mech-
anisms are discussed within a broader view of blood vessel growth and
differentiation (Risau et ah). The vascular endothelium, especially, the endo-
thelium of the capillaries, is involved in the transport not only of small
molecules but also of macromolecules such as albumin. The transport of
macromolecules, from the lumen of the blood capillaries to the interstitial
fluid, is effected by plasmalemma vesicles. In such transcytosis, albumin can
serve as a vehicle for fatty acids and other passenger molecules as well as for
metal ions. The vesicles functioning in transcytosis are differentiated micro-
domains, which can be maintained only in the polarized endothelium, pre-
sumably with the aid of cell-cell interactions and of serum factors (Palade
and Milici).
Unlike the preceding topics, which deal with vertebrate differentiation,
the next topic regards a Drosophila gene, decapentaplegic (dpp) which is
involved in pattern formation. Genes like dpp sometimes are called home-
otic since, when mutated, they can cause one part of an organism to trans-
form toward a corresponding homologous part. The protein specified by
dpp, to date, is the only member of the transforming growth factor-ß family
PREFACE XXI
of proteins discovered in invertebrates. The nearest vertebrate relative of
the dpp product is the bone morphogenesis protein BMP-2A. The dpp
product appears to function, at the cell-cell level, through interaction with a
transforming growth factor-type receptor (Gelbart). Frequently, the protein
products of homeotic genes function through quite a different mechanism at
the level of a single cell.
Part IV discusses homeotic systems regulated intracellularly, with the
characteristic involvement of sequence-specific DNA-binding proteins.
Homeotic genes in Drosophila have a long history beginning with the dis-
covery of bithorax, but a detailed understanding of their expression had to
await the availability of suitable molecular biological methods. Since home-
otic genes typically act early in the programming of the embryonic develop-
ment of an organism, they can be regarded as master regulatory genes.
Three such genes, bithorax, antennapedia, and engrailed, and their ex-
pression are discussed. Certain regions of these and related genes share a
remarkable degree of homology in the form of the homeo box. Similar se-
quences are found in other invertebrate genomes as well as in avian and
mammalian DNAs. The original mutant of engrailed has a striking homeotic
effect in that the posterior wing margin transforms toward a mirror image of
the anterior wing margin. The engrailed gene contributes to the control of
segmentation, a graphic instance of pattern formation in embryogenesis
(Lewis). The unusual structures of the engrailed gene promoter and of the
phosphoprotein gene product have been determined. The gene product of
engrailed, like those of similar genes, is thought to act intracellularly as a
sequence-specific DNA-binding protein that regulates transcription. It is
suggested that the engrailed gene directly orchestrates a subset of other
genes whose role is to elaborate the developmental program of the cell
(Kornberg et al.). Another gene, even-skipped, which affects segmentation in
Drosophila, controls morphogenesis by regulating the expression of en-
grailed and by also regulating its own expression (Hoey, Frasch, and
Levine).
In the mouse, there are two genes with sequence homology to the en-
grailed gene and the related invected gene of Drosophila. The region of
homology, containing a centrally located homeo box, can code for 107 amino
acids, of which 78 are identical in all four genes. There is evidence that the
two mouse genes are expressed early in embryogenesis during periods when
fundamental cell lineage decisions are made and when the basic embryo
body plan is established (Martin and Joyner). Structural and functional analy-
ses of murine homeo box genes have revealed highly distinct temporal and
spatial expression profiles. The intriguing possibility exists that similar mo-
lecular mechanisms underlie the generation of cellular diversity and pattern
formation in many different organisms (Gruss et al.).
Part V describes two differentiation systems thought to involve sequence-
xxii PREFACE
specific DNA-binding proteins in conjunction with small molecules. In the
developing chick limb, pattern formation depends on a gradient of a diffusi-
ble morphogen released from the zone of polarizing activity. This mor-
phogen is concluded to be all-frans-retinoic acid. Implantation, at the appro-
priate site in ovo, of all-irans-retinoic acid-containing Dowex beads can
mimic the eifects of the zone of polarizing activity. The morphogen is indi-
cated to act, in a transcriptional induction process, together with a sequence-
specific DNA-binding protein belonging to the superfamily of steroid recep-
tors (Eichele and Thaller).
An in vitro differentiation system that seems to depend on eifects of small
molecules is represented by a pluripotent human intestinal epithelial cell
line that can be caused to diiferentiate in culture. Phenotypes resembling
terminally differentiated absorptive cells appear in highly polarized con-
fluent cell monolayers when galactose is substituted for glucose as the main
carbon source in the culture medium. Apparently, a readily utilizable carbon
source (glucose) does not permit such differentiation, whereas a restrictive
carbon source (galactose) does. During differentiation of cells grown in the
presence of galactose, there is a considerable increase in the rate of synthesis
of villin, a calcium-regulated actin-binding cytoskeletal protein present in
the brush border of the absorptive cells. The increased rate of villin syn-
thesis is indicated to result from an increased rate of transcription, sugges-
tive of derepression, although a possible stabilization of villin mRNA has
not been ruled out (Louvard et ah),
It is hoped that the paracrine and autocrine molecular mechanisms consid-
ered in this volume contribute to an appreciation of the warp and woof of
differentiation as well as of normal and abnormal cellular growth and to an
integration of the findings flowing from the various fields of this inter-
disciplinary area. As documented here, the research is in a highly active
state, and accelerating advances are foreshadowed.
It is a pleasure to acknowledge the advice and help of Dr. G. Blobel, Dr.
H. Green, Dr. H. Hanafusa, Dr. E. B. Lewis, Dr. D. Louvard, Dr. H. L.
Moses, Dr. G. E. Palade, Dr. M. Rodbell, and Dr. J. J. Van Wyk.
We are grateful for the fine support of the College of Physicians and
Surgeons (P & S) of Columbia University, without which this volume would
not have reached fruition, and for the continued interest of Dr. D. F. Tapley.
This volume was developed from a P & S Biomedical Sciences Symposium
held at Arden House on the Harriman Campus of Columbia University.
Anthony R. Bellve
Henry J. Vogel