Department of Biology, Duke University, Durham, NC 27708, USA
e-mail: hfn{at}duke.edu
SUMMARY
The growth of a cell or tissue involves complex interactions between genes, metabolism, nutrition and hormones. Until recently, separate lines of investigation have concentrated in isolated sections of each of the many independent levels of growth control; the interactions within and between the diverse pathways that affect growth and size at the cellular, tissue and organismal level were little understood. However, new insights into the control of growth are now emerging in the context of signalling, ageing, evolution, cancer and nutrition. In particular, it is becoming clear that the insulin signaling network is a key player that integrates not only metabolism and the response to nutrition, but also the regulation of cell death, ageing and longevity, as well as the regulation of growth and body size.
Development is a continual and progressive iteration of pattern formation and growth. The control of patterning has traditionally received the greatest attention within the field of developmental biology, and in the past two decades we have come to understand a great deal about the mechanisms that underlie pattern formation in systems ranging from embryos to butterfly wings, and from plant roots to fly eyes. The control of growth and size has been more difficult to address, largely because the growth of a cell or tissue is not the result of a single discrete process, but is under the control of a great diversity of signaling mechanisms, and involves complex interactions between genes, metabolism, nutrition and hormones.
Understanding the control of growth and size requires a separate
understanding of the control of cell growth, cell size, cell division, tissue
and organismal size, as well as an understanding of how the processes that
regulate these separable events interact with each other. Recent advances in
our knowledge of these various processes were the subject of discussion at the
Arolla Workshop 2003, the theme of which was Growth Control in
Development and Disease, and which was held in the small resort town of
Arolla in the Swiss Alps in the latter half of August. The five-day meeting
was organized into topical sessions that dealt with growth in the context of
evolution, signaling, ageing, cancer and nutrition. The majority of talks
achieved excellent integration either within or between topics, and allowed
the participants not only to revel in the details of a great diversity of
processes, but also to develop insights into how these many kinds of `trees'
are beginning to fit together to form a `forest'. It is impossible to
highlight the entire diversity of interesting topics discussed at this
workshop in the space allowed for this review, so I will focus on the more
salient themes that emerged, but note that many of the participants at this
meeting have contributed chapters to a new book by Hall and Raff, which can be
consulted for additional reviews (Hall and
Raff, 2004).
A central role for insulin signaling
The role of insulin in carbohydrate metabolism is well understood; however, in recent years it has also become clear that altered insulin signaling can have profound effects on growth, body size, reproduction and the biology of ageing (Fig. 1). Insulin-like molecules have been identified in vertebrates and invertebrates, and include the mammalian insulins and IGFs, the Drosphila DILPs, the Caenorhabditis insulins and the lepidopteran bombyxins. The insulin signaling pathway is becoming increasingly well understood, and a substantial fraction of the talks and posters at this meeting were devoted to elucidating the structure and function of its many components. I decided the best way to summarize this information was to assemble it into a single diagram that illustrates the network of pathways by which insulin signaling can affect cell growth, proliferation and apoptosis (Fig. 2). This figure also shows how the insulin network interacts with other important pathways that affect cell growth and cell death, such as the signaling pathways that involve MAPK, JNK, Dpp and DIAP1 (Drosophila Inhibitor of Apoptosis 1).
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The interaction between TSC (tuberous sclerosis complex, a tumor suppressor
gene), TOR (target of rapamycin) and the small GTPase Rheb (Ras homologue
enriched in brain), has now been studied in Drosophila as well as in
mouse cells (Garami et al.,
2003; Oldham and Hafen,
2003
; Saucedo et al.,
2003
; Zhang et al.,
2003
). As reported by Duojia Pan (Southwestern Medical Center,
Dallas, USA) and Thomas Radimerski (University of Zurich, Switzerland),
overexpression of Rheb, or loss-of-function of TSC1/TSC2, indirectly induces
phosphorylation of S6K and 4E-BP, and inhibits PKB activation, and this
results in aberrant growth of Drosophila. These signaling molecules
are members of a complex regulatory network, immediately downstream of
PIP3, that appears to converge on S6K, and which is emerging as one
of the principal mediators in the stimulation of transcriptional activity and
cytoplasmic growth. The regulation of TOR is surprisingly complex as it
requires interaction with other proteins for activity, and these proteins
produce both rapamycin-sensitive and -insensitive complexes that have
non-overlapping functions (Loewith et al.,
2002
; Kim et al.,
2002
). In addition, Michael Hall and co-workers (University of
Basel, Switzerland) have shown that, in yeast, activation of the Ras/cAMP
signaling pathway confers a pronounced resistance to rapamycin. It appears
that Ras/cAMP signaling is a novel effector pathway for TOR that affects
ribosome biogenesis and is thus intimately involved in the regulation of cell
growth (Schmelzle et al.,
2003
).
Several investigators have shown that insulin signaling inhibits Forkhead
transcription factors of the FOXO class (which include DAF-16 in
Caenorhabditis). FOXO molecules have a large number of
phosphorylation and acetylation sites that affect their activity.
Phosphorylation by AKT results in the sequestration of FOXO in the cytoplasm
and causes its effective inactivation. Boudewijn Burgering (University Medical
Center, Utrecht, The Netherlands) and Anne Brunet (Harvard Medical School,
Boston, USA) described how stress (such as oxidative stress and heat shock)
induces different patterns of phosphorylation and acetylation, and triggers
the relocalization of FOXO to the nucleus and the transcription of genes that
enhance resistance to stress (Burgering and
Kops, 2002; Tran et al.,
2002
). Different patterns of phosphorylation and acetylation
appear to be involved in altering the effects of FOXO (such as resistance to
stress, regulation of apoptosis and organismal longevity), presumably by
altering the targets of this transcription factor.
Many of the players in the insulin-signaling network are members of
families of homologous genes. Depending on the cell type, and on the specific
alternative sets of family members that are activated, the network can affect
either cell proliferation, cell growth or cell death. The precise pathways by
which these effects are accomplished are still under investigation, and the
way in which insulin signaling interacts with the many other effectors and
inhibitors of growth, proliferation and apoptosis is still largely unknown.
The transcriptional regulator Myc, along with the various transcriptional
regulators that it interacts with in different contexts (e.g. Max, Mad, Sin3),
appears to be a key mediator of many of these responses
(Iritani et al., 2002).
Several members of the insulin-signaling network are tumor suppressor genes or
proto-oncogenes, and the network may thus play a crucial role in
carcinogenesis.
Nutrition and growth
The role of insulin signaling in diabetes and obesity was highlighted by Morris White (Harvard Medical School, Boston, USA). Insulin signaling through IRS2 is involved in coordination of growth and metabolism in the hypothalamus and the pancreatic ß-cells. Selective disruption of IRS2 signaling in the hypothalamus and ß-cells in mice results in animals that are obese and develop diabetes. Restoration of IRS2 expression in the ß-cells reverses the diabetes, but not the obesity. This work indicates that insulin/IRS2 signaling in the hypothalamus appears to be important for the regulation of food intake.
Nutrients are required for growth, but it is clear that in multicellular
organisms, such as mice and flies, they do not directly stimulate cell growth.
The action of nutrients such as amino acids and carbohydrates is clearly
indirect, and affects growth through the stimulation of insulin signaling
(Britton et al., 2002;
Ikeya et al., 2002
). Nutrition
affects insulin signaling in two ways: it can modulate the synthesis and
secretion of insulin and insulin-like ligands, and it can affect the activity
of molecules within the insulin-signaling network. The sites of synthesis of
the insulins are diverse. They can be produced by neurosecretory cells, islet
cells, intestinal cells and fat body depending on the species, and within a
species like Drosophila melanogaster, different members of the DILP
(Drosophila Insulin-Like Protein) family are produced in different
tissues (Brogiolo et al.,
2001
), although the functional significance of this site-diversity
is not yet clear. The pathway through which nutrition modulates the synthesis
and secretion of insulin-like signaling molecules is not yet understood.
In addition to neurosecretory DILPs, the fat body of Drosophila
produces a signal that regulates the growth of internal tissues in response to
nutrition (Britton and Edgar,
1998). Recent work, reported by Pierre Leopold (University of
Nice, France), has shown that an amino acid transporter gene slimfast
(slif) mediates the sensation of amino acid levels in the hemolymph.
Loss-of-function mutations in slif mimic amino acid starvation, and
result in slow growing larvae and miniature adults. Conditional loss of
slif function in the fat body alone produces exactly the same
effects, indicating that the sensory mechanism for amino acid availability
resides within the fat body. SLIF appears to act by modulating insulin
signaling in the fat body at the level of the TSC/TOR proteins, and not at the
level of PI3K.
The fat body regulates the growth of peripheral tissues by means of a
secreted cofactor for DILP, which is orthologous to the acid labile subunit
(ALS) of the IGF-binding protein complex. The fat body suppresses PI3K
signaling in endoreplicating peripheral tissues (these are tissues in
Drosophila that grow through polyploidization without cell division),
in response to amino acid starvation, through a pathway involving ALS
signaling. Imaginal disks (which grow by both cell growth and cell division)
are relatively insensitive to this inhibitory signal, presumably because they
are able to produce and secrete their own DILPs. The fat body amino acid
sensor and response system does not act via the modulation of DILP secretion
from the brain, but appears to act directly on peripheral tissues at the level
of PI3K (Colombani et al.,
2003).
Ageing, longevity and cell death
Insulin signaling is not only involved with the regulation of growth and
size, but recent work has shown that it also has profound effects on the
longevity of an organism. Some of the mechanisms by which this occurs are
beginning to be understood. Overexpression of FOXO family members, for
example, extends life span. This may be related to the fact that FOXO is
intimately involved in the response to stress, as noted above. Cynthia Kenyon
(University of California, USA) reported that in C. elegans,
reduction of DAF-2 (a receptor substrate for insulin signaling) and DAF-16 (a
FOXO ortholog) expression significantly increases life span. When the genes
that are upregulated in such long-lived animals are knocked down by RNA
interference (RNAi), they cause a decrease in longevity in the experimental
worms. Conversely, RNAi-induced restriction of the genes that are
downregulated in long-lived worms causes an enhancement of lifespan
(Murphy et al., 2003).
Evidently, insulin signaling affects the expression of many kinds of genes,
and these can have both positive and negative effects on lifespan.
Georg Halder (Anderson Cancer Center, Houston, USA) and Iswar Hariharan
(Massachussets General Hospital Cancer Center, Boston, USA) reported on a
novel protein complex, using Salvador as a scaffold for the proteins Warts and
Hippo, which is involved in the restriction of cell growth and proliferation,
and in the promotion of apoptosis. This complex affects apoptosis by
inhibiting DIAP1 activity, and this allows for the activation of caspases
(Kango-Singh et al., 2002;
Harvey et al., 2003
;
Wu et al., 2003
). However, the
mechanism by which the Salvador/Hippo/Warts pathway is activated is not yet
known.
Perhaps the most interesting finding of recent years is that caloric
restriction significantly increases life span in yeast, worms,
Drosophila and mammals. If food intake is restricted by feeding these
organisms only 70% of what they would voluntarily take in, their life span can
be extended by 10-20%. The mechanism by which longevity is achieved is not
known, but it has been ascribed to a decrease in oxygen consumption and a
corresponding decrease in the accumulation of oxidants and other reactive
species that induce cellular and molecular damage. In addition, it has been
suggested that the elevation of SIR2 during caloric restriction enhances gene
silencing because it deacetylates histones, and that this activity enhances
genomic stability and extends life by preventing inappropriate gene expression
(Lin et al., 2000). Linda
Partridge (University College London, UK) and her collaborators reported a
most remarkable finding to emerge from studies on longevity in
Drosophila. By initiating dietary restriction at different times in
adult life they found that two days after the initiation of restriction, the
survivorship of previously fully fed flies was the same as those that had been
subject to long-term dietary restriction
(Fig. 3). This was true no
matter how late in life dietary restriction was initiated, which suggests that
it is never too late to adopt a good habit. These results indicate that
dietary restriction does not increase longevity by simply reducing the
accumulation of cellular and molecular damage. In Drosophila at
least, dietary restriction appears to extend life span by somehow reducing the
short-term risk of death (Mair et al.,
2003
).
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Higher-level regulation of growth and size
The profound effects of insulin signaling on tissue and body size are
revealed by numerous experimental alterations of portions of the
insulin-signaling network. Inactivation of Chico (in addition to increasing
longevity) reduces body size of Drosophila by some 50%, due to a
decrease in cell size and cell number. Overexpression of DILP2 causes
gigantism and, again, these effects are due to changes in both cell size and
cell number (Brogiolo et al.,
2001). Mutations in downstream genes, such as those for PI3K,
TSC1/2, TOR, FOXO and D6K, also cause changes in cell size or cell
proliferation and affect overall body size.
Although much of the recent interest in the regulation of growth and size
has focused on the details of intracellular signaling mechanisms, it is clear
that the control of size and shape of multicellular tissues, organs and bodies
must be extracellular, through mechanisms that somehow obtain and integrate
information at a scale that is relevant to the dimensions of the object being
regulated. Some of these signaling mechanisms are not molecular, but
mechanical. Donald Ingber (Harvard Medical School, Boston, USA) showed that
strain in the extracellular matrix can be transmitted to elements of the
cytoskeleton, which can activate both cAMP and G protein-coupled signaling
pathways. Such strain-induced signals can affect checkpoints in the cell cycle
and cell proliferation, and can cause cell fates to switch between growth and
apoptosis (Ingber, 2003). In
this way, the cytoskeleton acts as a key integrator of the extracellular
mechanical and structural context during development, and can coordinate
growth, size and shape at the tissue level.
A centralized control of growth is indicated by the fact that insulin
signals are produced by discrete endocrine sources (either glandular or
neurosecretory cells) in response to systemic signals such as nutrition. In
addition, growth is also regulated by a variety of centrally regulated
developmental hormones, such as growth hormone in vertebrates and ecdysone in
insects. Fred Nijhout (Duke University, Durham, USA) pointed out that, in
insects, the final body size of the adult is determined by the size at which a
larva begins to secrete the hormones that initiate the metamorphic molt. The
neurosecretory prothoracicotropic hormone (PTTH) stimulates secretion of the
steroid hormone ecdysone. This hormone activates gene expression, which
results in the cessation of feeding and the switchover from larval to pupal
commitment. The stimuli that induce the secretion of PTTH and ecdysone are
diverse, and some appear to be evolved adaptations to particular modes of life
(Nijhout, 2003). Some of these
stimuli are mechanical rather than molecular. Abdominal stretch reception, for
example, appears to be the initial stimulus for the cessation of growth in the
Hemiptera. These receptor neurons send action potentials to the brain where
they stimulate the secretion of PTTH. In the beetle Othophagus
taurus, by contrast, nutrient restriction triggers PTTH secretion. Larvae
of the tobacco hornworm Manduca sexta are somehow able to sense their
size and at a well-defined critical size they cease secretion of juvenile
hormone (which represses PTH and ecdysone secretion). When juvenile hormone
disappears, the PTTH/ecdysone endocrine cascade is initiated, and this, in
effect, fixes the final body size of the individual
(Nijhout, 2003
).
Postscript
Great advances have been made in recent years in understanding the cellular
and molecular details of insulin signaling, and its role in the control of
growth. But it is also clear that much remains to be done. Some details of
signaling networks are still obscure, particularly in regard the mechanisms by
which switches between alternative outcomes (cell division, growth, apoptosis)
are made. Cells and organisms are complex things, and there are many ways in
which their growth can be disrupted experimentally and genetically.
Unfortunately, it is not always clear whether and how such disruptions reflect
the normal regulatory mechanisms that modulate growth in the intact system.
Finally, there are many independent pathways that affect growth and size at
the cellular, tissue and organism level
(Fig. 2), and until now there
has been little effort expended to understand how these many control
mechanisms interact (Stern,
2003). Many informal discussions at the Arolla meeting revolved
around the need to develop deeper insight into these more integrative aspects
of the control of growth.
REFERENCES
Brazil, D. P. and Hemmings, B. A. (2001). Ten years pf PKB signaling: a hard Akt to follow. Trends Biochem. Sci. 26,657 -664.[CrossRef][Medline]
Britton, J. S. and Edgar, B. A. (1998).
Environmental control of the cell cycle in Drosophila: nutrition
activates mitotic and endoreplicative cells by distinct mechanisms.
Development 125,2149
-2158.
Britton, J. S., Lockwood, W. K., Cohen, S. M. and Edgar, B. A. (2002). Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2,239 -249.[Medline]
Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R. and Hafen, E. (2001). An evolutionarily conserved function for the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11,213 -221.[CrossRef][Medline]
Burgering, B. M. and Kops, G. J. (2002). Cell cycle and death control: long live Forkheads. Trends Biochem. Sci. 27,352 -360.[CrossRef][Medline]
Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J. and Leopold, P. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114, 1-20.[Medline]
Garami, A., Zwartkruis, F. J. T., Nobukuni, T., Joaquin, M., Roccio, M., Stocker, H., Kozma, S. C., Hafen, E., Bos, J. L. and Thomas, G. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Molec. Cell 11,1457 -1466.[Medline]
Hall, M. N. and Raff, M. (2004). Cell Growth: Control of Cell Size. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (in press).
Harvey, K. F., Pfleger, C. M. and Hariharan, I. K. (2003). The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114,457 -467.[Medline]
Hill, M. M. and Hemmings, B. A. (2002). Inhibition of protein kinase B/Akt implications for cancer therapy. Pharmacol. Therap. 93,1 -9.[CrossRef][Medline]
Howitz, K. T., Bitterman, K. J., Cohen, H. Y., Lamming, D. W., Lavu, S., Wood, J. G., Zipkin, R. E., Chung, P., Kisielewski, A., Zhang, L.-L., Scherer, B. and Sinclair, D. (2003). Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature (in press).
Ikeya, T., Galic, M., Belawat, P., Nairz, K. and Hafen, E. (2002). Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12,1293 -1300.[CrossRef][Medline]
Ingber, D. E. (2003). Mechanosensation through
integrins: cells act locally but think globally. Proc. Nat. Acad.
Sci. USA 100,1472
-1474.
Iritani, B. M., Delrow, J., Grandori, C., Gomez, I., Klacking,
M., Carlos, L. S. and Eisenman, R. N. (2002).
Modulation of T-lymphocyte development, growth and cell size by the Myc
antagonist and transcriptional repressor Mad1. EMBO J.
21,4820
-4830.
Kango-Singh, M., Nolo, R., Tao, C., Verstreken, P., Hiesinger, P. R., Bellen, H. J. and Halder, G. (2002). Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila. Development 129,5719 -5730.[CrossRef][Medline]
Kim, D. H., Sarbassov, D. D., Ali, S. M., King, J. E., Latek, R. R., Erdjument-Bromage, H., Tempst, P. and Sabatini, D. M. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110,163 -175.[Medline]
Lin, S.-J., Defossez, P.-A. and Guarente, L.
(2000). Requirement of NAD and SIR2 for life-span
extension by calorie restriction in Saccaromyces cerevisiae.Science 289,2126
-2128.
Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J. L., Bonenfant, D., Oppliger, W., Jenoe P. and Hall, M. N. (2002). Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Molec. Cell. 10,457 -468.[Medline]
Mair, W., Goymer, P., Pletcher, S. D. and Partridge, L.
(2003). Demography of dietary restriction and death in
Drosophila. Science 301,1731
-1733.
Murphy, C. T., McCarroll, S. A., Bargmann, C. I., Fraser, A., Kamath, R. S., Li, H. and Kenyon, C. (2003). Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424,277 -284.[CrossRef][Medline]
Nijhout, H. F. (2003). The control of body size in insects. Dev. Biol. 261, 1-9.[CrossRef][Medline]
Oldham, S. and Hafen, E. (2003). Insulin/IGF and target of rapamycin signaling: a TOR de force in growth control. Trends Cell Biol. 13,79 -85.[CrossRef][Medline]
Saucedo, L. J., Gao, X., Chiarelli, D. A., Li, L., Pan, D. J. and Edgar, B. A. (2003). Rheb promotes cell growth as a component of the insulin/TOR signaling network. Nat. Cell Biol. 5,566 -571.[CrossRef][Medline]
Schmelzle, T., Beck, T. and Hall, M. N. (2003). Activation of the RAS/cAMP pathway suppresses a TOR deficiency in yeast. Mol. Cell. Biol. (in press).
Stern, D. (2003). Body-size control: how an insect knows it has grown enough. Curr. Biol. 13,R267 -R269.[CrossRef][Medline]
Tran, H., Brunet, A., Grenier, J. M., Datta, S. R., Fornace, A.
J., DiStefano, P. S., Chiang, L. W. and Greenberg, M. E.
(2002). DNA repair pathway stimulated by the forkhead
transcription factor FOXO3a through the Gadd45 protein.
Science 296,530
-534.
Wu, S., Huang, J. B., Dong, J. X. and Pan, D. J. (2003). hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114,445 -456.[Medline]
Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A. and Pan, D. J. (2003). Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Nat. Cell Biol. 5,578 -581.[CrossRef][Medline]