The control of growth

H. Frederik Nijhout

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, 2004Go).

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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Fig. 1. Insulin signaling affects many aspects of metabolism and cell physiology, with diverse and profound consequences for health and disease.

 



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Fig. 2. A compilation of some of the principal pathways and interactions discussed at the Arolla Workshop 2003. The interactions depicted here undoubtedly do not occur simultaneously, nor are all of them present in all cell types or in all species. Many of the proteins depicted are encoded by multigene families, and different homologs may be expressed in different contexts and can have different effects. Membrane associations and cytoplasmic/nuclear localizations are not indicated. Dotted arrows indicate indirect multi-step or presumptive pathways.

 
The binding of an insulin-like ligand to an insulin receptor (InR) results in the activation of phosphoinositide 3-kinase (PI3K), and this stimulates the synthesis of the lipid activator phosphatidylinositol (3,4,5)-triphosphate (PIP3). This stimulation is reversed by the action of PTEN (phosphatase and tensin homolog deleted on chromosome ten), which acts as an effective inhibitor of insulin signaling. PIP3 induces membrane attachment, and subsequent phosphorylation, of PKB (protein kinase B, a proto-oncogene, also known as AKT). PKB has long been implicated in the regulation of metabolism, glycogen synthesis, transcriptional regulation and apoptosis in many cell types, as well as in tumorigenesis (Brazil and Hemmings, 2001Go; Hill and Hemmings, 2002Go). At least some of the effects of PKB are achieved through the inhibition of TSC and FOXO.

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., 2003Go; Oldham and Hafen, 2003Go; Saucedo et al., 2003Go; Zhang et al., 2003Go). 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., 2002Go; Kim et al., 2002Go). 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., 2003Go).

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, 2002Go; Tran et al., 2002Go). 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., 2002Go). 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., 2002Go; Ikeya et al., 2002Go). 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., 2001Go), 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, 1998Go). 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., 2003Go).

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., 2003Go). 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., 2002Go; Harvey et al., 2003Go; Wu et al., 2003Go). 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., 2000Go). 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., 2003Go).



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Fig. 3. Age-specific mortality rates of adult female Drosophila under different feeding regimes. Upper curve shows results from fully fed adults. Dietary restriction was imposed from day 1 (lower curve), day 14 (blue curve) or day 21 (red curve) onward. Within 4 days of switching to dietary restriction the mortality rate became indistinguishable from those of flies of the same age that had been maintained on dietary restriction since the beginning of adult life (see Mair et al., 2003Go). Curves are 3-day moving averages. Modified with permission from Mair et al. (Mair et al., 2003Go).

 
For some time it has been suggested that red wine has beneficial physiological effects, and somehow enhances health and lifespan. The active principle in red wine that extends lifespan in yeast has now been shown by David Sinclair (Harvard Medical School, Boston, USA) and his collaborators to be the diphenol resveratrol. In yeast, resveratrol mimics the effects of dietary caloric restriction by stimulating members of the SIR2 family of deacetylases, also known as sirtuins (Howitz et al., 2003Go). SIR2 negatively regulates the tumor suppressor gene p53, and the overall effect on longevity may be achieved by the inhibition of inappropriate apoptosis.

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., 2001Go). 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, 2003Go). 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, 2003Go). 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, 2003Go).

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, 2003Go). 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.

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