Hh, Hedgehog; Shh, Sonic hedgehog; Ihh, Indian hedgehog; Dhh, Desert hedgehog; TM, Transmembrane spanning receptor; Disp, (D12) receptor; 12-pass SSD TM Dispatched protein; Drosophila Ptc/mammal Ptch1 receptor(12 TM)), 12-pass Tm Patched core hh protein containing Sterol-sensing domain (SSD); Ihog proteins, Interference hedgehog co-receptor with Ptc; Smo receptor, 7-pass TM Smoothened protein; GPCR, G protein-coupled receptor; Boi, Brother of Ihog in Drosophila; Boc, Brother of Cdo in mammals; (Ig)-like domain, Immunoglobulin-like domain; FN-III, Fibronectin type 3-like domain; IFT, Intraflagellar transport protein; Arrb2, Arrestin β2; HSC, Hedgehog signaling complex; TF, Transcription Factor; Ci-FL(Ci-155A), Cubitus interrupts full-length zinc-finger TF in Drosophila (155 kb intact active form of the Ci TF); Ci-75 R, 75 kb cleaved processed form of Ci TF; Gli- FL (Gli-A), Full length activator form of Krüppel-like zinc-finger TF in mammals ;Targets for Hedgehog signalling; Gli R, Cleaved and repressor form of Gli TF; p16 (INK4a), tumour suppressor; TGFβ, Transforming growth factorbeta; BMP, Bone morphogenetic proteins; FGF, Fibroblast growth factors; MSC, Mesenchymal stem cell; PPARγ, Peroxisome proliferator-activated receptor gamma; GATA2 and 3, Anti-adipogenic transcription factors; CEBPα, CCAAT/enhancer binding protein alpha; SREBP-1c, Sterol Regulatory Element Binding Protein 1c; Bax, Pro-apoptotic relative; BNDF, Brain-derived neurotrophic factor; VEGF, Vascular endothelial growth factor; LDL, Low density lipoprotein; VLDL, Very low density lipoprotein; HDL, High density lipoprotein; Kif7, Kinesin family member 7; Cos2, Costal2 atypical kinesin-like protein (KIF7 in vertebrates); Drosophila Boi and mammalian Cdo and Boc, Members of the Ihog family; Fu, Fused; Su(fu), Suppressor of fused; PKA, Protein kinase A; CKI, Casein kinase I; GSK3, Glycogen synthase kinase 3; SCF complex, Skp; Cullin; F-box containing complex for ubiqutination; P, Phosphate group; ER, Endoplasmic reticulum.
Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases.
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The mechanisms that orchestrate the development of a multicellular organism from a single cell have fascinated people for centuries. We are still a long way from truly understanding the robust programs that allow building of our specialised organs that regulate digestion, respiration, and haemodynamics, and guard against invasion by other unsolicited organisms. However, in the past three decades, many of the genes that play a role in patterning our bodies have been identified 1–4 and we have learned much about the mechanisms involved in development by characterising the nature of these patterning genes and the way they work together 4–7. A major component of the developmental systems creating the body during gestation is Hedgehog. In this essay we introduce the hedgehog pathway and argue that this morphogenetic system is not only of principal importance at the start of life, but that its action prevents the deterioration of the body during adult life 7–10. Its shutdown is associated with senescence 11 of the body, and makes the body vulnerable to diseases of the elderly. Murine models of increased hedgehog signalling are available, e.g. through the heterozygous knock-out for Patched (see below); however, until now, these have mainly been explored to pinpoint the role of enhanced hedgehog signalling in cancer. As hedgehog may be the first example of an anti-geriatric signal in the body, it should also prove highly interesting to investigate this in animal models of age-related disease.
General principles of hedgehog signal transduction
Hedgehog signalling is one of the highly conserved pathways in metazoan organisms, though a comparison between, Drosophila and Homo sapiens, for example, reveals variations in signalling details 12. As stated, it is one of the key pathways directing morphogenesis, and this property seems critically dependent on the ability of cells to show hedgehog concentration-dependent responses that result in alternative cell fates 12, 13. How concentration gradients of hedgehog are precisely established is not completely resolved. Hedgehog morphogens are a family of lipidated ligands, which in vertebrates include sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh), but all hedgehog signals act through the same receptors and same signal transduction cascades. After synthesis as pro-morphogens, hedgehogs undergo autocatalytic proteolytic processing; they acquire a covalent addition of cholesterol to the C-terminal domain and a cysteine palmitoylation at the N-terminal domain 14, all resulting in a highly hydrophobic protein. The subsequent release of the hedgehog molecule for intercellular signalling is an active process involving binding of the Dispatched translocator to the cholesterol moiety 15. The function of hedgehog as a long-range morphogen is evidently not hampered by its highly hydrophobic character, and thus mechanisms ensuring its solubilisation must be operative 16, 17. Various modes have been proposed to account for its transport through the aqueous environment. These include the formation of micelle-like multimeric complexes 14 in which the hydrophobic parts of the molecules are centralised via their lipophilic tails, transport by lipophorins (Drosophila) or lipoproteins (mammals) and attached to the surface of large membrane-sheathed vesicles together with retinoic acid (Fig. 1) 17, 18. However, the absolute and relative importance of these alternative mechanisms remains unclear, and long-range transport of hedgehog largely remains an unanswered question.
Hedgehog engages target cells in a complex fashion. Its transmembrane (TM) receptors include the 12-pass lipid bilayer transferring receptor Patched (Ptc in Drosophila and Ptch1/PTCH1 in mammals), the Interference hedgehog (Ihog) co-receptor (Table 1) and Smoothened, a G protein-coupled (Smo) receptor 19. Unligated Ptc inhibits signalling by repressing Smo, but hedgehog binding to Ptc leads to Ptc internalisation and release of further signal transduction that is Smo dependent (Fig. 1). Ihog, which has been best characterised in Drosophila 20, is composed of an immunoglobulin (Ig)-like domain and a fibronectin type 3 (FN-III)-like domain 21. The Ihog receptor binds to the hedgehog proteins and enhances alleviation of Ptc-dependent repression of the Smo receptor, possibly in conjunction with Boi/Boc (Brother of Ihog in Drosophila, brother of Cdo in mammals) 22. Ptc inhibits Smo via production of a 3-hydroxy steroid and by controlling Smo trafficking 18. In mammals, cells use the primary cilium for signal activation. In the absence of the hedgehog ligand, the inactive Smo receptor is located outside the cilium and inhibited by the active inhibitory receptor Ptch. After ligand binding and following Ptch degradation, the active Smo receptor translocates to the cilium in a process involving the intraflagellar transport (IFT) protein in conjunction with kinesin II (Kif3 family) and β-arrestin-2 (Arrb2) (Fig. 1). Further signalling occurs through a structure called the hedgehog signalling complex (HSC), also apparently located at the primary cilium in mammals, or the microtubule skeleton in general in Drosophila. The main targets for hedgehog signalling are the Ci (Drosophila) or Gli (mammals) transcription factors, which constitute the principal modulator(s) of the hedgehog-driven transcription. Ci (Gli) proteins act either as activators of transcription (the full-length Ci-155 in Drosophila and Gli2/3A in mammals) or as repressors of transcription (the cleaved Ci-75 in Drosophila or Gli2/3R in mammals), the hedgehog signal transduction converting transcriptional repression to activation by inhibiting proteolysis-favouring Ci/Gli phosphorylation (Fig. 1). The resulting transcriptional changes are critically important for pattern formation during embryogenesis, but, as we shall argue in this essay, remain of cardinal importance during adult life as well, and mediate anti-geriatric gene transcription 12.
|Microtubule||Primary cilium||Primary cilium|
|Ptc||Ptch 1||PTCH 1|
|Ci-155||Gli 3 A FL||GLI 3 A FL|
|Ci-75||Gli 2/3 R||GLI 2/3 R|
|Cos2||Kif 7||KIF 7|
Possible functionality of hedgehog in the ageing process
Generally speaking, following development and a juvenile phase, animals enter the reproductive phase. Following exhaustion of the reproductive potential, however, a long phase of gradual decline may follow before the organism dies. For humans in particular, it is the evolutionary forces that drive and counteract ageing 23. Generally, it involves a reduced capacity to regenerate tissues (including the immune system) comprising their functionality, a reduced capacity to combat genotoxic damage, resulting in a sharp increase in cancer incidence following the end of the reproductive and offspring-rearing phase, and the accumulation of sclerotic and fibrotic lesions in the body, especially in the vasculature. In humans, this phenomenon is associated with a wide range of pathologies, including senile dementia, atherosclerotic disease and osteoporosis 24. Earlier concepts of ageing were based on the idea that the body invests a certain amount of energy in mounting defences against, especially, oxidative stress, depending on the life span needed for efficient reproduction. However, over time damage accumulates and, after a certain tipping point, senescence quickly gathers pace. New data from mice genetically modified to exhibit reduced anti-oxidative defences and from studies in exceptionally long-lived animals, have questioned this notion 25. Although ageing is an extremely complicated process, it now appears that reduced capacity to cope with external stress and a reduced potential to mount regenerative responses to insults drive the geriatric process. In turn, this reduced functional and regenerative capacity is the consequence of reduced size and agility of the various stem cell compartments in the body. For instance, increasing evidence shows that central molecular players in Alzheimer's disease influence the generation of new neurons, and noteworthy alterations in neurogenesis take place earlier than the onset of hallmark lesions or neuron loss 26. The rapid pace of neurodegeneration and dementia is attributed to a reduced activity in neuronal stem cell compartments, resulting in inadequate neuronal and glial replenishment following injury. Evidence has been presented that similar mechanisms also play a role in benign senile dementia 27. In the immune system, e.g. a reduced capacity of the haematopoietic stem cell compartment to generate especially lymphoid cell types may be involved in the increased propensity to succumb to infectious disease 28. If, however, reduced functionality in the somatic stem cell compartments is responsible for ageing, the factors that govern the behaviour of this compartment might well be important to control the pace of ageing. It would thus be interesting to perform experiments in which experimental rodents are subjected to treatment with Smo agonists and subsequently investigated for the size of the various stem cell compartments. As hedgehog is a major regulator of stem cell function, this would suggest that the morphogen could guide ageing through influencing the stem cell compartment. The function of hedgehog in anti-ageing-related diseases is depicted in Fig. 2.
Hedgehog signalling and the stem cell niche
Maintenance of the body requires continuous replenishing of the organs with fresh effector cells, which are typically produced from a small stem cell pool in the specific organs. Ageing and the associated diminished functionality of organs is often attributed to the diminished size and action in the stem cell population, compromising the capacity of the organs to withstand mechanic and xenobiotic insult. Many stem cell niches require hedgehog signalling 10, 29, 30, especially in tissue derived from the ectodermal lineage (e.g. in the central nervous system or in the skin) 31 but there is also good evidence that hedgehog is required in the haematopoietic stem cell niche and others 32. The role of hedgehog as an important factor for maintaining stem cells is illustrated by the importance attributed to this signalling system for maintaining cancer stem cells, and inhibitors of hedgehog signalling are being proposed as tools to test the cancer stem cell pathway 33. Among the mechanisms employed by hedgehog signalling to maintain the stem cell phenotype is suppression of p16 (INK4a). A fragment of the GLI2 transcription factor directly binds and inhibits the p16 promoter, and stem cell senescence is associated with the loss of GLI2 34. Indeed, p16 is a well-established mediator of cellular senescence 35 and its expression rises with age in many tissues, as does the accumulation of dysfunctional senescent stem cells 36, providing a direct clue as to how hedgehog signalling can counteract ageing through the stem cell compartment. Thus, hedgehog can counteract senescence by maintaining the stem cell phenotype, but conversely this property of hedgehog also probably contributes to its role in promoting the process 37. It is important to note that, often, other components of morphogenetic code 38 mainly act as antagonists of stem cell functions. Members of the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) family of morphogens, for instance, mainly act as antagonists of stemness, as evident from the expression of BMP antagonists in many stem cell niches to protect the stem cells from the action of these morphogens 39, or the requirement for BMP antagonists such as noggin in the medium of stem cell-derived organoid cultures 40. In addition, fibroblast growth factor (FGF) family members often tend to favour differentiation over stem cell expansion throughout the body 41. In this sense, BMP and FGF-like morphogens are more pro-geriatric than anti-geriatric substances. Wnt ligands and ligands stimulating Notch signalling are often potent in promoting stem cell maintenance and expansion, but differ from hedgehogs in that they are essentially short-range paracrine factors. Thus hedgehog seems unique in its capacity to confer long-range signalling stimulatory activity on the stem cell compartment.
Hedgehog signalling maintains versatility in the skeleton
Hedgehog, by influencing stem cell functionality, can be found in the bone. With advancing age, the condition of bones deteriorates. Osteoporosis, especially in elderly women, is a major health problem. During skeletogenesis, Shh and Ihh provide positional information and initiate or maintain cellular differentiation programs, regulating the formation of cartilage and bone, and they are of particular importance for osteoblast generation 42, 43. During adult life bone mass is lost, and this has been attributed to a shift in the balance of osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation 44–47. In rodent adipocytes, the hedgehog signalling pathway (mainly Shh) interferes with adipocyte differentiation through an unknown mechanism. Hedgehog favours the osteogenic differentiation over adipogenic differentiation by acting as inhibitor of the peroxisome proliferator-activated receptor gamma (PPARγ), which in turn mediates the anti-adipogenic transcription factors GATA2 and 3 45, 48, 49. In addition, in bone, hedgehog signalling directly interferes with the transcription of CCAAT/enhancer binding protein (CEBP)α and sterol regulatory element binding protein (SREBP)-1c, which mediate adipogenesis. Conversely, osteogenic transcription factors are induced. In diabetic patients, high glucose interferes with Shh signalling and Shh-induced bone regeneration, explaining the association between the hyperglycaemia and osteoporosis 50. This highlights the important role of continued hedgehog signalling during life for maintaining skeletal strength. Evidence has also been provided that decreasing levels of Shh in the bone with advanced age correlate with impaired osteogenesis and is thus critical for osteoporosis in the elderly 11. In this sense, the continued action of hedgehog on skeletal bone stem cell compartments represents a good example of how the action of this hormone can counteract the consequences of ageing.
Hedgehog signalling contributes to the protection of the brain against neuron loss
Another example of how hedgehog signalling can counteract ageing is found in the nervous system. Increasing age is accompanied by a loss of mental faculties that is attributed to the loss of neuronal cells and associated reduced plasticity in cerebral functioning. Neurons in general are robust cells, but the central nervous system requires constant replenishing with neurons from neuronal stem cell compartments. It is evident from Alzheimer's disease that defects in the stem cell compartment are important factors in the progressive neuron loss, but active hedgehog signalling is generally important for maintaining neurons in adults 51. In apparent concordance, also in Alzheimer's disease, hedgehog has been proposed as a molecule counteracting the disease process through increased stem cell activity 52, and persistent hedgehog signalling is necessary for neuronal stem cells to acquire their identity during development 53. Thus, the evidence that continued hedgehog signalling through the stem cell compartment plays a cardinal role in enabling the brain to regenerate neurons throughout life seems compelling.
It seems that the capacity of neurons to activate transcriptional programs associated with increased robustness against chemical, nutritional or ischemic stress is sensitive to external cues, and that hedgehog signalling, apart from its action in maintaining neuronal stem cell populations, plays an important role here 54. In primary cultures of cortical neurons, oxidative insult results in Shh production. As inhibition of hedgehog production exacerbates apoptosis and neurotoxicity, whereas artificial hyperactivation reduces neuronal cell death, it appears that Shh is an important endogenous protective hormone for the cortical neuronal compartment 54. Similar results were also reported for spinal neurons 47. Mechanistically, Shh responses to improve survival of neurons seem kaleidoscopic and complex. Increased activation of survival pathways, induction or expression of the anti-apoptotic mitochondrial protein Bcl-2 with concomitant decreased expression of its pro-apoptotic relative Bax, enhanced expression of trophic factors such as BNDF and VEGF, and direct inhibition of cell stress-sensing pathways have all been reported. This may indicate that increased survival of post-mitotic cells is a bona-fide effect of hedgehog signals. As ageing involves a reduction in size of such post-mitotic compartments, this again represents an important anti-geriatric signal.
Neurodegenerative diseases, such as Parkinson disease 55, represent serious health problems. Thus, although treatment with pharmacological tailored hedgehog agonists will undoubtedly be associated with an increased risk of malignant transformation, the possible gains in brain function through enhanced stem cell function and decreased propensity to further neuron loss may still be considered a valid therapeutic gain to offset the risk in the future.
A final example of how loss of hedgehog may cause ageing-related phenomena is found in the skin. Reduced skin elasticity is one of most obvious effects of advancing age. Hedgehog is an important morphogen here, maintaining the basally located stem cell compartment, as evident for instance from the frequent development of basal cell carcinoma in patients having only one functional allele of Patched 56. Furthermore, a decreased capacity for skin repair is associated with reduced activation of hedgehog signalling in the bulge cells, an important population of stem cells in the skin. Finally, in a model for aged skin, juvenile markers can be induced by lentiviral-mediated overexpression of Gli1 57. Thus, in many locations in the body, bona-fide evidence for a role hedgehog signalling in combating ageing through modulation of the stem cell compartment is available.
Hedgehog counteracts stress-related lesions
From the above, it is clear that hedgehog directs tissue rejuvenation through trophic actions on the stem compartment throughout the body, and may be the only long-range signalling molecule to do so. Functionality in providing a rejuvenating signal in the body is further supported by the role that hedgehogs play in counteracting the effects of chronic insult to the body. During life, the body accumulates fibrotic and atherosclerotic lesions. Although such lesions can already be detected in young individuals, beyond the reproductive phase the number, particularly of atherosclerotic lesions, suddenly increases, because of an apparent incapacity to control and counteract growth of such lesions by compensatory responses. There is evidence that hedgehog provides important signals to the tissues that such compensatory responses should be mounted, so that hedgehog's function as an anti-geriatric signal lies not only in the dynamics of stem cell function but also in how the body deals with the metabolic demands conferred by ageing 7, 58.
Hedgehog signalling limits atherosclerotic disease
Atherosclerosis is one of the main manifestations of advanced age and a major cause of the associated aetiology. It has a close association with other energy metabolic diseases including metabolic syndrome and type 2 diabetes. Different pathways are involved in their pathophysiology, including the immune system and coagulation system. Lipoproteins have different effects in this process. Low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) have a stimulatory role in atherosclerosis, whereas high-density lipoprotein (HDL) has a protective effect on the blood vessels, which is counterintuitive in view of the fact that both are important carriers of cholesterol 59. In this context it is important to note that sterol metabolism is closely related to hedgehog signalling 11, 16, 18, 60, 61. Hedgehog is sterolated and hedgehog derivatives mediate the inhibitory function of Ptch receptor on the Smo receptor in the absence of the hedgehog molecule 62. Thus, sterol disorders affect the hedgehog pathway and development, either by improper sterolation of the hedgehog protein or by a reduced responsiveness of the cells to the hedgehog proteins. In the circulation, the transport of the highly hydrophobic hedgehog molecules in Drosophila occurs via lipophorin and in mammals VLDL is loaded with hedgehog (Fig. 3) in the adipocyte compartment. There has also been extensive speculation on the notion that LDL also carries hedgehog, but hard data are lacking 17, 63, 64. The therapeutic benefits of hedgehog in vascular disease are particularly evident from studies involving Shh myocardial gene therapy in counteracting experimental chronic myocardial ischaemia 32, 65, 66, whereas experimental peripheral ischaemia benefits from ectopic expression of Shh 67. The effects here are not large enough to indicate that the atherosclerotic process per se is inhibited but that hedgehog signalling mediates shunting angiogenesis in addition to effects on apoptosis, as described above for neurons. Conversely, genetically or pharmacologically interfering with hedgehog signalling compromises endothelial functionality and that precedes ageing-related diminished function of the vasculature 9, 67. By contrast, an ischaemic insult by itself provokes substantial production of hedgehog 32, 66. These effects of hedgehog improving endothelial function are manifold, but in particular increased secondary production of angiogenic factors, increased survival signalling of endothelial cells through phosphatidylinositol-3-OH-kinase and recruitment of endothelial precursor cells have been well documented. In toto, the evidence that hedgehog signalling counteracts age-related changes in the endothelial compartment is compelling, and thus hedgehog production represents an important anti-geriatric signal here, although the effects do not really prevent the atherosclerotic process but help in limiting the damage inflicted.
Hedgehog signalling is one of the most highly evolutionary conserved pathways in the metazoan body, with a pivotal importance during embryogenesis, but with abundant activity afterwards. Importantly, though, an accumulating body of evidence suggests that its remaining functionality has not only an important role in tissues repair, but is also involved in the maintenance of the tissues and preventing their senescence. In this regard hedgehog may act as an anti-geriatric signal, and its reduced expression associated with advanced age may, in fact, be a trigger for ageing.