Serotonin: a new player in the regulation of bone remodeling

Vijay K. YADAV, PhD
Department of Genetics and
Patricia DUCY, PhD
Department of Pathology
Columbia University Medical
Center, New York, NY

Serotonin: a new player in the regulation of bone remodeling

by V. K. Yadav, P. Ducy, and G. Karsenty,USA

Serotonin is a bioamine synthesized in the brain and gut that regulates diverse functions from mood to gastrointestinal tract motility. This diversity in serotonin function(s) is achieved through one or several of its 14 distinct receptor(s) expressed on the target cells. The emerging concept that brain- and gut-derived serotonin regulate bone remodeling in opposite manner has revealed novel mechanism(s) by which bone mass is regulated and maintained. Advances in our understanding of serotonin synthesis, receptor activation, and participation in distinct regulatory networks demonstrate a role for serotonin in osteoblast and osteoclast functions. This review focuses on this new “expanded serotonin biology” and discusses how drugs targeting serotonin synthesis or signaling can be harnessed for treating low-bone-mass diseases.

Medicographia. 2010;32:357-363 (see French abstract on page 363)

Skeleton in vertebrates serves multiple mechanical, hematopoietic, and endocrine functions.1 In order to perform its functions properly, skeleton continuously renews itself through a homeostatic process known as bone remodeling—a process carried out by osteoblasts and osteoclasts to maintain a fine balance between bone formation and resorption.1 Bone remodeling occurs constantly and simultaneously in numerous parts of skeleton and the maintenance of a normal, healthy skeletal mass depends on continuous exchange of information taking place among osteoblasts, osteoclasts, osteocytes, constituents of the bone matrix, and other organs.1 Therefore, understanding what factors are influencing bone mass in the context of other signals is important. The fact that osteoporosis is a heritable trait provides an opportunity to use modern molecular genetics to obtain mechanistic insights that were previously unobtainable. If we could find genetic variants with known or at least tractable functions that are unequivocally associated with osteoporosis, we might be able to build up a picture of what sorts of biological factors determine why some people are more susceptible to osteoporosis than others.

Lrp5: a multifaceted molecule

The low-density lipoprotein receptor (LDLR)-related protein (Lrp)-5 is part of a subset of the LDLR family of cell surface proteins.2,3 Since its cloning in 1998, Lrp5 has taken biologists to voyages of discoveries from lipoprotein clearance to glucose homeostasis to bone remodeling. Not surprisingly, it has been shown to bind to multiple ligands and activate a multitude of downstream cascades in distinct cell types to regulate different processes (Figure 1). In hepatocytes, Lrp5 binds apolipoprotein E (ApoE) and plays a role in the hepatic clearance of ApoE-containing chylomicron remnants, a major plasma lipoprotein carrying diet-derived cholesterol.4,5 In pancreatic islets, Lrp5 regulates insulin secretion and consequently Lrp5-deficient animals are glucose intolerant.6 Consistent with its role in glucose homeostasis, the LRP5 gene is mapped within the region (IDDM4) linked to type 1 diabetes on chromosome 11q13.7 LRP5 is also the gene responsible for osteoporosis- pseudoglioma (OPPG) syndrome and high-bone-mass (HBM) syndrome in humans due to an isolated change in bone formation.8-10

The main question surrounding Lrp5 biology, since its identification as the cause of OPPG, has been to define how its absence can cause the developmental onset of blindness and postnatal onset of osteoporosis characterizing this disease.8-10 Several recent studies have now shed11 new light on the mechanisms associated with these two functions of Lrp5. Indeed, ample studies have conclusively demonstrated that Lrp5 uses the Norrin and Wnt signaling pathways during embryogenesis to regulate vascularization in the eyes.12-14 That dysregulation of Wnt signaling plays a role in the development of blindness in a Lrp5-dependent manner fuelled interest in this signaling pathway, leading to the identification of critical Wnt-dependent mechanisms involved in controlling early differentiation of osteochondroprogenitor cells during embryogenesis as well as osteoblast and osteoclast functions.11,15-17 Some of these targets have already made it to preclinical trials, viz, sclerostin.18 However, and to our dismay, using an unbiased microarray approach, we serendipitously identified that the mechanism through which Lrp5 loss- and gain-of-function mutations regulate bone formation is by regulating serotonin production in the gut.19 This dual role of Lrp5—one developmental (directly dependent on Wnt signaling in the eye) and the other postnatal (relying on the indirect effect of gutderived serotonin on bone cells)—is consistent with the multifunctionality of Lrp5, which participates in a wide variety of signaling cascade(s).

Figure 1
Figure 1. Lrp5 ligands and targets.

Lrp5 binds to numerous ligands and regulates a wide variety of processes through different mechanisms. Structures not to scale.

We should emphasize that the fact that the deletion of Lrp5 in osteoblasts progenitors or mature osteoblasts did not result in a discernible effect on bone mass in our studies does not exclude, however, that Lrp5 could play a role, in a Wntdependentmanner, or not, in regulating the response of osteocytes to mechanical loading.20-21 Further studies analyzing, in parallel, mice deficient in Lrp5 globally as well as conditionally in osteocytes will be pivotal to address this specific point.

Ever-expanding tenets of serotonin biology

Serotonin (5-hydroxytryptamine) was discovered in 1948 as a factor causing vascular contractions, hence the name of the molecule serotonin (L, serum + Gk, tonos, tone).22 Since then serotonin biology has expanded exponentially and it is now recognized as a pivotal regulator in many central and peripheral functions.23 Serotonin is generated through an enzymatic pathway in which L-tryptophan is converted into L-5-OHtryptophan by an enzyme called tryptophan hydroxylase (Tph); this intermediate product is then converted to serotonin by an aromatic L-aminoacid decarboxylase.24,25 There are two Tph genes that catalyze the rate-limiting step in serotonin biosynthesis: Tph1 and Tph2. Tph1 is expressed mostly, but not only, in enterochromaffin cells of the gut and is responsible for the production of peripheral serotonin.23 Tph2 is expressed exclusively in raphe neurons of the brainstem and is responsible for the production of serotonin in the brain.25 Remarkably, serotonin does not cross the blood–brain barrier; therefore it should be viewed from a functional point of view as two distinct molecules depending on their site of synthesis.24 Brainderived serotonin (BDS) acts as a neurotransmitter, while gut-derived serotonin (GDS), till now, has only been appreciated as an autocrine/paracrine signal that regulates mammary gland biogenesis, liver regeneration, and gastrointestinal tract motility.23,26,27

Figure 2
Figure 2. Gut-bone endocrine axis: potential molecular targets for therapeutic interventions.

Lrp5 regulates synthesis of serotonin by enterochromaffin cells in the gut that is then released into the circulation. In the blood it is taken up mostly in the platelets
and a small amount (≈10% to 12%) is present outside (free) these cells. These free serotonin levels are increased under pathological conditions resulting in decreased
osteoblast proliferation and bone formation through the Htr1b-CREB signaling pathway. Nodes in the pathway that are amenable to therapeutic interventions are
highlighted. Structures not to scale.
Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); CREB, cAMP response element binding (protein); Htr1b, 5-hydroxytryptamine receptor 1B; HTT, serotonin
transporter; Lrp5, low-density lipoprotein receptor (LDLR)-related protein (Lrp)-5; Tph1, tryptophan hydroxylase–1; Trp, tryptophan.

Regulation of bone formation through gut-derived serotonin

Our work on the mechanism(s) underlying OPPG and HBM led us to identify Lrp5 as one of the regulators of GDS (Figure 2).19 Conditional inactivation of Lrp5 and Tph1 in the gut cells identified that GDS functions as a hormone that directly inhibits osteoblast proliferation and bone formation.19 We reasoned that if GDS was acting as a hormone one or several of its 14 receptors must be expressed on osteoblasts. Indeed, primary osteoblasts expressed three serotonin receptors: Htr1b, 2a, and 2b. Mice with either global deletion of Htr2a or osteoblast-specific deletion of Htr2b did not display skeletal phenotypes; however, that was not the case for mice with global or osteoblast-specific deletion of Htr1b gene.19 These latter animals displayed a high bone mass phenotype, although of lower magnitude than the one displayed by the mice expressing HBM mutation of Lrp5 (G171V) in the gut, suggesting that there may be, yet to be identified, mediators of the HBM mutations. Nevertheless, the fact that the HBM phenotype of Htr1b-deficient animals was similar in magnitude to one of the mice that had suppressed levels of GDS demonstrated that it is through Htr1b receptor that GDS regulates bone formation.19 Htr1b is a Gi-protein–coupled receptor and, consistent with its role in neurons, it inhibited cAMP production and phosphokinase A (PKA)-mediated cAMP response element binding protein (CREB) phosphorylation in primary osteoblasts. These results identified that a cAMPPKA- CREB pathway regulates osteoblasts proliferation and bone formation.19 Yet, this signaling pathway in the osteoblasts is utilized by many other receptors, and future studies would need to dissociate how this selectivity of Htr1b action on osteoblast proliferation is achieved.

Negative association of peripheral serotonin levels with bone mass in humans

The identification of a gut-derived serotonin-bone endocrine axis (Figure 2) begged the question of its biomedical importance in humans. Modder et al28 analyzed serum serotonin levels in a population-based sample of 275 women and related these to bone mineral densities (BMD) at distinct skeletal sites and bone microstructural parameters. They found that serum serotonin levels were inversely associated in these women with body and spine areal bone mineral density (aBMD) as well as with femur neck total and trabecular volumetric bone mineral density (vBMD).28 Moreover, multiple LRP5 mutations associated with decreased BMDs have been analyzed and all published studies thus far show that these mutations are associated with a 2-to-4 increase in serum serotonin levels.19,29 Conversely, analysis of two HBM patients in the US as well as a recent study of 9 HBM European patients, who harbor the T253I gain-of-function mutation of LRP5, showed that their serotonin concentrations in platelet-poor plasma were significantly lower compared to sex- and age-matched controls.19,30 Collectively, these studies performed in different continents by different investigators, provide convincing evidence to support a physiological role for circulating serotonin in negatively regulating bone formation in humans related to one it plays in mice.

Brain-derived serotonin: an expected player in the regulation of bone mass

In our quest to understand the serotonin regulation of bone mass in vertebrates, we then inactivated Tph2, the gene that catalyzes the rate-limiting step in the biosynthesis of BDS. The absence of serotonin in the brain resulted in a severe low-bone-mass phenotype affecting the axial (vertebrae) and appendicular (long bones) skeleton.31 This phenotype was secondary to a decrease in bone formation parameters (osteoblast numbers and bone formation rate) and to an increase in bone resorption parameters (osteoclast surface and circulating Dpd levels).31 Hence, BDS is a positive and powerful regulator of bone mass accrual acting on both arms of bone remodeling.31

While we were doing these studies we noticed, upon opening the abdominal cavities, that Tph2-deficient animals had a dramatic decrease in their adipose mass.31 This prompted us to analyze in great detail their energy metabolism phenotype. The decrease in their fat mass was due, in part, to the fact that these mice ate less and spent much more energy compared to their wild-type littermates.31 This observation was not entirely surprising since serotonin is known to play important roles in many other physiological processes. However, what caught our attention was the fact that the three most notable phenotypes of adult Tph2-deficient animals ie, decrease in bone mass, decrease in appetite, and increase in energy expenditure are a mirror image of what is observed in mice that lack leptin.32,33

Leptin is an adipocyte-derived hormone that regulates many functions, viz, appetite, energy expenditure, bone mass, etc.34-39 Studies in the last 16 years have highlighted a more complete neural and neurochemical circuit diagram for the leptin regulation of these functions.34-36 These neural circuits involve many distinct neuronal populations in the brain, including neurons of arcuate, Ventromedial, and lateral hypothalamus, and neurons of the nucleus tractus solitarius (NTS) etc.34-36,40 Three correlative experiments suggested that leptin might signal in the serotonin neurons, among others, to regulate some of its downstream functions. First, the leptin receptor (ObRb) is expressed on serotonin neurons located in the raphe nuclei of brainstem, where BDS is produced, and is functional.31,41 Second, serotonin neurons project to the key hypothalamic nuclei responsible for the regulation of appetite, energy expenditure, and bone mass.42 Third, patients on selective serotonin reuptake inhibitors (SSRIs) have been reported to have changes in their appetite and bone mass.43,44 To explore that leptin might utilize serotonin as one of its downstream mediators to regulate these three functions, we inactivated the leptin receptor in different nuclei of the hypothalamus or in the serotonergic neurons of the brainstem.31 Mice lacking ObRb either in Sf1-expressing neurons of the ventromedial hypothalamus (VMH) nuclei or in Pomc-expressing neurons of the arcuate (ARC) nuclei had normal sympathetic activity, bone remodeling parameters, and bone mass; they also had normal appetite and energy expenditure, and when fed a normal diet, did not develop an obesity phenotype.40,45 In contrast, mice that lack ObRb in Sert-Cre positive serotonin neurons (ObRbSERT-/-) developed a high bone mass phenotype; they also had an increase in appetite and displayed low-energy expenditure. As a result, ObRbSERT-/- mice, when fed a normal diet, developed an obesity phenotype. These genetic studies demonstrated that leptin signals, in part, in the serotonin neurons of the brainstem to regulate, bone mass, appetite, and energy expenditure (Figure 3). The identification of serotonin as one of its mediators adds to the list of the multitude of messengers (viz, dopamine, melanocortins, etc) utilized by leptin in the brain to affect peripheral functions.31,34-36

The demonstration that a leptin-dependent central control of bone mass, appetite, and energy expenditure occurs, among other neural relays, through its ability to inhibit serotonin production, raised questions about the location and identity of serotonin receptors on hypothalamic neurons mediating these functions. Double fluorescence in situ hybridization and nuclei- specific gene inactivation experiments revealed that serotonin promotes bone mass accrual through Htr2c receptors expressed on the VMH nuclei, while appetite was promoted through Htr2b and Htr1a receptors expressed on ARC nuclei of the hypothalamus. Further analysis revealed that Htr2c receptor expression on VMH nuclei is en route to the sympathetic center of the brain, while Htr1a and Htr2b achieve their functions on appetite most likely through modulation of melanocortin signaling (Figure 3). These studies emphasized that with respect to the bone mass and energy metabolism effects of leptin signaling in the brain, a systems approach involving anatomically distinct neural elements will provide a more complete explanation of leptin actions in the brain.

Gain of function in serotonin signaling and bone mass

Our loss of function studies with GDS and BDS dissociated the role played by peripheral and central serotonin signaling in the regulation of bone mass.19,31 As with any other study, these studies raised many more questions than they answered. For instance, what effect would an increase in serotonin signaling have on the bone mass? SSRIs are a class of drugs that do exactly that.46 Based on these effects, this class of drugs is prescribed to cure many psychiatric disorders associated with diminished serotonin signaling and their therapeutic actions are diverse, ranging from efficacy in the treatment of depression to obsessive-compulsive disorder, panic disorder, bulimia, and other conditions. The plethora of biological substrates, receptors, and pathways for serotonin are candidates to mediate not only the therapeutic actions of SSRIs, but also their side effects.47 In a cohort of 5008 communitydwelling adults, Richards et al44 revealed that patients that were taking SSRIs had increased risk of hip fractures. Because SSRIs readily cross the blood–brain barrier, it raised the question as to the site of action of these drugs to produce their deleterious actions on bone.

Figure 3
Figure 3. Neuronal relays underlying leptin regulation of bone mass, appetite, and energy expenditure.

Leptin inhibits release of brainstem-derived serotonin, among other neuronal relays, which favors bone mass accrual and appetite through its action on hypothalamic neurons. Serotonergic neurons are in blue; ARC is in green; NTS is in orange; and VMH is in purple. Structures not to scale.
Abbreviations: ARC, arcuate; NTS, nucleus tractus solitarius; PVH, paraventricular hypothalamus; VMH, ventromedial hypothalamus.

Several approaches have been used in the past to understand this deleterious effect of SSRIs on bone mass. Gustaffson et al,48 using naïve rats as a model of serotonin effect on bone mass, analyzed site-specific alterations in the long bone when rats were injected daily with serotonin. These authors reported a decrease in trabecular bone mass and an increase in cortical thickness in long bones. The negative influence of serotonin injections on the trabecular bone mass in their study is consistent with our and earlier mouse genetic studies. We reported that mice harboring a loss of function mutation for Lrp5 gene have increased levels of GDS and a low bone mass at vertebral sites.19 Battaglino et al49 tested the direct effects of SSRIs on bone mass and they consistently observed an increase in trabecular bone mass in these animals.49 These latter results, given the effects of SSRIs in humans, were surprising at the time they were reported, but with the advancement of knowledge related to serotonin signaling in the brain and periphery we can today explain these results. Likely the observed effects were due to the fact that, under the conditions tested in their study, SSRIs were having more profound influences on BDS, a positive regulator of bone mass. Warden et al,50 taking another approach for a model of chronic use of SSRIs, reported that mice that lack serotonin transporter (Htt-/- mice) have decreased bone mass at both cortical and trabecular sites.

Their study is consistent with Richards et al44 and other clinical reports that show that patients taking SSRIs often have a decrease in bone mass. Surprisingly, Htt-/- mice have undetectable levels of serotonin in their blood and a twofold reduction in brain serotonin content (VKY, unpublished observations). The low bone mass observed in Htt-/- mice would suggest that BDS compared to GDS has a dominant role in the overall regulation of bone mass through serotonin. Indeed, analyses of mice lacking both the Tph1 and Tph2 genes display a low bone mass phenotype demonstrating that despite accounting for >5% of total serotonin pool in the body, BDS dominates in the overall regulation of bone mass.31 Since SSRIs cross the blood–brain barrier, and osteoporosis is only observed when they are taken in the long term, development of SSRIs with selective central actions would be worth exploring in the future for curing depression while minimizing their side effects on bone.

Therapeutic implications of serotonin regulation of bone mass

The richness and complexity of the serotonin modulation of bone mass discussed in this review provide both a pharmacologic opportunity and a challenge. On the one hand, the involvement of specific serotonin receptors on osteoblasts and hypothalamic neurons provides an opportunity to pharmacologically target these specific receptors for the treatment of osteoporosis. On the other hand, the fact that each of these serotonin receptors participates in multiple physiologic processes presents a challenge, since even a drug targeting a single serotonin receptor is likely to have effects on multiple body systems. For example, although Htr2c agonists may be used to increase bone mass through its effect in the brain, their clinical use would be limited by their effects on other organ systems, such as sympathetic tone or melanocortin signaling.31,51 Fortunately, the system is less complex and more amenable to therapeutic interventions in the periphery. Since the effect of GDS, a negative regulator of bone formation, is dominant there, one would be able to suppress its levels mildly in order to avoid side effects of drugs targeting the receptors directly. This way one would be able to maintain basal level of signaling in other systems dependent on serotonin while at the same time getting the therapeutic outcome in sensitive systems such as bone, which responds robustly to >50% modulation in peripheral serotonin levels.19

As GDS is a potent inhibitor of osteoblast proliferation and bone formation, we tested the contention that pharmacologically suppressing GDS would be able to prevent, or cure, gonadectomy- induced bone loss. Serendipitously, we came across an inhibitor that was inhibiting peripheral serotonin production without having any detectable effect on brain serotonin content.52 This is, and will be, a prerequisite for any drug that is going to target serotonin synthesis or signaling, as brain serotonin has opposite influence on bone mass accrual and in fact is beneficial to bone. The drug, LP533401, a Tph1 inhibitor, was effective in preventing and even curing osteoporosis in mice and rats at an oral dose of less than 25 mg/kg/day through an isolated increase in bone formation.53 The effect of Tph1 inhibitors on bone mass establishes that inhibition of GDS biosynthesis can rescue ovariectomy-induced osteoporosis in the mouse through an anabolic mechanism. These studies further validate the role of GDS as a regulator of bone formation and provide foundation for the development of other molecules that target the Tph1/Htr1b/osteoblast pathway for the treatment of low bone mass diseases, either alone or in combination with other existing therapies (Figure 2).

Future studies would be necessary to investigate four specific issues: First, the absolute threshold levels at which suppression in peripheral serotonin signaling is anabolic to the bone. Second, to analyze in more detail plasma- vs serumvs platelet-derived serotonin in the regulation of bone mass. Third, to thoroughly characterize any toxicity or side effects the drugs that target this pathway might have on any of the functions of other peripheral organs. Fourth, and most importantly, if these types of drugs can be used to treat low bone mass conditions associated with specific genetic mutations in mouse models of human diseases such as osteoporosis pseudoglioma.

As research on the role of serotonin and its receptors in bone physiology progresses, the difficulty of these challenges will become clearer. In the process we will likely discover new therapeutic targets for osteoporosis treatments as well as gain a better understanding of the beauty and complexity of bone biology. _

This work was supported by a NIH grant (DK85328) and a Rodan fellowship from IBMS to VKY. I apologize to numerous researchers whose work I was unable to discuss due to space constraints.

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Keywords: serotonin; gut; bone; osteoblast; osteoclast