Abstract 1 (NFATc1), which is the master osteoclast regulator.4,5

Abstract

Melatonin,
a hormone produced by the pineal gland and peripheral tissue cells has been
widely studied for its role in photoperiodism. Recently, melatonin gets great
attention for its effects on bone homeostasis. Numerous studies documented that
melatonin promotes osteoblast differentiation and bone maturation, but a exact
role of melatonin in osteoclast differentiation is still elusive. Based on the
evidence that melatonin significantly reduced receptor activator of nuclear
factor-?B
ligand (RANKL)-induced bone resorption via suppression of nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-?B)
activity, the focus of this review mainly describes how melatonin interferes
with canonical NF-?B signaling pathway contributing to osteoclastogenesis.

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The review pointed out the two most significant molecules?Myeloid differentiation primary response
88 (MyD88) and TNF receptor-associated factor-?B
ligand 6 (TRAF6), which account for the melatonin-mediated suppression of NF-kB.

Though many studies related biological function of melatonin to its inhibition
of the proteasome, lack of evidence shows the direct interaction between
melatonin and proteasome. It seems melatonin mainly affects ubiquitin ligase (e.g.,
TRAF6) rather than proteasome to inhibit the ubiquitin-proteasome system.

Interestingly, the anti-osteoclastogenic effect of melatonin is reported to be
independent of melatonin receptors on cell membrane. In addition to previously
reported properties of melatonin, this review proposes another aspect of how
melatonin influences osteoclastogenesis.

 

Introduction

Osteoclastogenesis

Osteoclasts differentiate from
monocyte-macrophage lineage cells. They are multinucleated giant cells with function
of bone-resorbing. Receptor activator of nuclear factor-?B
ligand (RANKL), proinflammatory cytokines (TNF-?,
IL-1), and lipopolysaccharide (LPS, which is recognized for TLR4) induce
osteoclast differentiation and activation.1,2,3 RANKL, which is
produced by osteoblasts and bone marrow stromal cells, binds to RANK on
osteoclast precursor cells followed by receptor oligomerization and recruitment
of signaling adapter molecules such as TNF receptor-associated factor-?B
ligand 6 (TRAF6). Subsequent TRAF6 and transforming growth factor
beta-activated kinase 1 (TAK1) association activates the downstream signaling
pathways of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B)
and induces expression of nuclear factor of activated T-cell cytoplasmic 1

(NFATc1),
which is the master osteoclast regulator.4,5 Interference with these
signaling pathways may prevent excessive osteoclast formation and pathological
bone loss.

 

Melatonin

Melatonin
is found in almost every living creature including bacteria, plants, and
vertebrates. The main function of melatonin is known for regulating circadian
light-dark cycles of the body, with the peak level of melatonin secreted in the
darkness.6 In addition to its capacity for time keeping, recent
experiments have reported the various and unexpected functions of melatonin.7,8
Melatonin is able to influence a vast number of cellular proteins involved in a
variety of physiological processes including bone homeostasis.9,10,11
The regulation of osteoclasts activity of melatonin is physiologically
plausible since the level of melatonin in blood decreases with age, especially
in post-menopausal women. Thus, the decreased melatonin level could be involved
in the onset of osteoporosis in the elderly.12,13 Melatonin has been
demonstrated to promote osteoblast differentiation and bone maturation,13,14
but a direct role of melatonin on osteoclast differentiation is still elusive.

Considering the demonstration that melatonin significantly reduced
RANKL-induced bone resorption via suppression of NF-?B
activity,9,10,15 the review will make a further discussion on how
melatonin affects the pathological osteoclastogenesis.

 

Discussion

NF-?B and the ubiquitin-proteasome system

NF-?B
is a set of multifunctional transcription factors that regulate expression of
genes involved in numerous normal cellular activities, including
osteoclastogenesis, inflammation, and immunity.16,17,18 The NF-?B
activation pathways are broadly classified as the canonical and non-canonical
pathways, depending on whether activation involves I?B?
degradation or not. When the ligand binds to IL-1 receptors (IL-1R) or
Toll-like receptors (TLRs), triggering subsequent recruitment of the Myddosome
complex, which consists of myeloid differentiation factor 88 (MyD88), interleukin-1
receptor-associated kinase-4 (IRAK4), and interleukin-1
receptor-associated kinase-1 (IRAK1).19,20,21 IRAK1 is
phosphorylated by IRAK4 and then associates to activate TRAF6 (E3 ubiquitin
ligase).20,22 TRAF6 functions together with the E2 ubiquitin ligase
to conjugate polyubiquitin chains to substrates, including
TRAF6 itself.22,23 Ubiquitinated TRAF6 will recruit TAK1-binding
protein-2 (TAB2) and activate the TAB2-associated
TAK1 kinase, which then phosphorylates inhibitor of ?B kinase ? subunit (I?K?)
at two serine residues within the activation loop, leading to activation of I?K
complex.22,24,25 Some genetic experiments have proved that I?K?,
but not I?K?, phosphorylates inhibitor of NF-?B
(I?B?)
proteins, targeting I?B? for polyubiquitination and subsequent degradation by the
26S proteasome, thus results in activation of NF-?B.22,26,27

 

TRAF6 promotes NF-?B in RANKL-induced osteoclastogenesis

NF-?B
is a critical intracellular signaling molecule regulating RANKL-induced NFATc1
expression.10,28, Yu et al.29 identified the relationship
between NF-?B and osteoclasts by the evidence that NF-?B
inhibitors will suppress osteoclasts and overexpression of NF-?B
proteins, thus, reducing osteoclasts. Upon RANKL binds to the RANK receptor, TRAF6
will be recruited. TRAF6, associating with the ubiquitin E2 complex synthesize
and bind K63-linked polyubiquitin chains to NF-kappa-B essential modulator (NEMO)
and itself, leading to activation of I?K complex.22 I?K
complex induces phosphorylation and proteasome-mediated degradation of I?B?
(inhibitor of NF-?B), following the release of NF-?B.15,30
Several studies showed that deubiquitination of TRAF6 suppresses RANKL-induced
osteoclastogenesis.31,32,33

 

MyD88 mediates osteoclastogenesis in
IL-1R/TLRs signaling pathway

IL-1R
and TLRs belong to the same IL-1R/TLR superfamily and are believed to play
important roles in the bacteria-mediated bone loss in diseases especially
periodontitis.34,35 Studies have reported that IL-1 and TLR ligands
modulate RANKL-induced osteoclastogenesis by regulating NF-?B,
and downstream signaling, including NFATc1.4,36,37,38,39 According
to Horng et al.40, TLRs signaling via MyD88 associates with
TIRAP/Mal (another MyD88 adaptor family member), while IL-1R signaling requires
only MyD88. Both TLRs and IL-1R regulate RANKL-induced osteoclastogenesis via
MyD88.28,29 It is believed that MyD88 signaling has a pivotal role
in osteoclastogenesis via the NF-?B pathway because of the evidence that
MyD88 deficiency markedly inhibited RANKL expression.41

 

Melatonin suppresses TLRs-mediated MyD88
and downstream NF-kB pathway

Since
the main cause of degradation of IkB? is phosphorylation by I?K
complex (I?K?, I?B?, NEMO), it implies that melatonin may inhibit I?K
activity to prevent IkB? from degradation. Liang et al.42 proved that
melatonin significantly reduced the expression of I?K?.

Likewise, Lu et al.43 reported the similar consequence that
melatonin decreases the level of phosphorylation of I?K.

Nevertheless, it can probably be attributed to the fact that melatonin affects
upstream regulator of I?K resulting in the reduction of I?K.

There have been some studies demonstrated that melatonin suppresses
TLRs-mediated MyD88 protein expression which promotes the early activation of
NF-kB.44,45,46 Consequently, melatonin-mediated inhibition of MyD88
will reduce the downstream components of MyD88-dependent toll-like receptor
signaling pathway, including I?K, IkB?, and NF-kB.22,45

The difference between melatonin and
bortezomib

While
melatonin is gradually studied for its function related to the
ubiquitin-proteasome system,7,47,48 there has been another
FDA-approved synthetic proteasome inhibitor, bortezomib. Bortezomib is a
peptide boronic acid analog that reversibly inhibits the chymotryptic activity
of the 20S subunit of the proteasome.49 Some major proteins of
significance in cancer susceptibility include the transcription factor NF-?B,
tumor suppressor factor, p53, cell cycle regulator, p27, are controlled by the
ubiquitin-proteasome system and are targets of the proteasome inhibitor,
bortezomib.4 Each of these proteins has also been reported to be
influenced by the naturally-occurring indole, melatonin, suggesting the
hypothesis that melatonin is also a proteasome inhibitor.47,50,51,52
Park et al.53 demonstrated that melatonin inhibits proteasome
activity indeed. However, the mechanism of how melatonin and bortezomib
influence the ubiquitin–proteasome system may be different. Bortezomib has been
presented a model of the interaction with the ?5
subunit of the beta ring of the proteasome, while there is no direct evidence
that melatonin interacts with the ?5 subunit of the proteasome.47
It implicates that melatonin inhibits the ubiquitin–proteasome system in other
ways.

 

Melatonin affects ubiquitin ligase,
TRAF6 rather than proteasome to inhibit NF-?B

There
are numerous studies report that melatonin inhibits NF-?B
activity by blocking the proteasome-mediated degradation of IkB?.54,55,56,57,58
Yet, the main cause seems to be that melatonin influences ubiquitin ligase as
an inhibitory effect on proteasomes.9,47,48 In NK-?B
signaling pathway, Chuffa et al.45 showed melatonin downregulates
the level of TRAF6 (E3 ubiquitin ligase) which is essential for I?K
activation. As a result, I?K complex fails to phosphorylate and target I?B?
for polyubiquitination and subsequent degradation by the 26S proteasome. By
contrast, lack of evidence shows the direct interaction between melatonin and
proteasome. It seems melatonin mainly affects ubiquitin ligase rather than
proteasome to inhibit the ubiquitin-proteasome system, which is different from
the way bortezomib acts.

 

Melatonin receptors for
anti-osteoclastogenesis

Studies
have proven that presence of melatonin significantly reduced receptor activator
of RANKL-induced osteoclastogenesis.15 Generally, melatonin shows
its effects by melatonin receptor activation.59,60 However, Kim et
al.10 demonstrated that silencing of MT1/MT2 melatonin receptors in
osteoclast precursor cells failed to reverse the anti-osteoclastogenic effect
of melatonin. It indicates anti-osteoclastogenic effect of melatonin is
independent of cell membrane melatonin receptors.

Conclusion

This review focused on the effects of melatonin on canonical NF-kB
signaling pathway to interpret the reason why melatonin is capable of reducing
pathological osteoclastogenesis. There are two main target inhibited by
melatonin?MyD88 and TRAF6, which account for the melatonin-mediated suppression of
NF-kB.

MyD88 initiates an inflammatory response through the IL-1R/TLRs pathway,
involving RANKL-induced osteoclastogenesis. Importantly, LPS (bacterial
endotoxin) was recognized by TLR4, so MyD88 plays a crucial role in the
bacteria-mediated bone loss such as periodontitis. When the ligand binds to
IL-1R or TLRs, triggering MyD88 to activate TRAF6. Since MyD88 is an upstream
signaling mediator in IL-1R/TLRs-induced NF-?B pathway, MyD88 is believed to be a pivotal role in inflammatory
osteoclastogenesis. There have been many experiments proving that melatonin
suppresses MyD88 and its downstream components, including I?K, IkB?, and NF-kB.

TRAF6 (E3 ubiquitin ligase) participates in both IL-1R/TLRs and RANK
signaling pathway to synthesize and bind K63-linked polyubiquitin chains to I?K complex and itself. Ubiquitinated TRAF6 will activate TAK1 kinase to
phosphorylates ubiquitinated I?K?, leading to activation of I?K complex. Activation of I?K complex is essential in
canonical NF-kB signaling pathway due to the phosphorylation of IkB? is accomplished by I?K complex. Once IkB? is phosphorylated, it will be the target for proteasome-mediated
degradation, leading to activation of NF-kB. While many studies claimed that
melatonin inhibits NF-?B activity by protecting IkB? from degradation by the proteasome, the outcome can possibly be
attributed to melatonin-mediated inhibition of ubiquitin ligase, TRAF6. If
TRAF6 is inhibited, phosphorylation of IkB? and activation of NF-?B will consequently be
inhibited.

Notably, though melatonin receptors are expressed in osteoclast precursor
cells, melatonin seems to have its anti-osteoclastogenic effect independent of cell
membrane melatonin receptors. Yet, the review aims at the two most significant
and well-studied molecules in the NF-?B pathway which have been proved to be inhibited by melatonin. Whether
melatonin also directly influences another molecule in the NF-?B pathway is still uncertain. Further studies are required since the
crosstalk between different pathway cannot be ignored either. This review
expands the role played by melatonin in the regulation of bone resorption and
provides its therapeutic relevance in the inflammatory bone loss, e.g.,
periodontitis and rheumatic diseases.

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