Several members o f the TRP channel superfamily are beginning to attract
increasing interest in the field o f bone function research due to emerging
putative roles in osteoblast proliferation, mineralisation and differentiation.
The highly calcium-selective channels TRPV5 and TRPV6 are involved in
bone growth. TRPV5 appears to be essential for normal bone turnover and it
has been shown that TRPV5 knockout (TRPV5_/~) mice have ‘reduced bone
thickness’ (Hoenderop et al., 2003b) due to the diminished active Ca2+
resorption by the kidney nephrons, for which TRPV5 has been called the
‘gatekeeper’ o f transepithelial renal Ca transport. The same knockout mouse
model shows increased osteoclast cell numbers and cell size, but much
diminished resorptive activity compared to TRPV5+/+ mice (van der Eerden et
al., 2005). Therefore TRPV5 is required in the kidney for renal Ca2+ resorption
and in osteoclasts for bone resorption. The second highly Ca2+ selective TRP
channel, TRPV6, which shares more than 70% homology with TRPV5, has
been shown to have a role in cell proliferation, and is upregulated particularly
in later stage prostate and breast tumours that are metastasising (Schwarz et al.,
2006; Prevarskaya et al., 2007). It has been implied by Nijenhuis et al. (2003a)
that as TRPV6 expression has been identified in osteoblasts, this channel might
play a role in mineralisation. TRPV5 and TRPV6 channels may be targets for
the well-known bone regulator l,25(OH)2D3 or vitamin D3 (Hoenderop et al.,
et al.. 2002) as TRPV5 expression was stimulated by 17p-oestradiol in
ovariectomised rats.
TRPM7 has been shown by Elizondo et al. (2005) to be important for normal
skeletogenesis in the dwarf zebrafish model, in which the mutant nutria!12462
has different tissue expressions o f TRPM7 compared to wild-type.
Consequently the nutria112462 mutant zebrafish is phenotypically much smaller
and differently shaped to the wild-type dwarf zebrafish. Abed and Moreau
(2007) found that in human osteosarcoma cell lines MG63, SaOS-2 and U-
2 0 S , TRPM7 was important for osteoblast proliferation. Cells treated with
siRNAs targeted at TRPM7 showed 60 - 75% reduction in proliferation
compared to untreated TRPM7-4normal’ cells.
The cold-sensitive channel TRPM8 is upregulated in a variety o f malignant
tumours including prostate, breast, colon, lung and skin (Tsavaler et al., 2001;
Zhang et al., 2004; Zhang et al., 2006), many o f which metastasize to bone. In
chapter 4, the expression o f TRPM8 in human osteoblast-like cell lines was
shown to be low. The expression o f TRPM8 in prostate cancer is known to be
linked to androgen receptor expression - the tumour becomes more aggressive
and begins to metastasize at the same time as androgen-sensitivity o f the
tumour is lost, and TRPM8 expression is down-regulated (Zhang et al., 2004).
The transition from TRPM8 high-expression to low-expression may be
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F U N C T IO N A L P R O P E R T IE S O F T H E TRPV 1 R E C E P T O R IN O S T E O B L A S T S
5.7. /. / Current evidence fo r TRPV1 fu n ctio n in bone
TRPV1 has been shown to be expressed in osteoblasts and osteoclasts and
appears to have several functional roles:
Proliferation and mineralisation
TRPV1 activation is known to be sensitive to pH and reduced pH (increased
number o f protons) increases the activity o f this channel by potentiation (Ryu
et al., 2003). Bone is a pH sensitive tissue and small changes in pH may have
consequences for bone mineralisation or absorption. A decrease in extracellular
pH in vitro from 7.4 to 6.9 abolishes mineralisation by osteoblasts, and is
associated with an 8 X reduction in osteoblast alkaline phosphatase production
(a marker for mineralising osteoblasts) and a significant rise in the expression
o f matrix Gla protein, which is a calcium-binding protein that inhibits
mineralisation (Brandao-Burch et al., 2005). The same change in conditions
stimulates osteoclasts to maximum resorptive activity, whereas at pH 7.4
resorptive activity is minimal (Arnett, 2008). The author (ibid) argues that
TRPV 1 might well be the cellular sensor for pH in osteoblasts and osteoclasts,
and therefore has a vital role in normal bone turnover.
Differentiation
The association between decreased bone mineral density and increased bone
marrow or trabecular bone adipocyte content is well known, and occurs with
aging (Parfitt et al., 1992; Rozman et al., 1989), non-weight bearing
al., 1985), ovariectomy-induced osteoporosis (Martin & Zissimos, 1991) and
general osteoporosis o f all ages (Gimble et al., 1996; Meunier et a l 1971;
Nuttall et al., 1998). It is presumed that adipocyte formation takes place at the
expense o f osteoblast maturation and that these cell types have a common
ancestor (Gimble et al., 1996; Thompson et al., 1998). The mechanisms that
control the differentiation o f osteoblast and adipocyte precursors are poorly
understood, but clearly pharmacological intervention could provide a future
treatment for pathologies.
An emerging putative role for TRPV 1 in bone is in differentiation. It has been
shown that TRPV1 activation by capsaicin prevents adipogenesis in 3T3-L1-
preadipocytes (a non-bone precursor) and that this effect is lost when TRPV 1 is
knocked-down (Zhang et al., 2008). Furthermore, the authors also showed that
during regular adipogenesis in 3T3-LI-preadipocytes, TRPV1 expression is
downregulated, but exposure to capsaicin maintains TRPV 1 expression levels.
Therefore, it is clear that TRPV1 expression and activation is pivotal in
determining whether precursor cells differentiate into adipocytes. These very
interesting findings o f the above two studies beg further investigation, with the
obvious and important next step being to determine whether TRPV 1 activation
in osteoblastic-precursors can discourage adipogenesis and promote
osteoblastogenesis. Osteoblast-precursor differentiation has clear implications
for osteogenesis and diseases such as osteoporosis (Griffith et al., 2008;
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F U N C T IO N A L P R O P E R T IE S O F T H E TRPV 1 R E C E P T O R IN O S T E O B L A S T S
5.1.2 Cannabinoids as TRPV1 ligands
5.1.2.1 Cannabinoid receptors are fu n ctio n a l in bone
Firstly, it is pertinent to recognise that the expression o f cannabinoid receptors
CBj or CB2 influences bone cell function. CBi receptors have been shown to
be involved in the regulation o f bone mass and bone turnover, but CB]
receptors are expressed only at low levels in osteoblastic precursors, mature
osteoblasts and in osteoclasts (Tam et al., 2006) but high expression is found in
trabecular bone sympathetic nerve fibres. As noradrenaline release from these
nerve fibres decreases osteoblastic activity but increases osteoclastic bone
resorption, the likely mechanism o f bone regulation by CBi receptor
stimulation is downstream sympathetic signalling (Bab et al., 2008; Bab &
Zimmer, 2008; Tam et al., 2008). In contrast, CB2 receptors, which are also
expressed only at very low levels in osteoblast precursors, increase in
expression as cells mature into true osteoblasts in line with increasing
expressions o f markers o f osteoblast activity including alkaline phosphatase
(Zhou et al., 2004), parathyroid hormone receptor (Zhang et al., 1995) and
runt-related transcription factor 2 (RUNX2) (Araujo et al., 2004; Bab &
Zimmer, 2008). CB2 also shows high expression in osteoclasts (Bab et al.,
2008; Ofek et al., 2006). CB2 stimulation by the endogenous cannabinoid
anandamide results in increased osteoblastic activity and reduced osteoclastic
absorption, both directly and by increased osteoblastic RANKL expression,
thereby increasing bone mass, whereas CB2 ; mice display significantly
5.1.2.2 Cannabinoid ligands as TRPV1 channel agonists
It is evident that the endocannabinoid system plays an important part in the
regulation o f bone mass and metabolism, but it is becoming clear that
endocannabinoids - in particular anandamide - can be endogenous activators
o f TRPV1 and may therefore partake in a different mechanism for bone
regulation. Smart et al. (2000) recognised that anandamide is a full agonist of
TRPV1 producing similar inward currents to those induced by capsaicin, and
evoking [Ca2+]j increases in the same manner. The potency o f anandamide at
the TRPV 1 receptor was weaker than that o f capsaicin, and was approximately
20x weaker than its binding affinity at the cannabinoid receptor (Smart et al.,
2000; Devane et al., 1992).
The chemical structure o f anandamide resembles that o f many vanilloids
(Pertwee, 1997; Szallasi & Blumberg, 1999) and one o f the vanilloids, olvanil,
has been shown to inhibit the facilitated diffusion o f anandamide via carrier
proteins, thus maintaining high levels o f anandamide for CB receptor
activation (Di Marzo et al., 1998). In addition to TRPV1 activation by
anandamide, a second endocannabinoid ligand, palmitoylethanolamide
(Pertwee, 1997), has been shown to activate TRPV1, but is less potent than
anandamide at the same receptor site (Smart et al., 2000). Figure 5.1 shows the
structural similarities between some selected vanilloids and an
C H A P T E R 5:
F U N C T IO N A L P R O P E R T IE S O F T H E TRPV 1 R E C E P T O R IN O S T E O B L A S T S
C apsaicin
O lvanil
A nandam ide
Figure 5.1 Chemical structures of vanilloids and an endocannabinoid
5.1.3 Chapter hypothesis, aims and experimental strategies
Given the evidence, the hypothesis o f this chapter is that the TRPV1 channel
has a functional role in (i) the proliferation, (ii), mineralisation and (iii)
differentiation o f osteoblasts.
The aims o f this chapter are:
• to test whether known TRPV1 agonists and antagonists, and also
cannabinoid ligands, have any effect on the growth (proliferation) of
MG63 cells, using haemocytometry and MTS dye absorption
measurements.
• to test whether TRPV 1 agonists and antagonists affect the mineralisation
o f human SaOS-2 and mouse 7F2 osteoblasts.
• to test whether the differentiation o f mouse 7F2 osteoblast precursors into
either mature osteoblasts or adipocytes is affected by TRPV1 and
cannabinoid ligands.
• to investigate the effects o f capsaicin and other TRPV1 ligands on the
isolated guinea pig ileum, as a model for the short-term effects o f
capsaicin and other TRPV1 ligands, including anandamide and other
C H A P T E R 5:
F U N C T IO N A L P R O P E R T IE S O F T H E TRPV 1 R E C E P T O R IN O S T E O B L A S T S