Originally posted by thegame46 on IBE's forum:
Thyroid hormone (L-tri-iodothyronine; T3) has major effects
on Ca2l homeostasis in heart and skeletal muscle [1,2]. One of the
most striking effects is the increased speed of muscle relaxation
in hyperthyroidism and the decreased relaxation rate in
hypothyroidism [3-6]. In the last decade it has become clear
through work by ourselves and others that T3-dependent changes
in the sarcoplasmic reticulum (SR) underlie these phenomena. T3
administration in vivo causes an increase of the amounts of Ca21
pumps (Ca2+-ATPase) and SR, thereby increasing the rate of
Ca2+ removal from the sarcoplasma as well as the capacity for
Ca2+ storage [7-11]. As a consequence of the increased Ca2+
release and re-uptake (Ca2+ cycling) the metabolic rate is
stimulated in skeletal muscle by consuming more ATP, which
contributes to the well known thermogenic effect of thyroid
hormone
This process works continuously because the cell has Ca++ channels that operate solely on the concetratoin gradient. There fore the result is a faster cycleing of Ca++ ina nd out of the cell.
Thyroid hormone (T3) is a major determinant of the fast-type
sarcoplasmic-reticulum Ca2+-ATPase (SERCAl) level in skeletal
muscle [1-4]. This Ca2+-transporting protein is responsible for
the removal of Ca2+ from the cytosol during a contractionrelaxation
cycle. The T3-induced increase in SERCAl expression
is mainly responsible for the enhanced muscle relaxation rate
which is characteristic of the hyperthyroid status [5-7]. Evidence
that T3 regulates SERCAI expression in vivo at least partly at a
pre-translational level was provided in one of our previous
studies, in which it was shown that T3increases SERCAI mRNA
levels in rat soleus and extensor digitorum longus muscle
Please read the How T3 works before trying to understand thsi section
IGF-I production is controlled by growth
hormone (GH), which in turn is dependent on T3 for its
production [15]. Thus part of the influence of the thyroid status
on the SR might be derived from IGF-I action. To examine the
possible inter-relationship between the effects of T3 and IGF-I on
SR, we chose an in vitro cell culture model. The rat myogenic L6
cell line [16], which is not contaminated with other cell types and
has been shown to possess T3 nuclear receptors [17], was
considered to be suitable.
The results of this study show that, in L6AM cells (subcloned
from the L6 myogenic cell line) IGF-I is required for maximal
stimulation of Ca2+-ATPase net synthesis and Ca2+ uptake
activity by T3.
Factors regulating Ca2+-ATPase synthesis in vitro
There are still many gaps in the knowledge of the regulation of
Ca2+-ATPase synthesis in cultured skeletal muscle cells. It is,
however, well established that the rate of Ca2+-ATPase synthesis
sharply increases after fusion of myoblasts into multinucleated
myotubes [26-28]. Innervation, which determines the activity
pattern of muscle, and T, are major determinants of Ca2+-
ATPase activity in vivo [1,29]. It was shown earlier that
Ca2+-ATPase synthesis in vitro is stimulated after spontaneous
contractions in myotubes [30]. The present study shows that T3
is also able to stimulate Ca2+-ATPase synthesis in vitro. Thus the
effects of T3 and muscle contraction on Ca2+-ATPase synthesis
in vivo can be reproduced in vitro, at least qualitatively. How these
actions are related to the genomic expression of Ca2+-ATPase is
still an open question. Martonosi and co-workers found evidence
that changes in intracellular free Ca2+ could play a pivotal role in
the regulation of Ca2+-ATPase synthesis [31,32]. Thyroid status
and muscle contraction both strongly influence Ca2+ homeostasis.
Thus it cannot be excluded that the effects of T3 and muscle
contraction are at least partially mediated by changes in intracellular
free Ca2
In recent years it has become clear that growth factors play an
essential role in muscle differentiation, and the significance of
insulin-like growth factors among others for differentiation in
the L6 muscle cell line was convincingly demonstrated [14]. The
stimulation of Ca2+-ATPase by IGF-I and its potentiating effect
on T3-mediated stimulation as described here is a novel aspect of
the regulation of Ca2+-ATPase synthesis, at least in the L6
muscle cell line.
It should be noted that T3 and IGF-I, when both present,
stimulate Ca2+-ATPase synthesis to levels (approx. 15 pmol/mg
of protein) which are of the order of magnitude of the
concentrations found in rabbit soleus muscle (35 pmol/mg of
protein) [24] and in rat soleus muscle (40-60 pmol/mg of protein)
([33]; A. Muller, unpublished work). This is, however, still more
than 10-fold lower than the Ca2+-ATPase concentrations found
in fast extensor digitorum longus muscle of both species ([24,33];
A. Muller, unpublished work).
In spite of the lower degree of myotube formation in T3-treated cultures (55 % fusion), the stimulatory effect of T3 on net
Ca2+-ATPase synthesis was twice as large as the effect seen in the
presence of IGF-I (91 % fusion). The fusion process of myoblasts
and the synthesis of muscle-specific proteins, including Ca2+_
ATPase, coincide in many myogenic systems. According to the
results of Pinset & Whalen [34], these events are coupled in L6
cells. This is claimed not to be the case for Ca2+-ATPase and
myofibrillar proteins in primary myoblasts [35]. Although the
synthesis of Ca2+-ATPase was greatly accelerated upon fusion of
myoblasts and differentiation into myotubes, fusion was not
essential. In Ca2+-deprived medium myoblast fusion was inhibited
but Ca2+-ATPase synthesis, although delayed for 50 h, still took
place. To date it is not known whether T3 induces Ca2+-ATPase
synthesis in the L6AM cell line in both myoblasts and myotubes or
only in myotubes. In the latter case this would mean that the T3-
stimulated Ca2+-ATPase accumulation is 4-fold higher than the
IGF-I effect, since, as we mentioned previously, the percentage
fusion was approximately doubled in IGF-I treated cultures
compared with T3-treated ones. On the other hand, it would also
mean that the potentiating effect of IGF-I on T3 stimulation of
Ca2+-ATPase synthesis and Ca2+ uptake activity could be largely
ascribed to the IGF-induced increase in fusion (from 550% to
91 %). However, the experiments with cultures consisting of
more than 90 % myotubes (Figs. 7 and 8) show once more that the
combined effects of IGF-I and T3 on Ca2+-ATPase synthesis and
Ca2+ uptake activity are greater when they are added separately.
From this it can be concluded that increased fusion cannot be the
only mechanism by which IGF-I increases the effects of T3
Ca2+ uptake activity by T3 proceeded synchronously. Everts [36]
found that the Ca2+-ATPase content in soleus muscle from
developing rats increased significantly after I day of T3 treatment
and Ca2+ uptake activity increased only after 2 days. It was,
however, noted that this could be caused by differences in the
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A. Muller and others
sensitivity of the methods used. Two models of SR biogenesis
have been proposed: (1) the random incorporation model [27]
and (2) the growing point model [28]. The first model assumes
Ca2+-ATPase incorporation into multifunctional endoplasmic
reticulum (ER), which would gradually be transformed into a
monofunctional SR. The growing-point model assumes lateral
displacement of Ca2+-ATPase from the rough ER to fully
differentiated SR, with a growing point of SR in between the ER
and SR. In both models the possibility of a lag time between the
new synthesis of Ca2+-ATPase and its operation in SR, i.e. Ca2+
uptake, is not excluded. Our results do not support an appreciable
difference (at least 1 day) in the time sequence of Ca2+-ATPase
accumulation and Ca2+ uptake activity in L6AM cells.
Concluding remarks
For the present, the inter-relationship of the actions of T3 and
IGF-I in the L6AM muscle cell line can only be a matter of
speculation. Very recently we have found that fast Ca2+-ATPase
mRNA levels were elevated 8-fold by T3, relative to controls, in
differentiating myoblasts (A. Muller, unpublished work), which
is in agreement with a similar study in heart, where T3 increased
mRNA levels for slow Ca2+-ATPase [37]. These results suggest a
role for T3 at a level higher than protein turnover. This is also
possible for IGF-I, since it has been reported that insulin, acting
as an IGF-I analogue, enhanced stabilization of mRNA for
creatine kinase in the L6A1 muscle cell line [38]. On the other
hand, the relatively more prominent effect of IGF-I on Ca2+
uptake activity could point to an essential role for IGF-I in the
maturation of a functional SR membrane. Finally, since IGF-I
production is indirectly controlled by T3 (see the Introduction
section), the results of this study could be relevant for the thyroid
hormone control of SR function in vivo. Additional evidence for
this will, however, have to be provided from studies in primary
muscle cell cultures of neonatal rats
In a previous study with the L6 rat muscle cell line we
investigated the possible involvement of IGF-I in T3-stimulated
sarcoplasmic-reticulum development [12]. It was shown that not
only T3, but also IGF-I, elevated SERCAl levels (2.3- and 1.6-
fold respectively), and that T3 and IGF-I acted synergistically
(4.3-fold increase). The goal of the present study was to gain
further insight into the regulatory mechanism(s) by which T3 and
IGF-I enhance SERCAI expression in L6 myotubes.
The primary mechanism of T3 action in diverse cellular
processes involves an interaction of the hormone-receptor complex
with specific DNA sequences, leading to altered transcription
rates of target genes (reviewed in [13]). In addition, T3 may also
act at post-transcriptional levels. Evidence has been presented
that T3 increased the stability of growth hormone and malic
enzyme mRNA [14-16], whereas a negative influence of T3was
demonstrated on the stability of thyrotropin f-subunit mRNA
[17]. Additionally, it has been known for a long time that T3
enhances protein turnover in skeletal muscle by stimulating both
protein synthesis [18-20] and protein degradation [18,21].
IGF-I has a broad range of effects, comprising a general
stimulation of protein, RNA and DNA synthesis, as well as
induction of specific proteins involved in cell proliferation or
differentiation (reviewed in [22]). These effects are accomplished
after binding of IGF-I to the type 1 IGF plasma-membrane
receptor, followed by the activation of an intracellular signalling
system, which is not yet completely elucidated [23,24]. This
eventually results in transcriptional [25-27] or post-transcriptional
regulation, for example by increasing mRNA stability
The latter was also demonstrated in L6 muscle cells, in
which insulin (acting as an IGF-I analogue) increased the stability
of creatine kinase mRNA [30]. Furthermore, the increase in total
protein content that was induced by IGF-I in cultured muscle
cells was shown to be mediated partly through inhibition of
protein breakdown [31-33]. Taken together, these data illustrate
that T3 and IGF-I may regulate gene expression at various levels.
In this study we present evidence that T3 and IGF-I control
SERCAI expression at the transcriptional and post-transcriptional
level respectively, although not exclusively. Part of this
study has been presented in a preliminary form [34].
In a previous study with the L6 rat muscle cell line we
investigated the possible involvement of IGF-I in T3-stimulated
sarcoplasmic-reticulum development [12]. It was shown that not
only T3, but also IGF-I, elevated SERCAl levels (2.3- and 1.6-
fold respectively), and that T3 and IGF-I acted synergistically
(4.3-fold increase). The goal of the present study was to gain
further insight into the regulatory mechanism(s) by which T3 and
IGF-I enhance SERCAI expression in L6 myotubes.
The primary mechanism of T3 action in diverse cellular
processes involves an interaction of the hormone-receptor complex
with specific DNA sequences, leading to altered transcription
rates of target genes (reviewed in [13]). In addition, T3 may also
act at post-transcriptional levels. Evidence has been presented
that T3 increased the stability of growth hormone and malic
enzyme mRNA [14-16], whereas a negative influence of T3was
demonstrated on the stability of thyrotropin f-subunit mRNA
[17]. Additionally, it has been known for a long time that T3
enhances protein turnover in skeletal muscle by stimulating both
protein synthesis [18-20] and protein degradation [18,21].
IGF-I has a broad range of effects, comprising a general
stimulation of protein, RNA and DNA synthesis, as well as
induction of specific proteins involved in cell proliferation or
differentiation (reviewed in [22]). These effects are accomplished
after binding of IGF-I to the type 1 IGF plasma-membrane
receptor, followed by the activation of an intracellular signalling
system, which is not yet completely elucidated [23,24]. This
eventually results in transcriptional [25-27] or post-transcriptional
regulation, for example by increasing mRNA stability general effect of IGF-I on protein stability. The mechanism(s)
underlying the SERCA1 protein-stabilizing effect of IGF-I are as
yet unclear, but a suggestive observation was made in a previous
study of ours, in which an equal increase in SERCAI protein
induced by either IGF-I or T3 resulted in a larger increase in
sarcoplasmic-reticulum Ca2+-uptake activity in the case of IGFI
[12]. One might speculate that such increased formation of
functional sarcoplasmic-reticulum membrane is involved in the
prolongation of SERCAI protein half-life. As shown in Figure 3,
however, this effect of IGF-I is offset by T3. This is in accordance
with the less than proportional increase in SERCAI protein
relative to its mRNA in cultures treated with T3 alone. This
suggests that T3 increases the degradation rate of SERCAl
protein, which would be in line with studies reporting acceleration
of protein degradation by T3 [18,21]. However, our study does
not provide clear evidence that the half-life of the SERCAI
protein is decreased by T3, since the slight decrease in SERCAl
protein stability by T3 observed in this study did not reach
significance. Another possibility is that T3 diminishes the translation efficentcy of SERCA1 mRNA
The half-life of SERCAl protein was similar to the half-life of
SERCAI mRNA (ranging from 13.3 to 32.5 h). Although
proteins are usually more stable than the corresponding mRNA,
it is not uncommon that mRNA and protein turn over with an
equal rate [50]. Our study also presents some evidence that the
increased SERCAI mRNA levels observed in the presence of T3
and IGF-I were for a significant part due to post-transcriptional
regulation, i.e. stabilization of SERCAI mRNA. The SERCAI
mRNA half-life was twice as long in IGF-I + T3-treated cultures
compared with T3-treated cultures, which is in reasonable agreement
with the 1.6-fold greater increase in the SERCAI mRNA
levels by IGF-I + T3 compared with T3. T3 or IGF-I alone did not
affect the SERCAI mRNA half-life, which indicates that both T3
and IGF-I are involved in the process leading to the T3 + IGF-I
induced increase in SERCA1 mRNA stability. The mechanism
underlying this interaction between T3 and IGF-I is at present
unknown. Regarding the mechanisms operating in selective
stabilization of mRNA, a number of possibilities have been
proposed (reviewed in [51,52]), such as mRNA modification at
the 5-end or the 3-terminus, or protection of mRNAs against
RNAases by interaction with specific proteins. For instance,
evidence was obtained in a study with L6 muscle cells that the
increased creatine kinase mRNA stability that was induced by
insulin, here acting as an IGF-I analogue, was mediated by a
short-lived protein [30].
In the presence of T3 alone, a 3.4-fold increase in SERCAl
mRNA content was observed, which could not be attributed to
effects on SERCAI mRNA stability. This pointed to transcriptional
regulation, which was confirmed in the nuclear run-on
analyses. The 3-fold increase in the transcription initiation
frequency of the SERCAI gene induced by T3 strongly suggests
that enhanced transcription is the primary mechanism leading to
elevated SERCAI mRNA levels in the presence of T3. These
results confirm what a preliminary analysis in vitro of the
SERCAI promoter had already suggested. In that study, using
transient transfection assays, it was shown that the transcriptional
activity of the initial 2400 bp of the SERCAI promoter is
stimulated 3.5-fold by T3 [53]. Similar results have also been
reported for the T3 regulation of SERCA2a promoter activity in
transient transfection assays using cardiomyocytes, although
these results were not substantiated by run-on analyses [54]. With
respect to IGF-I regulation of SERCAI expression, our results
do not exclude the possibility of transcriptional regulation.
However, since the IGF-I induced increase in SERCAI mRNA
content is only small compared with the increase induced by T3, there is no need to postulate a major role of IGF-I in transcriptional
regulation of SERCAI expression.
In summary: this study suggests that T3 controls SERCA1
expression primarily by increasing the transcription frequency of
the SERCAI gene, whereas IGF-I seems to act predominantly at
post-transcriptional levels by enhancing SERCAI protein and
mRNA stability, the latter, however, only in the presence of T3.
