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Thyroid Research Unit, Division of Diabetes, Endocrinology and Metabolism, Departments of,Medicine, and Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, Minnesota 55455
Address all correspondence and requests for reprints to: J. H. Oppenheimer, M.D., University of Minnesota, Box 91-UMHC, Minneapolis, Minnesota 55455. E-mail oppen001{at}maroon.tc.umn.edu
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Abstract |
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gene preceded that of the
T3Rß gene. Although the absence of
T3 retarded the rate of accumulation of MBP
messenger RNA, the level ultimately attained was similar to that
reached in the presence of T3. This relationship
mirrored the pattern observed in the neonatal brain. Transient
transfection experiments showed that T3 regulates
MBP expression at the transcriptional level, but only for a limited
period during differentiation. These observations imply that the early
rise of MBP messenger RNA is T3 dependent,
whereas the terminal levels are maintained independently of
T3. Both the
T3-dependent and, surprisingly, the
T3-independent expression of MBP require the
presence of an intact T3 response element.
T3 receptor may regulate MBP expression in a
ligand-independent manner, or a nuclear factor other than
T3 receptor may bind to the
T3 response element of MBP to regulate terminal
gene expression. These findings support the use of differentiating
oligodendrocytes as a model of T3-induced brain
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies from our laboratory indicated that the rise in myelin basic protein (MBP) messenger RNA (mRNA) levels during brain development is sensitive to T3 and that the pattern of the rise parallels those of three other T3-sensitive mRNAs, Pcp2, calbindin, and the IP3 receptor (7). Further, immunohistochemical analysis demonstrated that oligodendrocytes, identified by MBP staining, also contain immunoreactive T3R that is restricted to the nucleus (8) consistent with a direct action of T3 to induce the rise in MBP mRNA. Farsetti et al. (9) defined the molecular basis for T3 regulation of expression of the MBP gene during development. Using transient transfection assays in NIH-3T3 cells, they identified a thyroid hormone response element (T3RE), an inverted repeat at position -186/-163 upstream of the start site of transcription. The presence of a T3RE is thus consistent with a direct action of T3 on transcription of the MBP gene during development.
The available evidence suggests a complex pattern of regulation of MBP expression by thyroid hormone during development. The ability of T3 to accelerate MBP expression becomes evident during the first postnatal week, and as a consequence, mRNA levels reach maximal levels by P15-20 (7). In the hypothyroid pup, there is a delay in MBP expression. However, by about P30 and despite the continued absence of T3, normal adult levels are achieved. These findings indicate that MBP gene expression is regulated during postnatal brain development by both T3-dependent and T3-independent mechanisms. In addition, more recent studies in our laboratory show that during the perinatal period, the MBP gene is completely refractory to the influence of thyroid hormone (10).
To define the mechanism responsible for the complex regulation of MBP gene expression by T3, it is desirable to study a cellular system that replicates the complex pattern of regulation observed in the developing rat brain. The in vitro culture of oligodendrocyte progenitor cells (O2-A cells) appeared to be a potentially useful model for carrying out such studies. The process of development of oligodendroglia from their progenitor cells in primary cell cultures from neonatal brain (11, 12) has been characterized by several laboratories and compared to developmental changes in situ (12, 13). The use of cell cultures to study gene expression offers advantages related to the ability to precisely control the cellular environment that are not possible in whole animal studies. For studies of the regulation of MBP gene expression during brain development, primary oligodendrocyte cultures have an additional advantage over transformed cell lines such as N20.1 (14), since the primary cultures retain their ability to differentiate. In the presence of medium containing very low levels of serum or chemically defined serum-free medium, the bipolar O-2A progenitor cells differentiate into oligodendrocytes with concomitant onset of expression of the myelin-associated proteins (11, 12, 15). Previous studies had shown that thyroid hormone facilitates oligodendroglial differentiation in culture (16). As in the brain, in vivo T3 stimulates MBP mRNA synthesis in developing cultured oligodendrocytes (16, 17). The present studies were undertaken to examine the developmental pattern of MBP expression in isolated differentiating oligodendrocytes with a view to defining the factors involved in T3-dependent and T3-independent MBP regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were
prepared by resuspending the cell pellet in fresh astrocyte growth
medium (AGM), a 50:50 mixture of Ham’s F-12 medium-DMEM containing
Pen-strep and 10% FBS, and immediately plating the cells at a density
of 3 x 104 cells/cm2 in
poly-L-lysine (PLL)-coated tissue culture dishes or
PLL-coated Tissue-Tek slides for immunocytochemistry. After 24 h,
the medium was switched to oligodendrocyte differentiation medium, as
described by Bottenstein and Sato (21), 50:50 DMEM H-16-Ham’s F-12
with 0.5% FBS cleared of T3 as described by
Samuels et al. (22), fatty acid free-BSA (0.66 mg/ml), ITS
(5 µg/ml insulin, 5 ng/ml selenium, and 5 µg/ml transferrin),
additional transferrin (45 µg/ml), T3 (30
nM), progesterone (20 nM), and putrescein (100
µM). The medium was changed every 2–3 days.
T3 (30 nM) was added to the
oligodendrocyte medium as indicated.
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Transfection
Cells were transfected with the -1300 MBP-Luc at the various
times indicated after removal of mitogens. Oligodendrocytes were
transfected with 30 µg lipofectin reagent (Life Technologies,
Gaithersburg, MD), and 2 µg plasmid DNA were used per 35-mm dish of
primary oligodendrocyte cultures. Cells were transfected overnight
(16–18 h) and washed, and fresh medium containing the various hormones
was added. Incubations were maintained for an additional 30 h.
Cells were washed twice in PBS, harvested by scraping in lysis buffer
(25 mM glycylglycine, 15 mM MgSO4,
4 mM EGTA, 1% Triton X-100, and 1 mM
dithiothreitol, pH 7.8), and frozen at -80 C to insure lysis before
preparing a cell extract for enzymatic assay. An internal control
plasmid, Rous sarcoma virus-chloramphenicol acetyltransferase (0.1
µg) was also cotransfected into the cells so that luciferase activity
could be normalized for transfection efficiency, as determined by CAT
activity. Assays of CAT (24) and luciferase activity (25) were
previously described.
Constructs
The 1300-base sequence of the upstream regulatory region of the
MBP gene including the start site of transcription was obtained by PCR
amplification of a larger 30-kilobase genomic fragment kindly provided
by L. Hood (26). Specific primers to allow amplification of the MBP
upstream region were synthesized on a PCR Mate 391 DNA Synthesizer
(Applied Biosystems, Foster City, CA). Reaction conditions were
previously described (27). All PCR products were sequenced before use
to identify random PCR mutations. The upstream region of the MBP gene
was ligated into the promotorless luciferase expression vector PXP2 to
create the -1300 MBP-Luc construct. Mutation of both pairs of GG
residues to TT in each half-site of the MBP T3RE
sequence in the -1300 MBP-Luc construct was performed using sequential
PCR reactions. Briefly, internal primers to the
T3RE sequence (-186 to -169) containing the GG
to TT mutation were synthesized along with anchor primers at the 5'-
and 3'-ends of the 1300-base sequence.
Immunofluorescent staining in cultured oligodendrocytes
The generation of T3R antisera used in
this study (anti-T3R-ß1,
anti-T3R-ß2, anti-TRa-1, and
anti-T3R-2) has been described previously (8).
O-2A progenitor cells were grown in Tissue-Tek chamber slides for
immunocytochemical studies. Cells were grown in oligodendrocyte medium
for 16 h, to allow adherence but not differentiation, or for 4
days, at which point the cells showed a distinct oligodendrocyte
morphology. Immunofluorescent staining of the cultures was performed as
previously described (8). All T3R antisera were
generated in rabbits (28). Mouse monoclonal anti-MBP (Boehringer
Mannheim) was used to confirm the identity of cells as O-2A progenitors
or mature oligodendrocytes. Secondary antibodies used were fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated
goat antimouse IgG (Cappel Laboratories, Durham, NC). Photomicrographs
of the stained cells were taken as previously described (28).
Northern analysis of T3R and MBP mRNAs in
cultured oligodendrocytes
Total RNA was extracted from cultured cells using the guanidine
thiocyanate procedure previously described (29) and stored at -80 C in
0.1 x SET to reduce RNA degradation. The relative masses of mRNAs
of interest were determined by Northern blot analysis as previously
described (30). Hybridizations were carried out overnight at 42 C with
2 x 106 cpm/ml random primer
32P-labeled (31) complementary DNA (cDNA) probes for
T3R-ß1,
T3R-1/T3R-2, and a
cDNA for MBP kindly provided by Dr. Arthur Roach (Division of Biology,
California Institute of Technology). The hybridized blots were
autoradiographed using Kodak XAR-5 film (Eastman Kodak Co., Rochester,
NY). The optical density of the bands was quantitated by
computer-assisted video densitometry (32). Values were corrected for
variations in loading using ethidium bromide staining of the 18S and
28S ribosomal bands as described by Correa-Rotter et al.
(33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a light
photomicrograph of an O-2A progenitor with a small cell body and thin
bipolar processes. During differentiation into oligodendrocytes, the
processes multiply, thicken, and become extensively branched to form a
web-like halo (Fig. 1B). A representative photomicrograph of
T3R-ß1 immunofluorescence in a mixed culture of
O-2A cells and oligodendrocytes is shown in Fig. 1C. Whereas the O-2A
progenitor cells isolated from newborn rat pups show an absence of
signal for T3R-ß1 (Fig. 1C, arrowhead) or T3Rß2 (data not
shown), the mature oligodendrocyte in the same field shows strong
nuclear signals for T3R-ß1 (Fig. 1C, arrow). Similarly, evidence of
T3R-ß2 was only present in the mature cells. In
contrast, when stained with a T3R-1 or
T3R-2 antiserum, strong nuclear fluorescence
was observed in both O-2A cells and oligodendrocytes (data not shown).
We have previously reported evidence of the expression of all four
T3R isoforms in mature oligodendroglia in brain
(8).
We also detected a very strong signal for MBP in the soma and web-like
processes of the mature oligodendrocyte (Fig. 1D) that is absent in
O-2A cells. These data indicate that differentiation of the
oligodendrocyte cell and expression of MBP are associated with an
increase in T3R-ß1 protein. This is consistent
with our earlier study that showed a lack of
T3R-ß1 immunoreactivity in fetal brains, but
strong nuclear signals in adult brains (28).
Northern blot analysis (Table 1) of RNA isolated from
O-2A cells and oligodendrocytes revealed differences similar to those
observed for the T3R proteins.
T3R-1 mRNA was present in both O-2A cells and
mature oligodendrocytes, consistent with the presence of a nuclear
immunofluorescent signal at both stages of development. The
T3R-ß1 and MBP mRNA signals were weak or not
detectable in the O-2A cells, but present in oligodendrocytes (Table 1). To gain a better understanding of the relationship between 1 and
ß1 T3R and MBP expression, Northern blot
analysis was performed on RNA isolated at different times during
oligodendrocyte development (Fig. 2). The O-2A cells
(day 0) contain predominantly T3R-1 and 2
mRNAs. Addition of the oligodendrocyte differentiation medium
containing 30 nM T3 to O-2A cells
resulted in a dramatic rise in T3R-ß1 and a
subsequent increase in MBP expression. The levels of
T3R-ß1 and MBP mRNAs achieve a maximum between
days 6–8, and remained elevated. This rise in
T3R-ß1 in primary culture during
differentiation mirrors the increases seen in the developing neonatal
brain previously reported by our laboratory (30).
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, lanes 1 and 2)
and MBP expression (Fig. 3B, lanes 7 and 8) associated with
differentiation. Upon removal of the mitogens and incubation with
T3 for 4 days, the O-2A cells undergo normal
differentiation, with a concomitant rise in
T3R-ß1 (Fig. 3A, lanes 3–6) and MBP expression
(Fig. 3B, lanes 9–12), as previously observed in freshly isolated
cells (Fig. 2). The experiments that follow employed O-2A precursor
cells previously stored in liquid nitrogen and treated with the
mitogens, as described, before developmental studies of MBP gene
regulation by T3.
Regulation of MBP expression by thyroid hormone and developmental
factors during differentiation of oligodendrocytes
As T3 regulates MBP expression in the
developing brain (7) and influences oligodendrocyte differentiation in
primary cultures (34), we examined its effects on endogenous MBP gene
expression (Fig. 4A). In the presence of
T3, MBP mRNA levels are maximal by 4 days. In the
absence of T3, the rate of accumulation of the
MBP mRNA is reduced with maximal levels of the MBP mRNA not attained
until days 8–10. As in the animal model, the levels of mRNA ultimately
achieved are similar in the presence and absence of
T3 (7). Unlike MBP mRNA, however, the
accumulation rate of T3R-ß1 mRNA was unaffected
by the absence of T3 (Fig. 4B), again consistent
with the in vivo data.
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). The T3RE first
identified by Farsetti et al. (35) is contained within this
promoter at position -184/-167. Progenitor O-2A/oligodendrocyte cells
were transfected at various times during differentiation. Progenitor
cells transfected on the first day in culture show low levels of -1300
MBP-Luc expression that are not influenced by the presence or absence
of T3 in the medium. Only a modest 1.8-fold
increase in expression was noted in cells transfected after 3 days in
culture in the absence of T3. Addition of
T3 to the medium of cells transfected on day 3,
however, resulted in a 5.4-fold increase in transgene expression.
Transfection of cells grown in defined medium containing
T3 on day 6 produced little further increase in
luciferase activity above that seen on day 3 in the presence of
hormone. Of interest was the finding that on day 6, -1300 MBP-Luc
expression following transfection in the absence of
T3 was elevated to approximately the same extent
as in cells cultured in the presence of T3. The
rise in -1300 MBP-Luc expression in the presence and absence of
T3 during oligodendrocyte development is similar
to that of the endogenous MBP gene (Fig. 4A). These findings again
reflect the pattern of T3-dependent and
T3-independent regulation observed in the intact
animal and confirm the report of Farsetti et al. (35) that
regulation of MBP gene expression by T3 is at the
transcriptional level. Our findings, however, clearly show that this
transcriptional regulation of MBP by T3 is
restricted to a defined period in cellular differentiation.
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). Expression of the
mutant construct was not different from that of the wild type when
transfected on day 1, when endogenous MBP gene expression is very low.
In contrast to the 5-fold increase in expression of the wild-type
-1300 MBP-Luc transfected on day 3, expression of the mutant remained
at a low level. Of particular interest are the results of transfections
into mature oligodendrocytes (day 6) when MBP gene expression is
maximal and independent of T3 (Fig. 6). Mutation
of the T3RE completely abrogated the anticipated
T3-independent expression observed for the
wild-type construct shown in Fig. 5. The luciferase activity of the
mutant T3RE construct remained relatively
unchanged during the entire period of cellular differentiation. Thus,
although T3 is not required to maintain the
expression of the MBP gene in mature oligodendrocytes, the
T3RE sequence clearly plays a critical role in
the developmental phase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results clearly show a striking resemblance between the
developmental MBP gene expression pattern in rat brain in
vivo and that observed in the cultured oligodendrocytes. Although
the time span during which development occurs in the whole animal is
substantially longer than that required for comparable development in
the cultured cell, the relative sequence of events is nearly identical
in both models. These studies indicate that during differentiation of
the O-2A progenitor cells to mature oligodendrocytes there is a rise in
the level of the T3R-ß1 isoform associated with
the appearance of MBP. The appearance of T3R-ß1
and MBP proteins in oligodendrocytes is related to increased expression
of the T3R-ß1 and MBP genes, as demonstrated by
the increase in their mRNA levels (Fig. 2).
T3R-1 and T3R2 mRNA
and protein are present in both the O-2A cells and the oligodendrocytes
at high concentrations. These data accord with our previous studies of
neonatal rat brain development (30). T3R-ß1
mRNA in the brain is barely detectable before birth (embryonic day 19),
then rises 40-fold to maximal levels by postnatal day 10. The rise in
T3R-ß1 mRNA is followed by an increase in MBP
mRNA starting on approximately postnatal day 5, with maximal expression
on days 15–20. T3R-1 and -2 are abundant
in fetal brain and change only modestly during development (30).
Interestingly, the addition of mitogens that prevent the O-2A cells
from differentiating also block expression of the
T3R-ß1 and MBP genes. Upon removal of the
mitogens from the medium, the normal pattern of
T3R-ß1 and MBP expression associated with
oligodendrocyte differentiation is initiated. Thus, there seems to be a
tight association among expression of the
T3R-ß1 isoform, differentiation of
oligodendrocytes, and the regulation of MBP expression. Whether this
indicates a special role for the ß1 isoform in mediating these
T3-initiated developmental changes remains
unclear. The current studies provide additional evidence supporting our
previous inference that T3 does not regulate the
expression of the T3R-ß1 gene during neonatal
development (30). This situation stands in contrast with the reported
T3 dependency of T3R-ß1
gene expression during amphibian metamorphosis (36).
The ability to transfect oligodendrocytes at various times during differentiation in culture with the -1300 MBP-Luc construct allowed us to examine both developmental and T3-mediated changes in MBP gene expression. Transfection studies indicate that regulation of transcription of the MBP gene is only transiently sensitive to T3 during oligodendrocyte differentiation, a finding consistent with the transient effect of T3 on the level of MBP mRNA in vivo during neonatal development. The present findings together with the results of our earlier studies reinforce the concept that potential target genes initially resistant to the action of the hormone subsequently become sensitive to hormonal regulation and finally are actively expressed even in the absence of T3.
There are several potential explanations for the lack of
T3 responsiveness in O-2A cells. Transfection
studies in NIH-3T3 cells indicate the MBP T3RE in
the context of its native promoter is preferentially activated by the
T3R-ß1 isoform (35). If MBP gene expression in
developing oligodendrocytes is T3R-ß1 isoform
specific, then expression of the MBP gene is not inducible in O-2A
cells due to the absence of T3R-ß1. However,
cotransfection studies in our laboratory using Neuro-2A cells
(unpublished data) and by Farsetti et al. in NIH-3T3 or
NG1108-15 cells (9) indicate that T3R-1 is
capable of transactivating constructs containing the MBP
T3RE sequence. As O-2A progenitor cells contain
high levels of T3R-1 mRNA, we must also
consider the possibility that the MBP gene is not activated by
T3R-1 in the O-2A cells due to the lack of a
necessary positive transacting factor, perhaps a thyroid hormone
receptor accessory protein (TRAP), or the presence of a transcriptional
repressor protein that blocks the actions of the
T3R-1. The finding of Barres et al.
(37) that replicating O-2A cells will differentiate in the absence of
thyroid hormone accords with our observation that full expression of
the MBP gene also ultimately occurs in the absence of
T3. The addition of T3
hastens morphological maturation, on the one hand, and target gene
expression on the other. It appears probable from the studies of Barres
et al. (37) that the effect of T3 on
morphological differentiation of oligodendroglia is contingent on
preceding cellular changes during the proliferative process. They found
that these cells acquired sensitivity to the influence of
T3 only after they had undergone several cycles
of cellular proliferation. The same may be applicable to
T3-induced MBP gene expression. The nature of the
cellular signal(s) that allows thyroid hormone to act remains to be
defined.
The T3-dependent activation of MBP gene expression in cells transfected on day 3 is consistent with the identification of a T3RE in the MBP gene by Farsetti et al. (9) and with the established role of T3 in supporting neuronal myelination. We speculate that the burst of T3-dependent induction of MBP gene expression is made necessary to meet a concomitant increase in myelin requirements resulting from the formation of new neuronal networks. The inability to synthesize the requisite amount of MBP at a particular stage of neurogenesis can, therefore, be presumed to have adverse functional and structural consequences despite the fact that the cell may eventually acquire the normal content of MBP mRNA.
A major question in understanding thyroid hormone-dependent regulation of development is whether T3 acts directly on a set of specific genes containing cognate TREs or whether T3 serves to trigger the expression of a gene coding for a mediator capable of regulating a specific set of secondary target genes. The possibility that T3 acts both directly and indirectly to affect cellular differentiation should be considered.
The T3-independent phase of MBP expression (day 6) is characterized by high levels of MBP gene expression achieved in the absence of T3. T3-independent activation of MBP expression is also evident in the rat, as MBP mRNA levels normalize in hypothyroid animals by 45 days of age (7). The loss of T3 sensitivity and the acquisition of T3-independent regulation by the oligodendrocyte explains why hypothyroidism in an adult animal does not result in the expected fall in MBP mRNA levels.
Transfection of a mutated T3RE MBP construct into developing oligodendrocytes predictably resulted in loss of the anticipated T3-dependent induction of expression. Unexpectedly, however, this mutation also prevented the T3-independent expression of the MBP transgene. One potential mechanism to account for the requirement of the T3RE sequence for T3-independent expression of the MBP gene in mature oligodendrocytes is modification of T3R activity that would allow it to transactivate as a ligand-independent transcription factor. Studies have indicated that members of the steroid/thyroid/retinoic acid superfamily, including the T3Rs, can function as ligand-independent transcription factors after activation of the dopamine receptor-linked adenylate cyclase system (38). As phosphorylation of the T3R has been demonstrated in several laboratories (29, 39), it seems plausible that phosphorylation could serve as a modifier of T3R activity in the adult brain to allow ligand-independent function. Another potential explanation would involve the activation of a specific TRAP in the adult brain that upon dimerization with the T3R allows the T3R-TRAP dimer to induce transcription without need for a ligand. Lastly, it is possible that transcription of the MBP gene could be driven by a transacting factor other than T3R induced in mature oligodendrocyte. If so, one would have to postulate that the hypothetical transcription factor would be capable of recognizing and binding to DNA sequences contained at least partially within the T3RE sequence of the MBP gene. Additional studies are clearly warranted to resolve these issues.
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Footnotes |
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Received September 9, 1997.
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2 in selected tissues: cerebellar Purkinje
cells as a model for ß1 receptor mediated developmental effects of
thyroid hormone in brain. Proc Natl Acad Sci USA 88:3887–3891This article has been cited by other articles:
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