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Molecular Endocrinology Laboratory, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Alan P. Farwell, M.D., Division of Endocrinology and Metabolism, Department of Medicine, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655. E-mail: alan.farwell{at}banyan.ummed.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The formation of neural networks in the developing brain is accomplished by the migration of the neuronal growth cone down specific, preprogrammed pathways to its target region, followed by the projection of axons toward its target cell (11, 12, 13). Signals derived from the extracellular matrix (ECM) are essential to the guidance of the migrating neurite to its target cell (11, 12, 13, 14, 15). Of particular interest is the ECM protein laminin, which plays a key role in neuronal migration, synapse formation, and cell survival (11, 12, 14, 15).
Laminin is synthesized and secreted by astrocytes, both in vivo (16, 17, 18, 19, 20) and in vitro (21, 22, 23, 24, 25). Laminin is deposited into the ECM and fixed on the cell surface through binding to specific transmembrane receptors known as integrins (26, 27, 28). The regionalization of laminin on the astrocyte surface is determined by the clustering of integrins bound to the microfilaments into macromolecular complexes known as focal contacts (14, 29, 30). It is the organization of laminin into specific patterns on the cell surface that provides directional cues to the elongating neurite (11, 12, 15). Indeed, in vitro studies have shown that neurons readily and preferentially migrate onto laminin-coated surfaces (15, 31, 32, 33, 34).
The appearance of laminin in the brain paranchyma is developmentally regulated and coincides with neuronal migration (11, 13, 15, 17, 18). Once the wiring network of the brain is established, laminin disappears from the brain paranchyma and is restricted to the basal lamina of the vasculature. We have shown that laminin is differentially expressed in the euthyroid and hypothyroid rat cerebellum (35). In the euthyroid rat cerebellum, laminin steadily increased from birth to postnatal days 8–10 and was concentrated in the molecular layer, through which the granular neurons must migrate to complete the cerebellum neuronal circuitry. By postnatal day 14, the granular neurons had all reached the inner granular layer, and laminin was restricted to the vasculature in the euthyroid rat. In the hypothyroid rat, laminin did not appear in the molecular layer until postnatal day 10 and disappeared by postnatal day 18 despite the presence of granular neurons that had yet to complete their migration.
In this study, we examined possible mechanisms for the in vivo differential expression of laminin by determining the effect of thyroid hormone on the expression and extracellular distribution of laminin in astrocytes in vitro. We show that laminin is rapidly synthesized and secreted in astrocytes independent of the presence of iodothyronines. In T4-treated astrocytes, laminin is bound to the astrocyte surface after secretion and is organized on the cell surface in discrete linear arrays. In T4-deficient and T3-treated astrocytes, laminin is primarily released into the medium after secretion; the small amount that remains bound to the astrocyte surface does so in a diffuse, disorganized pattern. Thus, the T4-dependent regulation of the extracellular distribution of laminin on the astrocyte surface suggests a mechanism by which this morphogenic hormone can influence neuronal migration in the developing brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture conditions
Rat type I astrocyte cultures were obtained by enzymatic
dispersion of neonatal rat brains (36). Cells were grown in a
humidified atmosphere of 5% CO2 and 95% air in 37 C in
DMEM, 15 mM sodium bicarbonate, 33 mM glucose,
1 mM sodium pyruvate, and 15 mM HEPES (pH 7.4)
with 10% (vol/vol) defined bovine calf serum. Culture medium was
changed three times weekly, and cells were subcultured (2–3 x
104 cells/cm2) when they reached confluence
(7–10 days) (37). Confluent cells from passages 1–3 containing more
than 95% astrocytes were used for experiments after incubation with
defined medium containing DMEM, 15 mM sodium bicarbonate,
33 mM glucose, 1 mM sodium pyruvate, 15
mM HEPES (pH 7.4), and 0.1% (wt/vol) BSA in the presence
and absence of 10 nM iodothyronines.
Immunocytochemistry
Confluent cultures of rat astrocytes were grown for 16 h in
defined medium in the presence and absence of 10 nM
iodothyronines, which achieves a free hormone concentration of about 60
pM and results in the maximal T4-mediated
effect on regulated processes in astrocytes, including actin
polymerization (38, 39) and type II iodothyronine 5'-deiodinase
activity (39, 40, 41, 42). Cells were collected by trypsinization, a
monocellular suspension was made by filtration through a 20-µm pore
size mesh, and cells were seeded onto glass coverslips (22 x 22
mm) coated with poly-D-lysine (10 µg/ml), an ionic glue
that allows attachment of cells independent of cell surface receptors.
After 3–36 h, cells were fixed to the coverslips with 4%
paraformaldehyde. Total cell-associated laminin was visualized by
permeabilizing the cells with iced methanol. Extracellular/secreted
proteins were identified by restricting access of the antisera to the
interior of the cell by staining unpermeabilized cells. Nonspecific
binding sites were blocked by incubation with BSA blocker (2 mg/ml BSA
in PBS) for 30 min at room temperature. Cells were then incubated with
antilaminin IgG (1:500 dilution) in BSA blocker for 1 h, and
immune complexes were visualized by incubation with an antirabbit IgG
conjugated with Texas Red. A Carl Zeiss Axioskop
microscope (New York, NY) equipped with an Olympus Corp.
OM4 camera (New Hyde Park, NY) and Kodak TMAX ASA 400 film
(Eastman Kodak Co., Rochester, NY) was used for image
acquisition. Coverslips were initially examined in a blinded fashion to
ensure unbiased evaluation of the immunocytochemistry results.
Western analysis: secretion of laminin into the medium
Confluent cultures of rat astrocytes were grown for 16 h in
defined medium in the presence and absence of 10 nM
iodothyronines and collected by trypsinization, and a monocellular
suspension was made by filtration through a 20-µm pore size mesh.
Cells were seeded out onto six-well tissue culture plates (
2 x
105 cells/well). Medium was collected from 3–24 h after
seeding. Secreted proteins in the medium were denatured in a boiling
water bath for 5 min, then applied to nitrocellulose via slot blot
under vacuum. Blots were blocked for 1 h with milk blocker [20
mM Tris-HCl, 0.1% (vol/vol) Tween-20, 5 g/ml powdered
milk, and 137 mM NaCl, pH 7.5], then probed for 1 h
at room temperature with antilaminin IgG (1:1000 dilution) in milk
blocker. Immune complexes were visualized by incubation with an
antirabbit IgG conjugated to horseradish peroxidase (1:2500 in blocker)
and developed with the Lumiglo chemiluminescent kit. Blots were
analyzed by scanning densitometry, and laminin was quantified by
comparison with results obtained with a standard curve that was run on
each blot.
Enzyme-linked immunosorbent assay (ELISA) analysis: deposition of
laminin into the ECM
Confluent cultures of rat astrocytes were grown for 16 h in
defined medium in the presence and absence of 10 nM
iodothyronines and collected by trypsinization, and a monocellular
suspension was made by filtration through a 20-µm pore size mesh.
Wells in 96-well ELISA plates were coated with
poly-D-lysine (10 µg/ml). Astrocytes were seeded into the
wells (1 x 106 cells/well) and grown for 24 or
36 h. Medium was then aspirated, and cells were burst by
incubation with distilled water and washed with PBS with 0.5%
(vol/vol) Tween-20. As shown by others, the remaining proteins attached
to the wells after hypotonic disruption represent the astrocyte-derived
ECM (43). Nonspecific binding sites were blocked by incubation with BSA
blocker (2 mg/ml BSA in PBS) for 1 h at room temperature
followed by incubation with antilaminin IgG (1:100 dilution) in BSA
blocker for 1 h. Controls included astrocyte proteins incubated
with either no antibody or only the secondary antibody, and wells were
coated for 2 h at room temperature with either BSA (10 µg/ml;
negative control) or EHS laminin (10 µg/ml; positive control). Immune
complexes were visualized by incubation with an antirabbit IgG
conjugated to horseradish peroxidase and developed with TMB solution.
Absorbance was read with an ELISA plate reader at 650 nm.
Metabolic labeling and immunoprecipitation
Analysis of the effects of iodothyronines on the production of
laminin by astrocytes was performed by incubating confluent cultures of
rat astrocytes with defined medium in the presence and absence of 10
nM iodothyronines for 16 h. Cells were collected by
trypsinization; resuspended in labeling medium containing the
35S label EZ-Tag (NEN Life Science Products-DuPont, Boston, MA), methionine-free DMEM, 0.1%
(wt/vol) BSA, and 10 nM iodothyronine; seeded onto tissue
culture flasks coated with poly-D-lysine (10 µg/ml); and
incubated for 6 h at 37 C. The medium, containing secreted
proteins, was collected. Cells were solubilized, and proteins deposited
into the ECM were removed from the flasks by incubation with 1%
(wt/vol) SDS, 10% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol,
and 62.5 mM Tris (pH 6.8) for 1 h at room temperature
(43). Proteins from the medium, cells, and ECM were combined and
further solubilized by the addition of 1% (vol/vol) Nonidet P-40, 0.4
M NaCl, 2 mM EDTA, 2 mM
phenylmethylsulfonylfluoride, and 50 mM Tris (pH 8) and
clarified. Solubilized proteins were incubated with antilaminin IgG
(1:400 dilution) in the presence and absence of 50 µg EHS mouse
sarcoma laminin for 2 h, and immune complexes were isolated with
immobilized rProtein A beads (Repligen) followed by centrifugation.
Isolated proteins were either counted or resolved on a 5% SDS-PAGE
slab gel, and labeled proteins were detected by autoradiography.
Specific laminin immunoprecipitation was determined by subtraction of
35S-labeled proteins immunoprecipitated by antilaminin IgG
preincubated with 50 µg EHS mouse sarcoma laminin (nonspecific
immunoprecipitation). Resolved proteins were also transferred to
nitrocellulose for 2 h at 20 mA and analyzed by Western blot with
antilaminin IgG as described above.
Statistical methods
Results are reported as the mean ± SE.
Statistical analysis was performed using single factor ANOVA.
Statistical significance was determined to be achieved at the
P < 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In permeabilized cells (Fig. 1
), which
identify both intra- and extracellular proteins, specific staining was
detected in the perinuclear space of astrocytes within 3 h of
attachment in all treatment groups. These observations show that
laminin is synthesized in all astrocytes attached to
poly-D-lysine. By 10 h, immunoreactive laminin was
diffusely distributed throughout the cell in all treatment groups. By
36 h, there appeared to be organization of staining into linear
arrays (arrow) in the T4-treated cells along
with continued staining in the perinuclear space. In contrast, no
linear arrays were observed in any of the thyroid hormone-deficient or
T3-treated cells. Instead, punctate clusters of staining
were present in both the thyroid hormone-deficient and
T3-treated cells at 36 h (arrows).
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is intracellular and which is extracellular using
routine immunofuorescence microscopy. To determine the effect of
thyroid hormone on the extracellular distribution of laminin,
immunocytochemistry was performed on nonpermeabilized cells to prevent
access of the antilaminin IgG to the intracellular compartment. There
was no detectable staining on the unpermeabilized cells at 3 h in
any of the treatment groups (Fig. 2),
confirming that the laminin IgG was indeed restricted from the
intracellular compartment in the nonpermeabilized cells and indicating
that the trypsin treatment removed any laminin attached to the
astrocytes before cell collection. Further, these data show that all of
the staining present in the permeabilized cells at this time point was
intracellular (Fig. 1). By 10 h, there was detectable staining on
the T4-treated cells in punctate clusters and in small
linear arrays (Fig. 2, arrows). In contrast, there was no
staining present on the thyroid hormone-deficient cells, and scant
punctate staining was present on the T3-treated cells at
10 h. By 24 h, there was an extensive meshwork of staining on
the T4-treated cells that was well organized into intense
linear arrays by 36 h. In the thyroid hormone-deficient cells,
there continues to be little detectable staining until 36 h, when
staining is present in a few punctate clusters (arrows). A
pattern similar to that for the thyroid hormone-deficient cells was
observed in the T3-treated cells at both 24 and 36 h,
with no linear arrays observed. These data indicate that astrocytes
organize laminin into specific linear patterns on the cell surface in
the presence of T4 but not in the presence of
T3 or in the absence of hormone.
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). Neither T4 or
T3 had any effect on astrocyte proliferation over the
ensuing 24 h (Fig. 3).
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is the effect of thyroid
hormone on the secretion of laminin into the medium by astrocytes.
Laminin was detected in the medium within 3 h of seeding onto the
tissue culture plate in all conditions. In the absence of thyroid
hormone or in the presence of T3, the amount of laminin
secreted into the medium increased linearly up to 24 h. In
contrast, the quantity of laminin secreted into the medium in the
presence of T4 was significantly less than that in the
absence of thyroid hormone or in the presence of T3 from
3–16 h after seeding. From 16–24 h, the quantity of laminin secreted
into the medium increased in all three treatment groups.
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is the effect of iodothyonines
on laminin deposition into the ECM. By 24 h, laminin had been
deposited by astrocytes into the ECM in all conditions. The laminin
content in the ECM of the T4-treated cells was
significantly greater than that observed in the absence of hormone or
in the presence of T3, although the differences were small
(10%). Over the next 12 h, laminin content in the ECM of the
T4-treated cells nearly doubled, whereas no significant
change was observed in the absence of hormone or in the presence of
T3. There was no significant difference between the amount
of laminin secreted into the ECM in the presence of T3 or
in the absence of hormone at either 24 or 36 h. These data
correlate with the immunocytochemical observations (Fig. 2), which show
the organization of laminin into intense linear arrays on the
T4-treated astrocyte by 36 h and indicate that
T4 increases the deposition of laminin into the ECM,
presumably by increasing the binding of this ECM protein to the cell
surface.
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and 5. However, the
immunocytochemical data in the permeabilized cells (Fig. 1) suggests
that the synthesis of laminin by astrocytes after cell attachment is
not altered by thyroid hormone. To clarify this and to determine
whether the altered release of laminin into the medium and deposition
onto the cell surface were due to altered production of this ECM
protein, we examined laminin protein expression by metabolically
labeling the cells after attachment to poly-D-lysine-coated
tissue culture flasks. Confluent astrocytes were incubated in defined
medium containing 10 nM T4, 10 nM
T3, or no hormone overnight, then collected, resuspended in
labeling medium, seeded onto poly-D-lysine-coated tissue
culture flasks, and grown for 6 h. Cells were solubilized and
combined with the medium and then spun to remove the insoluble
organelles. The supernatants were immunoprecipitated with an
antilaminin IgG and analyzed by SDS-PAGE. Preliminary studies indicated
that immunoprecipitation of EHS mouse sarcoma laminin standard
increased linearly from dilutions of 1:1000–1:500 and then plateaued
from dilutions of 1:500–1:100, indicating that excess antibody was
achieved at dilutions lower than 1:500 (data not shown).
Laminin consists of three chains: an 
(or A) chain of approximately
400 kDa and ß (or B1) and 
(or B2) chains of about 200 kDa each
(30, 45). Shown in Fig. 6 are the
35S-labeled proteins immunoprecipitated with the
antilaminin IgG (lane 1). The predominant signal is a band of proteins
of about 200 kDa. Immunoprecipitation of these labeled proteins was
completely blocked by preincubation of the antilaminin IgG with 50 µg
EHS mouse sarcoma laminin (Fig. 6, lane 2). Western analysis of the EHS
mouse sarcoma laminin (Fig. 6, lane 3) and the astrocyte proteins (Fig. 6, lane 4) immunoprecipitated with the antilaminin IgG showed a signal
at about 200 kDa (Fig. 6, lanes 3 and 4, arrow) that
corresponded to the ß- and -chains of laminin (22). The higher
molecular mass signal (400 kDa) detected in the EHS mouse sarcoma
laminin (Fig. 6, lane 3) and corresponding to the -chain (22) was
absent in the astrocytes (lane 4), consistent with the previous
observations that astrocytes do not produce the laminin -chain
(22, 23, 24, 46).
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. There was no significant difference in
the amount of laminin produced by astrocytes treated with
T4, T3, or no hormone. These data indicate that
attachment-stimulated synthesis of laminin by astrocytes is not
affected by thyroid hormone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(B2) chain of laminin inhibits neuronal
migration in the neonatal rat cerebellum (34) and in cerebellar explant
cultures (33) and blocks the elongation of retinal ganglion axons (19).
Thus, the absence of laminin-derived guidance cues results in
disordered neuronal pathfinding in vitro and in
vivo and would probably result in increased neuronal death caused
by neurites failing to reach their target destinations in the
developing brain, much like that observed in the cretinous brain. In this study, we show that thyroid hormone, specifically T4, regulates the ability of the cell to deposit and orient the ECM protein laminin into specific patterns on the surface of astrocytes. In T4-treated astrocytes, laminin is secreted and deposited in linear arrays upon the cell surface. In the absence of T4, what little laminin that is deposited on the cell surface is restricted to disorganized punctate clusters, and the remaining laminin secreted by the astrocyte is released into the medium. Neither the synthesis nor the secretion of laminin appears to be affected by thyroid hormone; thus, regulation of the extracellular distribution of laminin on astrocytes does not appear to be a transcriptionally mediated event. These data suggest that the ability of the astrocyte to provide laminin-derived cues to migrating neurites is markedly impaired in the absence of T4. These data taken together with the observation that the expression and regional distribution of laminin are delayed and diminished in the hypothyroid rat cerebellum (35) represent the first demonstration of a mechanism of action for thyroid hormone that can explain many of the morphological derangements observed in the cretinous brain.
The T4-dependent differential patterning of laminin on the astrocyte surface most likely results indirectly from the T4-dependent regulation of cytoskeletal-integrin interactions within the cell. The deposition of laminin on the astrocyte surface requires interactions between the laminin-binding transmembrane receptors known as integrins (26, 27, 28) and the filamentous actin microfilament network within the cell that allow clustering of integrins upon binding to laminin, forming strong focal contacts (14, 30). We have previously shown that T4 and its metabolite, rT3, dynamically alter microfilament organization in cultured astrocytes by regulating actin polymerization via a novel extranuclear mechanism of action (38, 42). The transcriptionally active thyroid hormone, T3, is approximately 100-fold less potent in promoting actin polymerization in astrocytes. The T4-dependent regulation of microfilament organization in the astrocyte, in turn, modulates the ability of integrins to cluster into focal contacts upon binding to laminin (44). The regionalization of integrins on the astrocyte surface is diffuse in the absence of thyroid hormone, whereas prominent focal contacts are observed in the T4-treated astrocyte (44).
Shown in Fig. 8 is a proposed mechanism
by which T4 could regulate the extracellular orientation of
laminin on the astrocyte surface. In the presence of T4,
laminin is fixed on the cell surface through binding to integrins,
which cluster into macromolecular complexes known as focal contacts
(Fig. 8, arrow) that are organized in a specific pattern
(14, 29, 30). The integrins are bound to the microfilaments within the
cell, which provide the locomotive force required for integrin
clustering. Indeed, the distribution of laminin on the astrocyte
surface of the T4-treated cell (Fig. 2, 24 and 36 h)
coincides with the distribution of integrins on the surface of the
T4-treated astrocyte attached to laminin (44). In contrast,
the microfilaments are disorganized in the T4-deficient
astrocyte (38) and are unable to allow integrins to cluster upon
binding to laminin (Fig. 8) (44). The absence of laminin deposited on
the surface of the thyroid hormone-deficient or T3-treated
cells (Fig. 2) correlates with the absence of integrins clustered into
focal contacts on the surface of the thyroid hormone-deficient
astrocyte attached to laminin (44). Thus, the inability of the
integrins to cluster into focal contacts after binding to laminin
prevents the T4-deficient cell from holding onto secreted
laminin and from forming linear patterns of laminin on the cell
surface. Although the T4-dependent regulation of
integrin-cytoskeletal interactions is a dynamic process, the regulation
of laminin distribution and organization on the cell surface occurs
more slowly, as the synthesis and secretion of laminin are the
rate-limiting events and are not affected by either T4 or
T3.
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The T4-dependent regulation of the organization of the
microfilaments in the astrocyte is a well characterized extranuclear
action of this morphogenic hormone (38). T4 dynamically
regulates the organization of the microfilaments via a mechanism that
requires neither transcription nor translation (38). Indeed, astrocytes
lack significant numbers of functional thyroid hormone receptors (TR)
(49, 50), and the transcriptionally active thyroid hormone,
T3, has little if any effect on microfilament organization.
The predominant (>95%) TR isoform in astrocyte cultures is the
non-T3-binding isoform c-erbA2 (49), which has been shown
to exhibit dominant negative activity in the presence of authentic
T3-binding receptors (51, 52, 53), although the degree of
dominant negative activity by c-erbA2 has been questioned
(54). However, only small quantities of TR1 (49) and TRß2 (55)
have been identified in cultured astrocytes and, even if the dominant
negative activity of c-erbA2 is weak, the approximately
100-fold excess of this isoform in astrocytes (49) is likely to render
the T3-binding TR isoforms transcriptionally inert. These
studies suggest that actions of thyroid hormone in astrocytes are not
mediated by TR (50).
If the T4-dependent regulation of laminin distribution plays an essential role in modulating brain development, the administration of T3 alone to a congenitally hypothyroid neonate would fail to restore normal brain development. While many T3-regulated genes have been reported (for review, see Refs. 56, 57), the data on the effects of T3 replacement alone on brain development are scarce. T4 is the iodothyronine of choice for thyroid hormone replacement in both animal and human studies. When reported, T3 is primarily used to make the animals thyrotoxic (58, 59, 60). Studies are ongoing in our laboratory to examine potential differential roles of T4 and T3 in brain development.
Consistent with previous results (22, 23, 24, 46), we found that rat
astrocytes produce a variant laminin that lacks the (A)-chain
present in basement membrane laminin in other tissues (Fig. 6). The
approximately 200-kDa laminin chains are rapidly synthesized and
secreted by astrocytes after the cells attach to
poly-D-lysine. The signaling mechanism that induces laminin
transcription after cell attachment is unknown at present. It is clear
that thyroid hormone does not alter this signal transduction pathway,
as production of the approximately 200-kDa laminin chains is equivalent
in the presence and absence of iodothyronines.
In summary, we have shown that T4 regulates the deposition and orientation of laminin on the surface of astrocytes. The T4-dependent regulation of the distribution of laminin on the surface of astrocytes during development provides a mechanism by which this morphogenic hormone can influence neuronal migration and development.
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Footnotes |
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Received May 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Adrenergic
receptor modulation of ß-adrenergic, adenosine and prostaglandin
E1 increased adenosine 3':5'-cyclic monophosphate levels in
primary cultures of glia. J Cyclic Nucleotide Res 4:15–26[Medline]
2) is due to
competition for binding sites on target genes. J Biol Chem 268:20904–209102 does not require binding to a thyroid
hormone response element. Mol Endocrinol 9:86–952. Role of the ninth heptad in dna
binding, heterodimerization with retinoid X receptors, and dominant
negative activity. J Biol Chem 271:28235–28242This article has been cited by other articles:
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