IGF-I Causes an Ultrasensitive Reduction in GH mRNA Levels via an Extracellular Mechanism Involving IGF Binding Proteins
Ty C. Voss1,
Maxfield P. Flynn and
David L. Hurley
Molecular and Cellular Biology Program (T.C.V., D.L.H.), Department
of Cell and Molecular Biology (M.P.F., D.L.H.), Tulane University, New
Orleans, Louisiana 70118
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ABSTRACT
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IGF-I-dependent decreases in endogenous GH mRNA expression were
studied in individual rat MtT/S somatotroph cells using in
situ hybridization. It was first shown that increasing
IGF-I concentrations (090 nM) decreased GH mRNA levels in
a ultrasensitive manner when averaged over the entire population, such
that the decrease occurred over a narrow range of IGF-I concentration
with an EC50 of 7.1 nM. The degree of
ultrasensitivity of the population average was expressed by calculating
the Hill coefficient (nA), which had a value of -2.0. GH mRNA levels
in individual dispersed cells from these cultures were then measured.
These results were first summed for all cells to show that the average
response of the population remained ultrasensitive (nA = -2.6,
EC50 = 8.1 nM). Then, parameters for
individual cells of the population were calculated using mathematical
modeling of the distribution of individual cell GH mRNA levels after
treatment with 090 nM IGF-I. Solution of the data from
the individual cells yielded a Hill coefficient (nI = -0.65) and
a heterogeneity coefficient (mI = -1.2) indicative of individual
cell responsiveness to IGF-I that was not ultrasensitive and very
heterogeneous. These results suggested that ultrasensitivity in the
population may likely be caused by an extracellular mechanism
regulating IGF-I concentrations, such as IGF binding proteins.
Increasing concentrations of long (Arg)3IGF-1, an
analog that binds the IGF type-1 receptor but not IGF binding proteins,
showed a linear inhibition of GH mRNA levels. Treatment with IGF
binding protein ligand inhibitor, an IGF-I analog that binds to IGF
binding proteins but not the IGF type-1 receptor, decreased GH mRNA
levels in the absence of exogenous IGF-I. Thus, IGF binding proteins
provide the extracellular sequestration of IGF-I necessary for the
precise and ultrasensitive regulation of GH mRNA levels in the entire
cell population, although expression within individual cells is
regulated in a graded fashion.
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INTRODUCTION
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REGULATION OF GH expression has been
extensively studied to understand the mechanisms that maintain normal
homeostatic control (1). GH is produced exclusively in
somatotrophs as the result of a developmental pattern of transcription
factor expression controlling cell type differentiation (2, 3). In the adult pituitary, GH transcription is controlled by
both positive and negative actions of hypophysiotropic factors as well
as feedback circuits from target tissues (4). IGF-I is
induced in the circulation by GH to mediate effects on peripheral
tissues and also serves as a negative feedback inhibitor of GH
production from the pituitary (5, 6). Inhibition by IGF-I
occurs through a reduction of the GH transcription rate, resulting in a
decrease of GH mRNA levels in primary pituitary cultures and
GH-producing cell lines (7, 8).
Rat MtT/S cells (9) have proved to be a useful cell
culture model of the somatotroph because they express the GHRH receptor
(10, 11) and regulate expression of the endogenous GH gene
by IGF-I and insulin (12, 13). The cellular signals
causing the decline in GH expression in MtT/S cells primarily involve
the PI-3 kinase pathway in transfected cells (13), but not
mechanisms involving transcription factor Pit-1 or MEK kinase
(12, 13). The decreased levels of GH mRNA in MtT/S cells
in response to IGF-I through IGF receptors (IGF-R) displayed
ultrasensitive or switch-like kinetics, with the reduction occurring
over a narrow range of IGF-I concentrations (12). Such a
switch-like response may be important in the physiological control of
GH. For example, an ultrasensitive response to IGF-I might result in
limited reduction of GH expression at low IGF-I concentrations, while
slightly higher concentrations cause dramatic inhibition. The
ultrasensitive response could arise by a variety of mechanisms but
appears to be specific to IGF-I because insulin inhibits GH mRNA
expression in a linear, nonultrasensitive manner (12).
The analysis of ultrasensitive cellular responses can be modeled after
the findings in single Xenopus oocytes (14).
Some of the mechanisms generating a switch-like response would have
ultrasensitive effects on the GH mRNA expression levels within each
individual cell in the population. Therefore, elucidating the mechanism
of ultrasensitive GH expression requires analysis of the kinetics of
IGF-I effects on GH mRNA in individual MtT/S somatotrophs. Although
individual endocrine cells have been studied to assess secretory
control (15) and expression of transfected genes
(16, 17, 18), regulation of endogenous GH mRNA expression
among individual somatotrophs has not been quantitatively assessed. Rat
MtT/S cells were derived clonally from an estrogen-induced pituitary
tumor (9), but it is unknown whether the population of
MtT/S cells is homogeneous in regard to GH mRNA level per cell. In this
study, therefore, the expression of GH mRNA of individual MtT/S cells
was measured using in situ hybridization, after which
mathematical analysis was used to determine responsiveness of the
individual cells in the population to IGF-I treatment. Further, GH
expression per cell was analyzed after treatments with proteins that
alter IGF binding protein activity. The results indicate that the
ultrasensitive regulation of GH mRNA is not established within the
individual MtT/S cells, but by the binding of IGF to its binding
proteins (19).
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RESULTS
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Analysis of GH mRNA Expression in Individual MtT/S Cells
To analyze GH expression in individual MtT/S cells, in
situ hybridization (ISH) to detect GH mRNA was performed on the
dispersed cells after culture (20). Hybridization
intensities were visualized by emulsion autoradiography using darkfield
microscopy. Figure 1
shows ISH signals
over individual MtT/S cells from cultures grown for 5 d with
complete medium containing serum (CM), or treated for 5 d with 90
nM IGF-I in CM. GH gene expression in MtT/S cells
declined after IGF-I addition with first-order kinetics determined
previously (12) with a measured half-life of 50 h, in
agreement with previously determined values in GC cells
(21). In the control culture, many cells expressed high
levels of GH mRNA as indicated by the high density of silver grains
over individual cells; however, some cells displayed reduced levels of
GH mRNA (Fig. 1A
). There appeared to be a reduction of GH mRNA per cell
detected using ISH after exposure to IGF-I (Fig. 1B
). In the presence
of IGF-I, many cells were identifiable only by eosin counterstain;
however, some individual cells retained higher levels of GH mRNA
expression.

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Figure 1. Darkfield Photomicrographs of Individual MtT/S
Cell GH mRNA Expression Assayed by ISH
Cultures were treated with CM with serum (panel A), or with 90
nM IGF-I (panel B), and then ISH was performed as described
on the dispersed cells. Reduced silver grains (bright
dots under darkfield illumination) in the emulsion indicate the
intensity of the hybridization of radiolabeled antisense GH riboprobe,
present over cell bodies (gray shapes) counterstained
with eosin.
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IGF-I Causes Switch-Like Suppression of Average GH mRNA
Expression
To quantify the expression of GH mRNA per cell in these cultures,
grain counts were performed on the cells after ISH. GH mRNA levels in
the MtT/S cells decreased after incubation for 5 d with increasing
concentrations of IGF-I (Fig. 2A
). The
decline was not linear; treatment with 1.65 nM IGF-I had a
slight effect on the GH mRNA levels, while a maximal effect was
observed after treatment with 22.5 nM IGF-I. This result
suggested that the response to IGF-I was ultrasensitive or switch-like
in nature. These results were from MtT/S cells grown on
poly-L-lysine-coated culture flasks to enhance resolution
during image analysis after ISH assays, which may account for the
slightly different hypersensitive response to IGF-I compared with that
previously reported in cells maintained in untreated cultureware
(12). From these GH mRNA ISH data, the Hill coefficient
(n), a measure of ultrasensitivity (22), was calculated to
be -2.7 (Fig. 2B
). The calculated EC50 for the
decline was found to be 8.1 nM. For comparison to previous
results (12), GH mRNA levels in an aliquot of cells used
for ISH were measured by ribonuclease protection assay (RPA). GH mRNA
levels in 1.5 µg of total MtT/S cell RNA quantified by RPA resulted
in an EC50 of 7.1, and n = -2.0, comparable
to the values from the ISH determination (Fig. 2B
).
Individual MtT/S Cell Response to IGF-I Is Heterogeneous and
Non-Switch-Like
The data collected from ISH for GH mRNA in the individual cells
were analyzed to determine the percentage of cells in the culture
vs. level of GH mRNA expression when treated with
concentrations of IGF-I between 0 and 90 nM (Fig. 3
). In the presence of serum but no IGF-I
(Fig. 3A
), the distribution of GH mRNA levels (on the x-axis of each
graph) and the percentage of MtT/S cells expressing (on the y-axis) are
very broad. There are approximately equal numbers of cells at each
expression level from maximal levels to 40% of maximum. This pattern
of cellular GH mRNA distribution was not altered by incubation with
1.65 nM or 4.95 nM IGF-I
(Fig. 3
, B and C). However, there was a change in the distribution of
expression after treatment with 9.9 nM IGF-I
(Fig. 3D
). The number of cells expressing high levels of GH mRNA (100%
to 60% of maximum) was reduced, and there was an increase in the
percentage of cells with low levels of GH mRNA (30% to 0% of
maximum). Analysis of MtT/S cells after treatment with 22.5, 45, or 90
nM IGF-I showed a further decrease in the number
of high GH expressing cells (Fig. 3
, EG). However, even after
treatment with as much as 90 nM IGF-I, some of
the cells in the population expressed GH mRNA at up to 50% of the
maximum (Fig. 3G
).

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Figure 3. Distribution of GH mRNA Expression among Individual
Cells Treated with Increasing Concentrations of IGF-I
GH mRNA levels in individual cells were measured by ISH after
treatment with increasing concentrations of IGF-I in the presence of
serum. The experimentally determined fraction of individual cells
expressing GH mRNA at a given level is shown in histogram form at the
indicated IGF-I concentration (AG). Results are the mean of
measurements from three cultures with error bars
denoting SEM. Histogram data were combined and used to
determine the best fit parameters for the inflection point, Hill, and
homogeneity coefficients for the response (H). The best-fit parameters
were then used to calculate the distribution of the response (I).
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Histogram data for the distribution of GH mRNA expression in the
individual cells were combined for all hormone concentrations tested
(Fig. 3H
) for further mathematical analysis to determine three values
needed to assess the ultrasensitivity of the response
(14). One value is the Hill coefficient for the individual
cells of the population (nI). Another value is the inflection point
(aI), which describes the point at which half-maximal response is
achieved in 50% of the cells, and the third is homogeneity coefficient
(mI), which describes the uniformity of the response of the population
to treatment. From interpolative mathematical analysis of the data in
Fig. 3H
, all three values were calculated. The value of aI was 4.5
nM, in good agreement with the EC50
measured previously (Fig. 2
). The Hill coefficient, nI, was -0.65; an
absolute value less than 1 indicates that ultrasensitivity is not
present. The homogeneity coefficient, mI, was determined to be -1.21;
this low absolute value indicates that the individual cells are not
responding uniformly (14).
Using these values that quantitatively describe the kinetics of
increasing IGF-I concentrations on GH mRNA levels in individual cells
in the population, the theoretical distribution of GH mRNA levels among
individual cells was calculated (Fig. 3I
). Confirming the validity of
the interpolated values, the calculated distribution of cellular GH
mRNA expression had the same broad distribution of intermediate GH mRNA
levels that was observed in the experimental data (compare Fig. 3
, H
and I).
IGF-I Analogs That Block Binding to IGF Binding Protein (IGFBP)
Prevent Ultrasensitive Responsiveness
Because the values of mI and nI suggest that the individual cells
do not display an ultrasensitive response, in contrast to the
switch-like response measured in the entire population, an
extracellular mechanism regulating the behavior was indicated.
Responsiveness to IGF-I has been shown to be modulated by the IGFBPs,
which prevent IGF-R activation by binding to free IGF-I molecules.
Several protein analogs have been produced that alter cellular
activation by IGF, and these were added to MtT/S cell cultures to
determine the effects on the ultrasensitive GH mRNA response to IGF-I
treatment.
Long (Arg3)IGF-I (LR3IGF-1)
is an IGF-I analog that binds to the IGF-R but not the IGFBPs
(23, 24). Quantification of the average endogenous GH mRNA
level was performed by RPA on MtT/S cultures to which increasing
concentrations of LR3IGF-1 or IGF-I were added
(Fig. 4
). Analysis by one-factor ANOVA
showed that the amount of GH mRNA in MtT/S cells treated with
increasing concentrations of LR3IGF-1 was
significantly reduced (F7, 16 = 13.4,
P = 0.0001; Fig. 4A
). Treatment with as little as 0.004
nM LR3IGF-1 significantly
reduced GH mRNA levels vs. controls (P <
0.05), and 2.6 nM LR3IGF-1
maximally reduced GH mRNA levels. As shown in Fig. 4B
, Hill plot
analysis indicated that the response is nonultrasensitive (nA =
-0.6), and that the EC50 for
LR3IGF-1 is 0.07 nM.
IGFBP ligand inhibitor (IGFBP-LI) is an IGF-I analog that increases the
concentration of free IGF-I by binding with high affinity to IGFBPs
(25). Therefore, MtT/S GH mRNA expression was analyzed by
RPA after IGF-I treatment in the presence of 1 µM
IGFBP-LI (Fig. 5
). Analysis by two-factor
ANOVA (IGFBP-LI concentration vs. IGF-I concentration)
indicated that IGFBP-LI and IGF-I concentrations interacted
significantly to change GH mRNA levels (F5, 32 =
24.9, P = 0.001; Fig. 5A
). Treatment with 1
µM IGFBP-LI caused a significant reduction of
GH mRNA levels vs. treatment without IGFBP-LI
(P < 0.05). Addition of increasing concentrations of
IGF-I in the presence of IGFBP-LI did not cause further reductions of
GH mRNA levels vs. IGFBP-LI alone. GH mRNA levels were not
significantly different in cultures treated with maximal concentrations
of IGF-I alone vs. cultures treated with IGFBP-LI
supplemented with increasing concentrations of IGF-I. Because the
results yielded a horizontal line, it was not possible to calculate an
nA or EC50 for IGFBP-LI.

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Figure 5. Effects of an IGF-I Analog That Binds to IGFBPs but
Not the IGF-R on GH mRNA Expression
Cells were incubated for 5 d in CM supplemented with the indicated
concentrations of IGF-I supplemented with 1 µM IGFBP-LI
( ) or IGF-I alone ( ). GH mRNA levels were measured in by f-RPA.
Results are the mean of measurements from three cultures and are
expressed as a percentage of the control with error bars
denoting SEM.
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DISCUSSION
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MtT/S Somatotrophs as a Model System for Ultrasensitive
Responsiveness
Rat MtT/S cells provide a model for analysis of somatotrophs
because they express high levels of GH but no detectable PRL
(9). These cells uniquely produce GHRH receptor mRNA, as
suggested by the stimulation of GH secretion by GHRH (9)
and confirmed by studies of the expression and regulation of GHRH
receptor mRNA (10, 11). IGF-I treatment of MtT/S cells has
been shown previously to inhibit expression from transfected constructs
under the control of the GH promoter (13). Subsequent
studies of endogenous GH gene expression revealed that this inhibition
is ultrasensitive, changing rapidly over a small range of IGF-I
concentrations. The switch-like responsiveness is specific for IGF-I,
because increasing insulin concentrations produced a linear reduction
in GH mRNA levels (12). To further explore the mechanism
of this ultrasensitive response, GH mRNA levels were measured in
individual cells by ISH. The use of ISH was validated by summing
individual measurements over the entire population and performing
calculations from this summation to compare with RPA measurements made
on the same cell population. The good agreement of RPA and ISH results
(Fig. 2
) provides evidence that ISH of individual cells provides an
accurate assessment of endogenous GH mRNA expression. Thus,
ultrasensitive responsiveness of MtT/S cells to IGF-I has now been
demonstrated by two different assays, validating the further use of the
ISH data to examine the mechanism of the ultrasensitive response.
Modeling Ultrasensitive Response Mechanisms
There are several possible explanations for the ultrasensitive
regulation of GH mRNA levels by IGF-I in MtT/S cells. An ultrasensitive
response could result from intracellular mechanisms that regulate the
formation of the GH transcriptional complex (26), that
control signal transduction pathways, or that inhibit GH expression by
some other mechanism (27). In particular, it has been
suggested that the transcriptional complexes assembled on a number of
genes are activated in a switch-like or all-or-nothing fashion (for
review, see Ref. 28). Thus, according to this
probabilistic model, if a gene is expressed in a cell, it is expressed
at the maximal level or not at all. Transcriptional complexes are thus
typically present in either on or off states, but only transiently in
the partially activated state (29). This switch-like
behavior may be due to stochastic accumulation of transcription factors
in the nucleus or the synergistic interaction of transcription factors
activating the gene (30, 31, 32). All of these switch-like
mechanisms may be extremely pertinent to terminal cell differentiation
(28), as exemplified by the MtT/S somatotrophs. Because
the activity of many transcription factors is regulated by cellular
transduction pathways that mediate extracellular hormonal signals,
these pathways could be the cause of switch-like or ultrasensitive
responses to increasing concentrations of hormones (for review, see
33).
To understand the mechanism(s) controlling cellular ultrasensitive
response, a mathematical model has been developed that allows
calculation of the ultrasensitivity of individual cells
(14). In this model, there are three parameters of
importance. The first is the Hill coefficient (nI), which is calculated
based on the distributions of individual cells with given levels of
response to increasing concentrations of a hormone. Another parameter,
termed the homogeneity coefficient (mI), accounts for differences in
individual cellular responses within the cell population as an
indication of the uniformity of cellular response. A third parameter,
termed the inflection point (aI), defines the concentration of hormone
required for half of the population of cells to respond half-maximally
(14). Coupled with ISH for GH mRNA, this mathematical
analysis for nI, mI, and aI was performed on individual MtT/S cells to
determine the origin of the ultrasensitivity of endogenous GH mRNA
expression in response to increasing concentrations of IGF-I.
Ultrasensitivity of Endogenous GH Production in Single Cells
The broad distribution of GH mRNA expression among individual
MtT/S cells in the absence of IGF-I (Fig. 3A
) shows individual cellular
heterogeneity. This was confirmed by calculation of the homogeneity
coefficient, which was found to be very low (mI = -1.2),
indicating that different cells within the population are not similar
in their response to a given IGF-I concentration. Further,
quantification of the Hill coefficient also showed that the cellular
response is not ultrasensitive, with an absolute value less than one
(nI = -0.65). Both of these values indicate that the individual
cellular response is not ultrasensitive, although the population is. If
ultrasensitivity in the population is generated by an intracellular
mechanism, then each individual cell is also expected to behave in an
ultrasensitive fashion (14). Therefore, for endogenous GH
mRNA expression in the MtT/S cells, the discrepancy between the kinetic
properties of the average response and the response at the level of the
individual cells suggests that the ultrasensitivity observed for the
average response is not caused by an intracellular mechanism. In fact,
the GH gene appears to be regulated at the level of individual cells in
a graded fashion similar to that found in a number of other
transcriptional model systems (34, 35, 36).
Ultrasensitive Response due to Sequestration of IGF by IGFBP
One possible extracellular mechanism for an average ultrasensitive
response is the presence of an IGF-I binding activity, sequestering
IGF-I and preventing the ligand from binding to the receptor. The
specificity of the switch-like effect for IGF-I, not insulin
(12), agreed with the binding selectivity of the IGF-I BP
superfamily (37). The affinities of IGFBPs for IGF-I are
greater than that of the ligand for the IGF-R, resulting in the
scenario that, although IGF-I is present in the medium, it is not
available to activate the receptor (38). However, if a
sufficiently high concentration of IGF-I is added to the medium, the
capacity of the binding proteins becomes saturated, and IGF-I will
begin to activate cellular IGF receptors. The free concentration of
IGF-I under these conditions will be much less than the total IGF-I
concentration, giving the result that the average response, even in a
system that is nonultrasensitive at the cellular level, will seem to be
switch-like in nature.
Such a mechanism is depicted in the two panels of Fig. 6
. The diagrams in panel A represent the
interactions among these factors at five different IGF-I
concentrations. In each diagram, free or bound ligand, binding protein,
or receptor indicate the interactions present at the total IGF-I
concentration. The situation is simple at 0 nM IGF-I. At
low IGF-I concentrations (5 nM), IGFBPs are in excess and
are avidly binding all available IGF-I. This large difference in
affinity compared with the IGF-R is still apparent at 10
nM, when the concentration of IGF-I in the culture
approaches the concentration of IGFBPs. From this point of equal
concentration, free IGF-I concentration and thus fractional receptor
binding will rise over a narrow range of IGF-I addition because
essentially all of the added IGF-I will be available to bind to
receptors. In other words, there is a resulting rapid increase in IGF-R
binding once all of the IGFBPs present in the extracellular environment
after IGFBPs become saturated with IGF-I. The rapid rise in receptor
binding occurs over the concentration range from 10 nM to
20 nM, saturating cellular receptors and maximally
inhibiting GH expression.
Figure 6B
is the mathematical representation of the different
components of this system. The graph shows the total concentration of
IGF-I on the x-axis. On the y-axis is the relative fraction of each of
the components of the system: IGF-I, IGFBPs, and IGF-Rs. As with the
diagrams in panel A, the modeling is based on an initial free IGFBP
concentration of 10 nM and incorporates the much higher
affinity of IGFBPs for IGF-I than of IGF-Rs (38). Addition
of IGF-I to the system increases the total IGF-I concentration on the
y-axis, causing a decrease in the free IGFBP (
) concentration.
However, the majority of IGF-I present in the culture is avidly bound
by the IGFBPs and is unavailable for binding to the receptor. However,
once saturation of the IGFBPs occurs at about 10 nM, the
levels of free IGF-I (
) rise rapidly and can activate cellular IGF-I
receptors (
). Mechanistically, although none of the individual
phenomena involved (total IGF-I concentration, IGF-I receptor binding,
or IGFBP binding) display ultrasensitive behavior, the effective
concentration of free IGF-I available to bind cellular receptors can be
modeled from the known behavior of these components. This modeling
shows that a switch-like response occurs, influenced primarily by the
high binding affinities of the IGFBPs, that is in close agreement with
the measured responses depicted in the present work.
To establish the role of IGFBPs in the mechanism of ultrasensitivity,
MtT/S cells were treated with LR3IGF-1, which
binds the type-1 IGF-R with affinity similar to that of IGF-I but has
negligible affinity for known IGFBPs (23, 24). Adding
increasing concentrations of LR3IGF-1 reduced GH
mRNA in a nonultrasensitive manner (Fig. 4
). This result indicates that
the IGF must be able to bind with high affinity with IGFBPs to create
the ultrasensitive inhibition of GH. From the results in Fig. 4
, treatment with LR3IGF-1 reduced the Hill
coefficient (nA) to an absolute value less than 1, evidence of the
linearity of the response. Thus, the sequestration of IGF-I by IGFBPs
is essential for the ultrasensitivity observed in the average response.
Another test of this sequestration was performed using IGFBP-LI, which
binds IGFBPs with high affinity but not the IGF-R, freeing IGF-I from
extracellular binding proteins (25). There was reduction
of GH mRNA levels after treatment with IGFBP-LI, in both the presence
and/or absence of added IGF-I. This decrease in MtT/S cell GH mRNA
levels appears to reflect an IGFBP-LI-dependent release of IGF-I
that is already present in CM. IGFBPs are secreted by a number of
tissues including the pituitary (39, 40). RNA extracted
from MtT/S cells was analyzed by RT-PCR amplification using primers for
IGF-BP 2 mRNA. A band of the predicted size was present, in levels
approximately 30-fold lower than GH used as a positive control reaction
(data not shown). Further assays for IGFBP activity are being performed
using Western ligand blotting (41).
Several aspects of the biology of the IGFBPs are in agreement with the
finding of regulation of GH expression in MtT/S somatotrophs. First,
IGFBPs have negligible affinity for insulin (42), in
agreement with the finding that insulin inhibits GH expression in a
nonultrasensitive manner in MtT/S cells (12). Second, some
IGFBPs may either positively or negatively modulate the actions of
IGF-I (43), as seen in the negative regulation described
in the present study. Finally, IGF-I and IGFBPs are present in the
serum-containing medium and may also be produced by pituitary cells
(39, 40, 44). Further experiments will be required to
determine the source and concentration of IGFBPs regulating MtT/S
expression.
Implications of Ultrasensitivity
The ultrasensitive regulation of GH mRNA expression by the IGFBPs
may be important in both normal pituitary cells and pituitary tumors.
For example, as somatotrophic tumors progress, circulating IGF-I
concentrations are increased, yet the tumors continue to produce GH. If
IGFBPs were concomitantly elevated, elevated IGF-I levels would not
alter GH expression due to sequestration from IGF receptors. Further,
the present study shows that a small population of the cells maintain a
high level of GH mRNA expression in the presence of 90 nM
IGF-I, greater than 10-fold the EC50 of the
average response. This subset of cells may represent the majority of GH
production when in vivo IGF-I levels are elevated during
somatotrophic tumor progression. The heterogeneity of GH production may
not be limited to tumor cells. IGF-I-dependent inhibition of GH
secretion from primary pituitary cells has also been shown to be highly
heterogeneous (45). Furthermore, transcriptional activity
of the evolutionarily related PRL gene has been shown to be highly
variable among individual primary pituitary cells (46),
although steady-state mRNA levels have not been quantified.
Taken together, these findings quantify the kinetics of endogenous GH
mRNA levels in individual cells and demonstrate that although MtT/S
cells are clonally derived, they have heterogeneous individual
responsiveness to IGF-I. Further, the ultrasensitivity observed in the
average response arises by an extracellular mechanism. IGF-I analogs
that changed the interactions with IGFBPs gave results consistent with
an extracellular mechanism of regulation via the binding of IGF by
IGFBPs. The sequestration of IGF-I by the IGFBPs, demonstrated in these
analyses of the MtT/S cells, represents a novel mechanism for
regulation of ultrasensitive control of cellular responses, and other
ultrasensitive systems may be regulated by a similar mechanism. With
the recent interest in compounds that influence IGFBP activity, such as
LR3IGF-1 (23, 24), IGFBP-LI
(25), and non-peptidyl compounds (47), it
will be essential to understand how ultrasensitive regulation is
applicable to the variety of targets of IGF-I action.
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MATERIALS AND METHODS
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Culture and Hormone Treatment of MtT/S Cells
MtT/S cells were maintained in 75-cm2
flasks using CM consisting of DMEM/F12 medium supplemented with 10%
horse serum, 2% FBS, and antibiotics as previously described
(12). For hormone treatment, cells were seeded with 5 ml
CM at a density of 100,000 cells per well in
poly-L-lysine-treated six-well plates (Falcon), and
incubated for 7 d at 37 C with 5% CO2 and
100% humidity. Human [Leu24, 59, 60,
Arg31] IGF-I (IGFBP-LI) protein was provided by
Dr. Nicholas Ling (Neurocrine Biosciences, San Diego, CA).
LR3IGF-1 and recombinant hIGF-1 were obtained
from Peninsula Laboratories, Inc. (Belmont, CA) and were
resuspended in 0.01 M sterile acetic acid and then diluted
with CM for use. Immediately before treatment, cells were washed three
times with 5 ml serum-free medium and then incubated for 5 d with
the indicated concentration of IGF-I, IGFBP-LI,
LR3IGF-1, or diluent.
Detection of GH mRNA by ISH
Cells were removed from poly-L-lysine-coated plates
by brief digestion with 0.05% Trypsin-0.5 mM EDTA
(Life Technologies, Inc., Gaithersburg, MD) and gentle
pipetting. Remaining cell-cell interactions were disrupted by passing
the suspension through a 20-µm nylon screen (Nitex, Sefar America,
Kansas City, MO) using centrifugation at 500 x g, for
3 min at 4 C. The medium was removed by aspiration, the cell pellet was
resuspended by gentle mixing with 500 µl 4% PBS-buffered
paraformaldehyde, and then cells were fixed for 1 h at 4C.
Pretreated microscope slides (SuperFrost Plus, Fisher Scientific, Pittsburgh, PA) were prepared by drawing a 3 x
8 grid on each slide (
25 mm2 per sample area)
with a hydrophobic PAP pen (RPI, Mount Prospect, IL). A 5-µl
aliquot of fixed single cell suspension was then added to each sample
area on the grid. Cells were bonded to the slides by heating at 45 C
until dry. Slides were then stored with desiccant.
Antisense rat GH DNA template was prepared as described
(12). SP6 RNA polymerase (Life Technologies, Inc.) was used to incorporate 35S-labeled
CTP (Amersham Pharmacia Biotech, Arlington Heights, IL)
into GH RNA probe at high specific activity (
4.5 x
109 cpm/µg). DNA template was removed from the
synthesis reaction by treatment with RNase-free DNase I (Ambion, Inc., Austin, TX). RNA was isolated by isopropanol precipitation
and resuspended in ultrapure water.
ISH and emulsion microautoradiography were performed as previously
described (20, 48). Cells mounted on slides were postfixed
in PBS containing 4% formaldehyde for 30 min at room temperature,
rinsed three times with PBS, and then permeabilized by incubation with
3 mg/ml Proteinase K (Life Technologies, Inc.) for 30 min
at 37C. After equilibration in triethanolamine buffer, positive charges
within the cells were blocked by incubation with acetic anhydride.
Cells were then postfixed in PBS-buffered 4% formaldehyde, rinsed
twice with PBS, dehydrated in an ascending series of ethanol solutions
(30%, 50%, 70%, 95%, and 99%), and finally air dried.
Hybridization solution consisting of 50% deionized formamide
(Life Technologies, Inc.), 1x SSPE buffer (150
mM NaCl, 10 mM
NaH2PO4, 1 mM
EDTA, pH 7.4; 5 Prime
3 Prime, Boulder, CO), 1x Denhardts solution
(0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% acetylated BSA; 5
Prime
-3 Prime), 10% dextran sulfate (5 Prime
3 Prime), 500
µg/ml yeast RNA (5 Prime
3 Prime), and 100 mM
dithiothreitol (Life Technologies, Inc.) was prepared.
Radiolabeled GH probe was then added to the hybridization solution at a
concentration predicted to saturate the target RNA as calculated by Cox
et al. (49). Hybridization solution (10 µl)
was then applied to each sample area (25 mm2).
RNase-free coverslips (HybriSlip, RPI) were applied to evenly
distribute hybridization solution over the cells. Slides were incubated
with hybridization solution for 14 h at 45C and 100%
humidity.
Coverslips were removed and the slides rinsed three times in 4x SSC
buffer. The slides were incubated in preheated RNase buffer [0.5
M NaCl, 10 mM TRIS base, 1 mM EDTA,
pH 8.0, and 25 µg/ml RNase A (5 Prime
3 Prime)] for 30 min at 37 C
to degrade single-stranded RNA, followed by incubation in RNase buffer
containing 10 mM dithiothreitol without RNase A for 30 min
at 37 C. After increasing stringency washes with 2x, 1x, and 0.5x
SSC containing 5 mM dithiothreitol for 15 min each at 37 C,
a final high-stringency wash was performed with 0.1x SSC with 5
mM dithiothreitol at 65 C for 30 min. The slides were then
dehydrated in ascending ethanol solutions and incubated twice for 5 min
in xylene.
Slides were dipped in Kodak NTB-2 autoradiographic
emulsion (Eastman Kodak Co., Rochester, NY), allowed to
air dry for 30 min, and then were stored with desiccant at 4 C in a
light-tight box. After exposure for 7 d, emulsion was processed
using Kodak D-19 developer and general-purpose fixer.
Slides were dehydrated by incubation in ascending concentrations of
ethanol and stained with eosin for 15 sec. Excess eosin was removed by
four rinses in 100% ethanol. After dehydration twice in xylene,
coverslips were mounted with Permount (Fisher Scientific).
Eosin-stained cells and associated silver grains were visualized using
an Optiphot-2 microscope fitted with a darkfield condenser and a 20x
magnification objective lens (Nikon, Melville, NY).
Photomicrographs of the cells were digitally captured using a CCD video
camera (Hamamatsu C2400; Nikon) and a Macintosh Quadra 950
computer (Apple Computer, Cupertino, CA). GH mRNA expression in
individual cells was measured by determining the density of silver
grains over the individual eosin-stained cells using NIH Image
software. Light intensity threshold was held constant at an empirically
determined level that selected silver grains but not
eosin-counterstained cells. A circular area of identical size (
20
µm diameter) was defined for each cell, and the area of silver grains
within the circular area was measured. For each area within the
24-position grid, measurements were taken from cells in nine adjacent
microscopic fields, and approximately 100150 individual cells were
measured in each of these 9 fields.
Detection of GH mRNA by Ribonuclease Protection Assay
Total RNA was isolated from treated MtT/S cultures using
single-step phenol/chloroform extraction (Biotecx, Houston, TX). GH
mRNA levels were measured in 1.5 µg total RNA by RPA using
fluorescent probes as previously described
(12).
Data Analysis
Hill coefficients were calculated from graphical analysis as
described previously (22). The mathematical equations
describing cellular ultrasensitivity (14) were used to
derive the parameters of aI and mI for the population using numerical
analysis as shown in full at the following web site:
http://www.tulane.edu/
hurley/derivation.htm. Numerical analysis was
performed on a Macintosh G3 computer using Mathematica 4 software
(Wolfram Research, Champaign, IL), SuperANOVA software (Abacus
Software, Berkeley, CA) and Systat for Windows 8.0 software
(SPSS, Inc. Chicago, IL) run with Virtual PC 2.0
(Connectix, San Mateo, CA).
 |
ACKNOWLEDGMENTS
|
---|
The authors appreciate the assistance of Dr. Wylie Vale (Salk
Institute), and Dr. Nicholas Ling (Neurocrine Biosciences), in
generously providing IGFBP-LI.
 |
FOOTNOTES
|
---|
Address requests for reprints to: David L. Hurley, Department of Cell and Molecular Biology, 1000 Stern Hall, 6400 Freret Street, Tulane University, New Orleans, Louisiana 70118-5698. E-mail:
dlh1000{at}tulane.edu
This work was supported by a grant from the Tulane/Xavier Center for
Bioenvironmental Research and National Science Foundation Career Award
IBN-9600805 to D.L.H. M.P.F. is the recipient of an Endocrine
Society Summer Research Fellowship.
These results were presented in part at the Annual meeting of The
Endocrine Society, June 2000, in Toronto Canada.
1 Current Address: Center for Research in Reproduction,
Department of Medicine, University of Virginia, Charlottesville,
Virginia. 
Abbreviations: CM, complete medium; IGFBP, IGF binding protein;
IGFBP-LI, IGFBP ligand inhibitor; LR3IGF-1, long
(Arg3)IGF-I; IGF-R, IGF receptor; ISH, in
situ hybridization; RPA, ribonuclease protection assay
Received for publication August 18, 2000.
Accepted for publication May 18, 2001.
 |
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