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Molecular Endocrinology 15 (9): 1549-1558
Copyright © 2001 by The Endocrine Society

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (0–90 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 0–90 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go 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. 1AGo). There appeared to be a reduction of GH mRNA per cell detected using ISH after exposure to IGF-I (Fig. 1BGo). 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.

 
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. 2AGo). 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. 2BGo). 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. 2BGo).



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  		Figure 2. Average GH mRNA Expression per Cell After Treatment with Increasing Concentrations of IGF-I

In panel A, GH mRNA levels were measured in cells by ISH ({circ}) or in the entire population by RPA ({blacksquare}). Results are the means from three cultures, are expressed as a percentage of the control. Error bars denote the SEM. In panel B, graphical Hill plot analysis was used to calculate the EC50 and Hill coefficient of the response with the ISH results ({circ}) or the RPA ({blacksquare}).

 
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. 3Go). In the presence of serum but no IGF-I (Fig. 3AGo), 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. 3Go, B and C). However, there was a change in the distribution of expression after treatment with 9.9 nM IGF-I (Fig. 3DGo). 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. 3Go, E–G). 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. 3GGo).



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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 (A–G). 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).

 
Histogram data for the distribution of GH mRNA expression in the individual cells were combined for all hormone concentrations tested (Fig. 3HGo) 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. 3HGo, all three values were calculated. The value of aI was 4.5 nM, in good agreement with the EC50 measured previously (Fig. 2Go). 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. 3IGo). 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. 3Go, 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. 4Go). 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. 4AGo). 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. 4BGo, Hill plot analysis indicated that the response is nonultrasensitive (nA = -0.6), and that the EC50 for LR3IGF-1 is 0.07 nM.



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  		Figure 4. Effects of an IGF-I Analog That Does Not Bind to IGFBPs on GH mRNA Expression

Cells were incubated for 5 d in CM supplemented with the indicated concentrations of LR3IGF-1 ({bullet}) or IGF-I ({blacksquare}). GH mRNA levels were measured in 1.5 µg total RNA 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 (A). The EC50 and Hill coefficient were determined by Hill plot (B) for the response to LR3IGF-1 ({bullet}) or IGF-I ({blacksquare}).

 
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. 5Go). 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. 5AGo). 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 ({blacksquare}) or IGF-I alone ({circ}). 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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. 2Go) 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. 3AGo) 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. 6Go. 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.



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  		Figure 6. Models for Generating Ultrasensitive Kinetics due to Sequestration of IGF-I by IGFBPs

In panel A, the effects of increasing IGF-I concentration from 0 nM (far left) to 20 nM (far right) are schematically depicted. The levels of free (black) and bound (gray) IGF-I and extracellular IGFBPs are changed in each panel for the given concentration. The resulting changes in cellular IGF-R binding show that an ultrasensitive change occurs once the capacity of the 10 nM IGFBP is saturated. The graph in panel B shows the predicted effects of increasing concentrations of total IGF-I on the y-axis: the free IGFBP ({circ}) concentration decreases, but 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 ({square}) rise rapidly and cause activation of cellular response via IGF-Rs in an ultrasensitive manner ({blacksquare}).

 
Figure 6BGo 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 ({circ}) 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 ({square}) rise rapidly and can activate cellular IGF-I receptors ({blacksquare}). 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. 4Go). 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. 4Go, 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 Denhardt’s 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 100–150 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. Back

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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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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