help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smink, J. J.
Right arrow Articles by van Buul-Offers, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smink, J. J.
Right arrow Articles by van Buul-Offers, S. C.
Endocrinology Vol. 140, No. 12 5876-5882
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor (IGF) II Induced Changes in Expression of IGF Binding Proteins in Lymphoid Tissues of hIGF-II Transgenic Mice

Jeske J. Smink, Johanna G. Koster, Brenda I. Hendriks-Stegeman and Sylvia C. van Buul-Offers

Department of Pediatric Endocrinology, University Medical Center Utrecht, Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Jeske J. Smink, Room kC.03.063.0, P.O. Box 85090, NL-3508 AB Utrecht, The Netherlands. E-mail: J.Smink{at}wkz.azu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overexpression of human insulin-like growth factor II (IGF-II) in transgenic mice does not result in increased overall body growth. The IGF-II overexpression, however, specifically causes growth of the thymus and not of the spleen. We address the question whether the observed differences in growth induction in lymphoid tissues by IGF-II can be related to differences in local IGF binding protein (IGFBP) production, using nonradioactive in situ hybridization and Northern blot analysis. IGFBP-2, -4, and -5 are expressed in both lymphoid tissues of normal mice. The spleen additionally expresses IGFBP-3 and IGFBP-6. IGFBP-1 expression was not detected. Although the expression pattern of the IGFBPs did not change upon IGF-II overexpression, the level of expression changed in a specific manner for each IGFBP. In both the thymus and the spleen of transgenic mice, IGFBP-2 and -5 gene expression was slightly increased, whereas the level of IGFBP-4 expression was not altered. In the spleen, IGFBP-6 expression was not altered by IGF-II overexpression, whereas IGFBP-3 expression was strongly increased. The differences in IGFBP expression, and the difference in response of these IGFBPs to IGF-II overexpression in thymus and spleen suggests an important role of these proteins in growth regulation of both lymphoid tissues. We speculate that an increase of IGFBP-3 expression together with changes in expression of other IGFBPs, inhibits IGF-II stimulated growth in the spleen by an autocrine-/paracrine pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors (IGF-I and -II) are potent mitogenic and differentiation promoting growth factors (1). Furthermore, the IGFs can inhibit cell death, induce differentiation and stimulate differentiated functions in several cell types (2). The IGFs play an important role in pre- and postnatal growth. In rodents, IGF-I is presumed to be important in pre- and postnatal growth, whereas IGF-II only seems to be important in prenatal growth (3, 4).

IGFs are produced in multiple tissues and can act both in an endocrine and autocrine/paracrine fashion. The activity of IGFs is regulated at various levels, resulting in a complex regulation of IGF bioactivity (2, 5). Their intracellular effects are mediated predominantly via the type I IGF receptor (6).

IGFs are present in the circulation and throughout the extracellular space bound to members of high affinity IGF binding proteins (IGFBPs). The availability of the IGFs for their receptor is modulated by these IGFBPs, of which six are cloned and characterized (7, 8). IGFBP genes are widely expressed in the developing tissues of rodents (9), sheep (10), and humans (11). The IGFBPs act mainly as autocrine and/or paracrine factors at or close to their sites of synthesis (2). The individual IGFBPs differ in their tissue distribution and may either inhibit or potentiate IGF activity. Furthermore, they differ in their IGF-binding capacity (2, 12). IGFBP-3 is the major serum carrier of IGFs, in a complex with an acid-labile subunit (ALS) (13).

Transgenic mice with recombinant human IGF-II under the control of the H-2Kb promoter show increased IGF-II serum levels, whereas overall body growth is not affected (14). The hIGF-II transgene is highly expressed in the two lymphoid tissues, the thymus and the spleen, causing thymic but no splenic growth (14, 15, 16).

Because there are indications that the IGFBPs regulate actions of locally produced IGFs in lymphoid tissues (5, 17), we studied the IGFBP expression in the spleen and the thymus of normal and the hIGF-II transgenic mice in situ. The observed differences in IGFBP expression and the difference in response to IGF-II overexpression suggest an important role of the IGFBPs in growth regulation. The IGFBP-3 gene, expressed in the spleen only, is most susceptible to IGF-II overexpression. We speculate that IGFBP-3 plays a prominent paracrine role with respect to the inhibition of the hIGF-II transgene bioactivity in the spleen, by either an IGF-dependent or IGF-independent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), as well as the digoxigenin-UTP, anti-digoxigenin Fab-fragments, nitro blue tetrazolium, 5-bromo-4-chloro-3-indolyl-phosphatase, blocking reagent, the Tripure isolation reagent and the Agarose Gel DNA extraction kit. Nylon membranes were purchased from QIAGEN (Westburg, The Netherlands). [{alpha}32P]dCTP (10 mCi/ml) and the RediPrime Random Primer labeling mixture were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Mouse IGFBP-1 to -6 complementary DNAs (cDNAs) were kindly provided by Prof. Dr. S. L. S. Drop and Dr. J. W. van Neck (Department of Pediatrics, subdivision of Pediatric Endocrinology, Rotterdam, The Netherlands). GAPDH cDNA was kindly provided by Dr. H. van Teeffelen (Department of Physiological Chemistry, Utrecht, The Netherlands).

Polyvinyl alcohol was obtained from Aldrich (Milwaukee, MI). Euparal mounting medium was purchased from Klinipath (Duiven, The Netherlands).

Mice and tissue preparation
hIGF-II transgenic mice were generated by introduction of a human IGF-II gene into FVB/N control mice as described in detail by Van Buul-Offers and colleagues (14). Throughout the study, the line designated 5'-74 (Tg-II), was used for our experiments. As controls normal FVB/N mice were used. The animals were kept under standardized laboratory conditions. The mice were killed by decapitation after ether anesthesia at the age of 4 weeks. Thymus and spleen were dissected, frozen in liquid nitrogen and stored at -80 C. The protocol received approval of the committee for Animal Experiments of the Medical Faculty, University of Utrecht.

Probes
Digoxigenin-labeled complementary RNA (cRNA) probes. Standard RNA synthesis reactions using T7- or T3-RNA polymerase were carried out using digoxigenin-UTP as substrate (18). cDNAs encoding human IGF-II (19) and mouse IGFBP-1, -2, -3, -4, -5, and -6 cDNAs, corresponding to amino acid position 100–133, 98–258, 137–204, 131–205, 88–182, 83–140 respectively (9), were used as templates for the synthesis of antisense and/or sense digoxigenin-labeled RNA probes.

[{alpha}32P]dCTP-labeled cDNA probes. Twenty nanograms of gel-purified inserts of plasmids containing mouse IGFBP-2, -3, and -5 cDNA, were radiolabeled with 50 µCi [{alpha}32P]dCTP, using random primed DNA labeling as described by the manufacturer.

In situ hybridization
Tissues used for in situ hybridization were fixed for 18 h in 4% (wt/vol) paraformaldehyde at 4 C, washed in PBS, dehydrated through a series of ethanol and embedded in paraffin.

Paraffin tissue sections (10 µm) were dewaxed, hydrated, rinsed in PBS and treated with proteinase K (0.07 U/ml) for 30 min at 37 C and subjected to an acetylation treatment (20). Sections were rinsed in 2 x SSC and kept in this solution until the start of the hybridization.

Hybridization was performed in a solution containing 50% formamide, 2 x SSC, 1 x Denhardt’s solution, 1 µg/µl yeast RNA and 10% dextran-sulfate and the digoxigenin labeled cRNA probe at a concentration of 500-1500 pg/µl. Sections were hybridized overnight at 53 C. After hybridization, sections were washed with 50% formamide in 2 x SSC at 53 C for 30 min and treated with RNase A (1 U/ml) for 30 min at 37 C. Subsequently, sections were rinsed in 2 x SSC, treated with 10% lamb serum for 30 min and incubated with sheep antidigoxigenin Fab-fragments coupled to alkaline phosphatase (1:500) for 2 h at room temperature.

Chromogenesis was performed with 0.38 mg/ml nitroblue tetrazolium and 0.19 mg/ml 5-bromo-4-chloro-3-indolyl-phosphatase in the presence of 6% (wt/vol) polyvinylalcohol (21), resulting in a blue precipitate. Sections were counterstained with nuclear fast red, dehydrated through a series of ethanol and mounted with Euparal.

Three to four different animals of each strain were used per analyzed IGFBP messenger RNA (mRNA) and for each probe the in situ hybridization was repeated 5 to 6 times.

Northern blot analysis
RNA extraction. Total RNA was extracted from frozen spleen and thymus from 4 weeks old normal (FVB/N) and hIGF-II transgenic mice, using Tripure solution reagent according to the procedures of the manufacturer, based on the single step acid guanidinium thiocyanate method (22).

Northern blot analysis. Twenty micrograms of total RNA was separated by electrophoresis in a 1% (wt/vol) agarose/2.2 M formaldehyde gel in 1 x 3-(morpholino) propanesulphonic acid (MOPS) buffer and transferred to a 0.2 µm nylon membrane and crosslinked by UV radiation (1200 µJ).

The membranes were prehybridized for 2 h at 60 C in a solution containing 0.1% SDS, 3 x SSC, 5 x Denhardt’s solution, 10% dextran-sulfate and 5% denaturated salmon sperm DNA. Hybridization was performed at 60 C overnight in the same solution, containing the [{alpha}32P]dCTP-labeled probe.

Following hybridization, the membranes were washed to a stringency of 0.2 x SSC, 0.1% (wt/vol) SDS. Membranes were analyzed using the GS-363 Molecular Imager (Bio-Rad Laboratories, Inc. Hercules, CA) and subsequently quantified using the Molecular Analyst software program, version 1.4 (Bio-Rad Laboratories, Inc.). The signals were also visualized by autoradiography on Fuji Photo Film Co., Ltd. RX x-ray film.

Tissues of at least three different animals of each strain were analyzed.

Statistical analysis
Data of the Northern blot analyses were analyzed with Student’s two-tailed test. A P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGFBP expression patterns and changes therein upon IGF-II overexpression
Thymus. Representative thymus sections of 4 weeks old normal and hIGF-II transgenic mice, analyzed with in situ hybridization using specific IGFBP probes, are shown in Fig. 1Go.



View larger version (157K):
[in this window]
[in a new window]
  		Figure 1. IGFBP expression in the thymus. Expression patterns of IGF-II and IGFBP-1 to -6 mRNA in representative sections of the thymus of 4-week-old normal (FVB/N) (A; D–J) and hIGF-II transgenic (B; K–M) mice, as analyzed by in situ hybridization, under brightfield illumination. C, A representative section of the transgenic thymus hybridized with an IGF-II sense RNA probe (IGF-IIs). Sections were hybridized with antisense digoxigenin-labeled cRNA probes, specific for human IGF-II and mouse IGFBP-1 to -6 as indicated. All sections were counterstained with nuclear fast red. The mRNA signal is shown as a blue precipitate under brightfield illumination. The signal of IGFBP-2 mRNA (D and G) is shown as a dark brownish precipitate, due to the used mounting medium. M, medulla; C, cortex. Magnification, 100 times. The black bar in the right corner refers to the actual size of 100 µm.

 
Control in situ hybridizations of thymus sections of transgenic mice with nonradioactive human IGF-II RNA probe showed very strong staining in the medulla and in a small number of cells in the cortex (Fig. 1BGo), whereas hardly any IGF-II transcripts could be detected in the thymus of FVB/N nontransgenic mice (Fig. 1AGo). As a control for specific hybridization, sections were hybridized with the corresponding sense RNA probes, which showed no signals (see e.g., Fig. 1CGo). In the thymus of normal mice no expression of IGFBP-1, -3, and -6 could be detected (Fig. 1Go, D–F). IGFBP-2, -4, and -5 transcripts were present, each showing a unique expression pattern (Fig. 1Go, G–J). Transcripts of IGFBP-2 were present in groups of cells, mainly at the boundary of the medulla and the cortex and, at a low level, in the medulla (Fig. 1GGo). IGFBP-4 was exclusively detected in the medulla (Fig. 1HGo). A similar expression pattern was seen for IGFBP-5 (Fig. 1JGo); however, the number of cells expressing IGFBP-5 was lower than for IGFBP-4. IGFBP-4 and -5 transcripts were detected in single positive cells and not in groups of positive cells as seen for IGFBP-2.

To study the influence of IGF-II overexpression on IGFBP expression, thymus sections of 4 weeks old hIGF-II transgenic mice were analyzed with in situ hybridization (Fig. 1Go, K–M). No expression of IGFBP-1, -3, or -6 could be detected (data not shown). The expression patterns of IGFBP-2, -4, and -5 in the transgenic thymus was similar to that found in the thymus of normal mice (Fig. 1Go, K–M). However, the level of expression was slightly increased for IGFBP-2, (cf. Fig. 1Go, G and K), and to a higher extent for IGFBP-5 (cf. Fig. 1Go, J and M). IGFBP-4 transcripts were present at the same level as in normal mice. Although the shown data suggests a lower level of IGFBP-4 mRNA in the transgenic thymus (Fig. 1LGo), most of the performed experiments showed no difference in levels of expression. However, the picture shown was chosen to illustrate the expression pattern.

Spleen. Representative spleen sections of normal and hIGF-II transgenic mice, 4 weeks of age, analyzed with in situ hybridization using specific mouse IGFBP probes are shown in Fig. 2Go.



View larger version (159K):
[in this window]
[in a new window]
  		Figure 2. IGFBP expression in the spleen. Expression and localization of IGF-II and IGFBP-2 to -6 mRNA in representative sections of the spleen of 4-week-old normal mice (A; C–D; G–J) and hIGF-II transgenic mice (B; E–F; K–M) by in situ hybridization, under brightfield illumination. Sections were hybridized with antisense digoxigenin-labeled cRNA probes, specific for human IGF-II and mouse IGFBP-1 to -6 as indicated. Sections were further treated as described in Fig. 1Go. WP, White pulp; RP, red pulp; MZ, marginal zone. Magnification, 100 times. The black bar in the right corner refers to the actual size of 100 µm.

 
Control in situ hybridizations of spleen sections of the transgenic mice with nonradioactive hIGF-II RNA probe showed high IGF-II transgene expression in the white pulp and a few positively stained cells in the red pulp and/or marginal zone (Fig. 2BGo). Spleen sections of the nontransgenic FVB/N control mice showed a very weak IGF-II expression in the red pulp (Fig. 2AGo). In the spleen of normal mice, no IGFBP-1 gene expression could be detected (data not shown). IGFBP-2, -3, -4, -5, and -6 mRNAs were present, each showing a unique expression pattern (Fig. 2Go, C, D, G–J). Control sections hybridized with the appropriate sense probes exhibited no signal (data not shown).

IGFBP-2 is expressed at a high level in groups of cells in the red pulp (RP) and at a very low level in the white pulp (WP) (Fig. 2CGo). IGFBP-3 transcripts were detected, at a low level, in the inner lining of the marginal zone (MZ) and in some cells in the white pulp, whereas no IGFBP-3 transcripts could be detected in the red pulp (Fig. 2DGo). IGFBP-4 transcripts were detected all through in the white pulp only (Fig. 2GGo). IGFBP-5 transcripts were detected in the white pulp in sparsely scattered cells and in cells concentrated around the veins. IGFBP-5 expression was also detected at a low level at the exterior part of the red pulp (Fig. 2HGo). At the exterior part of the red pulp, also a considerable amount of IGFBP-6 transcripts were detected, whereas lower amounts were detected in the other parts of the red pulp and in the white pulp (Fig. 2JGo).

To study the influence of IGF-II overexpression on IGFBP expression, spleen sections of 4-week-old hIGF-II transgenic mice were analyzed with in situ hybridization (Fig. 2Go, E, F, K–M). No expression of IGFBP-1 could be detected in the transgenic spleen (data not shown). Although the level of expression of some IGFBPs changed upon IGF-II overexpression, the pattern of expression remained the same, as was observed in the thymus. IGFBP-4 and -6 transcripts were detected at the same level as in normal mice (cf. Fig. 2Go, G and K, and J and M, respectively). On the contrary, the level of expression of IGFBP-2, -3, and -5 was increased compared with the expression level in the normal spleen. The expression of IGFBP-2 was slightly increased in the white pulp, whereas the level of expression in the red pulp was not altered (cf. Fig. 2Go, C and E). The expression of IGFBP-5, detected in the red pulp at the edge of the section and in the white pulp, showed an increase in level of expression (cf. Fig. 2Go, H and L). IGFBP-3 expression in the marginal zone and the white pulp was strongly increased (cf. Fig. 2Go, D and F).

Quantification of changes in IGFBP-3 expression
To quantify the changes in IGFBP-3 expression upon IGF-II overexpression, Northern blot analysis was performed. Total RNA was extracted from the spleen and the thymus of 4-week-old normal and hIGF-II transgenic mice and subsequently hybridized with a mouse IGFBP-3 [{alpha}32P]dCTP-labeled cDNA probe, a representative experiment is shown in Fig. 3AGo. This revealed a transcript of 2.6 kb (Fig. 3AGo), corresponding to an IGFBP-3 transcript length as described previously (23). This transcript was only present in the spleen of normal and hIGF-II transgenic mice (Fig. 3AGo, lanes 4 and 1, respectively) and not in the thymus of either mice strain (Fig. 3AGo, lanes 2 and 3, respectively). Quantification of the detected IGFBP-3 transcripts in the normal spleen (n = 3) and hIGF-II transgenic spleen (n = 5), after normalization with the GAPDH mRNA signal, showed a statistically significant increase in level of expression in the transgenic spleen of 1.75 ± 0.11 (Fig. 3BGo).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Quantitative analysis of changes in IGFBP-3 expression. Total RNA extracted from the spleen and the thymus of 4-week-old normal and hIGF-II transgenic mice was analyzed using Northern blot analysis. The membrane was hybridized with a cDNA probe specific for mouse IGFBP-3. The filter was subsequently hybridized with a GAPDH probe as a control for the amount of transferred RNA in each sample. Representative Northern blot data are shown in panel A. A, The RNA in lanes 1 and 4 were derived from the spleen; the samples 2 and 3 from the thymus. The RNAs in the lanes 2 and 4 were derived from normal mice; in lanes 1 and 3 from hIGF-II transgenic mice. B, Data obtained from Northern blots of the spleen, which have been quantified by densitometry. The open bar represents IGFBP-3 mRNA levels of the spleen of normal mice (FVB/N) and the cross-hatched bar of hIGF-II transgenic mice (Tg-II). The data are expressed as a percentage of the IGFBP-3 mRNA level detected in the spleen of normal mice, which has been attributed a value of 100%. Means ± SEM are given. The numbers of animals is FVB/N: 3; Tg-II: 5. **, P < 0.01 compared with the mean normal spleen value.

 
An increase in expression of IGFBP-2 and -5 in hIGF-II transgenic mice in the thymus and spleen is shown with in situ hybridization, although not as strong as seen for IGFBP-3 in the spleen. Quantification of IGFBP-2 and -5 expression using Northern blot analysis showed the presence of IGFBP-2 and -5 mRNA in the spleen and the thymus of normal and hIGF-II transgenic mice, however no statistically significant differences were detected (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Besides the endocrine actions of IGFs, these hormone peptides can exert actions locally in a variety of tissues and cell types. Tissue-specific regulation of the IGF-bioactivity may be accomplished by the presence of IGFBPs, which are differentially expressed in various tissues (2). The relative concentration of each IGFBP changes, depending on the physiological and pathological environment (12, 24). In hIGF-II transgenic mice, both the spleen and the thymus express the hIGF-II gene at a high level (14, 16), which might therefore induce a change in local IGFBP expression. Furthermore, it was shown that both IGF receptors are present in both organs, enabling IGF-II bioactivity (16). However, the thymus displayed growth in response to this IGF-II overexpression, whereas the size of the spleen was unaffected, although both organs displayed an increase in CD4+ T cells (15).

To answer the question whether the difference in response to IGF-II overexpression was related to differences in IGFBP expression, we studied the expression of the six IGFBP genes in both lymphoid tissues of normal and hIGF-II transgenic mice.

Normal IGFBP expression patterns in lymphoid organs
Each IGFBP gene has a specific, unique expression pattern in both lymphoid tissues, as shown in this study by nonradioactive in situ hybridization analysis. In both lymphoid tissues, IGFBP-2, -4, and -5 transcripts were detected. In addition to these genes, the spleen expresses the IGFBP-3 and IGFBP-6 gene. IGFBP-4 and -5 displayed a similar expression pattern in both tissues. On the contrary, IGFBP-2 transcripts showed a different localization, and were mainly present in the red pulp of the spleen and at the boundary of the medulla/cortex in the thymus. IGFBP-6 transcripts were mainly detected in the red pulp of the spleen and at a low concentration in the white pulp, colocalizing with IGFBP-2, -4, and IGFBP-5 transcripts. IGFBP-3 transcripts showed a completely different localization, and were present mainly in the marginal zone. Colocalization and differences in localization of certain IGFBPs was previously shown at the tissue level in human fetus, mouse kidney and mouse skeleton (11, 25, 26). In both the spleen and the thymus, the IGFBP transcripts are detected in mature lymphocytes, based on the localization and morphology of the positively stained cells. IGFBP-3 transcripts, however, are predominantly present in the macrophages of the marginal zone, based on staining results with an antibody specific for the detection of macrophages (MOMA) present in the marginal zone (Koster, personal communication), although a low expression in lymphocytes in the white pulp is also shown.

The observed differences in IGFBP expression in the spleen and the thymus of normal mice suggest a putative difference in mediating local IGF activity in these tissues.

IGF-II induced changes in IGFBP expression
Previous findings in the literature already suggested a direct relationship between IGFs and IGFBPs at the serum level (27, 28, 29, 30), especially of IGF-II on IGFBP-3 in Pit-1 deficient Snell dwarf mice (30). In this study, a direct relationship between IGF-II and a number of IGFBPs is shown at tissue level, varying in different tissues. In spleen and thymus, IGFBP-2 and -5 mRNA are positively correlated with IGF-II mRNA, as previously shown also at the protein level for IGFBP-2 in serum of PEPCK-IGF-II transgenic mice (27) and for IGFBP-5 in human serum (28). IGFBP-4 mRNA does not seem to be correlated with IGF-II mRNA levels, in accordance with data concerning the protein in human serum (29). IGFBP-6 expression, only present in the spleen, seems also not to be correlated with levels of IGF-II mRNA, as shown here in this study. The IGF-II-induced changes in IGFBP expression are summarized in Table 1Go.


View this table:
[in this window]
[in a new window]
  		Table 1. IGF-II-induced changes

 
Previous studies in IGF-I transgenic mice have shown an in vivo temporally and spatially relationship between IGF-I mRNA and IGFBP-5 mRNA, in the brain (31). This previous study with IGF-I transgenic mice and our study with IGF-II transgenic mice, indicate the possibility of tissue-specific direct regulation of the expression of IGFBPs by their ligands, the IGFs.

In the spleen, the IGFBP-3 gene seems to be most susceptible to IGF-II overexpression, which is confirmed by Northern blot analysis. hIGF-II transgenic Snell dwarf mice also show an increase in IGFBP-3 mRNA in the spleen and an increase in IGFBP-3 serum levels, compared with normal Snell dwarf mice (14; unpublished results). In these hIGF-II transgenic Snell dwarf mice no IGF-I is present (14, 24). Furthermore, IGF-I serum levels are not elevated in normal hIGF-II transgenic mice (14), indicating that the increase of IGFBP-3 in hIGF-II transgenic normal and Snell dwarf mice cannot be caused by IGF-I. In addition, IGF-II treatment of dwarf mice results in a pronounced increase of IGFBP-3 in serum (30).

Role of IGFBP-3 in growth regulation
As discussed above, the main difference between the spleen and the thymus in terms of regulation of IGFBP expression by IGF-II is the considerable increase in IGFBP-3 expression in the macrophages of the marginal zone of the spleen. The number of these macrophages in the marginal zone is not increased in the spleen of hIGF-II transgenic mice, indicating that the increase in IGFBP-3 expression is due to constitutive IGF-II expression and not due to changes in morphology of the marginal zone. The presence and up-regulation of IGFBP-3 is independent of GH and/or IGF-I, since similar results were obtained in hIGF-II transgenic Snell dwarf mice (unpublished results). The IGF-II-induced increase in IGFBP-3 expression can have effects on several mechanisms involving growth regulation. First, it has been demonstrated in vitro (32, 33, 34, 35) and in vivo (36), that IGFBP-3 can act as a negative growth regulator of cell proliferation by sequestering the IGF-II. Thus, the spleen-specific enhanced IGFBP-3 expression might inhibit the growth inducing effect of the IGF-II transgene in the spleen by sequestering IGF-II and thereby diminishing the IGF-II bioactivity. Observations in IGFBP-3 transgenic mice (37) seem to contradict this speculation, as in these mice an increase of IGFBP-3 serum levels results in growth of the spleen. However, no IGFBP-3 mRNA could be detected in the spleen of these mice, excluding high levels of IGFBP-3 in the spleen. Furthermore, the enhanced IGFBP-3 serum levels were of the binary complex. As this IGF-IGFBP-3 complex can cross the capillary endothelium, enhanced serum levels can transport more IGF-I or -II into the spleen causing enhanced IGF-bioactivity. Thus, in these animals, IGFBP-3 probably acts mainly by an endocrine pathway, instead of the proposed auto-/paracrine way of action in our transgenics.

Besides the growth-inhibitory effect of IGFBP-3 caused by regulating the availability of free IGFs, IGFBP-3 can also induce growth arrest (38, 39, 40) and apoptosis, mediated through a pathway independent of the IGF-IGF-receptor interaction (41). The relatively high concentrations of IGFBP-3 in transgenic spleen could have such an IGF-II independent activity.

Therefore, although the influence of other IGFBPs on the IGF-II bioactivity should not be neglected, the considerable increase in IGFBP-3 mRNA and selective expression in the spleen, suggest that this IGFBP plays a prominent role in the reduction of the growth promoting effect of IGF-II in the spleen.

In conclusion, in this study it has been shown that the thymus and the spleen display a tissue-specific IGFBP expression. In all likelihood, it is the difference in IGFBP expression that causes a difference in local IGF-II bioactivity, resulting in a strong growth promoting effect of IGF-II in the thymus and hardly any in the spleen. This suggests that the growth-inducing effect of IGF-II depends on its local environment.


   Acknowledgments
 
Thanks are due to Mrs. M. G. Gresnigt and Mr. R. J. Bloemen for technical assistance, to Mrs. I. van de Brink for taking care of the animals, to Dr. J. A. Koedam for critical reading the manuscript and to Mr. R. Hövig in help with photographic illustrations.

Received May 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cohick WS, Clemmons DR 1993 The insulin-like growth factors. Annu Rev Physiol 55:131–153[Medline]
  2. Jones J, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[CrossRef][Medline]
  3. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  4. Van Buul-Offers SC 1996 Insulin-like growth factor-II in the cycle of life. Biomed Rev 5:65–71
  5. Clark R 1997 The somatogenic hormones and insulin-like growth factor-I: stimulators of lymphopoieses and immune function. Endocr Rev 18:157–179[Abstract/Free Full Text]
  6. Baserga R, Hongo A, Rubini M, Prisco M, Valentinis B 1997 The IGF-I receptor in cell growth, transformation and apoptosis. Biochim Biophys Acta 1332:F105–F126
  7. Rajaram S, Baylink DJ, Mohan S 1997 Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev 18:801–831[Abstract/Free Full Text]
  8. Clemmons DR 1997 Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factors Rev 8:45–62[CrossRef][Medline]
  9. Schuller AGP, Zwarthoff EC, Drop SLS 1993 Gene expression of the six insulin-like growth factor binding proteins in the mouse conceptus during mid-and late gestation. Endocrinology 132:2544–2550[Abstract]
  10. Delhanty PJD, Han VKM 1993 The expression of insulin-like growth factor (IGF) II and IGF binding protein-2 genes during ovine development. Endocrinology 132:41–52[Abstract]
  11. Han VK, Matsell DG, Delhanty PJ, Hill DJ, Shimasaki S, Nygard K 1996 IGFBP mRNAs in the human fetus: tissue and cellular distribution of developmental expression. Horm Res 45:160–166[Medline]
  12. Mohan S, Baylink DJ 1996 Editorial: insulin-like growth factor (IGF)-binding proteins in serum. Do they have additional roles besides modulating the endocrine IGF actions? J Clin Endocrinol Metab 81:3817–3820[Free Full Text]
  13. Baxter RC 1995 Insulin-like growth factor binding proteins as glucoregulators. Metabolism 44:12–7[Medline]
  14. Van Buul-Offers SC, Haan K de, Reijnen-Gresnigt MG, Meinsma D, Jansen M, Oei SL, Bonte EJ, Sussenbach JS, Brande JL van den 1995 Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus. J Endocrinol 144:491–502[Abstract]
  15. Kooijman R, Van Buul-Offers SC, Scholtens LE, Schuurman H-J, Van den Brande JL, Zegers BJM 1995 T cell development in insulin-like growth factor-II transgenic mice. J Immunol 15:5736–5745
  16. Van der Ven LT, Roholl PJ, Reijnen-Gresnigt MG, Bloemen RJ, Van Buul-Offers SC 1997 Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II. Histochem Cell Biol 107:193–203[CrossRef][Medline]
  17. Van Buul-Offers SC, Kooijman R 1998 The role of growth hormone and insulin-like growth factors in the immune system. Cell Mol Life Sci 54:1083–1094[CrossRef][Medline]
  18. Melton D, Krieg P, Rebagliati M, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056[Abstract/Free Full Text]
  19. Jansen M, Van Schaik FMA, Van Tol H, Van den Brande JL, Sussenbach JS 1985 Nucleotide sequences of cDNAs encoding precursors of human insulin-like growth factor II (IGF-II) and IGF-II variant. FEBS Lett 179:243–246[CrossRef][Medline]
  20. Wilkinson DG 1992 In situ hybridization: a practical approach. In: Rickwood D, Hames BD (eds) The Practical Approach Series. Oxford, Oxford University Press
  21. De Block M, Debrouwer D 1993 RNA-RNA in situ hybridization using digoxigenin-labeled RNA probes: the use of high molecular weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium. Anal Biochem 215:86–89[CrossRef][Medline]
  22. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  23. Molnar P, Murphy LJ 1994 Effects of oestrogen on rat uterine expression of insulin-like growth factor-binding proteins. J Mol Endocrinol 13:59–67[Abstract]
  24. Van Buul-Offers SC, Bloemen RJ, Reijnen-Gresnigt MG, van Leiden HA, Hoogerbrugge CM, Van den Brande JL 1994 Insulin-like growth factors-I and -II and their binding proteins during postnatal development of dwarf Snell mice before and during growth hormone and thyroxine therapy. J Endocrinol 143:191–198[Abstract]
  25. Lindenbergh-Kortleve DJ, Rosato R, van Neck JW, Nauta J, van Kleffens M, Groffen C, Zwarthoff EC, Drop SLS 1997 Gene expression of the insulin-like growth factor system during mouse kidney development. Mol Cell Endocrinol 132:81–91[CrossRef][Medline]
  26. Wang E, Wang J, Chin E, Zhou J, Bondy CA 1995 Cellular patterns of insulin-like growth factor system gene expression in murine chondrogenesis and osteogenesis. Endocrinology 136:2741–2751[Abstract]
  27. Wolf E, Kramer R, Blum WF, Föll J, Brem G 1994 Consequences of postnatally elevated insulin-like growth factor-II in transgenic mice: endocrine changes and effects on body and organ growth. Endocrinology 135:1877–1886[Abstract]
  28. Mohan S, Libanati C, Dony C, Lang K, Srinivasan N, Baylink DJ 1995 Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids. J Clin Endocrinol Metab 80:2638–2645[Abstract]
  29. Honda Y, Landale EC, Strong DD, Baylink DJ, Mohan S 1996 Recombinant synthesis of insulin-like growth factor binding protein-4 (IGFBP-4): development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids. J Clin Endocrinol Metab 81:1389–1396[Abstract]
  30. Van Buul-offers SC, Reijnen-Gresnigt MG, Hoogerbrugge CM, Bloemen RJ, Kuper CF, Van den Brande JL 1994 Recombinant insulin-like growth factor-II inhibits the growth-stimulating effect of growth hormone on the liver of Snell dwarf mice. Endocrinology 135:977–985[Abstract]
  31. Ye P, D’Ercole J 1998 Insulin-like growth factor I (IGF-I) regulates IGF binding protein-5 gene expression in the brain. Endocrinology 139:65–71[Abstract/Free Full Text]
  32. Anelloz-Nicoud P, Binoux M 1995 Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). Endocrinology 136:5485–5492[Abstract]
  33. Cohen P, Lamson G, Okajima T, Rosenfeld RG 1993 Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth. Mol Endocrinol 7:380–386[Abstract]
  34. Kaicer E, Blat C, Imbenotte J, Troalen F, Cussenot Ol, Calvo F, Harel L 1993 IGF binding protein-3 secreted by the prostate adenocarcinoma cells (PC-3) differential effect on PC-3 and normal prostate cell growth. Growth Regul 3:180–189[Medline]
  35. Perkel VS, Mohan S, Baylink DJ, Linkhart TA 1990 An inhibitory insulin-like growth factor binding protein (In-IGFBP) from human prostatic cell conditioned medium reveals N-terminal sequence identity with bone derived In-IGFBP. J Clin Endocrinol Metab 71:533–535[Abstract]
  36. Van Buul-Offers SC, Reijnen-Gresnigt R, Bloemen R, Hoogerbrugge C, Van den Brande JL 1995 Co-administration of IGF-binding protein-3 differentially inhibits the IGF-I-induced total body and organ growth of Snell dwarf mice. Prog Growth Factor Res 6:377–383[CrossRef][Medline]
  37. Murphy LJ, Molnar P, Lu X, Huang H 1995 Expression of human insulin-like growth factor-binding protein-3 in transgenic mice. J Mol Endocrinol 15:293–303[Abstract]
  38. Mohseni-Zadeh S, Binoux M 1997 Insulin-like growth factor (IGF) binding protein-3 interacts with the type 1 IGF receptor, reducing the affinity of the receptor for its ligand: an alternative mechanism in the regulation of IGF action. Endocrinology 138:5645–6548[Abstract/Free Full Text]
  39. Velez-Yanguas MC, Kalebic T, Maggi M, Kappel CC, Letterio J, Uskokovic M, Helman LJ 1996 1{alpha}, 25-dihydroxy-16-ene-23-yne-26, 27-hexafluorocholecalciferol (Ro24–5531) modulation of insulin-like growth factor-binding-3 and induction of differentiation and growth arrest in a human osteosarcoma cell line. J Clin Endocrinol Metab 81:93–99[Abstract]
  40. Moerman EJ, Thweatt R, Moerman AM, Jones RA, Goldstein S 1993 Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts. Exp Gerontol 28:361–370[CrossRef][Medline]
  41. Rajah R, Valentinis B, Cohen P 1997 Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-ß1 on programmed cell death through a p53- and IGF-independent mechanism. J Biol Chem 272:12181–12188[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Chem.Home page
J. van Doorn, C. M. Hoogerbrugge, J. G. Koster, R. J. Bloemen, K. Hoekman, A. H. Mudde, and S. C. van Buul-Offers
Antibodies Directed against the E Region of Pro-Insulin-like Growth Factor-II Used to Evaluate Non-Islet Cell Tumor-induced Hypoglycemia
Clin. Chem., October 1, 2002; 48(10): 1739 - 1750.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Sato, M. Nakamura, D. H. Cho, S. J. Tapscott, H. Ozaki, and K. Kawakami
Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1
Hum. Mol. Genet., May 1, 2002; 11(9): 1045 - 1058.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smink, J. J.
Right arrow Articles by van Buul-Offers, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smink, J. J.
Right arrow Articles by van Buul-Offers, S. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals