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Endocrinology, doi:10.1210/en.2005-1241
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Endocrinology Vol. 147, No. 4 1632-1641
Copyright © 2006 by The Endocrine Society

Haplotype Insufficiency for Suppressor of Cytokine Signaling-2 Enhances Intestinal Growth and Promotes Polyp Formation in Growth Hormone-Transgenic Mice

Carmen Z. Michaylira, Nicole M. Ramocki, James G. Simmons, C. Kirby Tanner, Kirk K. McNaughton, John T. Woosley, Christopher J. Greenhalgh and P. Kay Lund

Departments of Cell and Molecular Physiology (C.Z.M., N.M.R., J.G.S., C.K.T., K.K.M., P.K.L.) and Pathology (J.T.W.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Cancer and Haematology Division (C.J.G.), The Walter and Eliza Hall Institute of Medical Research and the Cooperative Centre for Cellular Growth Factors, Parkville, Victoria 3050, Australia

Address all correspondence and requests for reprints to: Carmen Z. Michaylira, CB 7545, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545. E-mail: carmen_michaylira{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH may improve intestinal growth or function in patients with short bowel syndrome. Excessive trophic effects of GH or IGF-I may contribute to neoplastic growth or increased colorectal cancer risk in acromegaly. Identification of mechanisms that limit the tumorigenic potential of GH and IGF-I is desirable. Suppressor of cytokine signaling-2 (SOCS2) limits GH action on body and organ growth, but its role in GH action on intestine is unknown. We tested the hypothesis that SOCS2 limits GH-induced intestinal growth or neoplasia in vivo. GH-transgenic (GH-TG) mice were crossed with SOCS2 null mice to generate wild-type (WT) or transgenic (TG) mice with zero (HO-WT; HO-TG), one (HT-WT; HT-TG), or two (WT-WT; WT-TG) functional SOCS2 genes. No HO-TG mice were derived from crossbreeding. WT-WT, HT-WT, WT-TG, and HT-TG were compared. Body weight, small intestine and colon growth, and levels of jejunal IGF-I and sucrase-isomaltase mRNAs were assessed. Colon was analyzed for abnormal lesions. HT-WT did not differ from WT-WT. Compared with WT-TG, HT-TG had significantly increased body weight, small intestine growth, and local IGF-I expression and decreased sucrase-isomaltase expression. HT-TG colon spontaneously developed multiple hyperplastic and lymphoid polyps. GH-induced activation of STAT5 DNA binding activity was enhanced in intestine of SOCS2 null mice compared with WT control. Haplotype insufficiency for SOCS2 promotes trophic actions of GH in small intestine and promotes preneoplastic growth in colon during excess GH. Small variations in SOCS2 expression levels may significantly influence the outcome of therapeutic GH or acromegaly in intestine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EVEN THOUGH GH is approved as treatment for patients with short-bowel syndrome (SBS), the ability of exogenous GH to promote adaptive growth in intestine is controversial (1, 2). Numerous studies in patients with SBS treated with GH have reported variable results (3, 4, 5). In animal studies, enterotrophic actions of exogenous GH in intestine have primarily been observed in GH-deficient models such as hypophysectomized rats infused with GH (6, 7) or in models of GH excess such as GH-transgenic (GH-TG) mice with overexpression of GH transgenes (8). Studies in intact rats given total parenteral nutrition (TPN) identified postreceptor resistance to exogenous GH in the jejunal mucosa (9). In the TPN-fed animals, GH was shown to induce expression of suppressor of cytokine signaling-2 (SOCS2) in intestine. SOCS2 expression negatively correlated with intestinal mass in response to GH treatment, and in vitro studies demonstrated that SOCS2 inhibited proliferation of intestinal epithelial cell lines (10). The current studies aimed to assess the ability of SOCS2 to limit the trophic actions of GH in intestine in vivo. Mice with germ-line transmission of a mouse metallothionein-driven bovine GH gene (11), which we have previously characterized for small intestinal growth (8), were crossbred with mice having targeted disruption of the SOCS2 gene (12) to generate models that allowed us to assess the effects of SOCS2 deficiency on GH-transgene-mediated small intestinal growth.

SOCS2 belongs to a family of eight structurally related proteins, SOCS-1 to -7 and cytokine-inducible SH2-domain containing protein (CIS). These proteins contain an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a conserved 40-amino-acid motif on the C terminus referred to as the SOCS box (13). The SOCS box interacts with elongins B and C, which are members of the proteasome-degradation complex (14). SOCS may therefore serve to limit the duration of cytokine signaling by targeting activated signaling complexes for proteolytic degradation (14, 15). The phenotype of SOCS2 null mice suggests a role of SOCS2 in limiting the trophic actions of GH. Mice homozygous for SOCS2 gene disruption are 1.3–1.5 times the size of their wild-type (WT) littermates (12). The increase in weight becomes evident around 42 d of age, a time slightly later than the onset of the body overgrowth phenotype in the GH-TG mice (8, 11, 12). The increase in body weight in the SOCS2 null mice is associated with an increase in long bone length and a proportionate increase in the size of several organs (12). Although SOCS2 null mice have normal circulating levels of IGF-I, they do show characteristics of deregulated GH action, including increased local IGF-I production in some but not all organs studied, decreased production of major urinary protein in liver, and increased collagen deposition in the dermis (12). Recent studies showed enhanced body and organ growth responses to exogenous GH in GH-deficient SOCS2 null mice, providing direct evidence that SOCS2 limits GH action in vivo (16). The effect of SOCS2 on GH-induced intestinal growth was not analyzed in these studies.

Identifying novel mechanisms, which may limit the trophic effects of GH or its downstream effector IGF-I in the intestine, is of interest because considerable evidence implicates these factors in intestinal neoplasia (17). Patients with acromegaly, as a result of GH-secreting pituitary adenomas, have dramatically increased circulating levels of GH and IGF-I (18) and show an increased risk of developing precancerous polyps and colorectal cancer compared with normal individuals (19, 20, 21). The GH-TG mice used in these studies are a mouse model of acromegaly (22, 23). The present study therefore assessed the effect of partial deletion of SOCS2 on colon growth and examined colon for abnormal lesions and signs of dysplasia. Results from these studies demonstrate that haplotype insufficiency for SOCS2, associated with only a 40% reduction in normal SOCS2 expression levels, enhances the trophic actions of GH in small intestine and promotes formation of lymphoid and hyperplastic polyps in colon of GH-TG mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and genotyping
Derivation of mice with targeted disruption of one or both SOCS2 alleles was previously described (12). Mice homozygous for SOCS2 gene disruption (SOCS2 null mice) on a C57BL/6 background were provided by Drs. Douglas Hilton and Christopher Greenhalgh (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia) (12). Derivation of GH-TG mice that constitutively overexpress a bovine GH transgene comprising the entire bovine GH gene linked to a mouse metallothionein 1 promoter, was previously described (11). Hemizygous GH-TG mice on a C57BL/SJL background were originally provided by Drs. Richard Palmiter (University of Washington, Seattle, WA) and Ralph Brinster (University of Pennsylvania Veterinary School, Philadelphia, PA) (11). Mice homozygous for SOCS2 gene disruption (SOCS2-HO) and GH-TG mice were crossbred to generate SOCS2-HT/GH-TG (HT-TG) and SOCS2-HT/WT (HT-WT). A second round of crossbreeding (HT-TG x HT-WT) attempted to generate TG and WT mice with zero (HO-WT and HO-TG), one (HT-WT and HT-TG), or two (WT-WT and WT-TG) functional SOCS2 alleles. Genotyping for the WT or disrupted SOCS2 allele was performed on tail DNA by PCR with primers specific for the WT SOCS2 gene (sense, 5'-CGAGCTCAGTCAAACAGGTAGG-3'; antisense, 5'-GCTTTCAGATGTAGGGTGCTTCC-3') or for ß-galactosidase present in the disrupted allele (sense, 5'-GCAGACGATGGTCAGGATATCC-3'; antisense, 5'-GGATCGACAGATTTGATCCAGC-3'). Genotyping for the presence of the GH transgene was performed using primers specific for the bovine GH transgene (sense, 5'-TTGACACAAACATGCGCAGT-3'; antisense, 5'-GCACTTCATGACCCTCAGGTAC-3'). All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Effects of partial SOCS2 deficiency on body and intestine growth
Adult female mice (100–120 d) were studied to assess the effects of partial SOCS2 deficiency on body and intestinal growth. We focused on females because crossbreeding of SOCS2 null and GH-TG mice yielded greater numbers of female littermate pairs with appropriate genotypes for comparisons, and male HT-TG mice were required for breeding purposes. Body weights were monitored weekly between d 21 (weaning) and d 98. A sc injection of bromodeoxyuridine (BrdU) (200 mg/kg) (Sigma Diagnostic Inc., St. Louis, MO) was administered 90 min before mice were killed. For these studies, mice were anesthetized with sodium pentobarbital (200 µg/g) (Abbot Laboratories, Chicago, IL), the entire small intestine and colon were removed, and wet weight and length were assessed. Corresponding segments of jejunum were quick-frozen in liquid nitrogen and stored at –80 C for future analyses. Distal segments of jejunum (0.5 cm) were fixed in 4% formalin and paraffin embedded for morphometric measurements. Whole colon was placed on filter paper for support and opened longitudinally before fixing in 4% formalin. Fixed colon was later analyzed under a dissecting scope (Leica MZ 16 FA) to screen for abnormal lesions or screen for signs of dysplasia. A single observer, blinded to the genotype of the samples, counted the number of abnormal lesions. Colon was later paraffin embedded in Swiss-roll fashion for further histological examination.

Intestinal mass, villus height, and crypt depth
Frozen jejunum segments were thawed on ice, and a longitudinal cut was made along the entire segment. The segments were gently opened and scraped with a cold microscope slide resulting in a mucosal fraction and a submucosal/muscularis fraction (24). The wet weight of the mucosal fraction per unit length of jejunum was measured to assess the effects of partial SOCS2 deficiency on mucosal mass (mg/cm).

Paraffin-embedded samples of jejunum were sectioned at a thickness of 4 µm, placed on positively charged slides, and stained with hematoxylin and eosin (H&E). Crypt depth and villus height were measured in stained sections of jejunum by a single blinded observer. Measurements were performed using light microscopy and computer-assisted morphometry as described by Williams et al. (24). Six to 10 well-oriented villi and a similar number of crypts were measured per segment.

RNA extraction and Northern blot analysis
Total RNA was isolated from jejunum by the guanidine thiocyanate-cesium chloride method. Abundance of SOCS2, IGF-I, and sucrase-isomaltase mRNAs was assayed by Northern blot hybridization using 32P-labeled antisense cRNA or cDNA probes and methods detailed previously (25). The mouse SOCS2 probe was provided by Dr. Douglas Hilton (Walter and Eliza Hall Institute, Melbourne, Australia), and the sucrase-isomaltase probe was provided by Dr. Susan Henning (Baylor University, Houston, TX). Blots were reprobed for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (Ambion, Austin, TX) to control for RNA loading. Blots were scanned on a phosphorimager and abundance of specific mRNAs was quantified by ImageQuant software for Macintosh. Abundance of each mRNA examined was normalized to the abundance of control mRNAs.

Plasma IGF-I analysis
Plasma IGF-I levels were analyzed using the DSL-10–2900 ACTIVE mouse/rat IGF-I enzyme immunoassay kit (Diagnostic Systems Laboratories, Inc., Webster, TX) following the manufacturer’s instructions. Before assay, all samples were acid-ethanol extracted to remove IGF binding proteins.

Immunohistochemistry
Crypt proliferation was assessed by immunohistochemistry for BrdU using an immunostaining kit (BrdU staining kit; Zymed, San Francisco, CA) to label cells in S phase of the cell cycle based on incorporation of BrdU into DNA. Coded sections were scored under a light microscope to assess the number of BrdU-positive cells per crypt. Numbers of cells per crypt were counted so that data could be expressed as the fraction of total cells per crypt labeled with BrdU.

Because lymphoid polyps were observed in colon of HT-TG mice, immunohistochemistry was performed to assess the immune cell types present within the lesions. Formalin-fixed, paraffin-embedded colons were sectioned (4 µm) and placed on positively charged slides. Analysis for the presence of specific lymphocyte markers used the following antibodies: CD45R/B220 (B cell marker) (BD Biosciences, San Jose, CA), MAC-3 (macrophage marker) (BD Biosciences), and CD-3 (T-cell marker) (Dako, Carpinteria, CA). In brief, sections were deparaffinized in xylene, rehydrated in graded ethanols (95% for 4 min and 70% for 3 min) to distilled water, and rinsed in 0.05 M Tris buffer (pH 7.6). This was followed by methanol, peroxide block (30% peroxide 1:10 methanol for 10 min), and a rinse in water and Tris buffer (0.05 M Tris buffer, pH 7.6, for 3 min). Blocking was then performed with 2% fish gel (Sigma) in Tris before incubation with primary antibody (1:25 in 2% fish gelatin/Tris buffer, overnight, in a humid chamber at 4 C for 18–24 h). The following day, sections were washed in Tris (three times for 2 min each) and incubated in secondary antibody (biotinylated mouse antirat IgG 1/2{alpha}; BD Biosciences) (1:100) at room temperature for 90 min. Slides were incubated in avidin-biotin complex (Vectastain; Vector Laboratories, Burlingame, CA) for 75 min and labeling visualized using diaminobenzidine. Slides were counterstained in hematoxylin, dehydrated in ethanol, coverslipped, and analyzed with a light microscope.

Effects of SOCS2 deletion on signal transducer and activator of transcription 5 (STAT5) activation by GH ex vivo
Corresponding segments of small intestine (~8 cm) were isolated from SOCS2 null and WT mice. The segments were flushed with 1x PBS to remove luminal contents. One-centimeter segments were incubated at room temperature with serum-free medium with or without GH (10–7 M). After 30–90 min at room temperature, segments were Dounce homogenized (Wheaton Overhead Stirrer, Millville, NJ) in 1 ml ice-cold 1x Tris-buffered saline for nuclear protein extraction. Nuclei and nuclear proteins were extracted using standard methods (26). EMSAs were performed as previously described (26) on 30 µg protein using double-stranded oligomers corresponding to a consensus STAT5 binding sequence (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical analysis
Values are expressed as mean ± SEM. Absolute values were analyzed by two-way ANOVA to test for main effects of the GH transgene, SOCS2 gene deletion, or an interaction between GH transgene and SOCS2 deletion. Pair-wise comparisons were performed using Tukey’s test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Theoretically, crossbreeding of HT-TG and HT-WT should have yielded TG and WT mice with zero (HO-WT and HO-TG), one (HT-WT and HT-TG), or two (WT-WT and WT-TG) copies of SOCS2. Table 1Go compares the predicted and actual frequencies of different genotypes that were obtained. Surprisingly, no SOCS2-HO mice that were also GH-TG were obtained. The cross did, however, generate SOCS2-HT mice that were GH-TG. Because partial SOCS2 deficiency represents what may be considered small and more physiological reductions in SOCS2 expression, our analyses focused on WT-WT vs. HT-WT and WT-TG vs. HT-TG to assess whether haplotype insufficiency for SOCS2 altered intestinal growth effects of the GH transgene.


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  		TABLE 1. Frequency of genotypes generated from the SOCS2-HT/GH-TG (HT-TG) x SOCS2-HT/WT (HT-WT) cross

 
Effects of partial SOCS2 deletion on body growth
Figure 1Go shows growth curves for WT-WT, HT-WT, WT-TG, and HT-TG based on body weights measured between d 21 (weaning) and d 98 postnatally. Consistent with previously published reports (12), mice lacking one copy of SOCS2 but otherwise WT (HT-WT) showed no significant increase in body weight compared with their WT littermates (WT-WT). The GH-TG (WT-TG) mice showed the predicted increase in body weight at time points immediately after weaning (21–28 d) and continued to be significantly larger than their WT littermates. GH-TG mice lacking one copy of SOCS2 (HT-TG) did not show a significant change in body weight compared with their WT-TG littermates at time points immediately after weaning. These HT-TG mice, however, began to demonstrate a significant increase in body weight at around 49 d of age compared with WT-TG and continued to be significantly larger throughout the rest of the study period (Fig. 1Go).


Figure 1
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  		FIG. 1. Growth curves. Growth curves for female WT-WT ({triangleup}), HT-WT ({square}), WT-TG ({blacktriangleup}), HT-TG ({blacksquare}). Mouse body weights were measured weekly, and each point represents mean ± SEM; n = 6–10 per time point. WT-TG and HT-TG differed from WT-WT and HT-WT at all time points analyzed. a, P < 0.05 for HT-TG vs. WT-TG.

 
Loss of one copy of SOCS2 in GH-TG mice leads to increased growth in jejunum
Adult mice, 100–120 d of age, were analyzed to assess the effects of partial SOCS2 deficiency on body and intestinal growth. We chose to study adult mice because previous studies have indicated that in intestine, SOCS2 deletion plays a greater role in adult mice compared with young mice (unpublished results). As shown in Table 2Go, compared with their WT-WT littermates, adult WT-TG mice showed the expected increase in body, small intestine, and colon weight. Deletion of one copy of SOCS2 in HT-WT and HT-TG mice resulted in a 40% reduction in SOCS2 mRNA expression in intestine (Fig. 2Go). Endogenous SOCS2 expression was not altered in WT-TG and HT-TG vs. WT-WT and HT-WT mice. The HT-TG mice showed a significantly greater increase in body and small intestine wet weight compared with WT-TG mice having both copies of SOCS2. Deletion of one copy of SOCS2 led to a greater effect of transgene on body weight and small intestine weight compared with effects in WT-TG mice. There was also a trend for a greater increase in colon weight in HT-TG vs. WT-TG, although this increase did not reach statistical significance (Table 2Go).


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  		TABLE 2. Deletion of one copy of SOCS2 in GH-TG mice leads to increased body and small intestine weight

 

Figure 2
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  		FIG. 2. Reduced SOCS2 expression in jejunum of GH-TG mice lacking one copy of SOCS2. A, Representative autoradiograms of Northern blots probed for SOCS2 and GAPDH control mRNAs in jejunum; B, histograms show mean ± SEM of SOCS2 mRNA normalized to GAPDH mRNA; n = 4. a, P < 0.05 vs. other genotypes.

 
Partial SOCS2 deficiency results in increased mucosal growth and crypt cell proliferation in GH-TG mice
The effects of partial SOCS2 deficiency on mucosal mass, villus height, crypt depth, and crypt proliferation were measured in jejunum because previous observations demonstrated that the GH transgene increased these parameters in this bowel segment (8). Jejunal mucosal mass did not differ significantly in WT-WT and HT-WT but was increased in TG vs. WT mice, and this increase was greater in HT-TG mice than in WT-TG (Fig. 3AGo). Morphometric measurements of villus height and crypt depth were performed in jejunum to assess the effects of partial SOCS2 deficiency on the crypt-villus axis. HT-WT mice showed no significant difference in villus height and crypt depth compared with WT-WT mice (Fig. 3Go, B and C). Only the HT-TG mice showed a statistically significant increase in villus height and crypt depth compared with HT-WT (P < 0.001), and these parameters were greater in HT-TG than WT-TG (P < 0.001) (Fig. 3Go, B and C).


Figure 3
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  		FIG. 3. Effects of partial SOCS2 deficiency on mucosal mass, crypt depth, and villus height in jejunum of GH-TG mice. A, Histograms show wet mass (mg/cm) of jejunal mucosa for WT-WT, HT-WT, WT-TG, and HT-TG mice. Values are means ± SEM; n = 7. a, P < 0.05 for TG vs. WT control of the same SOCS2 genotype; b, P < 0.05 for HT-TG vs. WT-TG. B, Representative x4 bright-field microphotographs of H&E-stained jejunum from WT-WT, HT-WT, WT-TG, and HT-TG mice. C, Histograms show mean ± SEM of villus height and crypt depth compared with control of same SOCS2 genotype; n = 6–7. a, P < 0.05 vs. other genotypes.

 
Previous studies in adult GH-TG mice did not identify a difference in crypt cell mitoses compared with WT littermates (8). Consistent with these results, no significant change in percentage of BrdU-positive cells per crypt was observed in WT-TG mice compared with their WT-WT littermates. Deletion of one copy of SOCS2 from these transgenic animals, however, caused a small but significant increase in BrdU labeling, indicating that SOCS2 normally limits the proliferative effects of GH on intestinal epithelial cells (Fig. 4Go).


Figure 4
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  		FIG. 4. BrdU incorporation in jejunal crypt cells. A, Representative x15 bright-field microphotographs of BrdU-immunostained cells in jejunal crypts; B, histograms show mean number of BrdU-positive cells expressed as a fraction of mean number of cells per crypt total. Values are means ± SEM; n ≥ 6. a, P < 0.05 vs. other genotypes.

 
Increased local but not circulating IGF-I in GH-TG mice lacking one copy of SOCS2
To determine whether more pronounced GH-transgene-induced increases in jejunal growth in HT-TG vs. WT-TG mice were associated with an increase in circulating or locally expressed IGF-I, plasma IGF-I levels and jejunal IGF-I mRNAs were measured. As shown in Fig. 5AGo, the HT-WT mice showed no significant difference in plasma IGF-I compared with WT-WT mice. The WT-TG mice showed the predicted increase in plasma IGF-I compared with WT-WT mice. The HT-TG mice showed virtually identical plasma IGF-I levels as WT-TG mice, indicating that enhanced jejunal growth was not associated with more pronounced increases in plasma IGF-I (Fig. 5AGo). Figure 5BGo shows data on local levels of IGF-I mRNA expression in jejunum. Jejunal IGF-I mRNA levels did not differ significantly among WT-WT, HT-WT, and WT-TG. However, HT-TG mice showed a significant increase in local IGF-I expression (Fig. 5BGo). These results indicate that the increased trophic effects observed in intestine of HT-TG mice may be attributed at least in part to paracrine actions mediated by the increase in locally expressed IGF-I.


Figure 5
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  		FIG. 5. Plasma and jejunal IGF-I expression. A, Histograms show mean ± SEM of plasma IGF-I levels; n = 7. a, P < 0.05 vs. control of same SOCS2 genotype. B, Representative autoradiograms of Northern blots probed for IGF-I and GAPDH control mRNAs in jejunum (top) and histograms showing mean ± SEM of IGF-I mRNA normalized to GAPDH mRNA (bottom); n = 6. a, P < 0.05 vs. other genotypes.

 
Decreased sucrase-isomaltase expression in jejunum of GH-TG mice lacking one copy of SOCS2
Previous studies in Caco-2 cells have shown that SOCS2 overexpression results in increased sucrase-isomaltase expression and alkaline phosphatase activity (10), suggesting that SOCS2 may promote enterocyte differentiation. To determine the effects of partial SOCS2 deficiency on differentiation, sucrase-isomaltase mRNA was measured in jejunum. HT-WT mice showed no significant change in sucrase expression compared with WT, indicating that reduced SOCS2 expression alone has no effect on expression of this differentiation marker in the intestine of WT mice. In GH-TG mice, however, partial SOCS2 deficiency significantly decreased sucrase-isomaltase mRNA expression (Fig. 6Go). These results suggest that reduced SOCS2 expression in conjunction with GH excess results in diminished enterocyte differentiation.


Figure 6
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  		FIG. 6. Reduced sucrase-isomaltase in GH-TG mice with partial SOCS2 deficiency. A, Representative autoradiograms of Northern blots probed for sucrase-isomaltase and control GAPDH mRNAs in jejunum; B, histograms show mean ± SEM of sucrase-isomaltase mRNA normalized to GAPDH mRNA; n = 6. a, P < 0.05 vs. other genotypes.

 
Formation of aberrant lesions in colon of GH-TG mice lacking one copy of SOCS2
Acromegaly is associated with an increased risk of colorectal cancer (19, 20). Because the GH-TG mice used in these studies are a model of acromegaly, the colon was analyzed for the presence of aberrant lesions. No significant lesions were observed in WT-WT and HT-WT mice. A small number of possible lesions were identified under the dissecting microscope in colon of WT-TG mice but could not be histologically verified as polyps. The colon of HT-TG mice contained a large number of polyp-like lesions, which were primarily localized to the proximal colon (Fig. 7Go). Further histological examination demonstrated the presence of lymphoid aggregates as well as increased presence of hyperplastic mucosa or hyperplastic polyps in colon of HT-TG mice. To better characterize the lymphoid aggregates, immunohistochemistry studies using specific markers for T cells (CD-3), B cells (CD-45R/B220), and macrophages (MAC-3) were performed. The lymphoid aggregates showed no significant staining for MAC-3 or CD-3 (data not shown), whereas there was intense staining for CD45R/B220, indicating that these aggregates are composed primarily of B cells (Fig. 8Go). Lymphoid polyps also stained positively for BrdU, indicating ongoing B cell proliferation. Increased BrdU was also consistently observed in hyperplastic mucosa adjacent to polypoid lesions as shown in examples in Fig. 8Go.


Figure 7
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  		FIG. 7. Formation of colonic polyp-like lesions in GH-TG mice with partial SOCS2 deficiency. A, Representative x8 microphotographs of colon from WT-TG and HT-TG mice as visualized under a dissecting scope; B, histograms show mean number of polyp-like lesions present in colon. Values are mean ± SEM; n = 6–7. a, P < 0.05 vs. WT-TG and other genotypes.

 

Figure 8
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  		FIG. 8. Histology of colonic polyp-like lesions in HT-TG. A, Representative x10 bright-field microphotograph of H&E-stained hyperplastic polyp (top), serial section labeled with BrdU (middle), and immunohistochemistry showing presence of B cells in a nearby lymphoid aggregate but not within the hyperplastic polyp (bottom); B, representative x10 bright-field microphotograph of H&E-stained lymphoid aggregate (top), BrdU-labeled serial section showing the presence of proliferating cells within the immune aggregate (middle), and immunohistochemistry showing the presence of CD45R/B220-positive cells within the lymphoid polyp (bottom).

 
Increased STAT5 activation by GH in intestine of SOCS2 null mice
The effects of SOCS2 deficiency on GH signaling were addressed in small intestine of SOCS2 null and WT mice treated with exogenous GH. Activation of STAT5, a major downstream mediator of GH signaling, was assessed by EMSA. In response to GH treatment, the intestine of SOCS2 null mice shows enhanced binding of nuclear proteins to a STAT5 DNA binding sequence compared with WT control (Fig. 9Go).


Figure 9
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  		FIG. 9. Increased STAT5 activation in intestine of SOCS2 null mice treated with GH. A, Representative autoradiograms of EMSA for binding of nuclear proteins to a 32P-labeled STAT5 consensus sequence in small intestine of WT and SOCS2 null mice treated with GH or vehicle (V) for the indicated times at 0, 30, 60, and 90 min of treatment with GH; B, STAT5-response element (RE) binding activity in positive control jejunal extracts and competition with excess unlabeled STAT5-RE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies identified a novel effect of haplotype insufficiency for SOCS2 in modulating the actions of excess GH in the intestine. The results from these studies are relevant because GH is used in an increasing number of children to correct growth delay (27, 28) and is used clinically in patients with long-term TPN resulting from SBS (29). Results from the current studies suggest that the intestinal response to GH therapy may depend on the levels of expressed SOCS2.

In this study, we aimed to examine the effects of loss of one or both copies of SOCS2 on GH-transgene-dependent intestinal growth, because the GH-TG mice represent a model of acromegaly and we were interested in whether SOCS2 status impacts on normal or aberrant intestinal growth resulting from long-term GH excess. We were unable to generate GH-TG mice homozygous for SOCS2 disruption. This suggests that homozygous deletion of both SOCS2 genes and expression of the GH-TG is not compatible with embryonic survival. This was unexpected because the phenotypes of both the SOCS2 null and the GH-TG mice do not become evident until after weaning. When SOCS2 null mice were crossed with GH-TG mice overexpressing an ovine transgene, this also did not yield mice both homozygous for SOCS2 gene disruption and positive for the transgene (Greenhalgh, C. J., unpublished results). The mechanism by which GH-TG compromises survival of SOCS2 null embryos is unknown. Although body and organ phenotypes have been studied in GH-TG postnatally, there is no information about prenatal effects of GH-TG expression. However, recent studies suggest that both GH and SOCS2 play important roles in neurogenesis during embryonic development (30, 31), so it may be that the transgene is expressed in embryonic brain and, when combined with SOCS2 deficiency, has detrimental effects on the development of the central nervous system and compromises viability. This is speculative at present and will require further analysis. Nonetheless, the fact that combined GH overexpression and absolute SOCS2 deficiency appears to be embryonic lethal provides new evidence for interactions between GH and SOCS2 during embryonic development.

Studies in WT or GH-TG mice lacking one copy of the SOCS2 gene revealed significant effects of modest (40%) reductions in SOCS2 expression on body growth and intestinal responses to GH excess. This supports a concept that small, what may be considered physiological, variations in SOCS2 expression may profoundly impact the effects of GH on body or intestinal growth in clinical settings of GH therapy or situations of GH excess such as acromegaly. It is noteworthy that dual roles of SOCS2 as both an inhibitor and enhancer of GH action have been suggested. The potential enhancer role was based on small increases in body weight and weight of some organs in mice with widespread expression of supraphysiological levels of SOCS2 resulting from a ubiquitin promoter-driven SOCS2 transgene and findings that overexpressed SOCS2 bound to the GH receptor (32). Potential mechanisms suggested for these effects of SOCS2 overexpression were that supraphysiological levels of ectopically expressed SOCS2 may have a dominant negative effect to perturb the actions of endogenous SOCS2 or that overexpressed SOCS2 may perturb the actions of other SOCS family members that normally repress GH action (32). Because intestine was not examined in the mice that overexpress SOCS2, it could be of interest to generate crossbreeds of the GH-TG and SOCS2-overexpressing mice to assess how high levels of SOCS2 impact on phenotypic effects of GH excess in the intestine. However, the current studies demonstrating that small reductions in SOCS2 expression in SOCS2-HT enhance GH action strengthen the argument that endogenous SOCS2 generally serves as a negative modulator of GH action.

In small intestine, loss of one copy of the SOCS2 gene alone had no significant effect on growth or markers of differentiation. However, haplotype insufficiency for SOCS2 amplified the trophic effects of excess GH on jejunal mucosa based on major increases in jejunal mass, crypt depth, and villus height. The enhanced trophic effect of GH was also associated with decreased expression of sucrase-isomaltase, a marker of terminal differentiation of enterocytes. Thus, partial SOCS2 deficiency appears to favor a less mature epithelium. These results are consistent with previous observations in Caco-2 cells where SOCS2 overexpression resulted in increased sucrase-isomaltase expression and alkaline phosphatase activity (10). It is unlikely that SOCS2 is essential for enterocyte differentiation because the SOCS2 null mice do not display the dramatic phenotypes observed in mice with deletion in genes essential for differentiation such as Cdx-2 (33). Nonetheless, our previous in vitro findings and the current in vivo findings indicate that signaling pathways regulated by SOCS2 may interact with transcription pathways mediating sucrase-isomaltase expression. In this regard, it is of interest to note that the sucrase-isomaltase gene contains several potential STAT binding sites upstream of Cdx-2 binding sites known to regulate sucrase expression (10).

Partial SOCS2 deficiency in GH-TG mice resulted in increased crypt cell proliferation and local IGF-I mRNA expression in jejunum. Because IGF-I has well-established mitogenic effects on intestinal epithelial cells (34), the increase in crypt proliferation may be the result of increased action of locally expressed IGF-I.

The increased local IGF-I expression or trophic effects of the GH transgene observed in the intestine of HT-TG mice also may be the result of increased activation of early downstream mediators of GH receptor signaling. A likely candidate is increased activation of STAT5b. The growth phenotype observed in the SOCS2 null mice has been shown to require STAT5b (35). STAT5b has also been shown to be essential for GH-induced IGF-I gene expression in liver (36). Consistent with these previous observations, we observed evidence of enhanced STAT5 activation in intestine of SOCS2 null mice in response to exogenous GH treatment.

The presence of polyp-like lesions in the colon of GH-TG mice lacking one copy of SOCS2 indicates that SOCS2 plays an important role in limiting dysplasia resulting from GH excess in colon. Although hyperplastic polyps are not typically considered precancerous lesions, studies have demonstrated increased number of hyperplastic polyps in acromegalic patients who are also more susceptible to colorectal cancer (37). The presence of lymphoid aggregates is also of interest because recent studies have demonstrated an increased incidence of mucosal lymphoid aggregates in patients with colorectal cancer (38). The lymphoid aggregates observed in the HT-TG colon were primarily composed of B cells. A few cases of non-Hodgkin’s lymphoma have been reported in patients with acromegaly (39). Additionally, a study recently reported that induction of SOCS2 in B cell lymphoma cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent immunosuppressor, renders these cells less responsive to mitogenic stimulation (40). Additional studies assessing the role of SOCS2 in B cell pathogenesis may therefore be of interest. Because the mice used in these studies were relatively young, future studies will determine whether the colonic lesions observed in HT-TG mice progress to a more neoplastic phenotype in older animals. At present, little is known about the role of SOCS2 in tumorigenesis, but SOCS2 expression has been shown to be down-regulated in pulmonary adenocarcinoma (41). SOCS2 hypermethylation has also been observed in ovarian carcinoma cells and in patients with endometrial cancer (42, 43). Our studies indicate that additional analyses of the potential tumor repressor roles of SOCS2 are warranted.

GH is known to play an important role in postnatal growth. This is highlighted in the GH-TG mice, which begin to show a significant increase in body weight compared with WT littermates at points immediately after weaning (8). In these studies, we show that the increase in body weight shown by these transgenic mice is further enhanced by small reductions in SOCS2 expression resulting from disruption of one SOCS2 allele. These results are consistent with recent studies, which showed enhanced trophic effects of exogenous GH in GH-deficient mice lacking both copies of SOCS2 (16). However, the fact that haplotype insufficiency for SOCS2 enhances the growth response to GH excess supports a concept that small variations in SOCS2 expression may impact on body and intestinal phenotype in acromegaly. A novel finding of our studies is that the increase in body growth observed in the HT-TG mice did not manifest until the mice were 49 d of age. This indicates that small reductions in SOCS2 play an important role in limiting the trophic actions of GH in adult mice but has no effect at earlier stages in life. This is consistent with previous observations in SOCS2 null mice, where no significant effect on body weight was observed until the mice were 42–55 d of age (12). This finding is of interest given the current interest in the effects of the GH-IGF-I axis in aging and longevity. Future studies assessing the effects of SOCS2 on aging and longevity may, therefore, be of interest.

In conclusion, our findings in GH-TG mice with partial SOCS2 deficiency provide new evidence that endogenous SOCS2 normally limits intestinal growth and promotes enterocyte differentiation during GH excess in adult animals and that normal SOCS2 expression limits dysplasia in colon during GH excess. Together these studies provide new evidence that SOCS2 directly impacts on the actions of GH on intestine and suggest that the efficacy of GH therapy to promote intestinal growth in patients with SBS or intestinal abnormalities in acromegalic patients may be dependent on SOCS2 status.


   Acknowledgments
 
Outstanding technical assistance from C. Randall Fuller and Brooks Scull is gratefully acknowledged. We thank Katherine Kershaw and A. Christine Thomas for editorial assistance and assistance with figures.


   Footnotes
 
This work was supported by National Cancer Institute predoctoral research supplement 5 RO1 CA44684-14 (to C.Z.M.) and National Institutes of Health Grant DK-40247 (to P.K.L.). The study was facilitated by the histology and mouse cores of the Center for Gastrointestinal Biology and Disease (P30-DK-34987) and the DNA synthesis core of the Lineberger Cancer Center (CA 16086).

Disclosure summary: C.Z.M., N.M.R., J.G.S., C.K.T, K.K.M., and J.T.W. have nothing to declare. C.J.G. consults for AMRAD Corp. P.K.L. has previously consulted for Johns Hopkins University and consults for and has stock options with Saegis Pharmaceuticals.

First Published Online January 12, 2006

Abbreviations: BrdU, Bromodeoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GH-TG, GH-transgenic; GHR, GH receptor; H&E, hematoxylin and eosin; SBS, short-bowel syndrome; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TPN, total parental nutrition; WT, wild type.

Received September 30, 2005.

Accepted for publication December 28, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. Z. Michaylira, J. G. Simmons, N. M. Ramocki, B. P. Scull, K. K. McNaughton, C. R. Fuller, and P. K. Lund
Suppressor of cytokine signaling-2 limits intestinal growth and enterotrophic actions of IGF-I in vivo
Am J Physiol Gastrointest Liver Physiol, September 1, 2006; 291(3): G472 - G481.
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