Pituitary-Directed Leukemia Inhibitory Factor Transgene Causes Cushing’s Syndrome: Neuro-Immune-Endocrine Modulation of Pituitary Development

Hiroki Yano, Carol Readhead, Masahiro Nakashima, Song-Guang Ren and Shlomo Melmed

Department of Medicine Cedars-Sinai Research Institute University of California Los Angeles School of Medicine Los Angeles, California 90048


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leukemia inhibitory factor (LIF) regulates the mature hypothalamic-pituitary-adrenal axis in vivo. In vitro, LIF determines corticotroph cell proliferation and induces POMC transcription. To explore LIF action on pituitary development, transgenic mice expressing LIF driven by the pituitary glycoprotein hormone {alpha}-subunit ({alpha}GSU) promoter were generated. Transgenic mice exhibited dwarfism with low IGF-I (29 ± 9 ng/ml vs. wild type (WT) 137 ± 16 ng/ml; P < 0.001), hypogonadism with low FSH (0.04 ± 0.023 ng/ml vs. WT 0.63 ± 0.18 ng/ml; P < 0.001), and Cushingoid features of thin skin and truncal obesity with elevated cortisol levels (86 ± 22 ng/ml vs. WT 50 ± 14 ng/ml; P = 0.002). Their pituitary glands showed corticotroph hyperplasia, striking somatotroph and gonadotroph hypoplasia, and multiple Rathke-like cysts lined by ciliated cells. LIF, overexpressed in Rathke’s pouch at embryonal day 10, diverts the differentiation stream of hormone-secreting cells toward the corticotroph lineage and ciliated nasopharyngeal-like epithelium. Thus, inappropriate expression of LIF, a neuro-immune interfacing cytokine, plays a key role in the terminal differentiation events of pituitary development and mature pituitary function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary is an essential organ for mammalian homeostasis after birth. During pituitary ontogeny, distinct cell types differentiate in a time-dependent serial pattern involving a precise balance between proliferation and differentiation of individual cells orchestrated by cell-cell and/or epithelial-neuroectodermal-mesenchymal interactions mediated by soluble factors and/or extracellular matrix (1, 2, 3, 4, 5). During this process, pituitary cells express a variety of genes in spatial and temporal patterns and differentiate to different phenotypes by critically turning on or off specific genes through several transcriptional factors, such as Pit-1 (6, 7), Lhx3 (pLim/mLim-3) 98–100, Rpx (Hesx1) (11, 12), P-OTX (Ptx-1) (13, 14), and Prop-1 (15), to produce cytological and functionally distinct pituitary cells. These individual committed cells synthesize and secrete unique hormones in utero, including ACTH, somatotropin (GH), TSH, PRL, and gonadotropin (FSH and LH) (5, 16). These tropic hormones coordinate central regulation of metabolism, growth, reproduction, and behavior and also play crucial roles in peripheral organ development and function.

Leukemia inhibitory factor (LIF), a pleiotropic cytokine, inhibits embryonic stem cell differentiation and primitive ectoderm formation in vitro (17, 18, 19). LIF transgenic expression directed by a tissue-specific promoter switched pancreatic noradrenergic sympathetic to cholinergic innervation (20) and also caused thymic and lymph node morphological interconversion (21). In the pituitary, LIF and its receptors are recognized in human fetal corticotrophs and somatotrophs as early as 14 weeks of gestation (22). LIF also potently synergizes with CRH to enhance POMC transcription and ACTH secretion in vitro (23, 24). In vivo, intraperitoneal injection of lipopolysaccharide or interleukin-1 induces both LIF and LIF-receptor gene expression in the murine hypothalamus and pituitary, concomitantly with ACTH induction (25, 26, 27). This immune-neuro-endocrine interface is abrogated in mice harboring a disrupted LIF gene (28, 29). Pituitary-directed LIF transgenic mice, utilizing a rat GH promoter, exhibit dwarfism and persistent Rathke’s cysts (30). These findings lead us to hypothesize that LIF plays a key role in pituitary development, subserving both adenohypophyseal ontogeny and mature pituitary function.

In this paper, we demonstrate that early pituitary overexpression driven by the {alpha}GSU promoter (31, 32) causes thin skin, truncal obesity, corticotroph hyperplasia, and hypercortisolism, consistent with Cushing’s disease. These mice also exhibited central hypogonadism, dwarfism, and mild hypothyroidism, with gonadotroph, somatotroph, lactotroph, and thyrotroph hypoplasia. These findings strongly imply that neuro-immune-endocrine interfacing molecules act as key players in terminal pituitary differentiating events, in addition to the known transcription factors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cushingoid Features and Growth Retardation of LIF Transgenic Mice
Eleven LIF transgenic mice, obtained from a total of 85 weaned pups, were indistinguishable from siblings at birth, but all except one exhibited severe growth retardation by 2 weeks after birth, and truncal obesity was evident by 1 month (Fig. 1cGo). Although wild-type (WT) males usually grow larger than females (33), no intergender weight differences were observed in the rotund LIF transgenic mice. Plasma IGF-I levels were extremely low in transgenic animals [transgenic (n = 6) 29 ± 9 (±SD) ng/ml vs. WT (n = 9) 137 ± 16 ng/ml, P < 0.001] (Fig. 1dGo). These mice had thin skin, and basal corticosterone levels were higher (n = 5) than in transgene-negative littermates (n = 10) (86 ± 22 ng/ml vs. 50 ± 14 ng/ml; P = 0.002). Corticosterone levels were incompletely suppressed by dexamethasone administration, indicating autonomous hypercortisolism with a Cushingoid phenotype (Fig. 2Go). Although subdermal fat appeared similar in WT and transgenic mice, more intraperitoneal fat was observed in LIF transgenic animals. Histologically, fatty tissue in transgenic mice consisted of both mature and brown fat. Adrenal glands of the rotund transgenic mice were similarly sized to WT littermates in spite of differences in body size. At necropsy, however, some adrenals obtained from transgenic mice were intensely hyperemic and larger than WT, supporting the biochemical observation of hypercortisolism.



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Figure 1. LIF Transgenic Mice Exhibited Dwarfism

a, Diagrammatic representation of genomic murine LIF and the transgene construct. b, Tail DNA was digested with XbaI and run on a 0.8% agarose gel. After transfer, the membrane was hybridized with a random-radiolabeled probe from the 670-bp EcoRI-BamHI mLIF cDNA fragment. Expected sizes of the genomic and transgenic fragments were 2.7 and 1.5 kb, respectively. M, marker; P, injected plasmid digested with XbaI as control. c, Pituitary-directed LIF transgene attenuates growth. LIF transgenic mice (10 weeks old) were rotund with symmetrical dwarfism and truncal obesity. Ten of 11 transgenic mice showed weight gain less than half of control weight gain, and male and female LIF transgenic mice were not different. Solid and dotted lines indicate linear regression of respective weights of male and female WT animals. d, Plasma IGF-I levels were attenuated in LIF transgenic mice (P < 0.001). Values shown are mean ± SD.

 


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Figure 2. LIF Transgenic Mice Exhibited Morphological and Biochemical Features of Hypercortisolism

a, Twelve-week-old transgenic male skin appendages were not affected, but transgenic dermis was thin and only attained the depth of the appendage layer. Collagen fibers were atrophic. LIF transgenic mice also showed increased brown and mature intraperitoneal fat. b, Basal plasma corticosterone levels were higher in the transgenic mice, and these levels were incompletely suppressed 4 h after dexamethasone administration (0.1 mg/kg body wt). Histological figures are hematoxylin and eosin stains of paraffin sections. Bars, 100 µm. Values shown are mean ± SD.

 
LIF Transgene Attenuates Gonadal Maturation and Thyroid Function
Although breeding was attempted between LIF transgenic and WT mice, no pregnancies were obtained. Reproductive organs of both dwarf male and female animals were poorly developed (Fig. 3aGo). Testes and ovaries were markedly reduced in size and the uterus was thread-like. Transgenic testes exhibited arrested spermatogenesis at the first meiotic division and greatly reduced number of Leydig cells in the sparse interstitium. The transgene was not expressed in testis. In the ovary, no antral stage follicles nor ovarian corpora lutea were observed. Plasma FSH levels (n = 6) were less than 10% of transgene-negative littermates (n = 9) (0.04 ± 0.023 ng/ml vs. 0.63 ± 0.18 ng/ml, P < 0.001) (Fig. 3bGo). FSH is required for ovarian follicle maturation but not male fertility (34), suggesting that plasma LH levels must also be reduced in the mice (33). In the thyroid gland, some transgenic animals had tiny disorganized follicles whose interstitium was filled with brown fat, while others had a more normal thyroid (Fig. 4Go). Although total T4 levels were reduced (2.63 ± 0.99 µg/dl (n = 6) vs. 4.54 ± 1.40 µg/dl (n = 9), P = 0.021), wide variations of T4 were observed in transgenic mice.



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Figure 3. LIF Transgene Blocks Gonadal Maturation

a, Transgenic testes, ovaries, and uterus (12 weeks old). Although transgenic mice exhibited primary sexual differentiation, they had severely reduced gonadal development after birth. b, Circulating FSH levels in transgenic mice were markedly suppressed (P < 0.001). Histological figures are hematoxylin and eosin stains of paraffin sections. Bars, 100 µm. Values shown are mean ± SD.

 


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Figure 4. LIF Transgene Impairs Thyroid Development

a, Thyroid follicles of some transgenic mice were hypoplastic and disorganized while some appeared normal histologically. b, Total T4 was reduced in transgenic mice. Histological figures are hematoxylin and eosin stains of paraffin sections. Bars, 100 µm. Values shown are mean ± SD.

 
Cell Composition of LIF Transgenic Pituitary
The LIF transgenic pituitary was less than half the size of WT (Fig. 5aGo). Histologically, both posterior and intermediate lobes of the LIF transgenic pituitary were not altered; however adenohypophyseal hypoplasia was prominent in the anterior lobe, and many cysts with cell debris were observed. Cells lining these cysts were ciliated, and their contents were periodic acid- Schiff positive (data not shown), resembling upper airway epithelium. The original Rathke’s cleft showed little invagination as was previously seen in the GH promoter-directed LIF transgenic mice (30), and these multiple cysts were less contiguous with each other. All pituitaries from the 10 rotund transgenic animals appeared identical histologically. In the transgenic pituitary anterior lobe, somatotrophs producing GH and lactotrophs producing PRL were strikingly reduced in number, but individual GH-producing cells stained more intensely (Fig. 5cGo). The hypoplastic transgenic pituitaries could be attributed to the extreme hypoplasia of these two lineages, which normally represent 70% of the adenohypophyseal cell population. Gonadotrophs were also markedly decreased in all transgenic pituitaries. Interestingly, although TSH producing cells were reduced in number, there were some differences among transgenic pituitaries (data not shown), similar to the inconsistent findings in thyroid tissue and plasma T4 levels. The anterior pituitary lobe of transgenic mice comprised mainly clustered ACTH-producing cells. Corticotroph hyperplasia was absolute and not a reflection of hypoplasia of the other cell lineages. In transgenic animals, ACTH-immunopositive cells accounted for ~65% of anterior pituitary cells, while WT animals harbored ~13% ACTH-positive cells. In transgenic pituitaries, double immunostaining for LIF and {alpha}GSU showed many LIF immunostaining cells colocalized with {alpha}GSU-positive cells in the anterior lobe, although less LIF staining was observed in the WT anterior pituitary (Fig. 5dGo). Single LIF-positive immunostaining in the posterior lobe indicated endogenous LIF expression in both WT and transgenic pars posterior. The LIF and {alpha}GSU double-positive cells implied transgene expression driven by the {alpha}GSU promoter. Thus, in the transgenic pituitary, corticotroph hyperplasia, hypoplasia of other lineages, and multiple cysts lined by ciliated epithelium were associated with LIF overexpression.





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Figure 5. Morphology and Histology of the LIF Transgenic Pituitary

a, LIF transgenic pituitary (14 weeks) was hypoplastic and weighed less than half of WT gland. b, Hematoxylin and eosin staining. Although posterior and intermediate lobes were not affected by LIF transgene, the anterior lobe was dramatically altered with cellular hypoplasia and multiple cystic changes. Epithelium lining the cysts was ciliated, and cystic cavities were filled with PAS-positive content and occasional cell debris. c, Immunohistochemistry for GH, PRL, bLH, and ACTH of frontally sectioned pituitaries. Somatotrophs, lactotrophs, and gonadotrophs in the transgenic pituitary were markedly reduced in number. However, the LIF-transgenic anterior lobe was mainly occupied by ACTH-immunopositive cells. d, Double immunostaining of LIF (alkaline-phosphatase staining, blue) and {alpha}GSU (peroxidase staining, brown). Only posterior lobe cells, which served as a positive control for LIF immunostaining, were immunopositive for LIF in the WT, but many LIF-positive cells were recognized in the anterior and posterior lobes of the transgenic pituitary. P, Posterior lobe; I, intermediate lobe; A, anterior lobe.

 
Sensitivity and Selectivity of the {alpha}GSU Promoter Activity
Green fluorescent protein (EGFP) (35, 36, 37) cDNA was employed as a reporter by replacing mouse (m)LIF cDNA in the LIF-transgenic construct to confirm the {alpha}GSU promoter activity. Nine Southern-positive mice were obtained from a total of 43 pups. Both genders of these EGFP transgenic animals showed normal growth and reproduction patterns (data not shown). The distribution of EGFP expression was reproducible and identical in all lines, indicating specificity of the 4.6-kb {alpha}GSU promoter for transgenic expression. In the EGFP transgenic pituitary (Fig. 6aGo), green fluorescence was recognized in ectodermal cells lining Rathke’s pouch as early as embryonal day E10, reflecting the sensitivity of EGFP (32). With development of pars anterior, increased fluorescence was observed around areas of active cellular proliferation in the anterior wall of Rathke’s pouch and weak fluorescence in the wall of the infundibular recess (E12.5). Later in embryonal development (after E14.5), strong fluorescence was noted in the anterior lobe and rostral tip in a speckled pattern, and cell fluorescence of both Rathke’s cleft lining and the posterior lobe became weaker. Interestingly, almost all cells in the adult anterior lobe expressed EGFP, but no expression was evident in either posterior or intermediate lobes (Fig. 6bGo). Although high-intensity cells must be considered thyrotrophs or gonadotrophs because of their respective {alpha}GSU hormonal structural components, other lineages also expressed this exogenous fluorescent protein driven by the transgenic 4.6-kb {alpha}GSU promoter, suggesting that most anterior pituitary cell types might have a potential to express {alpha}GSU. As expected, no EGFP fluorescence was observed in skin, thyroid, adrenal, liver, adipose tissue, or testis.



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Figure 6. Transgene Expression by the Mouse 4.6-kb {alpha}GSU Promoter

Fluorescent microscopy of frozen sections showing pituitary ontogeny (a, saggital section). Fluorescence was identified in cells lining Rathke’s pouch as early as E10.5. Later, fluorescence spread in the anterior lobe, and Rathke’s pouch expression weakened. In the 12-week-old adult pituitary (b, frontal section), all anterior lobe cells exhibited EGFP fluorescence with no fluorescence in either posterior or intermediate lobes. R, Rathke’s pouch; If, infundiblum; P, posterior lobe; I, intermediate lobe; A, anterior lobe.

 
Comparison between LIF Transgenic and WT Pituitary Assessed by Tandem Transgene Expression
To compare development of LIF transgenic to WT pituitary, as well as to monitor transgene expression and penetration, LIF and EGFP tandem transgenic mice were also generated. As expected, these mice showed the same phenotype, including pituitary histology, as the LIF-alone transgenic animals. Bright EGFP fluorescence was recognized in the anterior lobe of the adult tandem transgenic mice (Fig. 7aGo) and distributed more widely than the LIF immunoreactivity observed in the LIF transgenic pituitary (Fig. 5dGo). The tandem transgenic pituitary also showed similar corticotroph hyperplasia as observed in the LIF transgenic (Fig. 7bGo), and many of these cells also exhibited EGFP fluorescence. EGFP fluorescence of the tandem transgenic pituitary at E14.5 was only recognized in scattered cells, while almost all cells expressed EGFP in EGFP-alone transgenic pituitary (Fig. 7cGo). Lhx3 and Pit-1 expression was suppressed at E14.5 in the tandem transgenic pituitary. In contrast, in the LIF transgenic pituitary, POMC-expressing cells were more abundant than in the EGFP-alone transgenic pituitary.



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Figure 7. Developmental Effect of {alpha}GSU-Directed LIF Transgene

a, Fluorescent microscopy of the tandem transgenic pituitary (12 weeks old). Many cells exhibited bright fluorescence, indicating transgenic LIF expression. b, POMC expression in the 12-week-old EGFP-alone and tandem transgenic pituitary. The tandem transgenic pituitary exhibited corticotroph hyperplasia by in situ hybridization, similar to the ACTH immunohistochemical results in the LIF-alone transgenic pituitary. c, LIF transgene altered embryonic pituitary transcription factor expression at E14.5. The tandem-transgenic pituitary exhibited decreased Lhx3 and Pit-1 transcripts, in addition to {alpha}GSU itself (EGFP fluorescence). In contrast, the LIF transgene manifested abundant POMC accumulation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During pituitary ontogeny, five phenotypically distinct hormone-producing cell types are believed to arise from a common lineage (5, 13, 16, 32, 38, 39). The common progenitor cells originate in Rathke’s pouch, an invagination of oral ectoderm (16, 40). In the mouse, the initial hormonal evidence of pituitary organ commitment commences with expression of {alpha}GSU. Subsequently, POMC transcripts appear at E12, bovine (b)TSH at E12–13, bLH and bFSH at E15, and GH and PRL finally emerge at E16–17 (40, 41). In the LIF transgenic pituitary, the LIF product could divert progenitor cells to differentiate from Lhx3-dependent cell lineages (gonadotroph, thyrotroph, somatotroph, and lactotroph) to a Lhx3-independent cell lineage (42) (Fig. 8Go). Embryonic pituitary Lhx3 expression was, in fact, markedly attenuated in transgenic animals, supporting this hypothesis. From this cytological turning point, corticotrophs appear to develop and flourish in response to LIF translated from the transgene.



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Figure 8. Schematic Illustration of Divergent Pituitary Development of LIF Transgenic Mice

In the pituitary, LIF blocks the stream to Lhx3 and subsequent Pit-1-dependent cell lineages. Directly or indirectly, LIF stimulates the corticotroph lineage and also diverts pituitary progenitor cells to nasal epithelial cells. Solid arrows depict normal pituitary ontogeny (5 13 15 32 38 39 ).

 
CRH is mitogenic for corticotroph cells both in vitro and in vivo (43, 44), and LIF potently synergizes with CRH to enhance POMC transcription and ACTH secretion (23, 24, 27). LIF, in fact, induces a switch of proliferating corticotroph tumor cells (AtT20) toward a more differentiated phenotype (44). In the stressed adult pituitary, abundant LIF was recognized in the hypothalamus and pituitary (25, 26). In fact, several of the hormonal features of these transgenic mice resemble a neuro-endocrine stress response, whereby ACTH is elevated and gonadotropin and TSH are suppressed. Thus, LIF-induced hypercortisolemia may also have secondary inhibitory effects on the reproductive and thyroid axes.

Interestingly, although somatotroph, lactotroph, and gonadotroph hypoplasia was manifest, the thyrotroph was variably affected in the LIF transgenic lines. This observation might support the notion of a Pit-1-independent pathway for thyrotroph ontogeny (45). Pit-1 mRNA expression was, in fact, virtually undetectable in embryonic transgenic animals. Paradoxically, in the embryonal period, however, {alpha}GSU promoter activity in LIF transgenics appeared weaker than WT (Fig. 7cGo), possibly caused by the LIF construct itself. Even in this circumstance of a delicate balance between embryonal LIF and {alpha}GSU promoter activity, LIF driven by {alpha}GSU causes striking pituitary maldevelopment. However, the adult tandem transgenic pituitary showed abundant EGFP fluorescence, suggesting that the adult anterior hypophysis appears to manifest a different expression pattern than embryonic pituitary. In the transgenic animals, the widespread but low adenohypophyseal expression of {alpha}GSU is consistent with the observation that most pituitary tumor types express a low abundance of immunoreactive {alpha}-subunits regardless of their cell type. Alternatively, the presence of a GH-polyadenylation site within the transgene may also have altered the tissue-specific pattern of pituitary {alpha}-subunit expression (46).

While LIF stimulates the corticotroph lineage, it also causes progenitor cells to differentiate into ciliated cells, similar to the embryological differentiation of original ectodermal cells to ciliated columnar cells in the nasal cavity. Furthermore, invaginated ciliated cysts, rather than multiple cysts, were also observed in the GH promoter-driven LIF transgenic (30), supporting the notion that LIF causes some pituitary progenitor cells to follow an abnormal differentiation pathway. In the {alpha}GSU promoter-driven transgenic mice reported here, however, LIF overexpression occurred earlier (E 9.5) and showed more diffuse multiple cysts in the pituitary anterior lobe than the GH promoter-driven transgenic mice (E17.5), implying that progenitor cells are distributed diffusely at an early stage of pituitary development and are contiguous with Rathke’s cleft at later stages. Several additional differences in the two LIF-transgenic models, including cell-specific expression (GH vs. {alpha}GSU) and the intrapituitary concentrations of local LIF achieved, may also determine their differing phenotypes.

The observed failure of gonadal development presumably results from gonadotroph failure as evidenced by gonadotroph hypoplasia and suppressed FSH levels. Dwarfism is due to the lower number of pituitary somatotrophs as also evidenced by low IGF-I levels, but could also be partially attributed to the lack of thyroid hormone effect on GH transcription (47, 48, 49) or the hypercortisolism (50). The thin skin may be attributed to persistently high levels of corticosteroids, and fat deposition may be a result of both hypercortisolism and hypothyroidism. Thus, early pituitary overexpression of LIF inhibits development of gonadotrophs, lactotrophs, and somatotrophs, but favors differentiation and over-proliferation of corticotrophs. LIF is known to be a powerful cytokine mediating both the differentiation or dedifferentiation of committed cells (17, 18, 19, 20, 21) as well as regulating POMC transcription (22, 23, 24, 25, 26, 27, 28, 29). In the developing pituitary, LIF inhibits gonadotroph, thyrotroph, lactotroph, and somatotroph lineages, induces corticotroph development, and directly or indirectly converts some pituitary cells to ciliated epithelium. These findings strongly imply that, in addition to transcription factors, a soluble neuro-immune interfacing factor, such as inappropriately expressed LIF, regulates terminal differentiating events during pituitary ontogeny.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Construction and Creation of Transgenic Mice
The plasmid containing -5000 to +43 kbp of the mouse {alpha}GSU promoter (a kind gift from Dr. E. C. Ridgway, University of Colorado) was digested with KpnI and HindIII. For LIF transgenic mice, this 4.6-kbp fragment was ligated directly into pBluescriptII SK(-) containing mouse LIF cDNA and bovine GH polyadenylation signal between the EcoRI and NotI sites. For EGFP transgenic mice, mLIF cDNA was replaced by EGFP cDNA (pEGFP-1, Clontech Laboratories, Inc., Palo Alto, CA). For tandem transgenic mice, the insert for EGFP transgenic mice including the {alpha}GSU promoter, EGFP cDNA, and poly-A signal was ligated into the NotI-SacII site just after the poly-A signal of the plasmid for LIF-alone transgenic mice, using blunt-end and ligation (KpnI site of insert of EGFP transgenic; NotI site of plasmid for LIF transgenic) with SacII ligation. After being cut with KpnI and NotI or SacII, DNA inserts were separated from vector sequences by a sucrose-gradient. The fractions containing the inserts were dialyzed. Purified DNAs were microinjected into fertilized egg pronucleus, and the eggs were transplanted to oviducts of pseudopregnant mice to obtain transgenic mice. Genomic DNA digested with XbaI from tail biopsy of pups was screened by Southern blot analysis using a probe of EcoRI-XbaI fragment of mLIF cDNA or BamHI-NotI of EGFP.

Histology
After fixation in 4% paraformaldehyde, each tissue was dehydrated, embedded in paraffin, and serially sectioned at 4 µm. After deparaffinization, specimens were stained with hematoxylin-eosin and used for immunohistochemistry. The specimens were immersed in 0.3% H2O2/methanol and preincubated with 1% skim milk/PBS. Sections were incubated with primary antibodies (antihuman ACTH, 1:500 dilution (Dako Corp., Carpinteria, CA), antihuman GH, 1:500; antirat PRL, 1:500; antirat {alpha}GSU, 1:200; antirat bLH, 1:200; antirat bTSH 1:200 (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Rockville, MD); antihuman LIF, 1:100 (Biodesign Int., Kennebunk, ME) for 6 h at 4 C. After washing with 0.05% Tween 20/PBS, slides were incubated with polymerized secondary antibodies (Dako Envision system, Dako) or peroxidase-conjugated anti-guinea pig antibody (Dako) for bLH, and AEC (Dako) was used as the chromogen. Hematoxylin counter stainings were then performed, except when used for double staining. For double immunostaining, slides were washed in 0.1 M glycine HCl, pH 2.2, at 37 C overnight before the secondary reaction, and BCIP/nitroblue tetrazolium was used for the colorimetric reaction. Negative controls were prepared by replacing the primary antibody with nonimmunized rabbit serum.

Dexamethasone Suppresses Test and Hormone Measurements
Five LIF transgene positive mice and 10 negative litters were kept in unstressed conditions overnight and treated with water-soluble dexamethasone (Sigma, St. Louis, MO) ip at a dose of 0.1 mg/kg body wt immediately after collection of a basal blood sample (0900 h). Blood sampling was then performed after 4 and 24 h. Plasma corticosterone levels were measured in 10 µl plasma by RIA (ICN Biomedicals, Inc. Costa Messa, CA). The T4 and IGF-I RIAs were performed using Coat-A-Count Kit (Diagnostic Products Corp., Los Angeles, CA) and DSL 5600 (Diagnostic Systems Laboratories, Inc., Webster, TX), respectively, according to the manufacturer’s instructions. FSH RIAs were performed using kit components provided by the National Hormone and Pituitary Program after rat FSH iodination by iodogen.

Fluorescent Microscopy and in Situ Hybridization
Tissue was fixed in 4% paraformaldehyde, frozen in OCT compound (Sakura Finetechnical Co., Tokyo, Japan), sectioned at 8 µm, and dried overnight. After immersion in PBS, specimens were evaluated under fluorescent microscopy according to the manufacturer’s instruction (CLONTECH, Palo Alto, CA). Mouse Lhx3 cDNAs were created using pGEM-T vector systems (Promega Co., Madison, WI) by RT-PCR with WT mouse pituitary. Forward primer, 5'-TGCAAGGC-GGACTACGAAAC-3' and reverse primer, 5'-CAAGGCTCAAGTTGGTGTCT-3'. Mouse Pit-1 cDNA (51) was obtained from the ATCC. 35S-labeled cRNA probes for Lhx3, Pit-1, and POMC 26, were generated using RNA labeling kit (Amersham Co., Arlington Heights, IL). In situ hybridization was performed, as previously described (52), on 8 µm-sectioned frozen specimens, which showed EGFP fluorescence.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. D. Chen for assistance with the transgenic mice.


    FOOTNOTES
 
Address requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048-1865. E-mail: Melmd{at}CSHS.org

This work was supported by NIH Grants DK-50238 (to S.M.) and RR-12406 (to C.R.), and by the Doris Factor Molecular Endocrinology Laboratory.

Received for publication May 28, 1998. Revision received August 12, 1998. Accepted for publication August 14, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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