ectopic expression of the crf-binding protein: minor impact on hpa axis regulation but induction of...

Journal of Neuroendocrinology, 1998, Vol. 10, 483–491

Ectopic Expression of the CRF-binding Protein: Minor Impact on HPAAxis Regulation but Induction of Sexually Dimorphic Weight Gain

D. A. Lovejoy*, J.-M. Aubry, A. Turnbull, S. Sutton, E. Potter, J. Yehling, C. Rivier and W. W. ValeClayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla CA 92037, USA.

Key words: binding protein, CRF, glucocorticoids, HPA regulation, LPS, urocortin.

Abstract

Corticotrophin-releasing factor (CRF) and urocortin possess a high-affinity binding protein. Althoughthe CRF binding protein (BP) can sequester these ligands and inhibit their activity, the endogenousactivity of this protein is not understood. Therefore, transgenic mouse lines that over-express theCRF-BP were created. The transgene was constructed by ligating rat CRF-BP cDNA (1.1 kb)between a mouse metallothionein-I promoter (1.8 kb) and a nonfunctional human growth hormonegene sequence (2.1 kb) in a modified pBR322 plasmid and microinjecting the transgene intoC57BL/6×SJL hybrid ova. The transgene was expressed in 50% in both male and female progeny.All transgenic lines were maintained by crossing transgenic animals with wild-type C57BL/6 mates.Reverse-transcriptase (RT) PCR of the CRF-BP transgene showed that it is widely expressed notonly in the brain and pituitary, but also peripheral tissues including the liver, kidney and spleen.Transgenic animals of both sexes showed significant increases in weight gain as established byanalysis of variance; however, the weight gain profiles for each sex were distinct. High levels ofcirculating CRF-BP were detected in the transgenic animals, but the basal ACTH and corticosteronelevels were not significantly decreased compared to wild-type littermates. The hypothalamo–pituitary–adrenal (HPA) axis was stimulated by systemic inflammation induced withlipopolysaccharide (LPS). An expected increase in transgene expression was observed and wasaccompanied by a significant attenuation of ACTH secretion at 3 h after LPS injection in thetransgenic males but not the females. These data suggest that HPA axis regulation is significantlyaffected only with very high circulating levels of CRF-BP. Moreover, this work supports previousstudies that implicate CRF and urocortin in the regulation of appetite and the binding proteinexpression may play a sexually dimorphic role in regulating this and other responses.

Corticotrophin-releasing hormone (CRF ) plays multiple roles appetite (14). Urocortin mRNA and immunoreactivity havebeen detected in the pituitary suggesting it may play a rolein the neuroendocrine, autonomic and behavioural responses

to stress through actions in the brain and the periphery. CRF in corticotroph regulation (15).A 37 kDa CRF/urocortin binding protein (CRF-BP), wasis synthesized in the parvocellular division of the paraventric-

ular nucleus (PVN ) and is the primary mediator of hypothal- originally identified and purified from human plasma (16,17) and subsequently cloned from rat and human brain (18,amo–pituitary–adrenal (HPA) axis activation. CRF also

modulates food intake (1, 2), affects arousal and learning (3, 19) and more recently, mouse brain (20). This solublecysteine-bridged protein binds both CRF and urocortin with4) and alters blood pressure (5). In 1995, we reported the

existence of urocortin, a novel mammalian CRF-like peptide an Ki of about 0.2 nM (6, 7, 21). In situ hybridizationexperiments indicate that prominent sites of mRNA expres-related to the fish urotensins-I, in rat (6), and later in human

(7). Cellular transduction of CRF and urocortin is mediated sion include the cerebral cortex, limbic system structures suchas the amygdala, and several sensory relays and cell groupsby one of two subtypes of G-protein coupled receptors

(8–13). Although urocortin possesses greater affinity for in the brain stem (22). In the anterior pituitary, CRF-BP islocalized in the corticotrophs, and can inhibit CRF-inducedCRF-R1 than does CRF, it possesses an order of magnitude

greater affinity for CRF-R2 (6, 7). Urocortin is particularly ACTH secretion in rat anterior pituitary cells in vitro.In humans the CRF-BP is present in the circulation, withpotent in lowering mean arterial pressure (6) and suppressing

Correspondence to: D. A. Lovejoy, 3.614 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, ManchesterM13 9PT, UK.

© 1998 Blackwell Science Ltd

484 Over-expression of CRF-BP in transgenic mice

the liver and/or placenta as possible source of secretion (17). 26.48±0.65 g, n=21). In contrast, the transgenic mice pos-sessed significantly different variances between the sexes asCRF-BP therefore may protect the corticotrophs from

increased circulating CRF levels which prevail during the determined by the F-test. The weights of the transgenicfemales (28.72±1.52 g, n=19) and the transgenic malesthird trimester of human pregnancy (23). In rodents however,

the CRF-BP has not been detected in peripheral organs or (32.80±0.86 g, n=25) were significantly different (P=0.0210) although the difference between means was less thanin the systemic circulation and seems to be restricted to the

brain and pituitary. Thus a clear physiological role for that shown by the wild-type animals.CRF-BP has not been established. In this study, we createda transgenic mouse over-expressing the CRF-BP to evaluate Hypothalamic–pituitary–adrenal axis hormone levels inthe impact of increased CRF-BP gene expression and to CRF-BP transgenicsinvestigate whether chronically elevated plasma levels ofCRF-BP can have a significant effect on the HPA axis ACTH and corticosterone levels were measured in the morn-

ing 1 h after lights on (07.00 h), and in the afternoon 2 hregulation.before lights off (16.00 h). During the morning determination,there were no significant differences between wild-type orResultstransgenic males or females. In the afternoon, while therewere no significant differences in the ACTH levels in eitherPhenotype and weight gainsex due to the high variances, combining male and femaledata revealed an attenuation of the ACTH level in theMale and female transgenic mice bred successfully within the

12-month period investigated and their life expectancy was transgenic animals (83±15 ng/ml ) compared with the wild-type mice (125±20 ng/ml ). Females showed the expectednot different from the wild-type littermates. Transgenic

animals have no overt phenotype apart from a tendency to higher plasma corticosterone levels of 22±8 ng/ml (morning)and 105±15 ng/ml (afternoon) compared to the male valuesbecome overweight (Fig. 2). The weight difference becomes

significant in females that are 1 year old when a sudden of 7±2 and 47±10 ng/ml, respectively. There were no signi-ficant differences between the wild-type and transgenicweight gain occurs after 8 months. In males, although some

of the over-expressors are heavier at all time points measured, animals for either sex at both time periods. Moreover, thecombined data for both sexes also yielded a statisticallythe difference is not significant for two age-grouped animals.

However, two-way analysis of variance indicated a significant insignificant result.(P=0.0158) difference between the weight gain profiles oftransgenic and wild-type groups (Fig. 2). Females showed Plasma binding protein and hormone concentrationsa significant (P=0.0049) weight gain by the 12th month tothe point where the mean weight was statistically similar to The CRF-BP was detected by LIRMA in all transgenic

animals and was below the level of detection in the wild-typethe males (Fig. 2). Generally among adult animals(>6 months), there was a small but significant (P=0.0260; mice. Males possessed a plasma binding protein concentration

of 37.5±2.5 ng/ml (n=8) and the females, 36.4±2.7 ng/mlFig. 2) difference in the mean body weight between the wild-type (28.83±0.61 g) and transgenic animals (31.36±0.85 g) (n=8). Zinc (25 mM ) in the drinking water resulted in a

40% increase of circulating CRF-BP in males to a mean levelas assessed by the Welch-corrected t-test. The differencesbetween the two populations were particularly manifest of 52.5±5.3 ng/ml (n=12; level of significance P=0.0218

using a two-tailed Welch-corrected t-test). The binding pro-within the variances of the weight of each sex. Among wild-type animals, the variances of the sexes were statistically tein concentration in the females remained statistically

unchanged at 31.8±5.2 (n=14).similar, although the difference between their weights weresignificant (P<0.0001; males 31.30±0.72 g, n=20, females There were no significant differences of plasma oestradiol,

mMT-1(1.8kb)

rCRF-BP(1.1kb)

hGH(2.1kb)

EcoRI KpnI SmaI BamHI BamHI SmaI SmaI EcoRI

1.1 0.7 0.4 0.7 0.9 0.7 0.6

cDNA 246 bp

transgene 520 bp

3’5’

F. 1. Schematic representation of the transgene. The mouse metallothionein-I promoter was used in conjunction with 1.1 kb of the rat CRF bindingprotein cDNA, and the human growth hormone gene. The expression of the growth hormone gene was inhibited by a nonsense mutation in thecoding sequence. The horizontal arrows at the bottom indicate the PCR amplification products for the primers used to distinguish the transgenetranscript from the endogenous CRF-BP transcript. Note that for the transgene primer pair, the 3∞ primer lies within the hGH sequence producing afragment which can only be amplified by the transgene template. Selected restrictions sites are shown by vertical lines. The size of each fragmentin kb is indicated.

© 1998 Blackwell Science Ltd, Journal of Neuroendocrinology, 10, 483–491

Over-expression of CRF-BP in transgenic mice 485

Age (months)

0 2 4 8 12

B

6 10 14

40

30

20

10

Transgenic

Wild-type

Wei

gh

t (g

)

P = 0.0049

Females

Age (months)

0 2 4 8 12

A

6 10 14

40

30

20

10

Transgenic

Wild-type

Wei

gh

t (g

)

Males

Wild-type

C

Transgenic

40

25

5

0

Transgenic

Wild-type

Wei

gh

t (g

)

45

30

10

50

35

15

20

F. 2. Difference in weight gain in wild-type and transgenic mice. () Body weights of male mice at different ages. There is a significant difference(P=0.0158) in the weight gain profile between the transgenic and wild-type animals as defined by a two-way analysis of variance. There were nosignificant differences between each genotype at any one time point. () Body weights of female mice at different ages. Between 8 and 12 months ofage, females showed a marked and significant (P=0.0049) increase in weight. At 12 months, the weight of the transgenic females was not significantlydifferent from the males. . Frequency distribution of the weights of all animals 6–12 months in age. There is a significant (P=0.026) differencebetween the mean weights of the transgenic and wild-type animals of both sexes as determined by a Welch t-test. The number of individuals (n) foreach point varied between 3 and 11. See text for further details.

testosterone and growth hormone concentrations between heart, lung, kidney, spleen, adrenal glands and testes.Transgene cDNA could not be found in the pancreas.wild-type and transgenic animals (data not shown).Examination of the small intestine yielded ambiguous results(data not shown). In situ hybridization of the liver confirmedTissue distribution of CRF-BP transgenethat the transgene is highly expressed in this organ and mayrepresent a major source of CRF-BP secretion in the circula-The transgene was widely expressed in a variety of peripheral

tissues (Fig. 3), which is consistent with the reported expres- tion (Fig. 4). Examination of RNAs isolated from severalbrain regions showed transgene expression in olfactory lobes,sion of a metallothionein promoter-regulated transgene. A

clear RT-PCR and hybridization signal was obtained in liver, pituitary, forebrain and brain stem (Fig. 3).



F. 4. Photomicrographs of the in situ hybridization of transgene bind-ing protein mRNA in liver. () Wild-type; () Transgenic ( line 839). Adramatic increase in mRNA levels is evident. The cRNA probe wassynthesized from a 500 Pst I fragment of a full-length rat binding proteincDNA clone. Magnification 400×.

blank

testes

adrenals

kidney

spleen

pancreas

lung

heart

liver

brainstem

forebrain

pituitary

olfactory lobe

Wild

-type

Transg

enic

blunted ACTH response at 3 h compared with the controlF. 3. Southern blots of RT-PCR-amplified transgene fragments from animals (1310±223 pg/mL, n=4). A decrease in plasmaperipheral and neural regions of transgenic ( line 854) and wild-type

ACTH in transgenic females (245±121 pg/ml, n=5) wasmice. The primers spanned a region between residue 27 of the exon 2 toevident at 1 h, compared with wild-type animalsresidue 109 of exon 3 of the rat binding protein producing the expected

product of 246 bases. The primers cross-hybridized with the native mouse (578±271 pg/ml, n=4). Maximal differences occurredbinding protein and therefore could amplify both the transgenic and between the two groups at 2 h where 275±41 pg/ml (n=4)native binding protein, thus the wild-type tissues are shown for compar- was measured in the transgenic female serum andison. These data indicate the presence or absence of amplified binding

918±282 pg/ml (n=5) in the wild-type serum. At 3 h, theprotein after 40 cycles of amplification. The transgene was expressed intransgenic values matched those of the wild-type animals.most tissues examined.

A quadratic function was used instead of a linear modelas the former provided a better fit of the plasma corticosteroneInflammatory stress by lipopolysaccharide administrationdata. Analysis of covariance of the LPS-induced glucocort-icoid data indicated that there were no significant differencesAfter LPS injection, the concentrations of circulating

CRF-BP increased from 84±8 ng/ml at 1 h to a peak value between either the wild-type and transgenic males, or betweenthe wild-type and transgenic females. As expected, wild-typeof 123±8 ng/ml at 2 h in transgenic males. In females,

CRF-BP increased from 64±9 ng/ml at 2 h after injection to females showed a significantly greater (P=0.0002) increasein plasma corticosterone than their male counterparts. The105±4 ng/ml at 3 h. Thus the greatest difference between

males and females in the plasma CRF-BP concentrations females showed a peak response of 533.9±56.3 pg/ml (n=4)at 2 h whereas the wild-type males showed the greatestoccurred at the 2 h mark.

Analysis of covariance of the plasma ACTH concentrations response at 3 h with a peak plasma corticosterone concentra-tion of 287.5±25.7 pg/ml (n=4). A similar significant (P=indicated a significant (P=0.0118) effect due to the presence

of the transgene (Fig. 5). No significant differences in ACTH 0.0024) trend was noted among transgenic animals. Thefemales possessed a peak of 546.9±41.2 pg/ml (n=5) at 3 hprofiles could be ascribed to the sex of the animals. In males,

following the LPS injection, the transgenic animals and the equivalent males had a value of 311.6±39.0 (n=4) pg/ml at the same time.(566±122 pg/ml, n=5) showed a significantly (P=0.0172)



paraventricular nucleus (PVN) as an integral regulator ofbody weight and appetite control where the release of CRF-like peptides may have inhibitory effects on feeding behaviour(2, 24, 25). CRF may mediate its appetite-related actions bydirect effects on NPY regulation in the arcuate nucleus (26),or TRH (27) and galanin (28) within the PVN, activation ofextrahypothalamic catecholaminergic systems (29), hypothal-amo–hypophyseal–adrenal (HPA) axis activation (30), or bysympathetic nervous system activation (1, 31–33). Recently,Spina and colleagues (15) showed that urocortin is a morespecific and potent inhibitor of appetite than CRF. In wild-type animals, CRF-BP expression is limited to a few cells inthe arcuate and anterior paraventricular nuclei, althoughthere is an absence of CRF-BP expressing cells in the medialparvocellular region of the PVN (22). Thus, enhanced expres-sion of the CRF-BP in brain regions outside of the PVN mayact to usurp free CRF or urocortin preventing their actionson appetite control centres. However, CRF appears to inhibitfeeding and parasympathetic activation only during periodsof stress. Chronic reductions of the CRF mRNA by eitherantisense oligonucleotides (34) or transgenic targeted inac-tivation of the CRF gene (35) have no long-term effects onweight gain. Our investigation is distinct from previous studiesin that the over-expression of the binding protein wouldpresumably affect urocortin actions as well. Thus, the greatermean body weight and individual variation may have beendue to a reduced inhibition of appetite during stressful periodswith both urocortin and CRF actions induction. However,

Hours after LPS injection

Saline 1 2 3

2000

1500

1000

500

0

FemalesWild-type

TransgenicPla

sma

AC

TH

(p

g/m

l)

Saline 1 2 3

2000

1500

1000

500

0

Males

NS

NS

P=0.02

F. 5. Increase in plasma ACTH concentrations after LPS injection. A previous in situ hybridization and immunocytochemistry stud-significant (P=0.0118) increase in ACTH concentrations due to the ies (22, 36) have shown only limited areas of overlap forpresence of the transgene was determined by analysis of covariance when

CRF-BP and CRF or CRF-R1. Thus, it is possible that thethe data for both sexes were combined. The number of individuals inCRF-BP exhibits actions, synaptic or otherwise, independenteach group was either four or five. However, at 3 h, the transgenic male

group showed a significant increase over its matched wild-type group. of any association with CRF and urocortin. In addition,See text for additional details. CRF-BP may modulate the effects of other CRF-like peptides

as immunocytochemical activity and PCR sequences suggestthe existence of novel ligands [(37); Lovejoy, unpublishedDiscussionobservations].

In any case, activation of the HPA axis does not appearOver-expression of the CRF-BP promotes a small but signi-ficant sexually dimorphic weight gain in male and female to be involved in the physiology of the weight differences

between the two groups of animals. Chronically elevatedtransgenic mice, compared to their wild-type littermates.Expression of the binding protein was enhanced in not only glucocorticoid concentrations can inhibit growth by several

mechanisms including suppression of the neuroendocrine–in the brain and pituitary but also most major peripheralorgans including the liver. The plasma ACTH concentrations growth axis (38) attenuation of nutrient uptake (39), syn-

thesis of insulin-like growth factor binding proteins (40), andin the transgenic animals were not significantly differentfrom the wild-type animals despite vastly increased plasma extracellular matrix proteins (41). Indeed, chronic stress can

retard growth and development in humans (42). However,CRF-BP concentrations. Plasma corticosterone concentra-tions were similar in both transgenic and wild-type animals. in our investigation, neither the plasma glucocorticoid nor

growth hormone concentrations were significantly differentTransgenic animals showed a significantly decreased ACTHresponse to LPS challenge although corticosterone values between the transgenic and wild-type animals. Further, the

vastly lowered plasma glucocorticoid levels which occur inwere not affected. These data suggest that the CRF-BP couldparticipate in an HPA-independent mechanism of body mice with the CRF gene nullified, do not exhibit significant

weight changes from their wild-type counterparts (35).weight regulation. In addition, the high expression of bindingprotein in plasma and liver in these animals suggests they While the data presented suggest a relationship between

the binding protein and weight regulation, caution is advisedcould be a useful model to understand CRF, urocortin andthe binding protein physiology in humans. in interpreting the physiological significance of these results.

In these transgenic animals, the CRF-BP is expressed inThe difference in the body weight observed in transgenicanimals compared to the wild-type mice suggests that numerous ectopic locations. The expression of a transgene is

dependent upon the primary sequence and length of theCRF-BP is involved in the complex and multifactorial regu-latory factors that participate in the regulation of growth and promoter, 5∞ and 3∞ flanking regions, reporter gene, presence

and position of introns and site of chromosomal integration.body weight. Recently, a spate of studies implicate the



The mouse metallothionein-I (mMT-I) promoter is not a ether or LPS injection did not vary significantly as a functionof the oestrous cycle. Other mechanisms probably play a rolestrong inducer of reporter genes in the brain, but typically

will induce expression in liver, testes and intestine and to a in the sexual dimorphism of HPA activation. For example,in female rats, angiotensin-II evokes an ACTH release twicelesser extent, pancreas, kidney and heart (43). Thus, the

growth pattern of our transgenic animals may be regulated that observed in males, whereas the reverse is true of ACTHstimulation of insulin (60). While it may be expected thatin part by peripheral sites. Over-expression of the CRF-BP

in the liver, for example, may have implications on the the binding protein may have some effect on sex steroidsynthesis by reducing potential HPA inhibition, we found nosynthesis and release of the insulin-like growth factors (IGF ).

CRF and urocortin are recognized as ligands for the CRF-BP, significant differences between oestradiol in female and testo-sterone in male wild-type and transgenic animals.but as evidence accumulates for the existence of additional

ligands, their peripheral expression cannot be ruled out. In this investigation, we have attempted to examine someof the peripheral aspects of CRF BP-mediated physiology.Stimulation of the HPA axis with LPS did effect a signific-

ant attenuation of plasma ACTH concentrations in the However, it is clear that the brain represents a major site ofaction. Preliminary in situ histochemical analysis of thesetransgenic mice. However, LPS would have a secondary effect

by also inducing the transgenic expression to a greater level. transgenic animals suggests that the transgene is expressed inthe dentate gyrus of the hippocampus, habenulae, cerebellum,LPS can directly activate the mMT-I promoter in a metal

ion-independent manner (44). Moreover, metallothioneins various nuclei of the brain stem and the anterior lobe of thepituitary gland (Aubry, unpublished data). Recently, metallo-can be induced by a variety of cytokines including interleukins

1 and 6 and interferon (45). Glucocorticoids can also induce thioneins have been localized to astrocytes by immunohisto-chemical methods (61), suggesting that the transgene may bemouse, sheep and human MT-1 expression (46–48) as would

occur during LPS treatment. Thus, although HPA activity expressed in these cells as well. Behan and his associates (62)have shown that astrocytes express the binding protein andmay be attenuated in part by the sequestering of CRF and

urocortin by the binding protein, the mechanism by which appear to secrete greater quantities of the protein than doneurones. As astrocytic metallothionein is induced by cyto-this could occur is not clear. Linton and associates (48)

evaluated the inhibitory effect of the binding protein on the kines (61), our metallothionein promoter-linked binding pro-tein transgene may be particularly useful for studies ofbioactivity of CRF in the perfused pituitary cell column

system. The results suggested that in vivo, the ACTH-releasing inflammation and the brain.The general lack of overt phenotype of these animalsactivity of CRF of placental origin could be inhibited but

that of hypothalamic origin was not. The crucial factor suggests at least two possibilities. The CRF-BP may not playa major regulatory role under normal conditions as the miceappeared to be the time the binding protein and CRF were

allowed to react with each other before reaching the pituitary. in the initial part of the study were not stress-challenged. Onthe other hand, the basal expression of the binding proteinTherefore, in our model, it is likely that although the circulat-

ing levels of CRF-BP are chronically elevated, the interaction in the wild-type animals may have been sufficient to satisfyall physiological requirements, and that the transgenic over-with ‘pulses’ of CRF released in the hypothalamic–pituitary

portal system may not be sufficient to significantly neutralize expression was effectively redundant. Thus, in summary, thetransgenic expression of the CRF-BP in liver and subsequentCRF actions on corticotrophs. A significant blunting of

ACTH secretion is obtained only when circulating CRF-BP secretion into plasma suggests these animals may be appro-priate to investigate attributes of CRF-BP physiology inare induced to even higher levels, as we observed after LPS

injection. In this experimental condition, we cannot exclude humans. At present, we cannot rule out possible physiologicaleffects ectopically expressed CRF-BP in other organ systemsthe possibility that expression of the transgene in the pituitary

also participates to CRF ‘buffering’ by autocrine and/or may have on normal physiology. However, the bindingprotein does appear to have a role in weight regulation eitherparacrine mechanisms. Moreover, ACTH release may be

partially regulated by urocortin. Urocortin mRNA has been indirectly through scavenging free urocortin, CRF or addi-tional unidentified ligand, or perhaps directly via a novelobserved in the pituitary of rats (15). Therefore, the binding

protein may complex with urocortin upon release from the mechanism.nerve terminals and sequester it from corticotroph activation.

We could not identify a sexually dimorphic effect of theMaterials and methodsbinding protein on the HPA axis. Sexual dimorphism of HPA

activation is well documented in several species. Studies have Development of transgenic miceindicated that this effect is mediated, in part, by enhanced The CRF-BP transgene was constructed by ligating the 1.1 kb fragment of

the rat CRF-BP into a modified pBR322 plasmid between a 1.8 kb portionactivation of the HPA axis by oestrogens (49–51) and aof the mouse metallothionein-I promoter, defined by the EcoRI-SmaI cleavagesuppressive effect by androgens (52–55). In female rats,sites (63), and a 2.1 kb fragment of the human growth hormone gene renderedstress-induced ovarian CRF is present in its highest concentra-nonfunctional by a mutation in the coding sequence (Fig. 1). This portion of

tions (56) and the CRF-R1 receptor transcription activity in the human growth hormone gene which contains a polyadenylation signalthe PVN peaks during pro-oestrus (57) Moreover, the human sequence was included because the polyadenylation signal sequence used in

CRF-BP transcription was not included in the CRF-BP sequence. The ratoestrogen receptor protein may bind to an oestrogenicCRF-BP sequence was defined by the sequence between the respective 5∞ andresponse element-like sequence in the 5∞ flanking region of3∞ SmaI and BamHI sites of the rat CRF-BP (19). Transgenic mouse linesthe human CRF gene in vitro (58). However, Guo and were created by microinjecting the plasmid into C57BL/6×SJL hybrid ova

coworkers (59) reported that the plasma glucocorticoid rise (DNX, NJ, USA). A total of nine founder animals were available forsubsequent breeding. Once the transgene was shown to be expressed in F1associated with stress resulting from acute cold-swimming,



and F2 generations and there were no overt phenotypic abnormalities between 32P. The reaction consisted of 150 ng of oligonucleotide, c 32P-ATP and 10 Upolynucleotide kinase (Boehringer-Mannheim) in a buffer of 50 mM Tris-lineages, three (839, 850 and 854) lineages were used for subsequent experi-

mental analyses. HCl, 10 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol and 0.1 mMspermidine at pH 8.2. The reaction was incubated at 37 °C for 3 h and purified

Mouse care and PCR identification of transgenic mice on a G-25 Sephadex spin column (Boehringer-Mannhiem). The final activitywas 1.497×106 c.p.m. The 50% formamide prehybridization/hybridizationMice from all three transgenic lines were housed in the Salk Institute animalsolution consisted of 10× Denhardts reagent, 5× SSC, 50 mM sodiumfacility, fed with standard chow, and kept on a 12-h dark, 12-h light schedule.phosphate (dibasic) 200 mg/ml salmon sperm DNA and 0.1% SDS. DNA wasThey were weighed at 2, 3, 6, 8 and 12 months. All animal experiments wereUV cross-linked (Stratagene) and prehybridized for 90 min at 42 °C. Theapproved by the Salk Institute Animal Care Committee. To stimulate expres-probe was added to achieve a final activity of 1–2×105 cpm/ml and hybridiza-sion of MT-1/rCRF-BP transgene, 25 mM ZnSO4 was provided in drinkingtion continued overnight at 42 °C. The hybridized membranes were washedwater. The genotype of transgenic progeny were identified by PCR. Genomicfor 2 min in 2×SSC/0.1% SDS at ambient conditions with rotary agitationDNA was extracted from tail tissue by standard methods. The 5∞ primerthen washed again for 20 min under the same conditions. They were washed(5∞-AGG AAC TGG ACT GGA CAC-3∞) was designed to hybridize to thea third time at 55 °C for 20 min with constant agitation.CRF-BP portion of the transgene whereas the 3∞ primer (5∞-GCC AGC ACT

CTC CCT GCT-3∞) hybridized to the proximal region of the hGH geneBlood collection(Fig. 1) to provide an amplification product of 520 bp. As this was an

artificial construct, the primers did not produce any amplification in the Plasma was obtained by retro-orbital bleeding from animals that were housedcontrol animals. The PCR conditions were: 4 min at 94 °C, followed by 34 individually in covered cages overnight. Transgenic mice from all threecycles of 45 s at 54 °C, 75 s at 60 °C and 120 s at 72 °C. The amplification lineages, 6 months or older were randomized. Control mice were nontransgenicwas terminated by 10 min at 72 °C. All reactions were performed on an littermates or nontransgenic age- and sex-matched animals. The blood wasMJ Research PTC-100 thermal cycler. In most cases, confirmation of PCR- collected at 07.00 h and 16.00 h within 45 s of initial disturbance of the cage,identified mice was established by the presence of binding protein in plasma and samples were immediately placed on ice into tubes containing EDTA.(see below). Plasma was stored at −20 °C until assayed.

Reverse-transcriptase polymerase chain reaction (RT-PCR) Hormone analysesTransgenic ( lines 839 and 854) and wild-type mice were quickly anaesthetized Radioimmunoassays: Adrenocorticotrophin (ACTH), growth hormone (GH),and decapitated. Brains were rapidly dissected and divided into olfactory corticosterone, oestradiol and testosterone were measured using commerciallobes, pituitary, forebrain, diencephalon/midbrain, cerebellum and brain stem. immunoassay kits. Corticosterone (ICN Biomedicals, Costa Mesa, CA, USA),The heart, lung, liver, pancreas, spleen, duodenum, kidney, adrenal glands oestradiol and testosterone (Diagnostic Proxucts Corporation, Los Angeles,and gonads were also removed. The tissues were frozen in liquid nitrogen CA, USA) were determined by radioimmunoassay whereas immunoradi-then stored individually at −80 °C until needed. Total RNA from all tissues ometric procedures were utilized for ACTH and GH (Nichols Institute, Sanwas extracted using RNAzol (Biotecx, Houston, TX, USA). The tissue Juan Capistrano, CA, USA). Data were evaluated using a 4-parametersamples were homogenized by sonication in 10 vol of extraction solution analysis for the immunoradiometric assays and a weighted linear regressioncontaining mercaptoethanol. The homogenates were incubated on ice for for the radioimmunoassays. The ACTH and GH assays possessed an intra-15 min, then centrifuged at 10 000 g for 20 min at 4 °C. The precipitated RNA assay coefficient of variation of <2% and interassay cofficient of variation ofwas washed once and dried briefly (5 min) in a Savant Speed Vac centrifuge 6% and 5%, respectively. The steroid radioimmunoassay interassay cofficientsand rehydrated in 500 ml of DEPC-treated water. The RNA preparations of variation were 8% for corticosterone and 5% for testosterone whilewere incubated with 1 ml DNase (Promega) at 37 °C for 30 min then interassay variation was 7% in both cases.re-extracted with 1 vol of buffered phenol and an additional 1 vol chloroform/ Ligand immunoradiometric assays (LIRMA): CRF-BP was measured usingamylalcohol (4951 v/v) and precipitated in 100% ethanol. a ligand immunoradiometric assay as described previously (21) using recom-

A total of 10 mg of the RNA with 10 ng dT (12–18, 10 ng/ml ) in a mixture binant human CRF-BP as a standard and rabbit antihuman CRF-BP serum.of 80 mM Tris-HCl (pH 8) and 80 m KCl were used in the annealing Briefly, samples were diluted in a PBS buffer containing 0.25% BSA, 0.01%reaction preceding first-strand cDNA syntheses. The mixtures were heated to Triton X100, 25 mM EDTA, and [125I-D-Tyr0]-hCRF (64) in a total volume90 °C and allowed to cool to 65 °C when they were transferred to a 52 °C of 0.2 ml. Precipitation was accomplished using rabbit hCRF-BP antiserumwater bath for 150 min. Synthesis was achieved using 1 ml MMLV reverse at a final dilution of 155000 and a secondary sheep antirabbit IgG serumtranscriptase (US Biochemicals) 10 ml 5∞ RT buffer, 1 ml RNAase inhibitor with 4% polyethylene glycol as a coprecipitant. Precipitates were collected by(Promega) and 18 ml of sterile water. The synthesis was allowed to proceed centrifugation and counted in a gamma counter (Micromedic Systemsfor 60 min at 42 °C. The cDNA samples were stored at 20 °C. Horsham, PA, USA). Results were evaluated using a standard 4-parameter

A 5-ml aliquot of each cDNA sample was subjected to PCR using primers analysis on a IBM-XT computer (A.I Research, San Diego, CA, USA) withto GAPDH and the CRF-BP transgene to ensure that the RNA was not the AGC software. Intra- and interassay coefficients of variation were <2%contaminated with genomic DNA, and to assess the quality of the cDNA. and 6%, respectively.For the cDNA analysis, GAPDH primers (Genosys) consisted of 5∞ primer,5∞-CTG GAG AAA CCT GCC AAG TATG-3∞ and the 3∞ primer, 5∞CAC In situ hybridizationCCT GTT GCT GTA GCC ATAT-3∞ at a final concentration of 20 pmol/50 ml

Mice from transgenic lines 839 and 584 were anaesthetized with chloralto provide an amplification product of 227 bp. The CRF binding proteinhydrate (0.3%) ip at a dose of 0.2 cc per mouse. About 2 min later, they wereprimers spanned a region from residues 27–36 of exon 2 (sense: 5∞ TAC GACperfused with saline, followed by 4% paraformaldehyde in 0.1 M borateCCT TTC CTG CTT TTC AGC 3∞) to residues 102–109 of exon 3 (antisense:buffer. Tissues were stored overnight at 4 °C in fixative containing 10%5∞ GAA GAC CCC ACC CTG GCA GTC GAT 3∞). A product of 246 bpsucrose. Frozen sections (30 mm thick) were cut on a Reichert microtome andwas expected. As the primers spanned an intron the presence of a largerstored in antifreeze solution (30% polyethylene glycol, 20% glycerol, and 50%amplified product would indicate genomic DNA contamination. The reactionNa3PO4 (0.05 M) until use. Tissue sections were mounted onto gelatin/polyconsisted of 2 mM MgCl2 at pH 9. A concentration of 100 mM was used for-lysine-coated slides, and hybridizations were carried out using conditionsall dNTPs. Amplitaq (Perkin Elmer) was used for all routine PCRs. Thedescribed previously (22, 33). 35S labelled cRNA probes were synthesizedcycling programme consisted of an initial 5 min of 98 °C, followed by 94 °Cfrom a 500 bp Pst I fragment (including 350 bp of the 3∞ untranslated region),(1 min), 55 °C (1 min), 72 °C (2 min) for 30 cycles followed by a final extensionof a full-length rat CRF-BP cDNA.period of 72 °C for 10 min. The conditions were similar for the transgene

primers (described above) although 40 cycles were used instead. Primers wereInflammatory stresssynthesized on a Pharmacia oligonucleotide synthesizer.

Southern blots: The amplified products from the PCR were resolved on a Lipopolysaccharide (LPS, 2 mg/kg) was injected intraperitonally (i.p.) intoCRF-BP over-expressing (line 854) and wild-type littermates. The mice were1.5% metaphor (FMC ) agarose gel. The fragments were transferred to a

Hybond N membrane using a combined transfer and denaturing solution divided into four groups. Saline injected ip served as control treatment. Threedifferent groups were decapitated 1 h, 2 h and 3 h after LPS treatment andof 0.5 M NaOH and 1.45 NaCl for 4 h. A 33-mer oligonucleotide (5∞ GAG

TTC ATT ACC ATC CAC TAC GAC CAG GTC TCC 3∞) designed to blood was collected on EDTA for hormone measurements. The control groupwas sacrificed 2 h after saline injection.hybridize with exon 3 of the rat CRF binding protein was end-labelled with



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ectopic expression of the crf-binding protein: minor impact on hpa axis regulation but induction of...

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