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Endocrinology Vol. 146, No. 8 3438-3444
Copyright © 2005 by The Endocrine Society

Strain-Dependent Influences on the Hypothalamo-Pituitary-Adrenal Axis Profoundly Affect the 7B2 and PC2 Null Phenotypes

Juan R. Peinado1, Virginie Laurent1, Sang-Nam Lee, Bonnie W. Peng, John E. Pintar, Donald F. Steiner and Iris Lindberg

Department of Biochemistry and Molecular Biology (J.R.P., V.L., S.-N.L., I.L.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; Howard Hughes Medical Institute (D.F.S.), University of Chicago, Chicago, Illinois 60637; and Department of Neuroscience and Cell Biology (B.W.P., J.E.P.), University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Address all correspondence and requests for reprints to: Iris Lindberg, Ph.D., Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana 70112. E-mail: ilindb{at}lsuhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two null mouse models have previously been created to study the role of the prohormone convertase (PC2) and its helper protein 7B2; unexpectedly, the phenotypes of these two nulls differ profoundly, with the 7B2 but not the PC2 null dying at 5 wk. The genetic backgrounds of these two models differ, with the 7B2 null in a 129/SvEv (129) background and the PC2 null in a mixed C57BL/N6:129/SvEv (B6:129) background. Because background can contribute greatly to phenotype, we have here examined strain influence on the hypothalamo-pituitary-adrenal (HPA) axis and glucose levels in wild-type, 7B2 null, and PC2 null mice. Wild-type B6 and 129 mice differed in basal corticosterone and glucose levels. When 7B2 nulls were transferred onto the B6 background, they survived and showed greatly decreased circulating corticosterone and increased blood glucose levels, most likely due to the comparatively higher adrenal resistance of the B6 strain to ACTH stimulation. Circulating ACTH levels were increased over wild-type in the B6 7B2 null but did not reach levels as high as the 129 7B2 null. Conversely, when the mixed-strain PC2 nulls were bred into the 129 background at the N6 generation, they began to exhibit the Cushing’s-like phenotype characteristic of 129 7B2 null mice and died before 6 wk of age. Taken together, these results indicate that background effects are critical because they increase the phenotypic differences between the 7B2 and PC2 nulls and play a life-or-death role in the ACTH hypersecretion syndrome present in both 129 nulls.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROHORMONE CONVERTASE 2 (PC2) is a member of the eukaryotic family of subtilase serine proteases (reviewed in Refs. 1, 2, 3). PC2 is principally involved in the later processing stage of neuroendocrine precursors such as proopiomelanocortin (POMC) (2, 4, 5, 6) by cleaving its substrates at paired basic residues to generate mature active peptides. However, the presence of the small neuroendocrine acidic protein 7B2 is required in PC2-expressing cells, such as the intermediate lobe of the pituitary, to generate active enzyme (7). The creation of two null mutant mouse models for PC2 and 7B2 has confirmed that the lack of either PC2 or 7B2 results in deficient hormonal processing in vivo (7, 8, 9, 10). Indeed, 7B2 null mice develop a peculiar disorder of the hypothalamo-pituitary-adrenal (HPA) axis due to aberrant POMC processing (7). Thus, in the 7B2 null intermediate lobe, inactive PC2 fails to generate {alpha}MSH from ACTH, resulting in secretion of extremely high quantities of intact ACTH that in turn increase circulating levels of corticosterone and promote the development of a Cushing’s-like disease (10, 11). Whereas PC2 null mice accumulate enormous amounts of pituitary ACTH, this peptide is secreted at much lower levels into the bloodstream than in 7B2 nulls, and corticosterone levels are unaltered (12). It is possible that adrenal resistance to ACTH may also play a role in limiting steroid secretion in PC2 nulls. Furthermore, POMC synthesis is essentially eliminated in the anterior lobe of the 7B2 but not in the PC2 null (10), most likely because POMC synthesis in the anterior lobe is controlled through feedback inhibition by corticosteroid levels (reviewed in Ref. 13), and glucocorticoid levels are elevated only in the 7B2 null (10). The 7B2 null thus exhibits increased circulating ACTH, which is of intermediate lobe origin only. In contrast, circulating ACTH in the PC2 null arises from both pituitary lobes, is much less elevated compared with the 7B2 null, and occurs without loss of control of circulating glucocorticoid levels (10). These hormonal differences result in profound consequences in the phenotypes and fates of the PC2 and 7B2 nulls; the PC2 null is slightly runted, hyperproinsulinemic, and moderately hypoglycemic but otherwise relatively healthy, whereas the 7B2 null quickly develops a Cushing’s-like disease and dies at 5 wk of age (7).

It is possible that 7B2, which is more widely distributed than PC2 and indeed has been used as a neuroendocrine marker (reviewed in Ref. 14), may have physiological roles other than its interaction with PC2 that could contribute to a more severe phenotype; however, these extreme phenotypic differences are nonetheless perplexing. Because analysis of these nulls was not performed in mice of identical backgrounds [the PC2 nulls are in a 129/SvEv (129) and C57BL/N6 (B6) F2 hybrid background, whereas the 7B2 null is in a 129 background], we suspected that differences in background could contribute to differences in phenotype. Strain differences in pituitary intermediate lobe size have been noted between the B6 and 129 mice (15). Background-specific phenotypes have been observed in other mutant mouse models such as the Tay-Sachs and Sandhof disease models (16, 17) as well as in the epidermal growth factor receptor mutant mouse (18).

In the work presented here, we have examined the influence of strain on the HPA axis in wild-type (WT) animals as well as 7B2 and PC2 null mice. In addition to using the original 129 7B2 nulls and the original 129/B6 hybrid PC2 nulls, we bred PC2 and 7B2 nulls onto both the B6 and 129 backgrounds and evaluated phenotypic differences in circulating glucose, ACTH, and corticosterone levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Westphal et al. (7) generated the original 129 7B2 null line (Jackson Laboratories, Bar Harbor, ME), whereas Furuta et al. (8) generated the original mixed strain PC2 null mouse line. The 7B2 nulls in the 129 background and PC2 nulls in the 129:B6 hybrid were obtained by breeding 7B2 and PC2 heterozygote (HET) mice from each of these colonies; WT animals used as controls were obtained from the same crosses. The 7B2 nulls were bred into the B6 background by repeatedly backcrossing 129 7B2 HET female mice with commercially purchased inbred WT male B6 mice (Taconic Farms, Germantown, NY) as recommended (19). Fifth-generation (N5) 7B2 nulls then were bred with N5 B6 7B2 HETs, and the resulting animals were used for the experiments reported here. To obtain PC2 nulls in the B6 background, PC2 nulls in the 129:B6 hybrids were backcrossed for 10 generations into the B6 background using a similar strategy in the Steiner laboratory. Placement of the PC2 null on the 129 background was initiated by mating a PC2 HET female (50% 129 and 50% B6) with a WT 129 male. The female HETs produced were bred with male 129 mice. Sixth-generation (N6) HETs were then cross-bred to generate the 129 PC2 null animals used in the experiments described here.

The colonies were maintained in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care in cages containing one to three animals of the same age (within 1 wk), sex, and genotype. Animals were fed a LabDiet 50:15 mouse chow containing 11% fat. Animals were always killed between 4 and 6 wk of age. All protocols were approved by the Louisiana State University Health Sciences Center Animal Care Committee.

ACTH assay
Serum was prepared from trunk blood obtained from 4- to-5-wk-old animals (not anesthetized) and killed by rapid decapitation in a specially constructed mouse guillotine at approximately the same time of day (1030–1330 h). Sera were individually collected and stored at –70 C until use. Pituitaries were individually collected in microcentrifuge tubes; for ACTH analysis, pituitaries were homogenized using brief sonication in 250 µl of ice-cold 5 N acetic acid with 2 mg/ml BSA and frozen at –70 C before ACTH analysis. Fifty microliters of the serum or 10 µl of either a 1:200 (WT pituitaries) or 1:400 (null pituitaries) dilution of pituitary extract (prepared in Nichols kit dilution buffer; Nichols Institute, San Juan Capistrano, CA) were assayed in duplicate using the two-site Nichols Institute human ACTH1–39 assay kit. The method is specific for full-length ACTH1–39 and does not detect ACTH cleavage products.

Corticosterone assay
Sera were obtained as described for the ACTH assay from 4- to 5-wk-old animals. Corticosterone levels were determined using the ICN corticosterone assay (ICN Biomedicals, Costa Mesa, CA) according to the manufacturer’s instructions.

Glucose assay
Resting blood glucose levels (obtained between 1130 and 1330 h) were determined using a One Touch glucometer (Johnson & Johnson, New Brunswick, NJ). Trunk blood from rapidly decapitated mice was collected in 50-ml plastic tubes. An approximately 50-µl drop of blood was immediately applied to a test strip and the result recorded after 40 sec.

In situ hybridization of POMC
The in situ hybridization studies were carried out as described previously (10). B6 and 129 7B2 null mice were used for these studies. Sections were hybridized with 35S-uridine 5-triphosphate-labeled POMC cRNA. Controls for specificity of hybridization were carried out by pretreatment of brain sections with RNase A or the use of sense strand probes of the same size and specific activity. No specific labeling was observed in controls.

ACTH challenge
To ascertain possible differences in adrenocortical activity between 129 and B6 mice (obtained from Taconic), we performed ACTH challenge tests using different concentrations of ACTH in 5-wk-old male mice (0.2 or 1.0 µg/mouse, in 0.1 ml PBS containing 0.3% BSA) [ACTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)] (American Peptide Co, Sunnyvale, CA). Mice were blocked with a sc injection of 0.1 ml PBS containing 10 µg dexamethasone (Sigma, St. Louis, MO) 30 min before the ip injections of either ACTH or vehicle (PBS containing 0.3% BSA); six to seven mice per group were used. Serum was prepared from trunk blood obtained from mice killed by rapid decapitation 30 min after injection and corticosterone measured using the ICN kit.

Statistical methods
Data are expressed as mean ± SEM of the number of animals indicated in each table. Differences in value distribution were statistically validated using the two-tailed t test for unpaired data. Statistical analyses were performed using the program GraphPad Prism 3 for Windows (GraphPad Software, Inc., San Diego, CA). Differences were considered to be significant at values lower than P < 0.05. In all tables P is indicated for every statistical comparison. When differences in the parameters seemed to be due to the gender, additional statistical analyses (ANOVA with interaction analysis) were performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In an attempt to explain the phenotypic differences between the PC2 and 7B2 nulls, we felt it was important to address potential strain differences in blood glucose and HPA axis parameters (circulating ACTH and corticosterone levels) in both WT and mutant mice in their original strains. We also analyzed these parameters in the WT PC2 and 7B2 nulls crossed into the 129 and B6 backgrounds to assess possible strain contributions.

The ACTH hypersecretory state of the 7B2 null pituitary is maintained when the null is placed in a B6 background
Tables 1Go and 2Go depict the levels of pituitary ACTH and circulating ACTH respectively in WT mice as well as in 7B2 and PC2 nulls in the various backgrounds. In the case of WT animals 129 females exhibited decreased pituitary ACTH, compared with the other WT females. As expected, 129 7B2 and mixed-strain PC2 nulls exhibited extremely high concentrations of pituitary and circulating ACTH, compared with their corresponding WT animals. This condition was maintained when both nulls were placed in the B6 or 129 backgrounds (Tables 1Go and 2Go), although circulating ACTH was generally higher in the 129 strain. This latter observation indicates that the loss of either 7B2 or PC2 is responsible for the increase in pituitary ACTH accumulation and secretion. Circulating ACTH in 129 7B2 null mice was always higher than that of 129 PC2 nulls (Table 2Go) and was accompanied by a lower concentration of pituitary ACTH in 129 7B2 nulls when compared with 129 PC2 nulls (Table 1Go), possibly reflecting increased rates of secretion vs. retention of ACTH in the 129 7B2 null pituitary. This result is in accordance with the accumulation in PC2 null neurointermediate lobe of twice as many secretory granules than in the 7B2 null neurointermediate lobe (12).


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  		TABLE 1. Pituitary ACTH content in PC2 nulls, 7B2 nulls, and WT mice of different strains

 

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  		TABLE 2. Circulating ACTH levels in PC2 nulls, 7B2 nulls, and WT mice of different strains

 
The extremely high corticosterone levels characteristic of 129 7B2 nulls disappear when the 7B2 null is placed in the B6 background
Both male and female B6 WT animals displayed lower levels of circulating corticosterone when compared with 129 animals (Table 3Go). Although no obvious differences were detected between the adrenal sizes, B6 7B2 null mice exhibited more than 10-fold lower levels of circulating corticosterone than did 129 7B2 null mice (Table 3Go). The 129 7B2 null mice have previously been shown to exhibit adrenocortical hypertrophy accompanied by physiological signs of chronic hypercorticosteronism (7). PC2 nulls in 129-containing strains displayed elevated corticosterone levels when compared with B6 PC2 nulls, indicating that the 129 background may be more susceptible to the loss of both the PC2 and 7B2 alleles. Interestingly, corticosterone levels of 129 7B2 nulls were always higher than those of the 129 PC2 nulls. Taken together, these results suggest that background plays an important role in the production of the extremely high corticosterone levels of the 7B2 null.


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  		TABLE 3. Circulating corticosterone levels in PC2 nulls, 7B2 nulls, and WT mice of different strains

 
129 PC2 and 7B2 nulls display lower glucose levels than either null in the B6 strain
Given that 129 7B2 nulls succumb in part due to low glucose levels (11) and that the deletion of 7B2 is not lethal in the B6 background, we compared the blood glucose levels of WT animals with the various null models (Table 4Go). The 129 WT mice had lower glucose levels than B6 WT; 129 mice may thus be more susceptible to glucose-lowering mutations than are B6 mice. WT 129 females exhibited significantly lower circulating glucose levels than WT 129 males, although no differences were observed between the two sexes in 7B2 129 nulls. The B6 7B2 and PC2 nulls did not exhibit the very low glucose levels observed when either null was placed on the 129 background; moreover, both male and female 129:B6 PC2 null mice exhibited significantly lower glucose levels than their corresponding single-background B6 PC2 nulls. The glucose differences between the nulls in a 129 background and their respective WT controls indicate that background is a major determinant of glucose level in both nulls.


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  		TABLE 4. Glucose levels in PC2 nulls, 7B2 nulls, and WT mice of different strains

 
Adrenal resistance of WT mice to ACTH
To test adrenocortical responsiveness to ACTH stimulation in the 129 and B6 strains, mice were challenged with injections of synthetic ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) after a dexamethasone block. As expected, ACTH injection caused a dose-dependent increase in circulating corticosterone levels in both the 129 and B6 strains (Fig. 1Go). However, B6 mice showed a blunted response to ACTH stimulation when compared with 129 mice, supporting the idea that the adrenals of B6 mice are more resistant to ACTH stimulation than those of 129 mice.



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  		FIG. 1. Circulating corticosterone levels after ACTH challenge. Mice were pretreated with dexamethasone (10 µg/mouse sc) and 2 h later were challenged with either vehicle (0.1 ml PBS ip) or ACTH (0.1 ml PBS with either 0.2 µg or 1 µg/mouse ip). Blood samples were taken 30 min after ACTH injection (n = 6–7 per group). *, Significantly different from vehicle group, P < 0.0005, Student’s t test; **, significantly different from vehicle group, P < 0.0006; ***, significantly different from vehicle group, P < 0.0001.

 
B6 7B2 nulls express POMC in the anterior lobe of the pituitary
It has previously been shown that 129 7B2 null mice, but not PC2 null mice, lack POMC expression in the anterior pituitary (10), possibly because PC2 null mice do not manifest the high corticosterone levels responsible for suppression of POMC expression. To test this idea, we compared the expression of POMC in the anterior pituitary of B6 7B2 nulls, which also exhibit normal circulating corticosterone levels, with the expression of POMC in 129 7B2 nulls. The B6 7B2 null animals were found to abundantly express POMC mRNA in the anterior lobe (Fig. 2Go) supporting the idea that corticosterone suppression is responsible for the loss of anterior lobe POMC expression in the 129 7B2 null.



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  		FIG. 2. In situ hybridization histochemistry of POMC mRNA in mouse pituitaries. A, 129 7B2 null mice; B, B6 7B2 null mice. AP, Anterior lobe; IL, intermediate lobe.

 
The B6 background predominates strongly over the 129 background
As mentioned above, PC2 nulls in the 129 background die between 5 and 6 wk of age. During the process of breeding mixed-strain PC2 nulls into the 129 background, we monitored successive generations to assess the amount of 129 background that could result in restoration of Cushing’s-like disease symptoms. The first physical symptoms, such as thinning neck fur, were detected in the PC2 N5 null animals in the 129 background, although these symptoms only became apparent at older ages, such as 34 wk, and were not visible at earlier ages (Fig. 3AGo). The ACTH levels and corticosterone levels of these older animals resembled those of young 7B2 nulls in the 129 background (data not shown). Further breeding of the animals into the 129 background (N6) aggravated the Cushing’s-like disease symptoms (Fig. 3BGo) and resulted in morbidity identical with that found in 129 7B2 nulls. Conversely, one generation of breeding of 129 7B2 nulls into the B6 background resulted in complete protection from the lethal effects of the 7B2 mutation, supporting the protective effect of the B6 background. Neither PC2 nulls (Fig. 3CGo) nor 7B2 nulls placed in the B6 background (not shown) showed any symptoms of Cushing’s-like disease.



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  		FIG. 3. Representative images of PC2 nulls in the different backgrounds. A, A PC2 WT and null in the 129 background (N5). These 5-wk-old null animals are apparently healthy, although some symptoms of illness can be detected after 34 wk. B, A 5-wk-old PC2 WT and null in N6 129 background. The animals exhibit a strong Cushing’s-like phenotype at 5 wk, and all die between 5 and 6 wk. C, A 5-wk-old PC2 null in a B6 background.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The 7B2 and PC2 null mice represent two fascinating null models sharing the common trait of impaired ACTH homeostasis. However, they exhibit very distinct phenotypes, potentially due to contributions from the different backgrounds on which they were created. Because it is generally admitted that genetic background has a major role in determining mutant phenotype (reviewed in Refs. 20 , 21), in the work reported here, we attempted to determine both the effects of the missing alleles and the strain contributions to the 7B2 and PC2 null phenotypes. Thus, we studied different parameters of the HPA axis as well as the blood glucose levels in WT and nulls in mice on different backgrounds.

Strain-dependent differences in the HPA axis in WT mice
The analysis of circulating ACTH, corticosterone, and glucose in 129 and B6 WT mice and the mixed strain reveals distinct contributions of genetic background and sex to phenotype; for example, WT 129 mice exhibit significantly lower glucose levels than the B6 strain. It has been shown that mutations affecting the central nervous system (i.e. nitric oxide synthase) modify glucose parameters differentially in 129 and B6 mice (22). Together with other unknown parameters, strain-specific parameters such as higher resting glucose blood levels probably assist the survival of B6 PC2 nulls. Interestingly, 129 WT mice show sex-specific differences in certain parameters, with females exhibiting lower levels of glucose and slightly higher levels of corticosterone than males. These results imply that 129 animals are more susceptible to stress than are B6 mice in a sex-dependent manner, a hypothesis supported by the findings of lower pituitary ACTH levels in 129 females vs. males. The higher basal circulating ACTH and corticosterone levels of 129 mice may result in poor habituation of the corticosterone response during prolonged or repeated stress; stress experiments will be required to test this idea. Previous studies have shown that different rat strains show intrinsic HPA-related differences in reactivity to prolonged stress (23, 24). The 129 mice differ from other mouse strains in behavior (20, 21, 25, 26) and neuroanatomy (27, 28) and are usually considered passive (28). Consequently, 129 WT mice are likely to possess an HPA response very different from that of the B6 mice.

The 7B2 null and PC2 null phenotypes result from both allele loss and a background contribution
To assess the contribution of the 129 background to the 7B2 null phenotype, we compared several hormonal parameters of 7B2 null mice bred into both the 129 and B6 backgrounds. Our results indicate that the 129 7B2 null phenotype is a combination of both background features and allele loss effects. ACTH hypersecretion from the intermediate lobe, the major consequence of the loss of both 7B2 and PC2, appears to be greatly enhanced in the 129 background, suggesting strain-specific differences in pituitary control of secretion. ACTH hypersecretion also appears to more efficiently stimulate corticosterone production in 129 7B2 null mice than in B6 7B2 null mice, leading to the development of a severe Cushing’s-like disease only in 129 mice (7, 10). We hypothesize that certain aspects of the ACTH response/steroid synthetic pathway, from ACTH receptor expression to transcription factors controlling steroidogenesis, may be differentially controlled in 129 vs. B6 mice. Our finding of a blunted steroid response to ACTH challenge in WT B6 mice, compared with 129 mice, supports this idea. Lastly, regarding glucose levels, it is likely that the extreme hypoglycemia exhibited by 129 null animals, compared with B6 null mice, can enhance the effects of the Cushing’s-like symptoms (11), thus contributing to the mortality of the 129 7B2 null.

Because the PC2 null was originally made in a mixed background (129:B6) and expresses a phenotype distinct from that of the 129 7B2 null, we performed similar measurements as those described above, comparing mixed strain PC2 nulls with B6 and 129 PC2 nulls. Interestingly, the corticosterone levels of PC2 nulls in the mixed strain generally appeared to lie in an intermediate range between the 129 and B6 strains, indicating that strain is the greatest contribution to this measurement. The circulating corticosterone values in the three different PC2 nulls paralleled those of their respective WT controls, with a significant increase only in the case of the 129 male nulls, supporting the idea that the loss of PC2 itself does not increase steroid levels in any of these three backgrounds.

Glucose levels were low only in PC2 and 7B2 nulls in the 129 and 129:B6 strains when compared with WT animals. Thus, background appears to contribute along with the gene deletion to the decreased glucose levels of either null. These results are in accordance with the data of Furuta et al. (29), who demonstrated that impaired processing of proglucagon in the PC2 null results in the total loss of glucagon (but not of insulin), which negatively impacts blood glucose (29). Restoration of glucagon levels using an osmotic minipump was able to bring blood glucose back into the normal range (30). Our data confirm that blood glucose regulation is disturbed in 129 animals unable to generate glucagon. Interestingly, unlike the elevated ACTH/corticosterone phenotype, which is suppressed in the presence of B6 background, the hypoglycemic effects of PC2 ablation appear to be predominantly controlled by the 129 background.

We found that circulating ACTH levels of 129 PC2 nulls were not as high as those of 129 7B2 nulls, potentially indicating an allele-specific effect. However, during the process of breeding PC2 null mice into the 129 background, our third and fourth generations survived without any symptoms of disease, supporting the beneficial effect of the B6 background on phenotype. However, because we have not produced congenic 129 PC2 nulls, we cannot rule out the possibility that the very small amount of B6 background still remaining in the N6 129 PC2 nulls studied here could account for the difference observed in circulating ACTH levels, compared with 129 7B2 nulls. In addition, differences between PC2 and 7B2 nulls in the 129 strain could result from dominantly protective alleles inherited through the B6 background. In any case, our data showing gradual acquisition of susceptibility to allele loss as inbreeding proceeds into the 129 background support a dominant contribution of the B6 background over the loss of the PC2 allele observed in the mixed and B6-containing PC2 nulls.

Corticosterone levels in the different strains are directly related to POMC expression and ACTH secretion
We have previously observed using POMC in situ hybridization that POMC synthesis is essentially eliminated in the anterior lobe of the 7B2 but not the PC2 null (11). The data presented here indicate that anterior lobe POMC expression is recovered when the 7B2 null is placed in the B6 background. Because POMC synthesis in the anterior lobe is controlled by corticosterone levels (13), it is likely that the chronically elevated corticosterone level of the 7B2 129 nulls is responsible for the lack of POMC expression in this lobe. In agreement with this idea, adrenalectomy of 7B2 nulls restores POMC expression (10). The large alterations in circulating corticosterone produced by PC2 or 7B2 allele deletions in 129 mice may be related to the generally higher basal corticosterone level in 129 WT animals (when compared with B6 mice), indicating that corticosterone regulation may contribute to the lethal phenotype of PC2 and 7B2 129 nulls. Increasing evidence suggests an important interaction between circulating corticosterone and pituitary/brain dopaminergic systems (31, 32); our recent data suggest that in 129 7B2 nulls, increased blood corticosterone is associated with lower pituitary dopamine (10). The mechanism of this effect is obscure; it may potentially occur via an action on pituitary tyrosine hydroxylase (33) or by influencing dopamine release via neural mechanisms extrinsic to dopaminergic pathways.

The observed differences in basal corticosterone levels between 129 and B6 animals could play a role in the very different behaviors previously noted between WT and D2 receptor nulls in different backgrounds; 129 mice have been shown to exhibit markedly reduced scores for initiation of spontaneous movement, rearing, and rotarod performance, compared with B6 mice (34). A recent study (35) confirmed that 129 mice are less spontaneously active than B6 mice and react more strongly in a cued fear conditioning test.

Our data confirm that genetic background represents an important modifier of a major phenotypic defect of PC2 and/or 7B2 ablation: impaired processing of POMC, leading to increased blood levels of ACTH and corticosterone. Indeed, placement onto the 129 background can result in the early death of either the 7B2 or the PC2 null and an exaggerated difference between male and female animals. The study of the influence of background on the loss of the 7B2 and PC2 alleles provides insight into strain-specific hormonal regulatory mechanisms. Most likely due to strain-specific modifier genes, B6 7B2 null mice were able to maintain blood corticosterone homeostasis and exhibited some adrenal resistance to increased pituitary ACTH secretion, whereas 129 7B2 null animals could not accomplish this homeostatic task. Because susceptibility to PC2 loss did not appear until the sixth generation of breeding into the 129 background, it appears that a small amount of B6 background in PC2 nulls is sufficient to confer resistance to this mutation. Conversely, 7B2 nulls made congenic on the highly susceptible 129 background displayed a profoundly morbid phenotype, which was eliminated after a single cross onto the B6 background. It is interesting to note that the 7B2 and PC2 alleles both reside on chromosome 2 about 17 centimorgans apart; crosses of heterozygotes from the original strains result in double nulls with a sexually dimorphic pattern of corticosteronemia that is much less pronounced than the original 7B2 null but is more severe than the original PC2 null (Laurent, V., J. R. Peinado, and I. Lindberg, unpublished results).

In conclusion, our data suggest that whereas the lack of either the 7B2 or PC2 alleles can be lethal, or at least quite damaging, in certain backgrounds, strain-specific genetic contributions can considerably reduce the damaging effects. The manner in which B6-contributed modifier genes are able to influence the lethal phenotype of the 7B2 and PC2 nulls is an interesting topic for further study and is potentially relevant to the study of human susceptibility to Cushing’s disease.


   Acknowledgments
 
We thank Drs. Christoph Westphal and Philip Leder for providing the 7B2 founder animals for this study. We also thank Gregory Hubbard and Jan Schilling-Dufrene for assistance with animal handling.


   Footnotes
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants 49703 (to I.L.); Grant DA-08622 (to J.E.P.); National Institutes of Health Grants DK13914 and DK 20595; and a grant from Howard Hughes Medical Institute (to D.F.S.).

Present address for V.L.: Centre National de la Recherche Scientifique-ULP Unité Mixte de Recherche 7518, Laboratoire de Neurobiologie des Rythmes, Université Louis Pasteur, 12 Rue de l’Université, F-67000 Strasbourg, France.

First Published Online May 5, 2005

1 J.R.P. and V.L. contributed equally to this work Back

Abbreviations: B6, C57BL/N6; HET, heterozygote; HPA, hypothalamic-pituitary-adrenal; N5, fifth-generation; N6, sixth-generation; PC, prohormone convertase; POMC, proopiomelanocortin; WT, wild type; 129, 129/SvEv.

Received September 30, 2004.

Accepted for publication April 28, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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