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. 2010 Oct 27;5(10):e13654.
doi: 10.1371/journal.pone.0013654.

Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development

Amin Al-Shami  1 , Kanchan G Jhaver, Peter Vogel, Carrie Wilkins, Juliane Humphries, John J Davis, Nianhua Xu, David G Potter, Brenda Gerhardt, Robert Mullinax, Cynthia R Shirley, Stephen J Anderson, Tamas Oravecz
Affiliations

Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development

Amin Al-Shami et al. PLoS One. .

Abstract

Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.

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Conflict of interest statement

Competing Interests: The authors of this article are or have been employees of, and received stock options from, Lexicon Pharmaceuticals, Inc. All of the above does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Generation of Uch37 and Rpn13 mutant mice.
(A) Gene trap mutation and location of primers for genotyping strategy and expression analysis of the Uch37 and Rpn13 genes. Arrows and arrowheads indicate transcription start sites. Primers A–D were used to identify the insertion site by nested PCR in the Uch37 clone. Primers E and F flank the genomic insertion site in the Rpn13 gene and amplify a product for the Wt allele. The LTR2 primer, complementary to the gene-trapping vector, amplifies the mutant allele in conjunction with primer H. SA, splice acceptor sequence; LTR, viral long terminal repeat; Neo, neomycin resistance gene. (B) Genotypic analysis of mice at the Uch37 and Rpn13 loci was performed by screening genomic DNA isolated from embryos and tail biopsy samples respectively. (C) Indicated gene transcripts were detected in the designated tissues by RT-PCR using primers G and H. (D) IHC was performed using the designated mAbs on the indicated thymus sections. (E) Expression analysis of Rpn13, Uch37 (in 13.5 days old embryos) and their respective neighboring genes. Indicated gene transcripts were detected by RT-PCR.
Figure 2
Figure 2. Uch37-deficient embryos show defects in brain development.
Histological examination of 13.5 days old embryos revealed that, when compared to age-matched Wt embryos, Uch37−/− mice showed undeveloped lateral and third ventricles in forebrain area, with associated disorganized development of cortex and midbrain as well as undeveloped mesencephalic vesicle and fourth ventricles in midbrain, with disorganized development of cerebellum and hindbrain.
Figure 3
Figure 3. The expression pattern of the Rpn13 protein is cell-type specific.
IHC was performed using anti-Rpn13 mAb on the indicated tissue sections.
Figure 4
Figure 4. The effect of Rpn13 deletion on proteasome function is tissue-specific.
Indicated proteasome activities were measured in protein lysates prepared from the designated tissues (left panels), or in purified 26S preparations with or without epoxomicin as controls (right panels). For the tissue lysates, data were obtained from 7–19 samples of each genotype pooled from at least four independent experiments giving similar results. 26S controls included 3 samples for each treatment. Enzyme activities were normalized to the mean of the Wt or control values and are presented as mean ± SEM. Numbers above bars indicate P values compared to Wts.
Figure 5
Figure 5. Rpn13 deficiency affects body weight and composition.
Body weights (A) and composition (B) were measured for the indicated mice at the time points and intervals shown. Minimum 3, maximum 38 mice were studied at each time point. Numbers above bars indicate P values compared to Wts. (C) Representative images of CAT-scan analysis of 16-weeks old male mice with the indicated genotype. An additional male and a female Rpn13 −/− mouse were also analyzed along with Wt littermates and showed similar increases in fat deposits as marked on the image.
Figure 6
Figure 6. Absence of Rpn13 results in abnormal sexual development.
Testes and ovaries from mice of indicated genotype and age were subjected to H&E staining. Arrows point to the following features: 1. Maturing sperms in the tubular lumen of the Rpn13 +/+ testis which are absent in the Rpn13 −/− section, and Leydig cell clusters present in both sections of 12-weeks old mice; 2. Spermatids and spermatogonia in the Rpn13 +/+ testis are largely missing from Rpn13 −/− mice, and hypertrophic Sertoli cells in the Rpn13 −/− testes of 3-weeks old mice; 3. Primordial follicles with ova and granulose cells present only in the Rpn13 +/+ ovary, and replaced by interstitial stroma in the Rpn13 −/− ovary of 12-weeks old mice; 4. Ova which are numerous in Wt and few in Rpn13 −/− mice at 3-weeks of age.
Figure 7
Figure 7. Deletion of Rpn13 alters T-cell development.
(A) Hematopoietic cell profile of the indicated tissues harvested from mice with genotypes designated on the figure. Data were obtained from 4–5 month old mice (n = 15–31) and are presented as mean ± SEM. Numbers above bars indicate P values compared to Wt mice. Mono, monocytes; Neutro, neutrophils. (B) Representative picture of thymi from mice of indicated genotype demonstrating difference in size of the organs. (C) Representative flow cytometric plots show normal CD4/CD8 mAb staining patterns of thymocytes from mice of both genotypes. Samples from 10 additional mice of each genotype gave similar results.
Figure 8
Figure 8. Rpn13 is essential for normal hormonal homeostasis.
(A) Serum concentration of the indicated hormones was measured in the mice designated on the figure. Data were pooled from at least three independent experiments giving similar results. Minimum 4, maximum 24 mice were studied in each group, and only male mice were used for testosterone measurements. Data are presented as in Figure 7A. (B) Expression of GH receptor and control GAPDH in the testes of mice with the indicated genotype. Western blot analysis was performed with the antibodies designated on the figure.
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