Regulators of the Proteasome Pathway, Uch37 and Rpn13, Play Distinct Roles in Mouse Development

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 2/2 mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13 2/2 mice showed increased T-cell numbers, resembling growth hormonemediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13 2/2 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

Requirement for Class II Phosphoinositide 3-Kinase C2α in Maintenance of Glomerular Structure and Function▿

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

<div><p>Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.</p></div

Mst1^−/− mice exhibit decreased incidence and severity of CIA.

<p>(<b>A</b>) Mice of indicated genotype (n = 11–13) were immunized with CII in CFA and observed for clinical signs of arthritis at the time points depicted on the X axis. Histological scores of synovial inflammation and cartilage/bone erosion were analyzed on day 45 after immunization. Data are expressed as mean (± SEM); * (p<0.05) indicates significant differences in comparison to WT littermates (Mann–Whitney U test). Similar data were obtained in two additional independent experiments. (<b>B</b>) Representative pictures of histological and radiographic signs of arthritis in the same mice as in (A), obtained on day 45 after immunization with CII (left and middle panels). Arrows of the H&E-stained sections of paw joints point to severe synovial inflammation and cartilage erosion in the Mst1^−/− animals. Representative µCT images of subchondral bone changes characteristic of arthritis were taken on day 45 after immunization (right panels).</p

Analysis of T cell apoptosis and functional immune responses in Mst1^−/− mice.

<p>(<b>A</b>) Increased apoptosis of activated Mst1^−/− splenic T cells in vitro. Early apoptotic T cells were quantitated by flow cytometry in after stimulation with the indicated mAbs for 48 hrs (n = 5 per genotype). (<b>B</b>) Analysis of apoptosis in unstimulated MST^−/− T cells. Early apoptotic T cells were quantitated by flow cytometry in CD44^low and CD44^high subsets of freshly isolated splenocytes (n = 5 per genotype). (<b>C</b>) The percentages of naïve (CD62L^highCD44^low), effector memory (CD62L^lowCD44^high), and CD279-, CD25-, and CD69-positive splenic CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). (<b>D</b>) Cytokine production by splenic CD62L^highCD44⁻ CD4⁺ T cells from WT and Mst1^−/− mice (n = 5 per genotype) was examined 48 hrs after stimulation with mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>E</b>) Th1 and Th2 polarized cells were generated from naïve splenic CD62L^highCD44⁻ CD4⁺ T cells after in vitro culture in polarizing conditions for 5 days. The percentages of effector memory (CD62L^lowCD44^high) CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). The percentage of sub-G0/G1 apoptotic cells was determined by the BrdU/7-AAD Flow kit and flow cytometry (n = 5 per genotype) following restimulation in vitro with plate-bound CD3 mAb (5 µg/ml) for 48 hrs. (<b>F</b>) Mst1^−/− deficiency leads to decreased Ag-specific adaptive immune responses in vivo. Mst1^−/− and WT mice (n = 6 and 9, respectively) were immunized with OVA in CFA. On day 14, serum samples were analyzed for OVA-specific IgG1 and IgG2a concentrations. Values and statistical significance are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098151#pone-0098151-g002" target="_blank">Fig. 2</a> and are representative of at least two independent experiments. Pre, preimmune serum.</p

Altered immune responses of activated Mst1^−/− T cells in vitro.

<p>(<b>A</b>) Th1 and Th2 cytokine production by splenic T cells from WT and Mst1^−/− mice was examined 48 hrs after stimulation with Con A (2.5 µg/ml) or mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>B</b>) IgE concentration was measured by ELISA in serum samples of WT (n = 8) and Mst1^−/− mice (n = 10). (<b>C</b>) Deficient upregulation of lymphocyte activation markers on Mst1^−/− T cells. Splenic T cells were stimulated with plate-bound CD3 mAb (1 µg/ml) for 48 hrs, stained for CD69 and CD95 (Fas/APO-1), and analyzed by flow cytometry (n = 5 per genotype). (<b>D</b>) Suppression of Mst1^−/− T cell proliferative responses in allogeneic MLR in vitro. Splenic C57Bl/6-Albino/129SvEv (H-2b) Mst1^−/− and WT T cells (responder cells; 5 mice per genotype) were stimulated with the indicated numbers of MHC-mismatched irradiated stimulator cells obtained from Balb/c mice (H-2d). Proliferation of the responder cells was assessed by [³H]-thymidine incorporation after 90 hrs of culture. Values and statistical significance are expressed as in Fig. 2 and represent results of at least two independent experiments.</p

Delayed cell cycle progression of Mst1^−/− T cells in vitro.

<p>(<b>A</b>) Mst1^−/− and WT T cells were activated for 36 hrs with the indicated stimuli, pulsed with BrdU, and analyzed by flow cytometry (n = 5 mice per genotype). The representative dot plots and fractional values show cell subsets residing in the indicated phases of cell cycle. (<b>B</b>) Nuclear DNA content at different stages of the cell cycle was determined by FACS analysis of splenic T cells stimulated for 48 hrs with plate-bound CD3 mAb (1 µg/ml) and stained with propidium iodide. Histograms are representative of 5 samples per genotype. Values on bar graphs and statistical significance are expressed as in Fig. 2. Similar data were obtained in two additional independent experiments.</p