Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, Speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients. Citation Format: Chunli Wei, Yun Liu, Xiaoyan Liu, Jingliang Cheng, Jiewen Fu, Xiuli Xiao, Robb E Moses, Xiaotao Li, Junjiang Fu. The Speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing oncogene TWIST1 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-03.

<p>Supplementary Figure1-10, Table1-2 Figure S1. The relations of REGγ with Hippo-YAP signal pathway in colon cancer. A. The protein levels of Lats2 and p-Lats1 were unchanged in HCT116 and HT29 human colon cancer cells with REGγ knockdown. The expression of REGγ, Lats2 and p-Lats1 were measured by Western Blot. B. REGγ knockdown did not change the levels of p-YAP (S397) in HCT116 and HT29 human colon cancer cells. Figure S2. REGγ promotes degradation of Lats1. A. Silencing REGγ slowed down degradation of endogenous Lats1. REGγ sh-N or sh-R HCT116 cells were treated with cycloheximide (100μg/ml) for indicated time followed by Western Blotting. Quantitated results were plotted to indicate dynamic changes (Analysis of Variance, n=3, *p<0.05, **p<0.01, ***p<0.001). B. Ectopic expression of wild-type (WT), but not inactive mutant N151Y REGγ promoted the degradation of endogenous Lats1 in HEK293 cells. Figure S3. REGγ promotes YAP signaling via degradation of Lats1. A Silencing Lats1 restored the expressions of YAP target genes in HCT116 sh-R cells similar to those in HCT116 cells by RT-PCR analysis. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, **p<0.01, ***p<0.001). B. RNAi efficiently depleted Lats1 in HCT116 cells for experiments in A. HCT116 cells were harvested 72h after transient transfection of a control (Ctrl) or Lats1 small interfering RNA (siRNA) followed by Western blot analysis. C. Overexpression of Lats1 in HCT116 sh-N cells changed the expression of YAP target genes similar to the levels in HCT116 sh-R cells by RT-PCR analysis. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, *p<0.05, **p<0.01, ***p<0.001). D. Western blot analysis validated successful expression of exogenous Lats1 in experiments shown in C. E Figure S4. REGγ promotes proliferation of human colon cancer cells. A. REGγ deletion and YAP silencing inhibited cell proliferation of HCT116 human colon cancer cells to a similar extent. After 72 h of transfection with a control- siRNA or YAP-siRNA, cell viability was measured on days 1, 2, 3, 4 and 5 by using MTT assays. Western Blot analysis showing marked silencing of YAP expression in HCT116 cells after 72h transfection with control (Ctrl) or YAP small interfering RNA (siRNA) oligos. B. Silencing REGγ or YAP alone or in combination inhibited cell proliferation of HT29 human colon cancer cells. Cell viability was measured on days 1, 2, 3, 4 and 5 by MTT assays following RNAi for 72h. Western blot analysis demonstrated the knockdown efficiency. C Figure S5. Constitutive YAP fully reversed the retardation of tumor growth induced by REGγ depletion. A. Xenograft tumors were generated by injecting HCT116 sh-N, HCT116 sh-N+YAP (S127A), HCT116 sh-R and HCT116 sh-R+YAP (S127A) cells into dorsal flanking sites of nude mice. B Tumors were dissected and volumes were measured. Values were presented as the means {plus minus} SEM (two-tailed Analysis of Variance, n=3, *p < 0.05, **p < 0.01). C. The xenografts were harvested 30 days post-injection and analyzed by Western blotting with antibodies against YAP or REGγ. Data in this figure are representatives of three independent repeats. Figure S6. P65 strengths YAP transcription and its signal pathway A. Quantitative PCR analysis of YAP expression in HT29 cells treated with TNFα (20 ng/ml) or IL-6 (20 ng/ml). Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, ***p<0.001). B. HCT116 cells were treated with or without TNFα (20 ng/ml) for 3 hours and processed for ChIP assay using anti-YAP antibodies. Immunoprecipitated chromatin was analyzed by RT-PCR using the specific primers for Cyr61 promoter. C. and D. TNFα or IL-6 treatment enhanced the expression of YAP downstream positive regulatory genes (Cyr61, AREG) and decreased the expression of negative regulatory gene (Trail, DDT4) in HCT116 and HT29 cells. Cells were treated were treated with TNFα (20 ng/ml) or IL-6 (20 ng/ml) with or without Verteporfin (VP) for 3 hours and were analyzed by real-time PCR. Figure S7. YAP enhances p65 transcription and its signal pathway A. Quantitative PCR analysis of p65 expression in HT29 cells treated with/without Verteporfin (VP) or transfected with S127A mutant YAP (S127A-YAP) compared with the empty vector. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, **p<0.01). B. YAP activation increased the transcriptional activity of NF-κB in HT29 human colon cancer cells. NF-κB luciferase reporter activities were measured in sh-N, sh-N+YAP (S127A), sh-R and sh-R+YAP (S127A) cells. Data represent the means {plus minus} SEM (Analysis of Variance, n=3, ***P<0.001). C. and D. Figure S8. Clinical implication of REGγ in human colon cancer. A. A significant correlation among REGγ, YAP1 and RELA mRNA expression in 53 patients with ulcerative colitis. Pearson correlation coefficient was 0.737, 0.917 and 0.734, respectively. P<0.0001. B. The correlation of survival rate with p-p65 overexpression in 172 CRC patients. p=0.379. C. Diagrams summarizing the percentage of positive staining for each marker examined. Results were evaluated in a double-blinded fashion. D. High expression of REGγ was not related with patient age, patient gender, tumor size, tumor grade and tumor metastasis (p>0.05) Figure S9. A Model of crosstalk among REGγ, Hippo-Yap, and NF-κB. In human colon cancer cells, overexpression of REGγ promotes the degradation of Lats1, thus activating YAP signaling. YAP activation empowers a reciprocal positive regulation with NF-κB, leading to aberrant cell proliferation and development of inflammationassociated colon cancer. In contrast, cells with REGγ depletion are resistant to Lats1 inactivation, inhibiting YAP activation and its crosstalk to NF-κB to prevent tumor formation Figure S10. P53 was not affected by REGγ deficiency in human colon cancer cells. A. Western Blot analysis for REGγ and p53 in HCT116 sh-N and sh-R human colon cancer cells. β-actin served as a loading control. B. Western Blot analysis of p53 from mouse normal colon and tumor tissues of REGγ+/+ and REGγ-/- mice showed no marked changes. All experiments were repeated three times. Table 1. Sequences of primers used for Q-PCR Table 2. Sequences of primers used for ChIP</p>

The REG gamma-20S proteasome is an ubiquitin-and ATP -independent degradation system, targeting selective sub-strates, possibly helping to regulate aging. The studies we report here demonstrate that aging-associated REG gamma decline predisposes to decreasing tau turnover, as in a tauopathy. The REG gamma proteasome promotes degradation of human and mouse tau, notably phosphorylated tau and toxic tau oligomers that shuttle between the cytoplasm and nuclei. REG gamma-mediated proteasomal degradation of tau was validated in 3-to 12-month-old REG gamma KO mice, REG gamma KO;PS19 mice, and PS19 mice with forebrain conditional neuron-specific overexpression of REG gamma (REG gamma OE) and behavioral abnormalities. Coupled with tau accumulation, we found with REG gamma-deficiency, neuron loss, dendrite reduction, tau filament accumulation, and microglial activation are much more prominent in the REG gamma KO;PS19 than the PS19 model. Moreover, we observed that the degenerative neuronal lesions and aberrant behaviors were alleviated in REG gamma OE;PS19 mice. Memory and other behavior analysis substantiate the role of REG gamma in prevention of tauopathy-like symptoms. In addition, we investigated the potential mechanism underlying aging-related REG gamma decline. This study provides valuable insights into the novel regulatory mechanisms and potential therapeutic targets for tau-related neurodegenerative diseases.

Diabetic vascular endothelial impairment is one of the main causes of death in patients with diabetes lacking adequately defined mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. Here, we found that REGγ was upregulated in streptozocin (STZ)-induced diabetic mouse aortic endothelium in vivo and high glucose (HG)-treated vascular endothelial cells (ECs) in vitro. REGγ deficiency ameliorated endothelial impairment in STZ-induced diabetic mice by protecting against a decline in cellular glucose uptake and associated vascular ECs dysfunction by suppressing high mobility group AT-hook 2 (HMGA2) decay. Mechanically, REGγ interacted with and degraded the transcription factor HMGA2 directly, leading to decreased HMGA2 transcriptional activity, subsequently lowered expression of glucose transporter type 1 (GLUT1), and reduced cellular glucose uptake, vascular endothelial dysfunction, and impaired diabetic endothelium. Ablation of endogenous GLUT1 or HMGA2 or overexpressing exogenous HMGA2 in vascular ECs significantly blocked or reestablished the REGγ-dependent action on cellular glucose uptake and vascular endothelial functions of HG stimulation in vitro. Furthermore, exogenously introducing HMGA2 improved diabetic mice endothelial impairment features caused by REGγ in vivo, thereby substantiating a REGγ-HMGA2-GLUT1 pathway in diabetic endothelial impairment. Our findings indicate that modulating REGγ-proteasome activity may be a potential therapeutic approach for diabetic disorders with endothelial impairment.

Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients.

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional Kras G12D , p53 fl/fl LOF and Smad4 fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the Kras G12D , p53 fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3′UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

AbstractOver 85% of lung cancer patients harbor overt or subclinical metastases at diagnosis, and therefore most patients die of progressive metastatic disease despite aggressive local and systemic therapies. Somatic mutations in theSmad4gene have been found in non-small-cell lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generated a highly aggressive lung cancer mouse model bearing conditionalKrasG12D,p53fl/flLOF and/orSmad4fl/flLOF mutations. TheSmad4fl/fl;p53fl/fl; KrasG12D(SPK) mutant mice manifested a much higher incidence of tumor metastases than thep53fl/fl; KrasG12D(PK) mice. Molecularly, PAK3 was identified as a novel downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice was achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3’UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components suggests clinical use of Smad4 LOF as a potential marker for prognosis in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a marker of intestinal stem cells (ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5+ stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated β-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+ stem cells in the regeneration of intestinal epithelial cells. Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

Despite significant progression in the study of hepatocellular carcinoma (HCC), the role of the proteasome in regulating cross talk between mTOR signaling and glycolysis in liver cancer progression is not fully understood. Here, we demonstrate that deficiency of REGγ, a proteasome activator, in mice significantly attenuates DEN-induced liver tumor formation. Ablation of REGγ increases the stability of PP2Ac (protein phosphatase 2 catalytic subunit) in vitro and in vivo, which dephosphorylates PRAS40 (AKT1 substrate 1) and stabilizes the interaction between PRAS40 and Raptor to inactive mTORC1-mediated hyper-glycolytic metabolism. In the DEN-induced animal model and clinical hepato-carcinoma samples, high levels of REGγ in HCC tumor regions contribute to reduced expression of PP2Ac, leading to accumulation of phosphorylated PRAS40 and mTORC1-mediated activation of HIF1α. Interestingly, mTORC1 enhances REGγ activity in HCC, forming a positive feedback regulatory loop. In conclusion, our study identifies REGγ-PP2Ac-PRAS40 axis as a new layer in regulating mTORC1 activity and downstream glycolytic alterations during HCC development, highlighting the REGγ-proteasome as a potential target for personalized HCC therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. REGγ is emerging as 11S proteasome activator of 20S proteasome to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner. Here, we found that REGγ was significantly upregulated in the transverse aortic constriction (TAC)-induced hypertrophic hearts and angiotensin II (Ang II)-treated cardiomyocytes. REGγ deficiency ameliorated pressure overload-induced cardiac hypertrophy were associated with inhibition of cardiac reactive oxygen species (ROS) accumulation and suppression of protein phosphatase 2A catalytic subunit α (PP2Acα) decay. Mechanistically, REGγ interacted with and targeted PP2Acα for degradation directly, thereby leading to increase of phosphorylation levels and nuclear export of Forkhead box protein O (FoxO) 3a and subsequent of SOD2 decline, ROS accumulation, and cardiac hypertrophy. Introducing exogenous PP2Acα or SOD2 to human cardiomyocytes significantly rescued the REGγ-mediated ROS accumulation of Ang II stimulation in vitro. Furthermore, treatment with superoxide dismutase mimetic, MnTBAP prevented cardiac ROS production and hypertrophy features that REGγ caused in vivo, thereby establishing a REGγ–PP2Acα–FoxO3a–SOD2 pathway in cardiac oxidative stress and hypertrophy, indicates modulating the REGγ-proteasome activity may be a potential therapeutic approach in cardiac hypertrophy-associated disorders.

Aims: Intact intestinal epithelium is essential to maintain normal intestinal physiological function. Irradiation-induced gastrointestinal syndrome or inflammatory bowel disease occurred when epithelial integrity was impaired. This study aims at exploring the mechanism of procyanidin B2 (PB2) administration to promote intestinal injury repair in mice. Results: PB2 treatment reduces reactive oxygen species (ROS) accumulation and protects the intestine damage from irradiation. Mechanistic studies reveal that PB2 could effectively slow down the degradation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and it significantly triggers Nrf2 into the nucleus, which leads to subsequent antioxidant enzyme expression. However, knockdown of Nrf2 attenuates PB2-induced protection in the intestine. More importantly, PB2 also promotes leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive intestinal stem cells (Lgr5+ ISCs) driven regeneration via enhancing Wnt/β-catenin signaling, which depends on, at least in part, activation of the Nrf2 signal. Evidence from an injury model of intestinal organoids is similar with in vivo results. Correspondingly, results from flow cytometric analysis and luciferase reporter assay reveal that PB2 also inhibits the level of ROS and promotes Lgr5 expression in vitro. Finally, PB2 alleviates the severity of experimental colitis and colitis-associated cancer in a long-term inflammatory model via inhibiting nuclear localization of p65. Innovation: This study, for the first time, reveals a role of PB2 for intestinal regeneration and repair after radiation or dextran sulfate sodium-induced injury in mice. Conclusion: Our results indicate that PB2 can repress oxidative stress via Nrf2/ARE signaling and then promote intestinal injury repair.

For quite a long time, the 11S proteasome activator REGɑ and REGβ, but not REGγ, are known to control immunoproteasome and promote antigen processing. Here, we demonstrate that REGγ functions as an inhibitor for immunoproteasome and autoimmune disease. Depletion of REGγ promotes MHC class I-restricted presentation to prime CD8+ T cells in vitro and in vivo. Mice deficient for REGγ have elevation of CD8+ T cells and DCs, and develop age-related spontaneous autoimmune symptoms. Mechanistically, REGγ specifically interacts with phosphorylated STAT3 and promotes its degradation in vitro and in cells. Inhibition of STAT3 dramatically attenuates levels of LMP2/LMP7 and antigen presentation in cells lacking REGγ. Importantly, treatment with STAT3 or LMP2/7 inhibitor prevented accumulation of immune complex in REGγ−/− kidney. Moreover, REGγ−/− mice also expedites Pristane-induced lupus. Bioinformatics and immunohistological analyses of clinical samples have correlated lower expression of REGγ with enhanced expression of phosphorylated STAT3, LMP2 and LMP7 in human Lupus Nephritis. Collectively, our results support the concept that REGγ is a new regulator of immunoproteasome to balance autoimmunity.

Anaplastic thyroid cancer (ATC) is the most aggressive human thyroid malignancy, characterized by dedifferentiation and resistance to radioiodine therapy. The underlying mechanisms regulating ATC dedifferentiation are largely unknown. Here, we show that REGγ, a noncanonical proteasome activator highly expressed in ATC, is an important regulator of differentiation in ATC cells. Ablation of REGγ significantly restored expression of thyroid-specific genes, enhanced iodine uptake, and improved the efficacy of 131I therapy in ATC xenograft models. Mechanistically, REGγ directly binds to the TGF-β signaling antagonist Smad7 and promotes its degradation, leading to the activation of the TGF-β signal pathway. With gain- and loss-of-function studies, we demonstrate that Smad7 is an important mediator for the REGγ function in ATC cell dedifferentiation, which is supported by expression profiles in human ATC tissues. It seems that REGγ impinges on repression of thyroid-specific genes and promotion of tumor malignancy in ATC cells by activating the TGF-β signal pathway via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for allowing radioiodine therapy in anaplastic thyroid cancer patients with poor prognosis.

Lung cancer is one of the cancers with highest morbidity and mortality rates and the metastasis of lung cancer is a leading cause of death. Mechanisms of lung cancer metastasis are yet to be fully understood. Herein, we demonstrate that mice deficient for REGγ, a proteasome activator, exhibited a significant reduction in tumor size, numbers, and metastatic rate with prolonged survival in a conditional Kras/p53 mutant lung cancer model. REGγ enhanced the TGFβ-Smad signaling pathway by ubiquitin-ATP-independent degradation of Smad7, an inhibitor of the TGFβ pathway. Activated TGFβ signaling in REGγ-positive lung cancer cells led to diminished expression of E-cadherin, a biomarker of epithelial–mesenchymal transitions (EMT), and elevated mesenchymal markers compared with REGγ-deficient lung cancer cells. REGγ overexpression was found in lung cancer patients with metastasis, correlating with the reduction of E-Cadherin/Smad7 and a poor prognosis. Overall, our study indicates that REGγ promotes lung cancer metastasis by activating TGF-β signaling via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for lung cancers with poor prognosis.

AbstractPurpose: Colorectal cancer is one of the most commonly diagnosed cancers closely associated with inflammation and hyperactive growth. We previously demonstrated a regulatory circuit between the proteasome activator REGγ and NF-kappaB (NF-κB) during colon inflammation, known to be important in the development of colitis-associated cancer as well as sporadic colorectal cancer. How the inflammatory microenvironment affects the Hippo pathway during colorectal cancer development is largely unknown.Experimental Design: Here, we used REGγ-deficient colon cancer cell lines, REGγ knockout mice, and human colorectal cancer samples to identify the novel molecular mechanism by which REGγ functions as an oncoprotein in the development of colorectal cancer.Results: REGγ can directly interact with Lats1 and promote its degradation, which facilitates Yes-associated protein (YAP) activation in colon cancer cells. REGγ deficiency significantly attenuated colon cancer growth, associated with decreased YAP activity. Suppression of tumor growth due to REGγ depletion was overcome by constitutively active YAP. Surprisingly, reciprocal activation of the YAP and NF-κB pathways was observed in human colon cancer cells. REGγ overexpression was found in over 60% of 172 colorectal cancer specimens, highly correlating with the elevation of YAP and p65. Postoperative follow-up revealed a significantly lower survival rate in patients with concomitantly high expression of REGγ, YAP, and p-p65.Conclusions: REGγ could be a master regulator during colorectal cancer development to promote YAP signaling and reinforce cross-talks between inflammation and growth pathways, and REGγ might be a new marker for prognosis of colorectal cancer patients. Clin Cancer Res; 24(8); 2015–25. ©2018 AACR.

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGg, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGg’s function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGg exacerbates local inflammation and promotes a reciprocal regulatory loop with NFkB involving ubiquitinindependent degradation of IkBe. Additional deletion of IkBe restored colitis phenotypes and inflammatory gene expression in REGg-deficient mice. In sum, this study identifies REGg-mediated control of IkBe as a molecular mechanism that contributes to NFkB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury. DOI: 10.1038/ncomms10761 OPEN

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGγ, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGγ's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGγ exacerbates local inflammation and promotes a reciprocal regulatory loop with NFκB involving ubiquitin-independent degradation of IκBɛ. Additional deletion of IκBɛ restored colitis phenotypes and inflammatory gene expression in REGγ-deficient mice. In sum, this study identifies REGγ-mediated control of IκBɛ as a molecular mechanism that contributes to NFκB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury.

GSK3β regulates some functions of the brain, but the mechanisms involved in the maintenance of GSK3β protein stability remain ambiguous. REGγ, an important proteasome activator for ubiquitin-independent protein degradation, has been shown to degrade certain intact proteins and is involved in the regulation of important biological processes. Here we demonstrate that REGγ promotes the degradation of GSK3β protein in vitro and in vivo. With increased GSK3β activity, REGγ knockout (REGγ−/−) mice exhibit late-onset sensorimotor gating and cognitive deficiencies including decreased working memory, hyperlocomotion, increased stereotype, defective prepulse inhibition (PPI), and disability in nest building, at the age of 8 months or older. Inhibition of GSK3β rescued the compromised PPI phenotypes and working memory deficiency in the knockout mice. Also, we found an age-dependent decrease in the trypsin-like proteasomal activity in REGγ−/− mice brains, which may be reflective of a lack of degradation of GSK3β. Collectively, our findings reveal a novel regulatory pathway in which the REGγ-proteasome controls the steady-state level of GSK3β protein. Dysfunction in this non-canonical proteasome degradation pathway may contribute to the sensorimotor gating deficiency and cognitive disorders in aging mice.