Gasdermin A3 (Gsdma3) was originally identified in association with hair-loss phenotype in mouse mutants. Our previous study found that AE mutant mice, with a Y344H substitution at the C-terminal domain of Gsdma3, display inflammation-dependent alopecia and excoriation [Zhou et al. (2012) Am. J. Pathol. 180, 763–774]. Interestingly, we found that the newly-generated null mutant of Gsdma3 mice did not display the skin dysmorphology, indicating that Gsdma3 is not essential for differentiation of epidermal cells and maintenance of the hair cycle in normal physiological conditions. Consistently, human embryonic kidney (HEK)293 and HaCaT cells transfected with wild-type (WT) Gsdma3 did not show abnormal morphology. However, Gsdma3 Y344H mutation induced autophagy. Gsdma3 N-terminal domain, but not the C-terminal domain, also displayed the similar pro-autophagic activity. The Gsdma3 Y344H mutant protein and N-terminal domain-induced autophagy was associated with mitochondria and ROS generation. Co-expression of C-terminal domain reversed the cell autophagy induced by N-terminal domain. Moreover, C-terminal domain could be co-precipitated with N-terminal domain. These data indicated that the potential pro-autophagic activity of WT Gsdma3 protein is suppressed through an intramolecular inhibition mechanism. Studies on other members of the GSDM family suggested this mechanism is conserved in several sub-families.
Body weight regain often causes failure of obesity therapies while the underlying mechanism remains largely unknown. In this study, we report that immune cells, especially CD4+ T cells, mediate the 'memory' of previous obese status. In a weight gain-loss-regain model, we found that C57BL/6J mice with an obesity history showed a much faster rate of body weight regain. This obesity memory could last for at least 2 months after previously obese mice were kept at the same body weight as non-obese mice. Surprisingly, such obesity memory was abrogated by dexamethasone treatment, whereas immunodeficient Rag1-/- and H2A-/- mice failed to establish such memory. Rag1-/- mice repossessed the obesity memory when immune cells or CD4+ T cells isolated from previously obese mice were transferred. Furthermore, depletion of CD4+ T cells led to obesity memory ablation. Taken together, we conclude that CD4+ T cells mediate obesity memory and promote weight regain.
Abstract While apoptosis is essential for male germ cell development, improper activation of apoptosis in the testis can affect spermatogenesis and cause reproduction defects. Members of the MAGE-A (melanoma antigen family A) gene family are frequently clustered in mammalian genomes and are exclusively expressed in the testes of normal animals but abnormally activated in a wide variety of cancers. We investigated the potential roles of these genes in spermatogenesis by generating a mouse model with a 210-kb genomic deletion encompassing six members of the Magea gene cluster ( Magea1 , Magea2 , Magea3 , Magea5 , Magea6 and Magea8 ). Male mice carrying the deletion displayed smaller testes from 2 months old with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. Furthermore, we found that Magea genes prevented stress-induced spermatogenic apoptosis after N-ethyl-N-nitrosourea (ENU) treatment during the adult stage. Mechanistically, deletion of the Magea gene cluster resulted in a dramatic increase in apoptotic germ cells, predominantly spermatocytes, with activation of p53 and induction of Bax in the testes. These observations demonstrate that the Magea genes are crucial in maintaining normal testicular size and protecting germ cells from excessive apoptosis under genotoxic stress.
Abstract Gasdermin B (GSDMB) has been reported to be associated with immune diseases in humans, but the detailed molecular mechanisms remain unsolved. The N-terminus of GSDMB by itself, unlike other gasdermin family proteins, does not induce cell death. Here, we show that GSDMB is highly expressed in the leukocytes of septic shock patients, which is associated with increased release of the gasdermin D (GSDMD) N-terminus. GSDMB expression and the accumulation of the N-terminal fragment of GSDMD are induced by the activation of the non-canonical pyroptosis pathway in a human monocyte cell line. The downregulation of GSDMB alleviates the cleavage of GSDMD and cell death. Consistently, the overexpression of GSDMB promotes GSDMD cleavage, accompanied by increased LDH release. We further found that GSDMB promotes caspase-4 activity, which is required for the cleavage of GSDMD in non-canonical pyroptosis, by directly binding to the CARD domain of caspase-4. Our study reveals a GSDMB-mediated novel regulatory mechanism for non-canonical pyroptosis and suggests a potential new strategy for the treatment of inflammatory diseases.
Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.
Intervertebral disc degeneration (IDD) is associated with dysregulated expression of microRNAs (miRNAs). However, the precise molecular mechanisms underlying this disorder remain unclear. Therefore, we tested the hypothesis that miRNAs modulate IDD through effects on the IL-6/STAT3 signaling pathway, a potential regulator of IDD. The miRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing miRNA microarray and quantitative real-time PCR (RT-qPCR). Biological functions of differential expression miRNAs were further investigated using immunofluorescent staining. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 41 miRNAs that were differentially expressed in patients compared with controls. Following RT-qPCR confirmation, miR-98 was significantly downregulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain-of-function and loss-of-function studies, miR-98 was shown to significantly promote type II collagen expression in NP cells. Interleukin-6 (IL-6) was identified as a target of miR-98. Knockdown of IL-6 induced effects on NP cells similar to those induced by miR-98. In contrast, IL-6 treatment abrogated the effects induced by miR-98 upregulation. Moreover, miR-98 dramatically suppressed expression of STAT3 target gene, MMP2. IL-6 treatment antagonized this effect, whereas knockdown of IL-6 by IL-6 short hairpin RNA (shIL-6) induced inhibitory effects on the expression of p-STAT3 and its main target genes, similar to miR-98. The mRNA level of IL-6 was inversely correlated with that of miR-98 in degenerative NP tissues. These results suggest the downregulation of miR-98 could promote IDD through the IL-6/STAT3 signaling pathway. Our findings also highlight miR-98 as a novel hopeful therapeutic target for IDD.
Protein phosphatase 2A (PP2A) is a key negative regulator of phosphatidylinositol 3-kinase/Akt pathway. Previous study showed that, in the liver, the catalytic subunit of PP2A (PP2Ac) is closely associated with insulin resistance syndrome, which is characterized by glucose intolerance and dyslipidemia. Here we studied the role of liver PP2Ac in glucose metabolism and evaluated whether PP2Ac is a suitable therapeutic target for treating insulin resistance syndrome. Liver-specific Ppp2cα knockout mice (Ppp2cαloxp/loxp: Alb) exhibited improved glucose homeostasis compared with littermate controls in both normal and high-fat diet conditions, despite no significant changes in body weight and liver weight under chow diet. Ppp2cαloxp/loxp: Alb mice showed enhanced glycogen deposition, serum triglyceride, cholesterol, low density lipoprotein and high density lipoprotein, activated insulin signaling, decreased expressions of gluconeogenic genes G6P and PEPCK, and lower liver triglyceride. Liver-specific Ppp2cα knockout mice showed enhanced glucose homeostasis and increased insulin sensitivity by activation of insulin signaling through Akt. These findings suggest that inhibition of hepatic Ppp2cα may be a useful strategy for the treatment of insulin resistance syndrome.
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