Interaction of checkpoint kinase 1 and the X-linked inhibitor of apoptosis during mitosis

Senescence marker protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen-independently with aging. We have now characterized a unique property of SMP30, the hydrolysis of diisopropyl phosphorofluoridate (DFP), which is similar to the chemical warfare nerve agents sarine, soman and tabun. Hydrolysis of DFP was stimulated equally well by 1 mM MgCl₂, MnCl₂ or CoCl₂, to a lesser extent by 1 mM CdCl₂ but not at all by 1 mM CaCl₂. No ⁴⁵Ca²⁺-binding activity was detected for purified SMP30, suggesting that SMP30 is not a calcium-binding protein, as others previously stated. Despite the sequence similarity between SMP30 and a serum paraoxonase (PON), the inability of SMP30 to hydrolyze PON-specific substrates such as paraoxon, dihydrocoumarin, γ-nonalactone, and δ-dodecanolactone indicate that SMP30 is distinct from the PON family. We previously established SMP30 knockout mice and have now tested DFPase activity in their livers. The livers from wild-type mice contained readily detectable DFPase activity, whereas no such enzyme activity was found in livers from SMP30 knockout mice. Moreover, the hepatocytes of SMP30 knockout mice were far more susceptible to DFP-induced cytotoxicity than those from the wild-type. These results indicate that SMP30 is a unique DFP hydrolyzing enzyme in the liver and has an important detoxification effect on DFP. Consequently, a reduction of SMP30 expression might account for the age-associated deterioration of cellular functions and enhanced susceptibility to harmful stimuli in aged tissue.     

A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli

Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 °C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity.     

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish by administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.     

Senescence Marker Protein 30 Has a Cardio-Protective Role in Doxorubicin-Induced Cardiac Dysfunction

Background

Senescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction.

Methods and Results

SMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice at 12–14 weeks of age were given intra-peritoneal injection of DOX (20 mg/kg) or saline. Five days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of reactive oxygen species and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The numbers of deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive nuclei in the myocardium, apoptotic signaling pathways such as caspase-3 activity, Bax/Bcl-2 ratio and phosphorylation activity of c-Jun N-terminal kinase were increased in SMP30 KO mice and decreased in SMP30 TG mice compared with WT mice after DOX injection.

Conclusions

SMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.     

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.     

Senescence marker protein 30 protects intestinal epithelial cells against inflammation-induced cell death by enhancing Nrf2 activity

Senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases during senescence. SMP30 deficiency increases susceptibility to cytokine-induced apoptosis in the liver and to radiation-induced apoptosis in the small intestine. Furthermore, colonic epithelial cell death is associated with the severity of colitis. Therefore, in the present study, we investigated the function of SMP30 during intestinal inflammation. In SMP30 deficient mice, colitis was significantly exacerbated as demonstrated by increased mortality (p?=?0.001), body weight loss (p?=?0.0105 at day 8), rectal bleeding (p?=?0.0047 at day 8) and diarrhea (p?=?0.0030 at day 8), histological scores (ulcers, p?=?0.0002; edema, p?=?0.0125; leukocyte infiltration, p?=?0.0016) and productions of pro-inflammatory cytokines (IL-1α, p?=?0.0452; IL-6, p?=?0.0074; G-CSF, p?=?0.0036). In addition, greater proportions of apoptotic cells and lower levels of anti-apoptotic marker proteins (total PARP-1 and Bcl-2) were observed in the inflamed intestines of SMP30 deficient mice than in wild type controls. In vitro experiments on colonic epithelial cells showed that stable SMP30 expression inhibited but that SMP30 siRNA expression increased TNF-α-induced apoptosis. SMP30 inhibition decreased Nrf2 mRNA expression levels (p?<?0.0001), but SMP30 overexpression increased Nrf2 mRNA expression levels (p?=?0.0495). The underlying mechanism by which SMP30 protected cells appeared to be by inhibiting Nrf2 ubiquitination and Keap1 expression, and thus enhancing Nrf2 activity. Moreover, SMP30 deficiency increased the incidence of colitis-associated colon cancer as determined by increased mortality (p?=?0.0572) and average polyp number (p?=?0.0277). Collectively, these findings suggest that SMP30 protects intestinal epithelial cells from apoptosis and this can contribute to amelioration of colitis and colitis-associated colon cancer.     

衰老标记蛋白30 mRNA在癌细胞中的表达研究

目的:检测衰老标记蛋白(SMP)30 mRNA在不同癌细胞系中的表达情况,探讨其在不同细胞中的表达差异。方法:分别采用RT-PCR与荧光定量PCR检测SMP30 mRNA在正常肝细胞、肝癌细胞、胃癌细胞、乳腺癌细胞、宫颈癌细胞中的表达,并用SPSS13.0进行统计学分析。结果:SMP30 mRNA在所有被检测的细胞株中均有表达,在癌细胞中的相对表达量分别为肝癌细胞(0.926±0.340)、胃癌细胞(0.922±0.379)、乳腺癌细胞(0.614±0.356)、宫颈癌细胞(0.608±0.346),而在正常肝细胞中为0.175±0.158,显示SMP30 mRNA在癌细胞中的表达量较正常肝细胞中高(P0.05),且在肝癌细胞中的表达量比在其他癌细胞中更高。结论:SMP30 mRNA在癌细胞中的表达高于正常肝细胞,且在肝癌细胞中的表达高于其他癌细胞,具有临床应用价值。     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Leprdb/db Mice with Senescence Marker Protein-30 Knockout (Leprdb/dbSmp30Y/?) Exhibit Increases in Small Dense-LDL and Severe Fatty Liver Despite Being Fed a Standard Diet

Background/Aims

The senescence marker protein-30 (SMP30) is a 34 kDa protein originally identified in rat liver that shows decreased levels with age. Several functional studies using SMP30 knockout (Smp30^Y/−) mice established that SMP30 functions as an antioxidant and protects against apoptosis. To address the potential role of SMP30 in nonalcoholic fatty liver disease (NAFLD) pathogenesis, we established Smp30^Y/− mice on a Lepr^db/db background (Lepr^db/dbSmp30^Y/− mice).

Research Design/Principal Findings

Male Lepr^db/dbSmp30^Y/− mice were fed a standard diet (340 kcal/100 g, fat 5.6%) for 16 weeks whereupon the lipid/lipoprotein profiles, hepatic expression of genes related to lipid metabolism and endoplasmic reticulum stress markers were analyzed by HPLC, quantitative RT-PCR and western blotting, respectively. Changes in the liver at a histological level were also investigated. The amount of SMP30 mRNA and protein in livers was decreased in Lepr^db/dbSmp30^Y/+ mice compared with Lepr^db/+Smp30^Y/+ mice. Compared with Lepr^db/dbSmp30^Y/+ mice, 24 week old Lepr^db/dbSmp30^Y/− mice showed: i) increased small dense LDL-cho and decreased HDL-cho levels; ii) fatty liver accompanied by numerous inflammatory cells and increased oxidative stress; iii) decreased mRNA expression of genes involved in fatty acid oxidation (PPARα) and lipoprotein uptake (LDLR and VLDLR) but increased CD36 levels; and iv) increased endoplasmic reticulum stress.

Conclusion

Our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.     

Accelerated tubular cell senescence in SMP30 knockout mice

An experimental model with accelerated but not drastic renal senescence seemed useful to recognize the mechanisms of how kidney function deteriorates with age. Senescence marker protein-30 (SMP30), whose expression decreased with age and was sex-independent, is mainly expressed in hepatocytes and proximal tubular cells. Therefore, we established a SMP30 deficient strain of mice with a C57BL/6 background by gene targeting to investigate whether this molecule is involved in renal tubular cell senescence. Male SMP30 knockout (SMP30Y/-) mice and male wild-type (SMPY/+) mice (n=5) aged 12 months were examined histologically. Their tubular epithelia showed the deposition of lipofuscin and the presence of senescence-associated beta-galactosidase (SA-beta-GAL). However, no tubular cells were atrophic. In electron microscopy, SMP30-KO mice showed markedly enlarged lysosomes containing an electron dense substance. These are convincing hallmarks of senescence. We recognized the early manifestation of senescence hallmarks in SMP30-KO mice at 12 months old. Thus, this model represents the first report of a mouse strain that manifests accelerated ordinal senescence in a kidney after gene manipulation.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Induced pluripotent stem cells' self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium

Senescence marker protein-30 (SMP30) plays an important role in intracellular Ca²⁺ homeostasis. The aim of the present study was to investigate the effects of estrogens on liver apoptotic damage and changes in SMP30 expression induced by a high saturated fatty acid diet (HSFD). Ovariectomized mice (OVX) and sham-operated mice (SHAM) were randomly divided into five groups: SHAM fed a normal diet (SHAM/ND), SHAM fed HSFD (SHAM/HSFD), OVX fed ND (OVX/ND), OVX fed HSFD (OVX/HSFD) and OVX fed HSFD with 17β-estradiol (E2) supplementation using an implanted slow-release pellet (OVX/HSFD + E2). After 8 weeks, markers of endoplasmic reticulum (ER) stress and apoptosis, and levels of tumor necrosis factor-α (TNFα and SMP30 expression were investigated. Compared with SHAM/ND, OVX/HSFD mice showed significantly increased spliced X-box protein-1 (s-XBP1), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), glucose-regulated protein 78 (GPR78), C/EBP homologous protein (CHOP), cytosolic cytochrome c, caspase-3 activity, and TNFα, and significantly decreased SMP30. These differences in OVX/HSFD mice were restored to the levels of SHAM/ND mice by E2 supplementation. These results suggest that E2 supplementation attenuates HSFD-induced liver apoptotic death in ovariectomized mice by up-regulating SMP30.     

Ascorbate promotes carbon tetrachloride-induced hepatic injury in senescence marker protein 30-deficient mice by enhancing inflammation

The genetic deletion of the senescence marker protein 30 (SMP30) gene results in ascorbate deficiency and the premature aging processes in mice. Apparent liver injury of SMP30^−/− mice was less severe than those of wild type (WT) mice, upon chronic CCl₄ injection. The purpose of this study was to investigate the pathophysiology underlying the mild CCl₄ toxicity in SMP30^−/− mice. Along with the lower level of serum alanine aminotransferase, the livers of SMP30^−/− mice revealed a lesser glycogen depletion, a decrease in c-Jun N-terminal kinase (JNK)-mediated inflammatory signaling in parallel with tumor necrosis factor-alpha and interleukin-1 beta, inducible nitric oxide synthase and glutathione peroxidase, and the lower lipid peroxidation as compared to those of WT mice. CCl₄-induced proliferation, measured by the expression of proliferating cell nuclear antigen, was low in SMP30^−/− mice as compared with that of WT mice whereas the levels of p21 and Bax were comparable to those of the CCl₄-treated WT mice. Moreover, CCl₄ toxicity in ascorbate-fed SMP30^−/− mice was comparable to that of the CCl₄-alone treated WT mice, accompanied by an increase in the above mentioned factors. Conversely, ascorbate partly compensated for the CCl₄-induced oxidative stress in WT mice, indicating that sufficient ascorbate may be required for an antioxidant function under severe levels of oxidative stress. Our data suggest that the restoration of ascorbate-deficiency reverses a sluggish immune system into an activated condition by an increase in JNK-mediated inflammation and free radical cascade; thus leading to accelerated hepatic damage in SMP30^−/− mice.     

SMP30 deficiency in mice causes an accumulation of neutral lipids and phospholipids in the liver and shortens the life span

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with aging. SMP30-deficient (SMP30Y/-) mice are viable and fertile but lower in body weight and shorter in life span than the wild-type. In the electron microscope, hepatocytes from SMP30Y/- but not the wild-type mice at 12 months of age clearly contained many lipid droplets, abnormally enlarged mitochondria with indistinct cristae, and enlarged lysosomes filled with electron-dense bodies. In liver specimens from SMP30Y/- mice, the marked number of lipid droplets visible around the central vein increased notably in size and amount as the animals aged. Biochemical analysis of neutral lipids, total hepatic triglyceride, and cholesterol from SMP30Y/- mice showed approximately 3.6- and 3.3-fold higher levels, respectively, than those from age-matched wild-type mice. Moreover, values for total hepatic phospholipids from SMP30Y/- mice were approximately 3.7-fold higher than those for their wild-type counterparts. By thin-layer chromatography analysis, phosphatidylethanolamine, cardiolipin, phosphatidylcholine, phosphatidylserine, and sphingomyelin accumulations were detected separately in lipid extracts from SMP30Y/- mouse livers and provided results that strongly indicate the profound effect of an SMP30 deficiency on the metabolism of these neutral lipids and phospholipids. Conceivably, this abnormality of lipid metabolism is sufficient to curtail the life span of SMP30-deficient mice.     

Senescence marker protein 30 inhibits angiotensin II-induced cardiac hypertrophy and diastolic dysfunction

Background and objective

Senescence marker protein 30 (SMP30) is assumed to behave as an anti-aging factor. Recently, we have demonstrated that deficiency of SMP30 exacerbates angiotensin II-induced cardiac hypertrophy, dysfunction and remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac adverse remodeling in response to angiotensin II.

Methods

We generated transgenic mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. Transgenic mice and wild-type littermate mice were subjected to continuous angiotensin II infusion (800 ng/kg/min).

Results

After 14 days, heart weight and left ventricular weight were lower in transgenic mice than in wild-type mice, although blood pressure was similarly elevated during angiotensin II infusion. Cardiac hypertrophy and diastolic dysfunction in response to angiotensin II were prevented in transgenic mice compared with wild-type mice. The degree of cardiac fibrosis by angiotensin II was lower in transgenic mice than in wild-type mice. Angiotensin II-induced generation of superoxide and subsequent cellular senescence were attenuated in transgenic mouse hearts compared with wild-type mice.

Conclusions

Cardiac-specific overexpression of SMP30 inhibited angiotensin II-induced cardiac adverse remodeling. SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects and could be a novel therapeutic target to prevent cardiac hypertrophy and remodeling due to hypertension.     

Immunohistochemical localization of senescence marker protein-30 (SMP30) in the submandibular gland and ultrastructural changes of the granular duct cells in SMP30 knockout mice

Senescence Marker Protein-30 (SMP30) is a calcium-regulating protein that decreases in an androgen-independent manner as aging occurs. An enzyme-labeled antibody technique has demonstrated that SMP30 localized to the ducts (granular, intercalated, and striated ducts) of mouse submandibular glands. Immunoelectronmicroscopy demonstrated that the granular duct cells were strongly positive for SMP30, but that pillar cells in the granular duct were negative for the protein. In SMP30-knockout (KO) mice, the granular ducts were smaller in diameter. Swelling of mitochondria in the granular duct cells was observed; however, this phenomenon was not observed in the pillar cells. After administration of alpha-isoproterenol, a beta-adrenergic stimulant, a large numbers of small secretory granules were present in the granular duct cells and an expansion of the rough endoplasmic reticulum in SMP30-wild type (WT) mice; in contrast, little change was observed in SMP30-KO mice. These results suggest that SMP30 may be closely related to a signal transduction pathway in the granular duct cells of submandibular glands.     

Nuclear localization of senescence marker protein-30, SMP30, in cultured mouse hepatocytes and its similarity to RNA polymerase

Senescence marker protein-30 (SMP30), expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity. By immunohistochemistry and western blotting, we found that SMP30 was in both the nuclei and cytoplasm of cultured mouse hepatocytes. By a homology search, we found that a domain of the SMP30 sequence 51 amino acid residues long was 60-66% similar to bacterial and yeast RNA polymerases.