Anti-biofilm potential of human senescence marker protein 30 against Mycobacterium smegmatis.

Human senescence marker protein 30 (huSMP30) has been characterized as a multifaceted protein consisting of various enzymatic and cellular functions. It catalyzes the interconversion of L-gulonate and L-gulono-γ-lactone in the ascorbate biosynthesis pathway. Therefore, we hypothesized that it could be a potential anti-biofilm agent against pathogenic bacteria due to its lactonase activity. In order to corroborate this, the huSMP30 was recombinantly expressed, purified, and analyzed for its ability to inhibit Mycobacterium smegmatis biofilm formation, which showed a concentration-dependent inhibition as compared to the untreated control group. Further, in silico analysis was performed to redesign the huSMP30 with enhanced lactonase activity. Molecular docking analysis of the huSMP30 and lactone substrates facilitated the selection of three single amino acid substitutions (E18H, N154Q, and D204V), which were created using a PCR-based site-directed mutagenesis reaction. These mutant proteins and the wild-type huSMP30 were purified, and the effects on the enzymatic activity and biofilm formation were studied. The mutants E18H and D204V showed non-significant effects on specific lactonase activity, catalytic efficiency, and anti-biofilm property; however, the mutant N154Q showed significant improvement in the specific lactonase activity, catalytic efficiency, and inhibition in the biofilm formation. The protein stability analysis revealed that the wild-type huSMP30 and its designed mutants were stable at 37 °C for up to 4 days. In conclusion, the anti-biofilm property of the huSMP30 has been established, and an engineered version, N154Q, inhibits biofilm formation with greater efficiency. Human SMP30 is a versatile protein with multiple cellular and enzymatic functions, however, its anti-biofilm potential has not been explored. Our work presents the method to produce soluble and active huSMP30 in the E. coli expression system and establishes its role as an anti-biofilm agent against Mycobacterium smegmatis owing to its lactonase activity. Our results provide support for the future advancement of huSMP30 as a potential anti-biofilm agent targeting pathogenic Mycobacterium species.

Resveratrol Upregulates Senescence Marker Protein 30 by Activating AMPK/Sirt1-Foxo1 Signals and Attenuating H2O2-Induced Damage in FAO Rat Liver Cells.

Resveratrol (RSV) is a polyphenol with numerous biological functions, including anti-inflammatory, antioxidant, and anti-aging activities. The novel senescence marker protein-30 (SMP30) indicates aging, and it suppresses hepatic oxidative stress. However, the effects of RSV on SMP30 expression regulation remain unclear. We observed that RSV positively regulates SMP30 expression in rat hepatoma-derived FAO cells. However, this was abolished by Compound C and EX-527 that specifically inhibit AMP-activated protein kinase (AMPK) and Silent Information Regulator T1 (Sirt1), respectively. We predicted binding sites for AMPK, forkhead box protein O1 (Foxo1), and Sirt1 downstream molecules as possible SMP30 promoters using the JASPAR and UniProtKB databases. We identified a Foxo1 binding site in the promoter region of SMP30. Inhibiting Foxo1 with AS1842527 also decreased the RSV-induced upregulation of SMP30 expression. Moreover, RSV suppressed the substantial downregulation of SMP30 expression caused by oxidative stress and hydrogen peroxide (H2O2) and released accumulated lactate dehydrogenase. These results demonstrate that, as a novel food factor, RSV-induced upregulation of SMP30 by activating AMPK/Sirt1-Foxo1 signaling and may attenuates H2O2-induced oxidative damage. The findings of this study offer new perspectives of the anti-ageing properties of RSV.

Functional pleiotropy of calcium binding protein Regucalcin in signaling and diseases.

Regucalcin (Mr âˆ¼ 33.38 kDa) is a calcium binding protein, discovered in rat liver. In humans, gene for regucalcin is located on chromosome-11 (p11.3-q11.2) consisting of seven exons and six introns. The protein differs from other calcium binding protein in the way that it lacks EF-hand motif of calcium binding domain. It is also called as Senescence Marker Protein-30 (SMP-30) as previously its weight assumes to be 30 kDa and expression of this protein decreases with aging in androgen independent manner. Among vertebrates, it is a highly conserved protein showing gene homology in Drosophila, Xenopus, fireflies and others too. It is primarily expressed in liver and kidney in addition to brain, lungs, and skeletal muscles. Regucalcin acts as a Ca2+ regulatory protein and controls various cellular functions in liver and other organs. It suppresses protein phosphatase, protein kinase, DNA and RNA synthesis. Published evidences suggest regucalcin to be a reliable biomarker in various disorders of liver, kidney, brain and ocular. In over expressed state, it subdues apoptosis in cloned rat hepatoma cells and also induces hyperlipidemia and osteoblastogenesis by regulating various factors. Owing to the multi-functionality of regucalcin this review is presented to elaborate its importance in order to understand its involvement in cellular signaling during various pathologies.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Vitamin C Is Essential for the Maintenance of Skeletal Muscle Functions.

Vitamin C (L-ascorbic acid, VC) is a water-soluble antioxidant essential for collagen polymerization. Previously, we reported that long-term VC deficiency causes muscle atrophy and deterioration in physical ability using female senescence marker protein-30 (SMP30)-deficient mice with a lack of VC synthesis, which is similar to that observed in humans. To determine whether these findings also hold true for male SMP30-deficient mice, two-month-old male SMP30-deficient mice were divided into two groups: the VC-treated group (VC(+)) was administered 1.5 g/L VC, and the VC-untreated group (VC(-)) was supplied water without VC. The VC level at four weeks in the gastrocnemius muscles from the VC(+) and VC(-) groups was 205.7 ± 8.5 nmol/g tissue and 13.1 ± 0.6 nmol/g tissue, respectively. Thus, four weeks was enough to reduce the VC level in the skeletal muscle in the VC-untreated group. On the other hand, muscle weights of the gastrocnemius, soleus, plantaris, tibialis anterior, and extensor digitorum longus in the VC(-) group were significantly reduced by VC deficiency after twelve weeks. The physical endurance of the VC(-) group at eight weeks was markedly lower than that of the VC(+) group. The grasping strength and activity in the cage in the nocturnal phases of the VC(-) group were markedly lower at twelve and sixteen weeks than those of the VC(+) group. Interestingly, muscle atrophy and declined physical ability were completely restored with VC supplementation for twelve weeks after VC deficiency. Thus, VC is essential for maintaining skeletal muscle function in both male and female SMP30-deficient mice with a lack of VC synthesis.

Effects of 6-Month Square Stepping Exercise Intervention on Physical and Cognitive Competence, Regucalcin, and Body Composition in Older People: Study Protocol for a Randomised Control Trial.

BACKGROUND: Age-related changes negatively affect physical fitness, body composition, and executive function and produce a decrease in regucalcin level expression in blood. The square-stepping exercise (SSE) is a balance and lower-limb strength training programme used to prevent falls and stimulate cognitive function in older adults. This project aims to analyse the effects of SSE on executive function, regucalcin expression, fall prevention, body composition, and physical fitness in people over 65 years old. METHODS: A randomized controlled trial will be conducted. A total of 90 older people over 65 years old will be recruited and randomly assigned to 2 groups: experimental (n = 45) and control (n = 45). The experimental group will perform an SSE-based intervention for 6 months (2 times per week), while the control group do not follow any treatment. RESULTS: The main outcome will be balance, but other motor (body mass index, upper- and lower-limb strength, flexibility, and speed-agility) and cognitive variables (executive functions and attention) will be assessed. The expression of regucalcin levels will also be evaluated. Therefore, this project aims to analyse the effect of a 6-month SSE intervention on cognitive and motor competence, physical fitness, regucalcin levels, fall risk, and body composition in older people. If the intervention proves to be effective, it could be implemented in centres, entities, and associations specialized in elderly care.

Papel de la expresión de regucalcina en el tejido hepático: revisión sistemática / Role of regucalcin expression in liver tissue: systematic review

Objetivo: El propósito de este trabajo fue revisar la literatura científica con relación al papel de la expresión de la regucalcina (SMP30) en el hígado. Método: Se realizó una búsqueda bibliográfica en la base de datos PubMed. Se encontraron 89 artículos. Tras analizar su contenido y aplicar los criterios de inclusión y exclusión, un total de 9 artículos fueron incluidos. Resultados: Se determinó que la expresión de SMP30 es significativamente mayor en el hígado en comparación con otros tejidos como pulmones, bazo, miocardio, próstata y piel (P < 0.05). Se observó, tras obtener muestras de 137 pacientes (30 controles hepáticos normales, 10 con hepatitis B, 49 con cirrosis hepática y 48 con carcinoma hepatocelular (HCC)) que la expresión de SMP30 fue del 100% en todos los tejidos adyacentes al hígado, a excepción del HCC, que solo mostró el 81% de expresión de SMP30 en el hígado. Sobre las concentraciones séricas de SMP30 se observó que 3 grupos distintos mostraron concentraciones diferentes de SMP30: grupo control (1.72 ng/mL), pacientes con hepatitis crónica (3.76 ng/mL) y pacientes con insuficiencia hepática (5.46 ng/mL), teniendo los pacientes con insuficiencia hepática aguda una concentración significativamente mayor de SMP30 que el resto de grupos (P< 0.01), los pacientes con insuficiencia hepática también presentaron una concentración significativamente mayor que el grupo control de pacientes sanos (P< 0.01). Sobre la proliferación de células HepG2 se ha demostrado que la incorporación de manera exógena de SMP30 suprime la elevación del número de células HepG2, revelando así que la proliferación de las células HepG2 fue suprimida con los niveles fisiológicos de SMP30 presente en suero in vitro. Conclusión: La SMP30podría desempeñar un papel fundamental sobre la supervivencia en pacientes con HCC, así como, su posible funcionamiento como proteína protectora de la apoptosis en células HepG2.(AU)

Objective: The purpose of this work has been to review the scientific literature regarding the role of regucalcin expression in the liver. Method: A bibliographic search was carried out on the PubMed database. Eighty-nine articles were found. After analyzing their content and applying inclusion and exclusion criteria, a total of 9 articles were included. Results: It was determined that SMP30 expression is significantly higher in the liver compared to other tissues such as lungs, spleen, myocardium, prostate and skin (P < 0.05). It was observed, after obtaining samples from 137 patients (30 normal liver controls, 10 with hepatitis B, 49 with liver cirrhosis and 48 with hepatocellular carcinoma) that SMP30 expression was 100% in all tissues adjacent to the liver except for hepatocellular carcinoma (HCC), which showed only 81% of protein expression. On serum regucalcin concentrations it was observed, that 3 different groups with different concentrations of SMP30: control group (1.72 ng/ mL), patients with chronic hepatitis (3.76 ng/mL) and patients with liver failure (5.46 ng/ mL) patients with acute liver failure had higher concentrations of SMP30 than patients with hepatitis B (P< 0.01), as well as the serum concentrations of the latter showed to be higher than in healthy patients (P< 0.01). On the proliferation of HepG2 cells it has been shown that the addition of exogenous SMP30 suppresses the elevation of cell numbers, thus revealing that HepG2 cell proliferation was suppressed with the physiological levels of SMP30 present in serum in vitro. Conclusion: Regucalcin could play a key role in survival in patients with hepatocellular carcinoma, as well as its possible role as a protective protein for apoptosis in HepG2 cells.(AU)

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Vitamin C alleviates alcoholic liver injury by suppressing neutrophil infiltration in senescence marker protein 30-knockout mice irrespective of its antioxidant effects.

AIMS: Alcoholic liver disease (ALD) comprises an important component in chronic liver diseases, and its clinical significance has increased due to the high consumption of alcohol worldwide. Vitamin C is a potent antioxidant, and several previous studies have suggested that its therapeutic role in ALD is derived from its antioxidant role. However, its anti-inflammatory role in ALD remains to be elucidated. Especially, the relationship between vitamin C and infiltration of neutrophils in ALD has not been discussed to date. For the reason, the present study investigated the precise role of vitamin C in neutrophil infiltration in ALD. MAIN METHODS: In the present study, wild-type C57BL/6 and vitamin C-deficient senescence marker protein 30-knockout mice were pair-fed with a Lieber-DeCarli control or ethanol diet. Ethanol-fed groups were fed with increasing concentrations of EtOH (Lieber-DeCarli control diet for 5 days, 3% EtOH diet for a week, and 5% diet for 2 weeks) with or without vitamin C supplementation. KEY FINDINGS: Vitamin C dramatically attenuated the ethanol-mediated liver disease in the vitamin C-deficient ethanol-fed mice group by suppressing the infiltration of neutrophils accompanied by less CD68-positive cell infiltration. This attenuating role of vitamin C in neutrophil infiltration in the liver is associated with its protective effect for the ethanol-mediated intestinal damage in vitamin C-deficient ethanol-fed mice. SIGNIFICANCE: This study provides a novel possibility of vitamin C to be used as an anti-inflammatory therapeutic agent associated with neutrophil infiltration in ALD, thereby helping to establish strategies for attenuating ALD.

Senescence marker protein 30 (SMP30) protects against high glucose-induced apoptosis, oxidative stress and inflammatory response in retinal ganglion cells by enhancing Nrf2 activation via regulation of Akt/GSK-3ß pathway.

Senescence marker protein 30 (SMP30) is an aging-related protein that participates in the regulation of tissue damage under various pathological conditions. However, the role of SMP30 in mediating high glucose (HG)-induced injury of retinal ganglion cells (RGCs) has not been fully determined. We found that SMP30 expression declined during HG stimulation in RGCs. Cellular functional studies showed that the up-regulation of SMP30 dramatically prohibited HG-evoked apoptosis, oxidative stress and inflammatory response in RGCs. Mechanism research reported that SMP30 overexpression led to the enhancement of nuclear factor erythroid 2-related factor (Nrf2) activation in HG-stimulated RGCs. Moreover, SMP30 overexpression enhanced the phosphorylation of Akt and glucogen synthase kinase-3ß (GSK-3ß), and the suppression of Akt markedly abolished SMP30-mediated Nrf2 activation in HG-stimulated RGCs. Additionally, the suppression of Nrf2 substantially reversed SMP30-overexpression-induced anti-HG injury effects in RGCs. Overall, these findings suggest that SMP30 protects against HG injury of RGCs by potentiating Nrf2 through regulation of the Akt/GSK-3ß pathway. Our work underscores that SMP30/Akt/GSK-3ß/Nrf2 may exert a vital role in mediating the injury of RGCs during diabetic retinopathy.

Study of Cellular Senescence and Vitamin D Deficiency in Nonalcoholic Fatty Liver Disease and The Potential Protective Effect of Vitamin D Supplementation.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a pathological process characterized by excessive hepatic fatty deposition with possible involvement of vitamin D deficiency and cellular senescence. The aim of this study is to investigate the pathophysiologic role of vitamin D deficiency and cellular senescence in NAFLD development. Moreover, it aims to investigate the potential protective role of vitamin D supplementation. METHODS: This is an experimental Case/Control study. Forty-five male albino rats were enrolled in this study. Animals were divided into four groups: negative and positive control groups (10 for each group), a model of NAFLD (11) and vitamin D-treated NAFLD groups (14). At the end of the experiment, all rats were subjected to the following investigation; biochemical estimation of serum 25 hydroxycholecalciferol, senescence marker protein-30 (SMP-30), lipid profile and calculation of homeostatic model of insulin resistance (HOMA-IR). RESULTS: NAFLD group shows a significant increase in glucose, insulin levels, and HOMA- IR compared with both normal controls. This finding indicates the intimate association between insulin resistance and NAFLD pathogenesis. Moreover, it was found that NAFLD group shows a significant decrease in SMP-30 level compared with normal controls. While vitamin D-treated NAFLD group shows significant increased SMP-30 and decrease in HOMA-IR in comparison with nontreated NAFLD group. CONCLUSION: Vitamin D deficiency and increased cellular senescence are key features of NAFLD. Vitamin D supplementation could play a protective role, which needs further investigation including clinical human study.

Senescence Marker Protein 30 (SMP30): A Novel Pan-Species Diagnostic Marker for the Histopathological Diagnosis of Breast Cancer in Humans and Animals.

Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.

Influencia de la regucalcina en la pérdida de masa ósea / Influence of regucalcin on bone loss

Objetivo: El propósito de este trabajo ha sido revisar la literatura científica con relación al papel de la regucalcina en la pérdida de masa ósea. Método: Se realizó una búsqueda bibliográfica en la base de datos PubMed. Se encontraron 31 artículos. Tras analizar su contenido y aplicar los criterios de inclusión y exclusión, un total de 13 artículos fueron incluidos. Resultados: La disminución en el contenido de calcio femoral observado en ratones transgénicos con regucalcina se observó con el aumento de la edad, lo que sugiere que la pérdida ósea no se restaura con el modelado óseo. De la misma manera, se encontró que la adición de regucalcina con 1 a 100 nM estimulaba significativamente la actividad basal de NF-kB (P<0.01). Se apreció una disminución significativa en el contenido de ADN en los tejidos metafisarios femorales, con una mayor disminución en hembras que en machos, siendo estos valores: 3.3 mg/g pasaron a ser 2.6 mg/g en las hembras (P<0.01). La reabsorción ósea osteoclástica aumentó en ratones transgénicos con regucalcina machos y hembras con edad creciente. La regucalcina exógena revela efectos supresores sobre la osteoblastogénesis y la mineralización in vitro y que no tuvo efectos sobre la proliferación celular y la apoptosis en las células osteoblásticas en cultivos a corto plazo. Conclusión: La regucalcina desempeña un papel fundamental en el mantenimiento del homeostasis celular y la función de la respuesta celular en relación a la masa ósea.(AU)

Purpose: The aim of this work has been to review the scientific literature regarding the role of regucalcin in bone loss.Method: A bibliographic search was performed in the PubMed database. A total of 31 articles were used. After analyzing its content and applying the inclusion and exclusion criteria, a total of 13 articles were included.Results: The decrease in femoral calcium content observed in regucalcin transgenic mice decreased with increasing age, suggesting that bone loss is not restored with bone modeling. In the same way, it was found that the addition of regucalcin with 1 to 100 nM significantly stimulated the baseline activity of NF-kB (P <0.01). Likewise, there was a significant decrease in the DNA content in femoral metaphyseal tissues, with a greater decrease in females than in males, these values ​​being: 3,3 mg / g became 2,6 mg / g (P <0.01). Osteoclastic bone resorption increased in male and female transgenic regucalcin mice with increasing age. Also, exogenous regucalcin reveals suppressive effects on osteoblastogenesis and mineralization in vitro and that it had no effects on cell proliferation and apoptosis in osteoblast cells in short-term cultures.Conclusion: Regucalcin plays a fundamental role in the maintenance of cell homeostasis and the function of the cellular response in relation to bone mass.(AU)

Senescence marker protein 30 confers neuroprotection in oxygen-glucose deprivation/reoxygenation-injured neurons through modulation of Keap1/Nrf2 signaling: Role of SMP30 in OGD/R-induced neuronal injury.

Senescence marker protein 30 (SMP30) is a senescence marker molecule and identified as a calcium regulatory protein. Currently, SMP30 has emerged as a cytoprotective protein in a wide range of cell types. However, the role of SMP30 in regulating neuronal survival during cerebral ischemia/reperfusion injury remains unclear. In the present study, we aimed to investigate the biological function and regulatory mechanism of SMP30 on neuronal survival using a cellular model induced by oxygen-glucose deprivation/reoxygenation (OGD/R). The results showed that SMP30 expression was significantly decreased by OGD/R exposure in neurons. Functional experiments demonstrated that SMP30 overexpression significantly rescued the decreased cell viability and attenuated the apoptosis and reactive oxygen species generation in OGD/R-exposed neurons. By contrast, SMP30 knockdown exhibited the opposite effect. Mechanism research revealed that SMP30 overexpression contributed to the activation of nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) signaling associated with downregulation of Kelch-like ECH-associated protein (Keap1). Keap1 overexpression or Nrf2 silencing significantly reversed SMP30-mediated neuroprotection against OGD/R-induced injury. Overall, these findings demonstrate that SMP30 overexpression protects neurons from OGD/R-induced apoptosis and oxidative stress by enhancing Nrf2/ARE antioxidant signaling via inhibition of Keap1. These data highlight the importance of the SMP30/Keap1/Nrf2/ARE signaling axis in regulating neuronal survival during cerebral ischemia/reperfusion injury.

Senescence marker protein 30 inhibits tumor growth by reducing HDAC4 expression in non-small cell lung cancer.

BACKGROUND: Senescence marker protein 30 (SMP30), which plays a pivotal role as a suppressor protein in cell proliferation, among other regulatory actions, is a marker of aging that shows decreased expression during senescence. Decreased SMP30 has been identified in several human cancers, but its expression and role in human non-small cell lung cancer (NSCLC) remain unclear. METHODS: Using tumor tissue and matched adjacent normal tissue from 341 patients with resected NSCLC, we assessed SMP30 expression using immunohistochemical methods. The relationship between SMP30 expression and clinicopathologic characteristics was investigated by Kaplan-Meier survival analysis and multivariate analysis. Cell viability assay, colony formation assay, EdU incorporation assay and in vivo tumor xenograft models were also performed to investigate NSCLC cell proliferation using A549 and H1299 cell lines. Recombinant lentivirus-meditated in vivo gene overexpression and Western blot were performed to clarify the underlying molecular mechanism of SMP30 inhibiting NSCLC proliferation. RESULTS: SMP30 expression was frequently downregulated in NSCLC tissue, as compared with adjacent non-tumor tissue. Kaplan-Meier survival analyses revealed NSCLC patients with low SMP30 expression had a significantly worse overall survival (OS), with median OS of 18 vs. 67 months in high SMP30 expression group. SMP30 overexpression significantly inhibited A549 and H1299 cell proliferation both in vitro and in tumor xenografts and downregulated the expression of c-Myc and CyclinD1 protein. Moreover, Western blot analyses confirmed that SMP30 overexpression significantly inhibited the histone deacetylase 4 (HDAC4) level in NSCLC cells, and HDAC4 overexpression reversed SMP30-mediated NSCLC repression both in vitro and in vivo. CONCLUSIONS: SMP30 inhibited NSCLC proliferation by reducing HDAC4 expression, and SMP30 and HDAC4 may serve as new prognostic biomarkers and future therapeutic targets for NSCLC.

Blast Exposure Leads to Accelerated Cellular Senescence in the Rat Brain.

Blast-induced traumatic brain injury (bTBI) is one of the major causes of persistent disabilities in Service Members, and a history of bTBI has been identified as a primary risk factor for developing age-associated neurodegenerative diseases. Clinical observations of several military blast casualties have revealed a rapid age-related loss of white matter integrity in the brain. In the present study, we have tested the effect of single and tightly coupled repeated blasts on cellular senescence in the rat brain. Isoflurane-anesthetized rats were exposed to either a single or 2 closely coupled blasts in an advanced blast simulator. Rats were euthanized and brains were collected at 24 h, 1 month and 1 year post-blast to determine senescence-associated-ß-galactosidase (SA-ß-gal) activity in the cells using senescence marker stain. Single and repeated blast exposures resulted in significantly increased senescence marker staining in several neuroanatomical structures, including cortex, auditory cortex, dorsal lateral thalamic nucleus, geniculate nucleus, superior colliculus, ventral thalamic nucleus and hippocampus. In general, the increases in SA-ß-gal activity were more pronounced at 1 month than at 24 h or 1 year post-blast and were also greater after repeated than single blast exposures. Real-time quantitative RT-PCR analysis revealed decreased levels of mRNA for senescence marker protein-30 (SMP-30) and increased mRNA levels for p21 (cyclin dependent kinase inhibitor 1A, CDKN1A), two other related protein markers of cellular senescence. The increased senescence observed in some of these affected brain structures may be implicated in several long-term sequelae after exposure to blast, including memory disruptions and impairments in movement, auditory and ocular functions.

Decreased ascorbic acid biosynthesis in response to PMSG in the pre-pubertal female rat ovary.

Ascorbic acid (AA) is known to be an important antioxidant serving as a cofactor in collagen synthesis, and thus facilitates follicular growth in the ovary. Many studies have shown that AA is synthesized in the liver and transported to other organs including ovary, however, there is no direct evidence of ascorbic acid synthesis in the ovary. Hence, we examined the expression pattern of different proteins (SMP30/GNL and GULO) involved in the AA synthesis in pre-pubertal rat, which showed significant expression of these proteins, suggesting the synthesis of AA in the ovary. Accumulation of AA in the ovary during follicular growth has been well demonstrated. However, the effect of Pregnant Mare Serum Gonadotropin (PMSG) on the AA synthesis in the ovary has not been studied in detail. Hence to decipher the effect, different doses of PMSG were injected subcutaneously into the pre-pubertal female rats, and ovarian AA level was measured after 48 h. A significant increase in AA content was observed in PMSG treated animal groups. Further, to understand the mechanism underlying ovarian AA accumulation, the expression levels of SMP30/GNL and GULO genes were measured. Expression of both the genes was significantly suppressed, which suggested a lowered AA synthesis in the PMSG treated rat ovary. For further understanding, mRNA expression of AA transporters SVCT1 and SVCT2 encoded by SLC23A1 and SLC23A2 genes respectively were measured, which showed increased level of SVCT1 expression. These observations suggested that the increased AA content might not be due to increased synthesis of AA within the ovary but possibly due to increased uptake from blood during the stimulation of follicular growth.

High dietary vitamin C intake reduces glucocorticoid-induced immunosuppression and measures of oxidative stress in vitamin C-deficient senescence marker protein 30 knockout mice.

Vitamin C (VC) is a vital micronutrient for humans and some other mammals and also has antioxidant activity. Stress-induced elevation of glucocorticoid production is well known to cause immunosuppression. The present study evaluated the effect of high VC intake on glucocorticoid-induced immune changes in mice. Senescence marker protein 30 knockout mice with genetic VC deficiency were fed a diet containing the recommended VC content (20 mg/kg per d; 0·02 %VC group) or a high VC content (200 mg/kg per d; 0·2 %VC group) for 2 months, then dexamethasone was given by intraperitoneal injection. After administration of dexamethasone, the plasma ascorbic acid concentration decreased significantly in the 0·02 %VC group and was unchanged in wild-type C57BL/6 mice on a VC-deficient diet (wild-type group), while it was significantly higher in the 0·2 %VC group compared with the other two groups. In the 0·02 %VC and wild-type groups, dexamethasone caused a significant decrease in the cluster of differentiation (CD)4+ and CD8+ T cells among splenocytes as well as a significant decrease in IL-2, IL-12p40 and interferon-γ protein production by splenocytes and a significant decrease in T-cell proliferation among splenocytes. In the 0·2 %VC group, these dexamethasone-induced immunosuppression improved when compared with the other two groups. In addition, reduction in the intracellular levels of ascorbic acid, superoxide dismutase and glutathione in splenocytes by dexamethasone as well as elevation in thiobarbituric acid-reactive substances were significantly suppressed in the 0·2 %VC group. These findings suggest that high dietary VC intake reduces glucocorticoid-induced T-cell dysfunction by maintaining intracellular antioxidant activity.

Vitamin C and vitamin E double-deficiency increased neuroinflammation and impaired conditioned fear memory.

BACKGROUND: Vitamin C (l-ascorbic acid, VC) and vitamin E (α-tocopherol, VE) play important physiological roles as endogenous antioxidants in many tissues and organs. However, their roles in the brain remain entirely elusive. We established senescence marker protein 30 (SMP30)/α-tocopherol transfer protein (αTTP) double knockout (DKO) mice as a novel VC and VE double-deficiency model and examined the effect of VC and VE double-deficiency on brain functions. METHODS: DKO and wild-type (WT) mice were divided into the following two groups: mice in the CE (+) group were supplied with sufficient amounts of VC and VE and mice in the CE (-) group were deficient in both VC and VE. After 8 weeks of CE (+) or CE (-) treatments, a battery of behavioral experiments was conducted to analyze cognitive functions, including memory, through the Morris water maze and Pavlovian fear conditioning tasks. RESULTS: The plasma VC and VE levels in DKO-CE (-) mice and VE level in WT-CE (-) mice were almost completely depleted after 8 weeks of the deficient treatment. The behavioral study revealed that the general behaviors, including locomotor activity and anxiety level, were not influenced by the CE (-) treatment in DKO and WT mice. However, in the Pavlovian fear conditioning task, DKO-CE (-) mice showed impaired conditioned fear memory compared with that of DKO-CE (+) mice. Furthermore, increased mRNA expression was observed in inflammatory-related genes, such as IL-6, TNFα, F4/80, and Mcp-1, in the hippocampus of DKO-CE (-) mice. CONCLUSIONS: The findings of this study provide evidence that VC and VE deficiency led to impaired conditioned fear memory possibly caused by neuroinflammation in the brain.

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.