Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

BACKGROUND: Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. OBJECTIVES: We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. METHODS: Male wild-type (WT), global Nrf2-knockout (Nrf2-/-) and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or 100 ppm) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20 nM) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. RESULTS: Compared with relevant control mice, global Nrf2-/- or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100 ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20 nM) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N-acetyl-l-cysteine (2 mM) and mitoquinone mesylate (MitoQ; 0.2µM) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. CONCLUSIONS: This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Nrf2 Mitigates RANKL and M-CSF Induced Osteoclast Differentiation via ROS-Dependent Mechanisms.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to be a negative regulator of osteoclast differentiation, but the precise mechanisms have not yet been established. We examined the precise roles of Nrf2 in regulating antioxidants and reactive oxygen species (ROS) levels, especially the cytoplasmic and mitochondrial ROS during osteoclastogenesis in vitro. In the current study, we found that the absence of Nrf2 promotes osteoclast differentiation in bone-marrow-derived macrophages (BMMs) and RAW 264.7 cells. The receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) significantly lowered the levels of Nrf2 and its downstream antioxidant enzymes at mRNA and/or protein levels during osteoclast differentiation in the BMMs of mice and RAW 264.7 mouse leukemic monocytes. Compared to the wild-type cells, Nrf2-deficient cells exhibited heightened sensitivity to both transient RANKL-induced cytoplasmic ROS and prolonged RANKL and M-CSF-induced cytoplasmic and mitochondrial ROS accumulation. Furthermore, exogenous antioxidant agents, including N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and mitoquinone mesylate (MitoQ), exhibited substantial capability to suppress the elevation of ROS levels during osteoclast differentiation induced by Nrf2 deficiency, and they consequently inhibited osteoclast differentiation augmented by the lack of Nrf2. The activation of phosphorylated c-FOS resulting from elevated ROS promoted osteoclast differentiation. The inhibition of c-FOS blocked osteoclast differentiation, which was elevated by Nrf2-deficiency. Taken together, these data reveal that Nrf2 effectively decreased the accumulation of intracellular ROS and the phosphorylation of c-FOS during osteoclastic differentiation by regulating antioxidant enzymes and subsequently inhibited RANKL-induced osteoclast differentiation.

A novel germline frameshift mutation in the MLH1 gene in a patient with Lynch syndrome.

Lynch syndrome (LS) is an autosomal dominant inherited disorder, characterized by a predisposition to various cancers, mainly colorectal cancer (CRC). LS is caused by germline mutations in DNA mismatch repair genes i.e. mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6), and post-meiotic segregation increased 2 (PMS2). In this study, we report a novel germline frameshift mutation in the MLH1 gene [NM_000249: exon1: c.99dup p.(Glu34ArgfsTer4)] in a 34-year-old male patient with LS. This MLH1 alteration has never been reported in any database or any publications. The frameshift mutation in MLH1 gene [NM_000249: exon1: c.99dup p.(Glu34ArgfsTer4)] was confirmed by Sanger sequencing conducted on peripheral blood of the proband. Meanwhile, Sanger sequencing results revealed the proband's uncle was the carrier. As multiple downstream germline frameshift mutations of this variation are pathogenic, such as MLH1 M35fs, N38fs, and S44fs, it is predicted that MLH1 p.(Glu34ArgfsTer4) might be also pathogenic. Meanwhile, this MLH1 mutation p.(Glu34ArgfsTer4) is predicted to be disease-causing by the MutationTaster software, as the duplication c.99dupA introduced a premature stop codon early in the translation, resulting in a non-functional protein. This study may contribute to the mutational spectrum of MLH1 leading to LS.

A combination of methylation and protein markers is capable of detecting gastric cancer detection by combined markers.

Aim: This study aimed to validate a combination of mSEPT9, mRNF180 and CA724 for gastric cancer (GC) detection. Patients & methods: The performance of mSEPT9, mRNF180 and CA724 was examined in a prospective cohort study with 518 participants (151 with GC, 56 with atrophic gastritis, 87 with other gastrointestinal diseases and 224 with no evidence of disease). Results:mSEPT9, mRNF180 or CA724 alone detected 48.3, 37.1 and 43.1% of GC, respectively. The combination of mSEPT9 and mRNF180 detected 60.3% of GC, and the combination of all three markers detected 68.6% of GC. The detection sensitivity of mSEPT9 and mRNF180 was significantly higher for gastric body and in elder subjects. mSEPT9 was correlated with poorer GC survival. Conclusion: The combination of mSEPT9, mRNF180 and CA724 was adequately sensitive for GC detection. The blood mSEPT9 was predictive for GC prognosis.

Lay abstract Gastric cancer is the most common cancer type in the digestive system. In this study we developed an effective and convenient blood-based test to detect gastric cancer. This test combined a conventional protein test with a newly invented methylation test. We aimed to validate the test and examine its performance using a prospective cohort study. The study showed that the test has promising potential for noninvasive early detection of gastric cancer. We found that single protein or methylation markers detected a proportion of gastric cancer cases, while a combination of these markers exhibited maximal detection capability. Therefore we concluded that the combined test provided adequate efficacy and should be used as a strategy for future gastric cancer detection.

LncRNA ADAMTS9-AS1 knockdown restricts cell proliferation and EMT in non-small cell lung cancer.

A recent bioinformatics analysis identified long non-coding RNA antisense 1 ADAMTS9-AS1 as an independent prognostic marker in several tumors, including prostate cancer and bladder cancer. Nevertheless, the prognostic value and functional role of ADAMTS9-AS1 in non-small cell lung cancer (NSCLC) remain elusive. Here, we first found that the expression of ADAMTS9-AS1 was significantly upregulated in NSCLC tissues compared with adjacent normal tissues using quantitative real time PCR analysis. Clinically, we observed that ADAMTS9-AS1 expression was associated with TNM stage, lymph node metastasis and poor prognosis in NSCLC patients. By performing loss-of-function assay in A549 and 95D cells, our in vitro experiments further showed that knockdown of ADAMTS9-AS1 remarkedly suppressed cell proliferation, caused cell cycle G0/G1 arrest and apoptosis, and inhibited cell migration and invasion in NSCLC cells using CCK-8, colony formation, flow cytometry and transwell assays. Moreover, we found that ADAMTS9-AS1 knockdown downregulated the expression of CDK4, N-cadherin, Vimentin, but upregulated the expression of Bad and E-cadherin. In summary, our results revealed that ADAMTS9-AS1 may serve as a potential therapeutic target for the treatment of patients with NSCLC.

Small nucleolar RNA host gene 22 (SNHG22) promotes the progression of esophageal squamous cell carcinoma by miR-429/SESN3 axis.

BACKGROUND: It has been observed that lncRNAs have been taking part in many cancer progressions, including non-small cell lung cancer and gastric cancer. Meanwhile, lncRNA small nucleolar RNA host gene 22 (SNHG22) has been studied, taking part in the progression of ovarian epithelial carcinoma. However, we know little about the function of SNHG22 in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, we will explore the inner mechanism of SNHG22 in ESCC. Quantitative real-time PCR (qRT-PCR) assay was implemented in ESCC cells for detecting the expression of lncRNA, SNHG22, and miR-429. Also, functional experiments, including CCK8 and colony formation assay, were implemented to assess the growth of ESCC cells. Meanwhile, flow cytometry analysis was conducted to test the apoptosis of ESCC cells. The immunofluorescence (IF) assay and western blot were conducted to verify the autophagy of ESCC cells. RESULTS: Inhibition of SNHG22 was found that can inhibit the progression and promotes autophagy and apoptosis of ESCC cells. Meanwhile, as subcellular fraction assay and FISH assay found that SNHG22 mainly in the cytoplasm, miR-429 was found can bind to SNHG22 and SESN3 by RIP assay and luciferase reporter assay. SESN3 was found it can play the oncogene in ESCC cells. CONCLUSIONS: SNHG22 promotes the progression of ESCC by the miR-429/SESN3 axis.

The influence of exendin-4 intervention on -obese diabetic mouse blood and the pancreatic tissue immune microenvironment.

The aim of the study was to determine the influence of exendin-4 intervention on non-obese diabetic (NOD) mouse blood and the pancreatic tissue immune microenvironment. A total of 40 clean NOD mice were used in the study and randomly divided into 4 groups (n=10/group). The first group was blank control group D with normal saline intervention, and with different doses of exendin, i.e.,-4 2, 4 and 8 µg/kg/day. The three remaining groups were: i) Low-dose group A; ii) medium-dose group B; and iii) high-dose group C. Mice in the four groups went through intervention for 8 weeks. Their mass and blood glucose levels were tested each week. After 8 weeks, the mice were sacrificed, and mouse serum samples were reserved. The ELISA method was used to test peripheral blood (PB), IL-2, IFN-γ and IL-10 levels. Pancreatic samples were created. Immunohistochemistry was used to observe the infiltration degree of mouse pancreatitis and the local expression state of pancreatic IL-10. Mouse pancreatic tissues were suspended in pancreatic cell suspension. Flow cytometry was used to test the state of T-cell subsets CD4 and CD25. Mouse pancreatitis in control group D was mainly at grade 2and 3. Under a light microscope, it was observed that pancreatic cell morphology was in disorder, and the size and quantity of the pancreas was small. Mouse pancreatitis in the exendin-4 low-dose group A, medium-dose group B and high-dose group C was mainly at grade 0 and 1. Under a light microscope, it was observed that pancreatic cell morphology improved, the infiltration degree of lymphocyte was improved and pancreatic islet size was restored somewhat. Additionally, a few brownish granules were identified within the pancreatic sample cells in control group D. There were many brownish granules with deep color within the pancreatic sample cells in exendin-4 low-dose group A, medium-dose group B and high-dose group C. IL-10 immunohistochemistry scores in the low-dose group A, medium-dose group B and high-dose group C were 3.82±0.72, 4.34±0.86 and 4.81±0.94, respectively, and were higher than the score of 2.25±0.63 in control group D. CD4+CD25+T-cell proportions in mouse pancreatic tissues of low-dose group A, medium-dose group B and high-dose group C were 5.31, 5.53 and 5.74%, respectively, which were higher than that of the CD4+CD25+T-cell proportion (1.62% in control group D). The CD4+CD25high T-cell proportion in CD4+T-cells in group A, B and C increased. Compared with control group D, serum IL-10 levels in the exendin-4 low-dose group A, medium-dose group B and high-dose group C increased (P<0.05), while levels of IL-2 and IFN-γ decreased (P<0.05). Additionally, the difference of serum IL-10, IL-2 and IFN-γ levels in the low-dose group A, medium-dose group B and high-dose group C was of statistical significance (P<0.05). Exendin-4 intervention can increase quantities of CD4 and CD8+T cells in NOD mouse pancreases, with PB IL-10 expression and local expression of IL-10 in pancreatic tissues. It also can inhibit the expression of serum IL-2 and IFN-γ, regulate the organism immune microenvironment and prevent diabetes. CD4+CD25high T cells increase in NOD tumor infiltration lymphocytes mediated by exendin-4 intervention, which may be related to the fact that exendin-4 inhibits the lethal effect of CD8+T cells through contact among cells and eventually exerts immunosuppressive effect.