Decreased serum dependence in the growth of NIH3T3 cells from the overexpression of human nuclear receptor-binding SET-domain-containing protein 1 (NSD1) or fission yeast su(var)3-9, enhancer-of-zeste, trithorax 2 (SET2)

Nuclear receptor-binding SET-domain-containing protein 1 (NSD1), a culprit gene for Sotos syndrome, contains a su(var)3-9, enhancer-of-zeste, trithorax (SET) domain that is responsible for histone methyltransferase activity and other domains such as plant homeodomain (PHD) and proline-tryptophan-tryptophan-proline (PWWP) involved in protein-protein interactions in the C-terminal half of NSD1. To elucidate the function of NSD1 on cell growth, we overexpressed NSD1 in NIH3T3 cells. Cells overexpressing NSD1 grew in the presence of 2% serum, whereas vector transfected cells did not. Overexpression of the C-terminal half of NSD1 but not the N-terminal half of NSD1 also produced cell growth under low serum concentration. Furthermore, overexpression in NIH3T3 of Schizosaccharomyces pombe SET2 which has a SET domain but not PHD or PWWP domains conferred the reduced serum dependence. Thus, the SET domain of NSD1 is involved in cell growth by modulating serum dependence.     

Calmodulin‐like skin protein suppresses the increase in senescence‐associated β‐galactosidase induced by hydrogen peroxide or ultraviolet irradiation in keratinocytes

Calmodulin‐like skin protein (CLSP) is a secreted peptide that is produced by skin keratinocytes and some related epithelial cells. It has previously been shown that CLSP is recruited via the bloodstream into the central nervous system where it likely exerts a neuroprotective effect against toxicity related to Alzheimer's disease (AD) by binding to the heterotrimeric humanin receptor and activating intracellular survival signaling. However, it remains to be elucidated whether secreted CLSP shows a protective effect in the skin tissues. In the current study, using primary keratinocytes treated with hydrogen peroxide (H₂O₂) or exposed to ultraviolet (UV) irradiation as senescence models of keratinocytes, we addressed whether CLSP affects senescence in skin keratinocytes. We found that CLSP expression was upregulated by H₂O₂ or UV in keratinocytes. Furthermore, co‐incubation with recombinant CLSP reduced the increase in senescence‐associated β‐galactosidase‐positivity in keratinocytes that were induced by H₂O₂ or UV. These results suggest that CLSP may function as a senescence‐suppressing factor in keratinocytes.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Exogenous high‐mobility group box 1 protein prevents postinfarction adverse myocardial remodeling through TGF‐β/Smad signaling pathway

High‐mobility group box 1 (HMGB1) has been reported to attenuate ventricular remodeling, but its mechanism remains mostly unresolved. Transforming growth factor‐beta (TGF‐β) is a crucial mediator in the pathogenesis of post‐infarction remodeling. Our study focused on the effects of HMGB1 on ventricular remodeling, and explored whether or not these effects were depended upon the TGF‐β signaling pathway. Rats underwent coronary artery ligation. An intramyocardium injection of phosphate buffered saline (PBS) with or without HMGB1 was administered 3 weeks after myocardial infarction (MI). At 4 weeks after the treatment, HMGB1 significantly increased the left ventricular ejection fraction (LVEF) (P < 0.05), decreased the left ventricular end diastolic dimension (LVEDD; P < 0.05), left ventricular end systolic dimension (LVESD) (P < 0.05) and the infarct size (P < 0.05) compared with control group. The expressions of collagen I, collagen III, and tissue inhibitor of metalloproteinase 2 (TIMP2) were also decreased, while the matrix metalloproteinases 2 (MMP2) and MMP9 expressions were upregulated by HMGB1 injection (P < 0.05) compared with control group. No effect on TIMP3 was observed. Furthermore, TGF‐β1 and phosphor‐Smad2 (p‐Smad2) were significantly suppressed and Smad7 was increased in HMGB1‐treated group (P < 0.05) compared with control group, no effects on p‐Smad3 and p‐p38 were observed. HMGB1 also upregulated Smad 7 expression and decreased the level of collagen I on cardiac fibroblasts (P < 0.05). Silencing of Smad7 gene by small interfering RNA abolished the fibrogenic effects of HMGB1 on cardiac fibroblasts (P < 0.05). These finding suggested that HMGB1 injection modulated ventricular remodeling may function through the possible inhibition of TGF‐β/Smad signaling pathway. J. Cell. Biochem. 114: 1634–1641, 2013. © 2013 Wiley Periodicals, Inc.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.     

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1‐βTrCP2‐Cdc25A‐mediated mitotic entry to ensure neurogenesis

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase βTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of βTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1‐deficient neuroprogenitors in vivo. MCPH1 itself is degraded by APC/C^Cdh1, but not APC/C^Cdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1‐knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin‐proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.     

Targeting the ROS/PI3K/AKT/HIF‐1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2‐Deoxy‐d‐glucose

It is well established that cancer cells depend upon aerobic glycolysis to provide the energy they need to survive and proliferate. However, anti‐glycolytic agents have yielded few positive results in human patients, in part due to dose‐limiting side effects. Here, we discovered the unexpected anti‐cancer efficacy of Polydatin (PD) combined with 2‐deoxy‐D‐glucose (2‐DG), which is a compound that inhibits glycolysis. We demonstrated in two breast cell lines (MCF‐7 and 4T1) that combination treatment with PD and 2‐DG induced cell apoptosis and inhibited cell proliferation, migration and invasion. Furthermore, we determined the mechanism of PD in synergy with 2‐DG, which decreased the intracellular reactive oxygen (ROS) levels and suppressed the PI3K/AKT pathway. In addition, the combined treatment inhibited the glycolytic phenotype through reducing the expression of HK2. HK2 deletion in breast cancer cells thus improved the anti‐cancer activity of 2‐DG. The combination treatment also resulted in significant tumour regression in the absence of significant morphologic changes in the heart, liver or kidney in vivo. In summary, our study demonstrates that PD synergised with 2‐DG to enhance its anti‐cancer efficacy by inhibiting the ROS/PI3K/AKT/HIF‐1α/HK2 signalling axis, providing a potential anti‐cancer strategy.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

High‐fat diet‐induced dysbiosis mediates MCP‐1/CCR2 axis‐dependent M2 macrophage polarization and promotes intestinal adenoma‐adenocarcinoma sequence

High‐fat diet (HFD) is a well‐known risk factor for gut microbiota dysbiosis and colorectal cancer (CRC). However, evidence relating HFD, gut microbiota and carcinogenesis is limited. Our study aimed to demonstrate that HFD‐induced gut dysbiosis promoted intestinal adenoma‐adenocarcinoma sequence. In clinical study, we found that HFD increased the incidence of advanced colorectal neoplasia (AN). The expression of monocyte chemoattractant protein 1 (MCP‐1), CC chemokine receptor 2 (CCR2) and CD163 in CRC patients with HFD was significantly higher than that in CRC patients with normal diet. When it comes to the Apc^min/+ mice, HFD consumption could induce gut dysbiosis and promote intestinal carcinogenesis, accompanying with activation of MCP‐1/CCR2 axis that recruited and polarized M2 tumour‐associated macrophages. Interestingly, transfer of faecal microbiota from HFD‐fed mice to another batch of Apc^min/+ mice in the absence of HFD could also enhance carcinogenesis without significant body weight gain and induced MCP‐1/CCR2 axis activation. HFD‐induced dysbiosis could also be transmitted. Meanwhile, antibiotics cocktail treatment was sufficient to inhibit HFD‐induced carcinogenesis, indicating the vital role of dysbiosis in cancer development. Conclusively, these data indicated that HFD‐induced dysbiosis accelerated intestinal adenoma‐adenocarcinoma sequence through activation of MCP‐1/CCR2 axis, which would provide new insight into better understanding of the mechanisms and prevention for HFD‐related CRC.