Elevated ATF4 Expression in Odontogenic Keratocysts Epithelia: Potential Involvement in Tissue Hypoxia and Stromal M2 Macrophage Infiltration.

The aim of this study was to investigate the expression of the activating transcription factor 4 (ATF4) in odontogenic keratocysts (OKC), its association with hypoxia and M2-polarized macrophages infiltration, and its potential relationships with angiogenesis in OKC. The expression of ATF4, hypoxia-inducible factor 1α (HIF-1α), macrophage colony-stimulating factor (M-CSF), and receptor activator of nuclear factor κ-B ligand (RANKL) in OKC samples and normal oral mucosa (OM) was detected by immunohistochemistry. Meanwhile, microvessel density (MVD) was measured using antibody against CD31. M2-polarized macrophages were identified using double-staining for CD68+ and CD163+. The correlations of ATF4 with HIF-1α, M-CSF, and M2-polarized macrophages infiltration were determined by Spearman's rank correlation test and hierarchical clustering. Human immortalized oral epithelial cells (HIOECs) were used in in vitro experiments. Our data showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in the epithelium of OKC when compared with the OM. The expression of ATF4 was positively correlated with that of HIF-1α, M-CSF, MVD, and M2-polarized macrophages infiltration. Elevated expression of ATF4 in the epithelial lining of OKC may facilitate the M2 macrophages infiltration in response to hypoxia, leading to the development of OKC.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4'-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4.

The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPß:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPß and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg(-/-) mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg(-/-) mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.

Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions.

In Alzheimer's disease (AD) brain, exposure of axons to Aß causes pathogenic changes that spread retrogradely by unknown mechanisms, affecting the entire neuron. We found that locally applied Aß1-42 initiates axonal synthesis of a defined set of proteins including the transcription factor ATF4. Inhibition of local translation and retrograde transport or knockdown of axonal Atf4 mRNA abolished Aß-induced ATF4 transcriptional activity and cell loss. Aß1-42 injection into the dentate gyrus (DG) of mice caused loss of forebrain neurons whose axons project to the DG. Protein synthesis and Atf4 mRNA were upregulated in these axons, and coinjection of Atf4 siRNA into the DG reduced the effects of Aß1-42 in the forebrain. ATF4 protein and transcripts were found with greater frequency in axons in the brain of AD patients. These results reveal an active role for intra-axonal translation in neurodegeneration and identify ATF4 as a mediator for the spread of AD pathology.

Study on changes of clinical indicators and key proteins from fluoride exposure.

Few studies have evaluated the biomarker changes of fluoride exposure. In order to explore early and sensitive indicators, animal experiment was designed. Ninety-six healthy SD rats (48 males and 48 females) weighing approximately 60 g were randomly divided into six groups of 16 animals each by gender average. Control animals were supplied with distilled water only as group 1. Exposure groups' animals were treated with 2, 4, 8, 16, and 32 mg NaF/kg bw, respectively, as groups 2, 3, 4, 5, and 6. Our study found that contents of white blood cell (WBC), lymphocyte percentage (LYMPH%), lymphocyte (LYM), mean platelet volume (MPV), and platelet distribution width (PDW) increased significantly in high-fluoride-exposure groups (p < 0.05), which revealed that immune system may be interfered by high fluoride. Meanwhile, levels of alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and ALT/AST in groups 5 and 6 decreased significantly compared to those in control group (p < 0.05), as well as the concentration of uric acid (UA) in groups 3, 4, 5, and 6 exhibited the same trends (p < 0.05). On the contrary, the level of blood B2 microglobulin (BB2MG) increased significantly (p < 0.05) in groups 4, 5, and 6. Changes of ALT, AST, UA, and BB2MG suggested the functions of the liver and kidney be altered by fluoride exposure. At the same time, the ATF4 content decreased gradually with the increase of fluoride concentration; furthermore, a highly significant (r = -0.586, p < 0.01) negative relationship between ATF4 content and fluoride exposure level was found. Results meant that clinical indicators cannot act as indicators of high fluoride exposure, and it also suggested that protein ATF4 might be the early and sensitive indicator in epidemiologic study of high fluoride exposure.

Lentiviral-mediated expression of SATB2 promotes osteogenic differentiation of bone marrow stromal cells in vitro and in vivo.

Special AT-rich sequence-binding protein 2 (SATB2 ) acts as a potent transcription factor to promote osteoblast differentiation and bone regeneration. In this study, we first used lentiviral-mediated gene transfer of Satb2 into mouse bone marrow stromal cells (BMSCs) and investigated the capacity of SATB2 overexpression to promote osteogenic differentiation in vitro and in vivo. We found that LV-Satb2 -transduced BMSCs produced SATB2 protein and underwent rapid and marked osteogenic differentiation, as demonstrated by increased expression of osteoblastic genes, including runt-related transcription factor 2 (Runx2), transcription factor Sp7 (Sp7), activating transcription factor 4 (Atf4), and bone sialoprotein (Bsp), and increased alkaline phosphatase activity and Alizarin Red S staining. To analyze the induction of bone formation in vivo, LV-Satb2-transduced BMSCs were implanted into the hindlimbs of syngeneic mice, with ß-tricalcium phosphate as the scaffolding material. Four weeks after implantation, transduction with LV-Satb2 had greatly enhanced the formation of new bone. These data demonstrated the capacity of lentiviral-mediated SATB2 to promote the osteogenic differentiation of BMSCs in vitro and to enhance bone formation through a tissue-engineering technique that may be useful in bone-regenerative medicine.

Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms.

Angiogenesis is the hallmark of cancer, and development of aggressiveness of primary tumor depends on de novo angiogenesis. Here, using multiple in vitro and in vivo models, we report that osteopontin (OPN) triggers vascular endothelial growth factor (VEGF)-dependent tumor progression and angiogenesis by activating breast tumor kinase (Brk)/nuclear factor-inducing kinase/nuclear factor-kappaB (NF-kappaB)/activating transcription factor-4 (ATF-4) signaling cascades through autocrine and paracrine mechanisms in breast cancer system. Our results revealed that both exogenous and tumor-derived OPN play significant roles in VEGF-dependent tumor angiogenesis. Clinical specimen analysis showed that OPN and VEGF expressions correlate with levels of neuropilin-1, Brk, NF-kappaB, and ATF-4 in different grades of breast cancer. Consequently, OPN plays essential role in two key aspects of tumor progression: VEGF expression by tumor cells and VEGF-stimulated neovascularization. Thus, targeting OPN and its regulated signaling network could be a novel strategy to block tumor angiogenesis and may develop an effective therapeutic approach for the management of breast cancer.

The effect of centrifugal force on the mRNA and protein levels of ATF4 in cultured human periodontal ligament fibroblasts.

OBJECTIVE: The aim of this study was to examine the changes of ATF4 expression in cultured human periodontal ligament fibroblasts (hPDLF) after mechanical stimuli, and to investigate whether ATF4 is essential for the mechanical stress-induced hPDLF differentiation. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting were used to examine mRNA and protein levels of ATF4 expression in hPDLFs after application of centrifugal force. pMyc-ATF4 transfected cells were subjected to centrifugal force for 30min, and the changes of alkaline phosphatase (ALP) activity and osteocalcin (OCN), osteopontin (OPN), collagen I (COLI), bone sialoprotein (BSP) genes were measured to assess the differentiation of hPDLFs. RESULTS: The mRNA and protein levels of ATF4 increased shortly and then decreased rapidly towards its pre-pressure levels. Overexpression of pMyc-ATF4 exhibited a greater increase in ALP activity and all four osteogenic genes compared to the untransfected cells in response to the centrifugal force. CONCLUSION: Our results indicate that ATF4 is essential in response of hPDLFs to mechanical stress, resulting in increased differentiation of hPDLFs to osteoblast-like cells.