Research on the correlation between activating transcription factor 3 expression in the human coronary artery and atherosclerotic plaque stability.

BACKGROUND: Activating transcription factor 3 (ATF3) is an early response gene that is activated in response to atherosclerotic stimulation and may be an important factor in inhibiting the progression of atherosclerosis. In this study, we directly measured the expression of ATF3 and inflammatory factors in human coronary atherosclerotic plaques to examine the relationship between ATF3 expression, inflammation and structural stability in human coronary atherosclerotic plaques. METHODS: A total of 68 coronary artery specimens were collected from the autopsy group, including 36 cases of sudden death from coronary heart disease (SCD group) and 32 cases of acute death caused by mechanical injury with coronary atherosclerosis (CHD group). Twenty-two patients who had no coronary heart disease were collected as the control group (Con group). The histological structure of the coronary artery was observed under a light microscope after routine HE staining, and the intimal and lesion thicknesses, thickness of the fibrous cap, thickness of necrosis core, degree of lumen stenosis were assessed by image analysis software. Western blotting and immunohistochemistry were used to measure the expression and distribution of ATF3, inflammatory factors (CD45, IL-1ß, TNF-α) and matrix metalloproteinase-9 (MMP-9) and vascular cell adhesion molecule 1 (VCAM1) in the coronary artery. The Pearson correlation coefficient was used to analyse the correlation between ATF3 protein expression and inflammatory factors and between ATF3 protein expression and structure-related indexes in the lesion group. RESULTS: Compared with those in the control group, the intima and necrotic core in the coronary artery were thickened, the fibrous cap became thin and the degree of vascular stenosis was increased in the lesion group, while the intima and necrotic core became thicker and the fibrous cap became thinner in the SCD group than in the CHD group (P < 0.05). There was no or low expression of ATF3, inflammatory factors, VCAM1 and MMP-9 in the control group, and the expression of inflammatory factors, VCAM1 and MMP-9 in the SCD group was higher than that in CHD group, while the expression of ATF3 in the SCD group was significantly lower than that in CHD group (P < 0.05). In the lesion group, the expression of ATF3 was negatively correlated with intimal and necrotic focus thickness, positively correlated with fibrous cap thickness (P < 0.01), and negatively correlated with inflammatory factors, VCAM1 and MMP-9 (P < 0.01). CONCLUSIONS: The expression of ATF3 may be related to the progression and stability of atherosclerotic plaques, and may affect the structural stability of atherosclerotic plaques by regulating the inflammatory response, thus participating in the regulation of atherosclerotic progression.

Activating transcription factor 3 (ATF3) regulates cell growth, apoptosis, invasion and collagen synthesis in keloid fibroblast through transforming growth factor beta (TGF-beta)/SMAD signaling pathway.

The successful treatment of keloids is a great challenge in the plastic surgery field. Activating transcription factor 3 (ATF3) is discovered as an adaptive responsive gene, which plays a critical role in fibroblast activation. This study aimed to investigate the expression and biological role of ATF3 in the pathogenesis of keloids. ATF3 expression in normal skins and keloids was evaluated by real-time PCR, western blot and immunohistochemistry. Effects of ATF3 on cell growth, apoptosis, invasion and collagen production were evaluated in keloid fibroblast cells overexpressing or downregulating ATF3. ATF3 expression was significantly elevated in keloid tissues when compared with that of normal skins and parakeloidal skin tissues. Moreover, ATF3 promoted cell proliferation and collagen production in keloid fibroblast cells. Conversely, transfection with siRNA targeting ATF3 led to decreased cell viability and collagen synthesis via inhibiting transforming growth factor-ß1 (TGF-ß1) and fibroblast growth factor 2/8 (FGF2/8) production in keloid fibroblasts. ATF3 could reduce the apoptosis rate of keloid fibroblast cells. Molecularly, we found that ATF3 promoted BCL2 level and inhibit the expression of BCL2 associated agonist of cell death (Bad), Caspase3 and Caspase9 in keloid fibroblast cells. ATF3 also enhanced the invasive potential via upregulating the expression of Matrix Metalloproteinases (MMP) family members (MMP1, MMP2, MMP9 and MMP13). ATF3 could induce activation of TGF-ß/Smad signaling pathway in fibroblasts. Collectively, ATF3 could promote growth and invasion, and inhibit apoptosis via TGF-ß/Smad pathway in keloid fibroblast cells, suggesting that ATF3 might be considered as a novel therapeutic target for the management of keloid.

Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg), which results in enhanced cerebrovascular permeability and leads to short-term memory (STM) reduction. Previously, we showed that extravasated Fg was deposited in the vasculo-astrocyte interface and was co-localized with cellular prion protein (PrPC) during mild-to-moderate TBI in mice. These effects were accompanied by neurodegeneration and STM reduction. However, there was no evidence presented that the described effects were the direct result of the HFg during TBI. We now present data indicating that inhibition of Fg synthesis can ameliorate TBI-induced cerebrovascular permeability and STM reduction. Cortical contusion injury (CCI) was induced in C57BL/6J mice. Then mice were treated with either Fg antisense oligonucleotide (Fg-ASO) or with control-ASO for two weeks. Cerebrovascular permeability to fluorescently labeled bovine serum albumin was assessed in cortical venules following evaluation of STM with memory assessement tests. Separately, brain samples were collected in order to define the expression of PrPC via Western blotting while deposition and co-localization of Fg and PrPC, as well as gene expression of inflammatory marker activating transcription factor 3 (ATF3), were characterized with real-time PCR. Results showed that inhibition of Fg synthesis with Fg-ASO reduced overexpression of AFT3, ameliorated enhanced cerebrovascular permeability, decreased expression of PrPC and Fg deposition, decreased formation of Fg-PrPC complexes in brain, and improved STM. These data provide direct evidence that a CCI-induced inflammation-mediated HFg could be a triggering mechanism involved in vascular cognitive impairment seen previously in our studies during mild-to-moderate TBI.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

The expression of ATF3, MMP-2 and maspin in tissue chip of glioma.

This paper tested and analyzed the expression of ATF3 (activating transcription factor), MMP-2 (matrix metalloprotease) and maspin in tissue chip of glioma and its correlation with glioma advancement. Based on immunohistochemical staining, this paper selected 100 patients with glioma and 13 healthy persons to test the relative expression of ATF3, MMP-2 and maspin. The result witnessed 72.0% of ATF3 expression in glioma and 15.4% in healthy brain tissues with P<0.05; glioma had 76.0% of MMP-2 expression while healthy brain tissues only had 7.7% (P<0.05); but maspin expression with 53.0% in glioma was much lower than that with 100% in healthy tissues with P<0.05. If the pathological stage of glioma rose up, the expression of ATF3 and MMP-2 accordingly increased while maspin expression decreased. The correlation between ATF3 expression and MMP-2 expression was positive with r=0.553 and p<0.01; negative correlation between ATF3 expression and maspin expression was found with r=-0.457 and p<0.01; and the expression of MMP-2 and maspin were negatively related with r=-0.551 and p<0.01. According to the above results, it could be concluded that the expression of ATF3, MMP-2 and maspin did relate with each other. Besides, the high expression of ATF3 and MMP-2 as well as the low expression of maspin had great influence on glioma, playing a key role in glioma's occurrence, advancement, invasion and metastasis.

ATF3 expression is induced by low glucose in pancreatic α and ß cells and regulates glucagon but not insulin gene transcription.

The pancreas is critical for maintaining glucose homeostasis. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. There are major discrepancies in previous reports on pancreatic ATF3; therefore, its role in the pancreas is unclear. To better elucidate the role of ATF3 in the pancreas, we conducted in vitro studies using pancreatic α and ß cell lines, and also evaluated the use of ATF3 antibodies for immunohistochemistry. We determined ATF3 expression was increased by low glucose and decreased by high glucose in both αTC-1.6 and ßTC3 cells. We also showed that adenovirus-mediated ATF3 overexpression increased glucagon promoter activity and glucagon mRNA levels in αTC-1.6 cells; whereas, it had no effect on insulin promoter activity and insulin mRNA levels in ßTC3 cells. Although immunostaining with the C-19 ATF3 antibody demonstrated predominant expression in α cells rather than ß cells, ATF3 staining was still detected in ATF3 knockout mice as clearly as in control mice. On the other hand, another ATF3 antibody (H-90) detected ATF3 in both α cells and ß cells, and was clearly diminished in ATF3 knockout mice. These results indicate that previous discrepancies in ATF3 expression patterns in the pancreas were caused by the varying specificities of the ATF3 antibodies used, and that ATF3 is actually expressed in both α cells and ß cells.

In vivo RNAi screen for BMI1 targets identifies TGF-ß/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis.

In mouse and human neural progenitor and glioblastoma "stem-like" cells, we identified key targets of the Polycomb-group protein BMI1 by combining ChIP-seq with in vivo RNAi screening. We discovered that Bmi1 is important in the cellular response to the transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) and endoplasmic reticulum (ER) stress pathways, in part converging on the Atf3 transcriptional repressor. We show that Atf3 is a tumor-suppressor gene inactivated in human glioblastoma multiforme together with Cbx7 and a few other candidates. Acting downstream of the ER stress and BMP pathways, ATF3 binds to cell-type-specific accessible chromatin preloaded with AP1 and participates in the inhibition of critical oncogenic networks. Our data support the feasibility of combining ChIP-seq and RNAi screens in solid tumors and highlight multiple p16(INK4a)/p19(ARF)-independent functions for Bmi1 in development and cancer.

A novel model of combined neuropathic and inflammatory pain displaying long-lasting allodynia and spontaneous pain-like behaviour.

Many clinical cases of chronic pain exhibit both neuropathic and inflammatory components. In contrast, most animal models of chronic pain focus on one type of injury alone. Here we present a novel combined model of both neuropathic and inflammatory pain and characterise its distinctive properties. This combined model of chronic constriction injury (CCI) and intraplantar Complete Freund's Adjuvant (CFA) injection results in enhanced mechanical allodynia, thermal hyperalgesia, a static weight bearing deficit, and notably pronounced spontaneous foot lifting (SFL) behaviour (which under our conditions was not seen in either individual model and may reflect ongoing/spontaneous pain). Dorsal root ganglion (DRG) expression of Activating Transcription Factor-3 (ATF-3), a marker of axonal injury, was no greater in the combined model than CCI alone. Initial pharmacological characterisation of the new model showed that the SFL was reversed by gabapentin or diclofenac, typical analgesics for neuropathic or inflammatory pain respectively, but not by mexiletine, a Na(+) channel blocker effective in both neuropathic and inflammatory pain models. Static weight bearing deficit was moderately reduced by gabapentin, whereas only diclofenac reversed mechanical allodynia. This novel animal model of chronic pain may prove a useful test-bed for further analysing the pharmacological susceptibility of complicated clinical pain states.

Alterations in neuronal gene expression profiles in response to experimental demyelination and axonal transection.

The main pathological features of multiple sclerosis, demyelination and axonal transection, are considered to cause reversible and irreversible neurological deficits, respectively. This study aimed to separately analyze the effects of these pathological hallmarks on neuronal gene expression in experimental paradigms. The pontocerebellar pathway was targeted with either lysolecithin-induced chemical demyelination or a complete pathway transection (axonal transection) in rats. Transcriptional changes in the pontocerebellar neurons were investigated with microarrays at days 4, 10 and 37 post-intervention, which was confirmed by immunohistochemistry on protein level. A common as well as unique set of injury-response genes was identified. The increased expression of activating transcription factor 3 (Atf3) and thyrotropin-releasing hormone (Trh) in both injury paradigms was validated by immunohistochemistry. The expression of Atf3 in a patient with Marburg's variant of multiple sclerosis was also detected, also confirming the activation of the Atf3 pathway in a human disease sample. It was concluded that this experimental approach may be useful for the identification of pathways that could be targeted for remyelinative or neuroprotective drug development.

[Isolation of Id1 interaction protein in human prostate cancer].

OBJECTIVE: To isolate the inhibitor of DNA binding 1 (Id1) interaction protein and to determine the role and action mechanism of Id1 in human prostate cancer. METHODS: The expression vector pET-28a/Id1 was established and used as a bait to prey the interaction protein by pull-down assay. RESULTS: A clear interaction protein band was observed by SDS-PAGE, which was found to be activating transcription factor 3 (ATF3) by Western blotting. CONCLUSION: Id1 may play a role in human prostate cancer by interacting with ATF3.

Nucleolar enlargement, nuclear eccentricity and altered cell body immunostaining characteristics of large-sized sensory neurons following treatment of rats with paclitaxel.

Paclitaxel-induced sensory neuropathy is a problematic side-effect of cancer chemotherapy. Previous studies in rodents have shown paclitaxel treatment to have many effects on different parts of the peripheral nervous system, but those responsible for its bothersome clinical side-effects are still unclear. In the current study, we sought to obtain information about the involvement of sensory neurons in paclitaxel neurotoxicity at the level of the dorsal root ganglion. Rats were treated with a clinically relevant dose of paclitaxel (87.5mg/m(2) weekly for a total of nine doses) to induce a sensory neuropathy; then their L5 dorsal root ganglia were studied by morphometry and immunohistochemistry. Paclitaxel treatment was generally well tolerated, and slowed conduction velocity and prolonged conduction latencies in the peripheral sensory nerves without altering conduction in the central or motor pathways of the H-reflex arc. In the L5 dorsal root ganglion, nucleolus size and the number of neurons with eccentric nuclei were increased only in a subpopulation of dorsal root ganglion neurons with cell body cross-sectional areas greater than 1750 microm(2), which made up less than 10% of the total population. Paclitaxel treatment increased immunohistochemical staining for activating transcription factor-3 (ATF-3), c-Jun and neuropeptide Y (NPY) but only in a small percentage of neuronal cell bodies and mainly in those with large cell bodies. In conclusion, we have demonstrated that nucleolar enlargement, nuclear eccentricity, ATF-3, c-Jun and NPY are neuronal markers of paclitaxel-induced sensory neuropathy, however, these axotomy-like cell body reactions are infrequent and occur in mainly large-sized sensory neurons.

Activating transcription factor 3 is up-regulated in patients with hypospadias.

Hypospadias is a congenital anomaly of the genitalia characterized by abnormalities of the urethra and foreskin, with the urethral meatus located in an abnormal position anywhere from the distal ventral penile shaft to the perineum. Because the incidence of hypospadias is approximately 1/200-1/300 live male births, it is one of the most common congenital malformations, but its etiology is largely uncharacterized. Genomic analysis of hypospadic tissue indicated a potential role for activating transcription factor 3 (ATF3) in the development of this anomaly. ATF3 may be involved in homeostasis, wound healing, cell adhesion, or apoptosis, and normally it is expressed at a steady-state in quiescent cells. Additionally, it has been shown to be an estrogen-responsive gene, and the etiology of hypospadias may be related to in utero exposure to estrogenic or anti-androgenic compounds. We examined the expression of ATF3 in tissues from 28 children with hypospadias compared with 20 normal penile skin tissue samples from elective circumcision. Eighty-six percent of the hypospadias samples were immunohistochemically positive, compared with 13% of normal tissue samples. Seventy-five percent of hypospadias samples were positive from in situ hybridization, compared with 1% of circumcision samples. Our results indicate that ATF3 is up-regulated in the penile skin tissues of boys with hypospadias, suggesting a role for this transcription factor in the development of this abnormality. Because the etiology of hypospadias may include exposure to estrogenic compounds, the responsiveness of ATF3 to estrogen is also discussed.