Expression of optineurin isolated from rat-injured dental pulp and the effects on inflammatory signals in normal rat kidney cells.

We previously isolated rat 14.7K-interacting protein-2 (rFIP-2) from the rat-wounded pulp. The protein, homologous to human FIP-2, is known as optineurin and was initially identified as a novel tumor necrosis factor-α (TNF-α)-inducible protein, and more recently, as an autophagy receptor. However, the biological role of optineurin in dental pulp remains elusive. We hypothesized that optineurin has a crucial role in regulating molecular processes during pulp inflammatory responses induced by TNF-α. We examined the kinetics of optineurin expression in pulp inflammation. Optineurin localization and expression were examined using rat pulp fibroblasts. The cells were treated with pharmacological inhibitors for TNF-α-induced inflammatory signals or with hydrogen peroxide as apoptotic stimuli. Stable optineurin-knockdown cells (OPTN-KD cells) were established by transfecting normal rat kidney cells with a vector expressing optineurin-specific small interfering RNA. Cell proliferation and the profiles of cytokines and intracellular signaling molecules were examined using OPTN-KD cells stimulated by TNF-α. Optineurin was localized in the cytoplasm and then translocated into the nucleus upon apoptotic stimuli. Optineurin expression was increased by TNF-α and decreased by a specific inhibitor of c-Jun N-terminal kinase. The OPTN-KD cells secreted smaller amounts of monocyte chemotactic protein-1 (MCP-1) and intracellular MCP-1 mRNA, and cell proliferation was significantly increased. Apoptosis-related signaling molecules were downregulated in OPTN-KD cells. These results demonstrated that optineurin is a crucial molecule mediated by TNF-α, which induces the production of inflammatory factors and apoptosis signaling, suggesting the presence of signaling interactions between optineurin and a transcription factor for MCP-1.

Correction to: Expression of optineurin isolated from rat-injured dental pulp and the effects on inflammatory signals in normal rat kidney cells.

In the original publication of the article, one of the author name was published incorrectly as "Keisuke Yamashairo" and correct name should be "Keisuke Yamashiro".

Gene profiles during root canal treatment in experimental rat periapical lesions.

The purpose of this study was to profile gene expression in periapical lesions during root canal treatment (RCT). Periapical lesions were induced experimentally by exposing the pulp in Sprague-Dawley rats. After 3 wk, the animals received root canal filling (RCF) and were sacrificed 1 or 4 wk later. From the periapical tissues, total RNA was extracted and processed for cDNA-microarray analysis. The lesions were histologically and radiographically confirmed to expand 4 wk after pulp exposure (inflammation phase) and to stabilize 4 wk after RCF (healing phase). In approximately 30,000 genes on the microarray, 203 genes were up-regulated to more than 5-fold (e.g., IL-1beta), and 864 genes were down-regulated to less than 20% of baseline level (e.g., caspase 8) in inflammation phase. Compared with inflammation phase, we found that 133 genes were up-regulated (e.g., IL-1alpha) and 50 genes were down-regulated (e.g., defensin alpha5) in healing phase. Corresponding to the gene expression profiles, accumulation of IL-1alpha and IL-1beta was observed in the periapical lesions by immunohistochemistry. These gene profiles might be useful in diagnosing the healing process of periapical lesions.

Oligonucleotide array analysis of cyclic tension-responsive genes in human periodontal ligament fibroblasts.

Mechanical stress results in differential gene expression that is critical to convert the stimulus into biochemical signals. Under physiological stress such as occlusal force, human periodontal ligament fibroblasts (HPLF) are associated with homeostasis of periodontal tissues however the changes in response to mechanotransduction remain uncharacterized. We hypothesized that cyclic tension-responsive (CT) genes may be used to identify a set of fundamental pathways of mechanotransduction. Our goal was to catalogue CT genes in cultured HPLF. HPLF were subjected to cyclic tension up to 16h, and total RNA was isolated from both tension-loaded and static HPLF. The oligonucleotide arrays analysis revealed significant changes of mRNA accumulation for 122 CT genes, and their kinetics were assigned by the K-means clustering methods. Ingenuity Pathway Analysis was completed for HPLF mechanotransduction using 50 CT genes. This analysis revealed that cyclic tension immediately down-regulated all nuclear transcription factors except v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) reacting as an early responsive gene. In turn, transcription factors such as tumor protein p53 binding protein 2 (TP53BP2), and extra-nuclear molecules such as adrenergic receptor beta2 (ADRB2) were up-regulated after 1-2h, which may result in fundamental HPLF functions to adapt to cyclic tension. Subsequent inhibition assays using Y27632, a pharmacologic inhibitor of Rho-associated kinase (ROCK), suggested that HPLF has both ROCK-dependent and ROCK-independent CT genes. Mechanical stress was found to effect the expression of numerous genes, in particular, expression of an early responsive gene; FOS initiates alteration of HPLF behaviors to control homeostasis of the periodontal ligament.

Cloning and characterization of lipopolysaccharide-induced tumor necrosis factor alpha factor promoter.

We have recently identified lipopolysaccharide tumor-induced tumor necrosis factor alpha factor (LITAF) as a novel transcription factor controlling necrosis factor (TNF)-alpha expression in the human monocytic cell line, THP-1. To characterize the human (h) LITAF promoter, we isolated a 1.2-kb DNA fragment and followed this by a screening of human genomic DNA with a hLITAF cDNA probe. A 34-bp sequence domain located from nucleotides -74 to -43 in the hLITAF promoter exhibited the highest basal reporter gene activity; however, the activity was not elevated by lipopolysaccharide (LPS) stimulation. The sequence domain included a consensus sequence for hepatocyte nuclear factor (HNF)-3alpha, regulating the transcription of many kinds of genes. Interestingly, the DNA sequence position between -542 and -538 in the hLITAF promoter contained the CTCCC motif, which has been reported to act as a specific binding site for hLITAF protein. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of THP-1 nuclear factors to a 22 bp probe containing the CTCCC motif. In addition, hLITAF mRNA and nuclear hLITAF protein increased significantly in the THP-1 cells immediately after LPS stimulation. These results suggest that the consensus sequence for HNF-3alpha, or a nuclear binding protein to the CTCCC motif, may play an important role in regulating LPS-dependent LITAF transcription.

Transcriptional regulation of beta-defensin-2 by lipopolysaccharide in cultured human cervical carcinoma (HeLa) cells.

Human beta-defensin-2 (hBD-2) is an antimicrobial peptide with a broad spectrum of antimicrobial activity against bacteria, yeast and fungi. Here, we analyzed the transcriptional regulation of hBD-2 in cultured human cervical carcinoma (HeLa) cells with or without lipopolysaccharide (LPS). DNA from position -329 to -39 in the hBD-2 promoter region contained the consensus binding sites for transcription factors, one site for nuclear factor for IL-6 expression (NF-IL6) and two sites for nuclear factor-(kappa)B (NF-(kappa)B). Reporter gene assays for promoter activity revealed that the region had the highest level of responsiveness to LPS. Furthermore, mutations in both of the NF-(kappa)B binding sites caused a significant reduction of the responsiveness to LPS, whereas mutation in the NF-IL6 binding site resulted in an elevation of the basal promoter activity. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of HeLa nuclear factors to 60-bp probe containing the two NF-(kappa)B binding sites, suggesting that the sites were essential for the binding. Our results suggest that the two NF-(kappa)B binding sites contribute to LPS-mediated hBD-2 transcription while the NF-IL6 binding site represses LPS-independent hBD-2 transcription in the HeLa cells.

Aspects of interleukin-8 gene expression by gingival and dermal fibroblasts stimulated with interleukin-1beta or tumour necrosis factor alpha.

Fibroblast-derived interleukin (IL)-8 is thought to have an important role in the orchestration of immuno-participant cells infiltrating the skin and gingiva in response to continuously recurring bacterial infection. Therefore, the IL-8 gene expression should be under tight regulatory control and it might be temporally and spatially limited in inflammatory tissue. The purpose of this study was to examine the aspect of the IL-8 gene expression by fibroblasts stimulated with pro-inflammatory cytokines, IL-1beta and TNF-alpha. In situ hybridisation revealed that fibroblasts did not express IL-8 mRNA whereas keratinocytes and endothelial cells did in IL-1beta- or TNF-alpha-injected mice skin. However, cultured mouse dermal fibroblasts expressed not only IL-8 but also IL-1beta mRNA without stimulation by exogenous IL-1beta and TNF-alpha, and the expression was not enhanced by the exogenous cytokines. A similar result was obtained in late-passage human gingival fibroblasts. These results suggest that fibroblasts remain insensitive to IL-1beta and TNF-alpha so as to induce the IL-8 gene expression in non-inflammatory mice skin. Mouse dermal and late-passage human gingival fibroblasts in vitro are likely to be altered in phenotype into IL-8-producing cells along with the production of IL-1beta. In skin inflammation and periodontal diseases, fibroblasts may express the IL-8 gene even without an exogenous cytokine, IL-1beta or TNF-alpha, during their proliferation similar to the situation in our culture system.

Unique genes induced by mechanical stress in periodontal ligament cells.

OBJECTIVES: The aim of this study is to isolate mechanical stress-induced genes (MSGens) from human periodontal ligament (PDL) cells and to analyze profiles of the mRNA expression of these genes. BACKGROUND: Differential expression of genes in PDL cells under physiological stress such as occlusal force is thought to be orchestrated not only for the remodeling of PDL itself but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. METHODS: The cDNA from mechanical stress-applied human PDL cells was subtracted against the cDNA from static control cells. The subtracted cDNA was amplified by polymerase chain reaction (PCR) and cloned for further analysis. RESULTS: Among 68 independent clones isolated, 15 contained DNA fragments greater than 250 bp. Reverse Northern analysis revealed a marked induction of MSGen-15 and MSGen-28 mRNA expression in the mechanical stress-applied cells. However, little difference in the magnitude of expression for the other MSGens was detected between the stress-applied cells and the control cells. After nucleotide sequencing and the analysis of homology with known genes, five clones were identified; ribosomal protein S27 (MSGen-9), MRG 15 (MSGen-15), androgen-binding protein (MSGen-18), cathepsin H (MSGen-28), and cytochrome c (MSGen-47). Interestingly, it has been reported that MRG 15 is a novel transcription factor involved in the regulation of cell growth and senescence. The remaining 10 clones, classified into six sequence types, had no significant homology with any known genes. CONCLUSIONS: These results suggest that many known and unknown genes are expressed in response to mechanical stress in PDL cells, and that a transcription factor, MRG 15, may be responsible for molecular events in PDL cells under mechanical stress.

Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis: (chick limb bud/HoxD homeobox genes/ZPA factor/AER factor/in situ hybridization).

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.