Xiaoren Tang

LPS-induced TNF-α factor (LITAF)-deficient mice express reduced LPS-induced cytokine: Evidence for LITAF-dependent LPS signaling pathways

Previously we identified a transcription factor, LPS-Induced TNF-α Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo, we generated a macrophage-specific LITAF-deficient mouse (macLITAF−/−). Our data demonstrate that in macrophages (i) several cytokines (such as TNF-α, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF−/− compared with LITAF+/+ control macrophages; (ii) macLITAF−/− mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2−/−, -4−/−, and -9−/− and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-κB pathway. Furthermore, using a kinase array, p38α was found to mediate LITAF phosphorylation and the inhibition of p38α with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-α protein levels. Finally, macLITAF−/− macrophages rescued by LITAF cDNA transfection restored levels of TNF-α similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-κB pathway and that p38α is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.

Identification and functional characterization of a novel binding site on TNF-α promoter

Transcription of the tumor necrosis factor (TNF) gene is rapidly and transiently induced by lipopolysaccharide in cells of monocyte/macrophage lineage. Previous studies have suggested that in the mouse, multiple NF-κB/Rel-binding sites contribute to the TNF transcriptional response to LPS. But the role of these regulatory elements in transcriptional activation of the TNF-α gene in human monocytes remains unclear. Previously, a transcription factor, termed lipopolysaccharide-induced TNF-α factor (LITAF), was found to regulate TNF-α gene expression. However, the specific protein domain(s) of human (h)LITAF that interact with the hTNF-α promoter had not been identified. In this study, we identify by footprinting a sequence motif, CTCCC (−515 to −511), within the TNF-α promoter that binds to hLITAF. We also identify the region of hLITAF (amino acids 165–180) that was named peptide B and specifically mediates binding to the hTNF-α promoter. When THP-1 cells were stimulated with this peptide B, it was sufficient to induce TNF-α secretion. Induction of TNF-α transcription by LPS or peptide B depended on the presence of the −515 to −511 promoter region, which was found to be essential for hLITAF binding. Together, these findings help to clarify the mechanism of hLITAF/hTNF-α interaction and the manner by which hLITAF contributes to hTNF-α regulation in an attempt to design new pharmacological interventions to address TNF-related diseases.

Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP‐mediated transcription

The retinoblastoma protein (RB) recruits histone deacetylase (HDAC) to repress E2F‐mediated transactivation that plays a critical role in cell cycle regulation. RB is also involved in activation of expression of a number of tissue specific‐ and differentiation‐related genes. In this study, we examined the mechanism by which RB stimulated the expression of a differentiation‐related gene, the surfactant protein D (SP‐D), which plays important roles in innate host defense and the regulation of surfactant homeostasis. We demonstrated that RB specifically stimulated the activity of human SP‐D gene promoter. The RB family member, p107 but not p130, also increased SP‐D promoter activity. Activation by RB was mediated through a NF‐IL6 (C/EBPβ) binding motif in the human SP‐D promoter, and this sequence specifically bound to C/EBPα, C/EBPβ, and C/EBPδ. RB formed stable complexes with all three C/EBP family members. RB small pocket (amino acid residues 379–792), but not the C‐pocket (amino acid residues 792–928), was necessary and sufficient for its interaction with C/EBP proteins. Furthermore, we demonstrated that the complexes containing RB and C/EBP proteins directly interacted with C‐EBP binding site on DNA. These findings indicate that RB plays a positive, selective, and direct role in the C/EBP‐dependent transcriptional regulation of human SP‐D expression. J. Cell. Biochem. 83: 414–425, 2001.

p53 Short Peptide (p53pep164) Regulates Lipopolysaccharide-Induced Tumor Necrosis Factor-α Factor/Cytokine Expression

The p53 protein is a sequence-specific DNA-binding factor that can induce apoptosis or activate genes whose dysregulation is involved in cancer. By using serial analysis of gene expression technique, p53-induced genes (PIGs) have been identified, one of which was lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) factor (LITAF/PIG7). LITAF regulates the transcription of cytokines such as TNF-alpha. To further elucidate the role of p53 in LITAF expression, LITAF promoter activity was carefully dissected. In this study, we found that the element required for transcriptional activity is mainly located in the region from -990 to -500 of the LITAF promoter; the specific site required for p53 protein-DNA binding is located between -550 and -500. We also found that transient transfection of either a p53 short DNA sequence, called p53LFB12, or its corresponding 7-amino-acid synthetic peptide from amino acids 164 to 170 (K164Q165S166Q167H168M169T170), named p53pep164, significantly reduced LITAF promoter activity to 15% in p53-null H1299 cells. Transfection of p53pep164 into H1299 cells significantly down-regulated LPS-induced LITAF expression as well. Furthermore, transfection of p53pep164 into human monocytes resulted in down-regulation of nine proinflammatory cytokines, including TNF-alpha. We also found that the LPS-activated p53 is a short-lived protein, and that p53-orchestrated apoptosis occurs shortly after the initiation stage following LPS stimulation and lasts a short time. Once p53 levels return to baseline, the p53-mediated inhibition of LITAF is released, and LITAF-mediated cytokine production can proceed. The present finding proposes a novel link between p53 and the inflammatory processes and highlights potential interventional approaches to control p53-associated inflammatory processes.

Identification and Characterization of Kava‐derived Compounds Mediating TNF‐α Suppression

There is a substantial unmet need for new classes of drugs that block TNF‐α‐mediated inflammation, and particularly for small molecule agents that can be taken orally. We have screened a library of natural products against an assay measuring TNF‐α secretion in lipopolysaccharide‐stimulated THP‐1 cells, seeking compounds capable of interfering with the TNF‐α‐inducing transcription factor lipopolysaccharide‐induced TNF‐α factor. Among the active compounds were several produced by the kava plant (Piper mysticum), extracts of which have previously been linked to a range of therapeutic effects. When tested in vivo, a representative of these compounds, kavain, was found to render mice immune to lethal doses of lipopolysaccharide. Kavain displays promising pharmaceutical properties, including good solubility and high cell permeability, but pharmacokinetic experiments in mice showed relatively rapid clearance. A small set of kavain analogs was synthesized, resulting in compounds of similar or greater potency in vitro compared with kavain. Interestingly, a ring‐opened analog of kavain inhibited TNF‐α secretion in the cell‐based assay and suppressed lipopolysaccharide‐induced TNF‐α factor expression in the same cells, whereas the other compounds inhibited TNF‐α secretion without affecting lipopolysaccharide‐induced TNF‐α factor levels, indicating a potential divergence in mechanism of action.

Novel transcriptional regulation of VEGF in inflammatory processes.

Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well‐defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and osteoarthritis). Recently, we showed that transcription factors LITAF and STAT6B affected the inflammatory response. This study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF‐mediated processes. Cells cotransfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between −338 and −305 upstream of the transcription site) important in LITAF and/or STAT6B‐mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA‐mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction in VEGF mRNA levels and inhibits LPS‐induced VEGF secretion in mouse RAW 264.7 cells. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164‐soaked matrigel were implanted in whole‐body LITAF‐deficient animals (TamLITAF−/−), wild‐type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF−/− as well as in STAT6B‐silenced wild‐type animals compared with control animals. The present data demonstrate that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signalling pathways and may be a useful target in the treatment of VEGF diseases.

Kavain inhibition of LPS-induced TNF-α via ERK/LITAF

Kavain, an extract from the shrub Piper Methysticum, was recently reported to modulate TNF-α expression in both human and mouse cells via regulation of LPS-Induced TNF-Alpha Factor (LITAF). The purpose of the present study was to define the molecular pathway(s) associated with Kavain effects on TNF modulation. In vitro studies using WT mouse primary macrophages showed that Kavain significantly reduced E.coli LPS-induced TNF-α production but this effect was almost abrogated in LITAF-/- and ERK2-/- cells. Therefore we reintroduced the ERK2 gene in ERK2-/- cells and partially restored E.coli LPS-induced LITAF-mediated TNF-α production. The translocation of LITAF into to nucleus was found to be dependent on ERK2 S206 residue. Kavain inhibits LITAF/TNF-α expression via dephosphorylation of ERK2 in response to E.coli LPS. Finally, in vivo, Kavain had a significant anti-inflammatory effect on wild type mice that developed Collagen Antibody Induced Arthritis (CAIA), but only a minor effect in ERK2-/- mice also affected by CAIA. Based on these findings, we concluded that ERK2 may be the kinase upstream of LITAF with its Serine residue 206 being crucial for the regulation of LPS-induced TNF-α.

A PTP4A3 Peptide PIMAP39 Modulates TNF-Alpha Levels and Endotoxic Shock

Lipopolysaccharide (LPS) stimulation of macrophages initiates intracellular signaling pathways leading to activation of MAPK and its subsequent influence on cytokine production. We recently identified a LITAF-STAT6(B) complex regulated by p38 MAPK in response to LPS stimulation. However, the LPS-induced cascade in the p38/LITAF/TNF signaling pathway remains unclear. Here, we identified PTP4A3, a protein tyrosine phosphotase, as a novel negative regulator of LPS-induced LITAF/TNF-α production. PTP4A3 exerts its negative role by dephosphorylating p38α MAPK in response to LPS stimulation of primary macrophages. PTP4A3 expression is upregulated in primary macrophages. Further structure-function analysis revealed that a unique short peptide (PIMAP39) derived from PTP4A3 is capable of mimicking the functionality of full-length PTP4A3 to selectively dephosphorylate p38α and indirectly suppress LPS-induced LITAF-STAT6B complex when it is translocated from the cytoplasmic region to the nucleus of the cell. Treatment of mice with PIMAP39 significantly attenuates the severity of adverse host responses to LPS stimulation, and in some cases provides complete resistance to a lethal dose of LPS due to suppression of TNF-α production. All together, these results reveal a previously unrecognized role for the PTP4A3 pathway in response to LPS.

Novel regulation of CCL2 gene expression by murine LITAF and STAT6B.

Inflammation is a multifaceted process: beneficial as a defense mechanism but also detrimental depending on its severity and duration. At the site of injury, inflammatory cells are activated by a cascade of mediators, one of which is LITAF, a transcription regulator known to upregulate TNF-α. We previously showed that human LITAF forms a complex with human STAT6B, which translocates into the nucleus to upregulate cytokine transcription. To dissect the molecular implications of this complex, a murine model was developed and interactions between mouse STAT6B (mSTAT6B) and mouse LITAF (mLITAF) were analyzed. Both mLITAF and mSTAT6B expression were MyD88- and TLR ligand-dependent. Furthermore, mLITAF was found to mediate LPS-induced CCL2 gene transcription with the cooperation of mSTAT6B leading to CCL2 protein expression. In LITAF-deficient mice, mLITAF-mediated CCL2 production in macrophages was significantly reduced compared to the wild-type control animals. Mice knockdown for mSTAT6B by 6BsiRNA1 tail vein injection resulted in a decrease in serum TNF-α and CCL2 production. mLITAF/mSTAT6B complex is proposed to play a role in LPS-induced CCL2 expression and possibly other cytokines.

p53 Peptide Prevents LITAF-Induced TNF-Alpha-Mediated Mouse Lung Lesions and Endotoxic Shock

Abnormal and prolonged inflammatory reaction is seen in a wide variety of disorders, and high level of Tumor Necrosis Factor alpha (TNF-α) has been linked to these disorders. Therefore, modulation of TNF-α expression is important in the regulation of inflammatory disorders. In our previous study, we have shown that a transcription factor LPS-induced TNF factor (LITAF) significantly induces TNF-α production. Furthermore, we found that p53 and its synthetic peptide 162-motif specifically downregulate LITAF/TNF-α gene expression in human cells in vitro. Thus, in the present study, the role of p53 in regulating TNF-α-mediated inflammation was investigated. Our data showed that a synthetic peptide, named 162-motif, corresponding to this region functions independently from p53 to cause a significant suppression of TNF-α gene expression in mouse primary macrophages. The 162-motif, when delivered into cells and organs, reduces serum TNF-α level in mice and prevents TNF-α-induced lung lesions and endotoxic shock. Our findings highlight the regulation of LITAF/TNF-α by p53 and its short peptide 162-motif. These in vitro and in vivo observations serve to pave the way for pharmacotherapeutic approaches in the treatment of inflammatory diseases.