TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.     

NF-kappaB site interacts with Sp factors and up-regulates the NR1 promoter during neuronal differentiation

The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.     

Molecular regulation of MHC class I chain-related protein A expression after HDAC-inhibitor treatment of Jurkat T cells

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.     

Functional characterization of the promoter region of human TNFAIP1 gene

Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.     

Anp32e promotes renal interstitial fibrosis by upregulating the expression of fibrosis-related proteins

Acidic nuclear phosphoprotein 32 family member e (Anp32e) has been reported to contribute to early mammalian development and cancer metastasis. However, the pathophysiological role of Anp32e in renal interstitial fibrosis (RIF) is poorly understood. Here, we demonstrated that Anp32e was highly expressed in the region of RIF in patients with IgA nephropathy, unilateral ureteral obstruction (UUO) mouse kidneys, and Boston University mouse proximal tubular (BUMPT) cells when treated with TGF-β1; this upregulation was positively correlated with the total fibrotic area of the kidneys. The overexpression of Anp32e enhanced the TGF-β1-induced production of fibrosis-related proteins (fibronectin (Fn) and collagen type I (Col-I)) in BUMPT cells whereas the knockdown of Anp32e suppressed the deposition of these fibrosis-related proteins in UUO mice and TGF-β1-stimulated BUMPT cells. In particular, Anp32e overexpression alone induced the deposition of Fn and Col-I in both mouse kidneys and BUMPT cells without TGF-β1 stimulation. Furthermore, we revealed that the overexpression of Anp32e induced the expression of TGF-β1 and p-Smad3 while TGF-β1 inhibitor SB431542 reversed the Anp32e-induced upregulation of Fn and Col-I in BUMPT cells without TGF-β1 stimulation. Collectively, our data demonstrate that Anp32e promotes the deposition of fibrosis-related proteins by regulating the TGF-β1/Smad3 pathway.     

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells

Multi-drug resistance (MDR), an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC) transporters and activated Sonic hedgehog (Shh) signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX), we examined the effect and mechanism of norcantharidin (NCTD), a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S) and DOX-resistant (MCF-7R) cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.