Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA

Objective.Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors.Methods.A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family.Results.An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors.Conclusion.VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

Abstract 710: A Novel Mechanism by Which CPI-17 Is Down-regulated in Cultured Vascular Smooth Muscle Cells and Tissues by TNFα and in C57BL/6 Mice by Lipopolysaccharide

Objectives: Sepsis is characterized by a severe inflammatory response to infection, and its complications, including hypotension, can be fatal. It has long believed that hypotension in sepsis occur as a result of failure of the vascular smooth muscle cells (VSMC) to constrict. However, the molecular mechanism that links inflammation and decreases in vasoconstriction is largely unknown. Approaches and Results: CPI-17, a key regulator in vasoconstriction, was markedly decreased in mesentery arteries (MA) in mice injected with lipopolysaccharide (LPS). Incubation of cultured MA or VSMC with TNFα had similar effects on CPI-17 as LPS injection in mice. To identify transcriptional factors that respond to TNFα to suppress CPI-17 expression, we cloned a 1 kb murine CPI-17 promoter (-1,015 to +2 bp). Promoter deletion analysis revealed that a CPI-17 promoter region (-115 to -50 bp) was critical for TNFα to inhibit CPI-17 promoter activity. Bioinformatics analysis revealed this region contained 3 GC boxes that are putative binding sites for transcription factors SP1 and KLF4. Mutation of the 2nd and 3rd GC boxes, but not 1st GC box, abolished TNFα-mediated inhibitory effect on CPI-17 promoter activity. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) illustrated that SP1 and KLF4 bound to the 2nd and 3rd GC boxes, but not 1st GC box. Interestingly, TNFα had no effect on SP1 expression but suppressed SP1 binding to the CPI-17 promoter. In contrast, TNFα increased KLF4 expression and KLF4 binding to the CPI-17 promoter. Down-regulation of either SP1 or KLF4 by siRNA abolished inhibition of CPI-17 promoter activity by TNFα but the effects were opposite: SP1 siRNA decreased CPI-17 promoter activity whereas KLF4 siRNA increased CPI-17 promoter activity. Moreover, KLF4 up-regulation, but not SP1, was also found in MA in mice injected with LPS. KLF4 up-regulation by LPS preceded CPI-17 down-regulation and correlated with a decrease in vasoconstriction and hypotension. Conclusions: These studies reveal a novel mechanism by which CPI-17 is down-regulated by LPS/TNFα through SP1 and KLF4 in vascular wall and identify SP1, KLF4, and CPI-17 as new potential therapeutic targets for treatment of hypotension in patients with sepsis.

KONSTRUKSI VEKTOR EKSPRESI GEN HORMON PERTUMBUHAN LELE DUMBO (Clarias sp.) UNTUK PRODUKSI IKAN LELE LOKAL (Clarias batrachus) TRANSGENIK

Penelitian ini bertujuan untuk memperoleh konstruksi vektor ekspresi (pTarget) rekombinan yang mengandung sisipan gen hormon pertumbuhan ikan lele dumbo (Clarias sp.) dan promoter β-aktin ikan lele lokal (C. batrachus) dalam upaya pembuatan ikan lele lokal transgenik. Promoter β-aktin lele lokal (pCbBA) telah berhasil diisolasi dari hipofisa ikan tersebut dengan ukuran sekitar 1,7 kbp; dan memiliki elemen transkripsi CAAT box, TATA box, GC box, motif CarG, dan TGACG berdasar analisis program TF Bind. Penggantian promoter CMV (cytomegalovirus) yang terkandung dalam vektor ekspresi pTarget menggunakan dua enzim restriksi SgfI dan I-PpoI, menghasilkan fragmen DNA berukuran 6.083 bp (pTarget-GH lele dumbo) sebagai produk digesti. Fragmen pTarget-GH lele dumbo yang diligasi dengan promoter β-aktin lele lokal membentuk konstruksi By: Ibnu Dwi Buwono, Nono Carsono, Yuniar Mulyani, and M. Untung KurniaThis study aims to obtain an expression vector construct (pTarget) containing recombinant growth hormone gene insertion of African catfish (Clarias sp.) and β-actin promoter derived from walking catfish (Clarias batrachus) in order to produce transgenic local catfish. β-actin promoter of walking catfish (pCbBA) have been isolated from the pituitary of the fish with a size of about 1.7 kbp, and has a transcription element: CAAT box, TATA box, GC box, CarG, and TGACG motif based on analysis result of TF Bind program. Replacement of CMV (cytomegalovirus) promoter contained in the expression vector pTarget using restriction enzymes SgfI and I-PpoI, obtained a product of digestion with the fragment size of 6,083 bp (pTarget-GH African catfish). pTarget-GH fragments were ligated with the African catfish β-actin promoter to arrange a construct of pTarget-pCbBA-African catfish GH (7,783 bp) as transgenic walking catfish expression vector.

17β-Estradiol suppresses MHC class I chain-related B gene expression via an intact GC box

Major histocompatibility complex class I chain-related B (MICB) is a membrane-bound glycoprotein involved in both innate and adaptive immunity through its interaction with NKG2D receptors present on γδ T, αβ CD8+ T, and natural killer cells. Factors known to upregulate MICB expression include heat shock, viral or bacterial infection, and tumorigenesis, and here, we explored the effect of 17β-estradiol (E2) on MICB regulation. Physiological concentrations of E2 were found to suppress MICB mRNA and surface protein levels and this effect was antagonized by the antiestrogen ICI 182780. The inhibitory effect of E2 was also observed for other NKG2D ligands, MICA and ULBPs. Evaluation of promoter fragments from the common MICB*00502 allele revealed that inhibition of transcription by E2 required the GC box at −87. The electrophoretic mobility shift assay and supershift analysis established the presence of SP1, SP3, or estrogen receptor α recognition sites within the MICB promoter sequence and interaction of these factors in situ was confirmed by chromatin immunoprecipitation. We conclude that E2 upon forming a complex with its cognate receptor suppresses MICB expression through binding with SP1/SP3 sites within the MICB promoter GC box. These results suggest that the partial benefit of 17β-estradiol on autoimmune diseases may be mediated by reducing the immune NKG2D ligands like MICB.