Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.
Transforming growth factor- (TGF-) and interferon-␥ (IFN-␥Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smadbinding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-␥/YB-1 counteracts TGF-/Smad3. They also indicate that IFN-␥/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-␥ response element and through a cross-talk with the TGF-/Smad signaling pathway.
We have recently shown that two distinct prostaglandin (PG) E 2 synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE 2 biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE 2 synthase, the perinuclear enzymes thromboxane synthase and PGI 2 synthase generated their respective products via COX-2 in preference to COX-1 in both the A23187-induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD 2 synthase preferentially used COX-1 and COX-2 in the A23187-induced immediate and interleukin-1-induced delayed PGD 2 -biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A 2 . Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA 2 s at a moment when PG production takes place.Biosynthesis of prostaglandins (PGs) 1 through the cyclooxygenase (COX) pathway involves oxidation and subsequent isomerization of membrane-derived arachidonic acid (AA) via three sequential enzymatic reactions. The initial step of this metabolic pathway is the stimulus-induced liberation of AA from membrane glycerophospholipids by the action of phospholipase A 2 (PLA 2 ) enzymes, including cytosolic PLA 2 ␣ (cPLA 2 ; group IVA) and several secretory PLA 2 (sPLA 2 ) isozymes (groups IIA, IID, V, and X) (1-10). The released AA is sequentially metabolized to PGG 2 and then to PGH 2 by either COX-1 or COX-2. PGH 2 is then converted to various bioactive PGs (thromboxane (TX) A 2 , PGD 2 , PGE 2 , PGF 2␣ , and PGI 2 ) by the respective terminal PG synthases, which have different structures and exhibit cell-and tissue-specific distributions.Segregated utilization of COX-1 and COX-2 in the PG biosynthetic events has been demonstrated by a number of cell biological, pharmacological, and genetic studies (3,(11)(12)(13)(14)(15)(16)(17). Generally, the constitutive COX-1 is mainly utilized in the immediate PG biosynthesis, which occurs within several minutes after stimulation with Ca 2ϩ mobilizers, whereas the inducible COX-2 is an absolute requirement for delayed PG biosynthesis, which lasts for several hours after proinflammatory stimuli. When cells are first treated with proinflammatory stimuli and subsequently exposed to Ca 2ϩ mobilizers, the induced COX-2 can also promote the immediate response (priming response) (3, 18 -22). However, the precise molecular me...
The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tightjunctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.
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