The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor ␣ (ER␣) is also subjected to rapid ubiquitinproteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ER␣ proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ER␣ and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ER␣. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ER␣ are mechanistically separable functions of ER␣. We find that proteolysis of ER␣ correlates with the ability of ER␣ mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligandinduced receptor degradation which permits the uncoupling of estrogen-regulated ER␣ proteolysis and transcription.The ubiquitin-proteasome pathway contributes to the control of transcription through the ubiquitination and regulated degradation of multiple components of the transcriptional machinery (10, 34). Among these components is a large list of transactivators whose activity can be related to their proteolytic degradation. For many, particularly those that possess acidic activation domains, such as VP16 and c-myc, sequence elements essential for proteasome-mediated proteolysis reside within transactivation domains (33,43). Several members of the nuclear receptor superfamily are substrates for the ubiquitin-proteasome pathway (11,19,21,24,26,29,37,50,52,55), the first identified being estrogen receptor ␣ (ER␣) (1, 13, 36). ER␣ possesses two transactivation domains, AF-1 and AF-2, which reside in the N terminus and C terminus of the receptor, respectively. These activation domains are bridged by a conserved DNA binding domain and a hinge region responsible for receptor nuclear localization.The transcriptional activity of AF-2 is strictly ligand dependent, but the AF-1 is not; thus, AF-2 received much attention for analysis of the relationship between estrogen-stimulated proteolysis and transcription. AF-2 is highly structured, consisting of 12 ␣-helices that adopt an active conformation upon agonist binding, which exposes a hydrophobic surface where coactivator proteins bind (6). It has been shown that mutations of residues critical for AF-2-mediated transactivation disrupt proteolysis (27, 53). E6-AP, a ubiquitin ligase (35), and the TRIP1/Rpt6/SUG1 (28, 42) subunit of the 19S regulatory cap of the proteasome bind to ER␣ through the coactiva...
Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X 7 . In this regard, P2X 7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X 7 agonists ATP and 3′-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X 7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X 7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X 7 -mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ ERK2, p38, and JNK1/JNK2) phosphorylation, and that the antioxidants N-acetyl-cysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X 7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.
Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor P2RX7 on monocytic cells, which promotes the processing/release of pro-inflammatory mediators. Although the pro-inflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of pro-angiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that both transient and long-term P2RX7 activation promotes the robust release of VEGF from primary human monocytes. This VEGF release is calcium-dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that it may not only participate in inflammation and cell death, but that it is also likely to be important in the control of angiogenesis and wound repair.
Extracellular nucleotides can act as important intercellular signals in diverse biological processes, including the enhanced production of factors that are key to immune response regulation. One receptor that binds extracellular adenosine triphosphate released at sites of infection and injury is P2X 7 , which is an ionotrophic receptor that can also lead to the formation of a non-specific pore, activate multiple mitogen-activated protein kinases (MAPKs), and stimulate the production of immune mediators including interleukin family members and reactive oxygen species (ROS). In the present report, we have investigated the signaling mechanisms by which P2X 7 promotes monocytic cell mediator production and induces transcription factor expression/ phosphorylation, as well as how receptor-associated pore activity is regulated by intracellular trafficking. We report that P2X 7 stimulates ROS production in macrophages through the MAPKs ERK1/2 and the nicotinamide adenine dinucleotide phosphate oxidase complex, activates several transcription factors including cyclic-AMP response element-binding protein and components of the activating protein-1 complex, and contains specific sequences within its intracellular C-terminus that appear critical for its activity. Altogether, these data further implicate P2X 7 activation and signaling as a fundamental modulator of macrophage immune responses.
The nucleotide receptor P2X 7 is an immunomodulatory cation channel and a potential therapeutic target. P2X 7 is expressed in immune cells such as monocytes/macrophages and is activated by extracellular ATP following tissue injury or infection. Ligand binding to P2X 7 can stimulate ERK1/2, the transcription factor CREB, enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms, and the formation of a non-specific pore. However, little is known about the biochemistry of P2X 7 , including whether the receptor is N-linked glycosylated and if this modification affects receptor function. Here we provide evidence that P2X 7 is sensitive to the glycosidases EndoH and PNGase F, and that the human receptor appears glycosylated on N187, N202, N213, N241 and N284. Mutation of N187 results in diminished P2X 7 agonist-induced phosphorylation of ERK1/2, CREB, and p90 ribosomal S6 kinase, as well as decreased pore formation. In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X 7 agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation. Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function. To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X 7 N187A. This is the first report to map human P2X 7 glycosylation sites and reveal residue N187 is critical for receptor trafficking and function. P2X 7 ; N-linked glycosylation; cell surface expression; ERK1/2; macrophages The nucleotide ATP can act as an important extracellular signaling molecule that regulates multiple processes, including neurotransmission and immune response mediator production (1,2). ATP is released in millimolar concentrations from cells following infection or tissue injury and can stimulate cells in the microenvironment by binding to the P2 nucleotide receptors (3). The P2 nucleotide receptors have been divided into two major subfamilies: the G proteincoupled P2Y receptors and the ionotropic P2X cation channels (4). KeywordsThe cation channel P2X 7 is considered an important component of the inflammatory response (5). Activation of P2X 7 by extracellular nucleotides leads to the processing of interleukin-1β * To whom correspondence should be addressed: pbertics@wisc.edu | tel 608-262-8667 | fax 608-262-5253. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 June 8. and the production of reactive oxygen species through the NADPH oxidase complex (6,7). The P2X 7 receptor stimulates a number of downstream targets including mitogen activated protein kinases (MAPK) and several transcription factors, including cyclic-AMP response elementbinding protein (CREB) and activating...
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