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The pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The objectives of the study were to understand P2X(7) gene transcription; to identify the active promoter and the transcription initiation site (TpIS); and to begin understanding regulation of P2X(7) gene transcription. Experiments in vitro utilized normal and cancerous cultured human uterine cervical epithelial cells, and HEK293 cells overexpressing P2X(7)-luciferase reporters. Experiments in vivo used surgical specimen of normal and cancerous uterine cervix. Assays involved DNA, RNA, and protein techniques. (a) The P2X(7) TpIS was localized to adenine (+1) at nt 1683 of the human P2X(7) gene [GenBank Y12851]), with a TTAAA sequence at nt -32/-28 and an active promoter region within nt -158/+32. (b) P2X(7) transcription was found to be regulated by two enhancers located at nt + 222/+232 and +401/+573 regions downstream of the active P2X(7) promoter. (c) The putative enhancer regions formed four DNA-protein complexes. (d) P2X(7) transcription was found to be controlled by hypermethylated cytosines at cytosine-phosphodiester-guanosines (CpG) that cluster or co-localize with the enhancers' sites. (e) We identified nine CpGs as inhibitory cis elements, and three CpG sites that are hypermethylated in cultured cervical epithelial cells and in cervix epithelia in vivo. (f) In cancer cervical cells, the degree of hypermethylation of the CpG sites was greater than in the normal cervical cells. Expression of the P2X(7) receptor is controlled by hypermethylated CpGs that flank transcription enhancers located within a 547-nt region downstream of the promoter.

The P2X7 receptor regulates cell growth through mediation of apoptosis. P2X7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X7 mRNA stability. Overexpression of a reporter containing the full-length human P2X7 3′-untranslated region (3′-UTR) or reporters containing parts of the 3′-UTR-P2X7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3′-UTR-P2X7, which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3′-UTR-P2X7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3′-UTR-P2X7.

Objectives. To understand the potential role of P2X7 as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X7. Methods. Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25–75. Endpoint for P2X7 protein was average pixel signal density of tissue immunoreactivity with anti-P2X7 antibody. Endpoint for P2X7 mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3′ untranslated region (3′UTR)-P2X7 cDNA, using as endpoint steady-state luciferase mRNA levels. Results. Levels of P2X7 protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89– 100%, p

The most striking feature of a G protein-coupled receptor (GPCR) is its highly exclusive agonist specificity. This feature guarantees that a GPCR recognizes only its specific native agonist(s). In this study, we showed that two point mutations of N295S and L305Q enabled the AT(1) receptors to recognize multiple Ang II fragments. Similar to the well established constitutively active AT(1) mutant receptor N111G, the mutations of N295S and L305Q induced an increased production of basal inositol 1,4,5-phosphates in the absence of exogenous Ang II when expressed in HEK293 cells. Distinct from the N111G, however, is the fact that the increased basal activity disappeared in COS-7 cells because of the lack of endogenous Ang II fragments produced by the cells-a pseudo-constitutive activity. It is surprising that the Ang II analog [Sar(1),Ile(4),Ile(8)]Ang II and the native angiotensin II fragments Ang 1-7, Ang IV, and Ang 5-8, which are inactive in activating the wild-type receptor, activated N295S and L305Q. Results generated by lowering the Na(+) concentration suggest that the mutant N295S and L305Q may be trapped in neutral conformational states (R(N)). These data allow us to identify for the first time a novel pattern of GPCR mutations with a broad spectrum of agonist specificity, suggesting possible existence of functional GPCRs in nature that are activated through conformational "selection" rather than "induction" mechanisms.

Functional diversity of AT 2 receptor orthologues in closely related species. Background The most striking feature of life is biodiversity. However, mechanisms of biodiversity remain poorly understood, as most protein orthologues of different species are highly homologous in sequence and identical in function. Interestingly, recent evidence has demonstrated heterogeneity for a G protein-coupled angiotensin II (Ang II) type 2 (AT 2 ) receptor in both ligand binding and induction of arachidonic acid (AA) release. The present study investigated the properties of AT 2 receptors in closely related species. Methods AT 2 receptors cloned from human, rabbit, rat, and mouse were expressed in Chinese hamster ovary cells (CHO-K1), African green monkey kidney cells (COS-1), and human embryonic kidney (HEK)-293 cells and characterized in ligand binding and signal transductions. Critical residues in rabbit AT 2 receptor attributable to heterogeneity were examined using both gain-of-function and loss-of-function approaches with mutagenesis. Results The newly cloned rabbit AT 2 receptor exhibits distinct biochemical and biologic properties compared to its highly homologous orthologues (91% in overall amino acid sequence) of rat, mouse, and human. All these orthologues activate SH2 domain-containing phosphatase-1 (SHP-1) and show similar binding affinities for Ang II and AT 2 -specific ligands CGP42112A and PD123319. However, reducing agent dithiothreitol (DTT) inactivates the rabbit orthologue but potentiates the others in ligand binding, a hallmark of AT 2 versus AT 1 receptor subtypes. Most interestingly, rabbit AT 2 receptor, but not the other orthologues, induces AA release in various cell systems when stimulated by both Ang II and CGP42112A, the peptide antagonist. Mutagenesis studies and sequence analyses further indicate that residues His 106 , Asp 188 , and Thr 293 are responsible for the DTT inactivation and residues Val 209 and Val 249 are partially responsible for AA release. Conclusion These results deny the coexistence of an additional AT 2 subtype in rabbit proximal tubule cells and demonstrate for the first time the presence of functional diversity for closely related Eutherian orthologues of a G protein–coupled receptor (GPCR) that are more than 90% homologous in the amino acid sequence.

Transport of the essential amino acids arginine and lysine is critical for the survival of mammalian cells. The adaptive response to nutritional stress involves increased translation of the arginine/lysine transporter (cat-1) mRNA via an internal ribosome entry site (IRES) within the mRNA leader. Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2α. We show here that translation of the upstream ORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an active IRES. The IRES, whose activity is induced by amino acid starvation, is created by RNA-RNA interactions between the 5′ end of the leader and downstream sequences. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control.

The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged during amino acid starvation, suggesting a post-transcriptional mechanism of regulation. This mechanism was investigated by expressing a cat-1 mRNA from a tetracycline-regulated promoter. The cat-1 mRNA contained 1.9 kilobase pairs (kb) of coding sequence, 4.5 kb of 3'-untranslated region, and 80 base pairs of 5'-untranslated region. The full-length (7.9 kb) mRNA increased 5-fold in amino acid-depleted cells. However, a 3.4-kb species that results from the usage of an alternative polyadenylation site was not induced, suggesting that the cat-1 mRNA was stabilized by cis-acting RNA sequences within the 3'-UTR. Transcription and protein synthesis were required for the increase in full-length cat-1 mRNA level. Because omission of amino acids from the cell culture medium leads to a substantial decrease in protein synthesis, the translation of the increased cat-1 mRNA was assessed in amino acid-depleted cells. Western blot analysis demonstrated that cat-1 mRNA and protein levels changed in parallel. The increase in protein level was significantly lower than the increase in mRNA level, supporting the conclusion that cat-1 mRNA is inefficiently translated when the supply of amino acids is limited, relative to amino acid-fed cells. Finally, y(+)-mediated transport of arginine in amino acid-fed and -starved cells paralleled Cat-1 protein levels. We conclude that the cat-1 gene is subject to adaptive regulation by amino acid availability. Amino acid depletion initiates molecular events that lead to increased cat-1 mRNA stability. This causes an increase in Cat-1 protein, and y(+) transport once amino acids become available.

The P2X(7) receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X(7) receptor expression in normal and cancer human epithelial tissues. P2X(7) receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X(7) receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X(7) receptor, and P2X(7) mRNA levels were determined by real-time polymerase chain reaction. P2X(7) receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X(7) receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X(7) mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X(7) receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X(7) receptor precedes or coincides with neoplastic changes in those tissues.

Internalization of a G-protein–coupled receptor (GPCR) is essential to the desensitization, endocytosis, and signal transduction of the receptor. It has been the general view that conventional homologous internalization of a GPCR requires activation of the G-protein(s) coupled to the receptor. However, whether and how GPCR-mediated G-protein–independent signals trigger receptor internalization remains unknown, although G-protein–independent internalization has been reported. Here we show that an angiotensin II (Ang II) type-1 (AT 1 ) receptor mutant incapable of activating any G-protein still undergoes normal internalization. Substitution of Asp 125 with Ala and Arg 126 with Leu at the highly conserved DRY motif of the AT 1 receptor disabled the ability of the receptor to activate G-proteins, as shown by various Ang II binding studies, GDP–GTP exchange, and inositol phosphate production assays. Surprisingly, the mutant internalized normally in the presence of Ang II and transactivated the epidermal growth factor receptor (EGFR). Similar to the wild-type receptor, overexpression of a dominant-negative K220R mutant GRK2 diminished the internalization of D125A-R126L but not the transactivation of EGFR. These data indicate that G-protein–independent specific signals may also trigger homologous internalizations of the AT 1 receptor through β-arrestin–dependent and -independent pathways, suggesting a possible mechanism for G-protein–independent activation of G-protein–coupled receptor kinases (GRKs). This may represent a general mechanism for triggering GPCR internalization.

Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.

Transport of the essential amino acids arginine and lysine is critical for the survival of mammalian cells. The adaptive response to nutritional stress involves increased translation of the arginine/lysine transporter (cat-1) mRNA via an internal ribosome entry site (IRES) within the mRNA leader. Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2alpha. We show here that translation of the upstream ORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an active IRES. The IRES, whose activity is induced by amino acid starvation, is created by RNA-RNA interactions between the 5' end of the leader and downstream sequences. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control.

BackgroundThe study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7action.MethodsSkin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X7pore formation) were determined by confocal laser microscopy.Results(a) Co-application on the skin of the P2X7specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X7receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X7receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8.Conclusion(a) P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X7-dependent apoptosis is mediated by calcium influx via P2X7pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7receptor. (d) Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.