ABSTRACT
Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown as a receptor for protons. In the present study, we aimed to know whether OGR1 plays a role in insulin secretion and, if so, the manner in which it does. To this end, we created OGR1-deficient mice and examined insulin secretion activity in vivo and in vitro. OGR1 deficiency reduced insulin secretion induced by glucose administered ip, although it was not associated with glucose intolerance in vivo. Increased insulin sensitivity and reduced plasma glucagon level may explain, in part, the unusual normal glucose tolerance. In vitro islet experiments revealed that glucose-stimulated insulin secretion was dependent on extracellular pH and sensitive to OGR1; insulin secretion at pH 7.4 to 7.0, but not 8.0, was significantly suppressed by OGR1 deficiency and inhibition of G(q/11) proteins. Insulin secretion induced by KCl and tolbutamide was also significantly inhibited, whereas that induced by several insulin secretagogues, including vasopressin, a glucagon-like peptide 1 receptor agonist, and forskolin, was not suppressed by OGR1 deficiency. The inhibition of insulin secretion was associated with the reduction of glucose-induced increase in intracellular Ca(2+) concentration. In conclusion, the OGR1/G(q/11) protein pathway is activated by extracellular protons existing under the physiological extracellular pH of 7.4 and further stimulated by acidification, resulting in the enhancement of insulin secretion in response to high glucose concentrations and KCl.
Subject(s)
Glucose/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/metabolism , Animals , Immunohistochemistry , In Vitro Techniques , Insulin Secretion , Mice , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Receptors, G-Protein-Coupled/geneticsABSTRACT
Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.
Subject(s)
Breast Neoplasms/metabolism , Estrogens/pharmacology , Signal Transduction/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Biomarkers, Tumor/metabolism , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Profiling , Humans , Immunoprecipitation , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transforming Growth Factor beta/genetics , Tumor Cells, Cultured , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein.
Subject(s)
Antineoplastic Agents/pharmacology , Chromans/pharmacology , Colonic Neoplasms/metabolism , Cyclin E/antagonists & inhibitors , E2F2 Transcription Factor/antagonists & inhibitors , Oncogene Proteins/antagonists & inhibitors , PPAR gamma/agonists , Thiazolidinediones/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cyclin E/genetics , Cyclin E/metabolism , E2F2 Transcription Factor/genetics , E2F2 Transcription Factor/metabolism , Humans , Ligands , Oligonucleotide Array Sequence Analysis , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , PPAR gamma/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , Retinoblastoma Protein/antagonists & inhibitors , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Troglitazone , Wnt Proteins/metabolismABSTRACT
Recent studies have shed light on the ligand-dependent transactivation mechanisms of nuclear receptors (NRs). When the ligand dose is reduced, the transcriptional activity of NRs should be downregulated. Here we show that a ubiquitin-proteasome pathway plays a key role in turning off transcription mediated by estrogen receptor beta (ERbeta). ERbeta shows estrogen-dependent proteolysis, and its degradation is regulated by two regions in the receptor. The N-terminal 37-amino acid-region is necessary for the recruitment of the ubiquitin ligase, i.e., the carboxyl terminus of HSC70-interacting protein (CHIP), to degrade ERbeta. In contrast, the C-terminal F domain protects ligand-unbound ERbeta from proteolysis to abrogate proteasome association. Suppression of CHIP by interfering RNA inhibited this switching off of receptor-mediated transcription when the ligand dose was reduced. Our results suggest that after ligand withdrawal, the active form of the NR is selectively eliminated via ligand-dependent proteolysis to downregulate receptor-mediated transcription.
