Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells.

This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,ß-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase.

Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. We have previously reported that major cytotoxic factors in the gas phase of cigarette smoke are ACR and MVK. ACR and MVK induce cell damage by reactive oxygen species generation via protein kinase C and NADPH oxidases, and antioxidants, such as glutathione (GSH) and N-acetylcysteine (NAC), can effectively suppress their cytotoxic activities. In this study, we attempted to elucidate the molecular mechanism(s) for suppression of ACR- and MVK-induced cytotoxic activities by these antioxidants. GSH, NAC, L- and D-cysteines completely suppressed cell damage induced by gas phase extract of cigarette smoke. The results of HPLC and mass spectrometry showed that GSH and NAC directly reacted with ACR and MVK. Cysteines and cysteine derivatives suppressed ACR-induced GAPDH carbonylation, a representative protein for carbonylation. The current results suggest that GSH, NAC, and cysteines directly reacted with ACR and MVK, and suppressed these unsaturated carbonyl compounds-induced cell damage by inhibition of protein carbonylation.

Ca2+ signal is involved in endothelin-1-induced internalization of endothelin type A receptor expressed in Chinese hamster ovary cells.

Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

SMOC1 is essential for ocular and limb development in humans and mice.

Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice.

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

Endothelin receptor signaling: new insight into its regulatory mechanisms.

The endothelin (ET) system consists of two G protein coupled-receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca(2+) concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca(2+) entry and receptor-operated Ca(2+) entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca(2+) sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1-induced Ca(2+) mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca(2+) signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.

An essential role for RAX homeoprotein and NOTCH-HES signaling in Otx2 expression in embryonic retinal photoreceptor cell fate determination.

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Different binding property of STIM1 and its novel splice variant STIM1L to Orai1, TRPC3, and TRPC6 channels.

Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum (ER) Ca(2+) sensor to control ER Ca(2+) levels. A recent study has shown that STIM1L, a new splice variant of STIM1, is expressed in various tissues of rodent and in human skeletal muscle, and that the interaction of STIM1L with actin filament allows rapid activation of store-operated Ca(2+) entry (SOCE) mediated through Orai1 channels. Here, we characterize mRNA expression and function of human STIM1 and STIM1L, and compare their binding property to Orai1 functioning as store-operated Ca(2+) channels (SOCCs), and TRPC3 (transient receptor potential canonical 3) and TRPC6 channels functioning as endothelin type A receptor (ET(A)R)-operated Ca(2+) channels (ROCCs). Although mRNA for STIM1 was ubiquitously expressed in human tissues, STIM1L was detected only in skeletal muscle. STIM1L augmented thapsigargin- and endothelin-1-induced SOCE more strongly than STIM1 in human embryonic kidney 293 cells stably expressing ET(A)R, whereas, it tends to suppress ET(A)R-operated Ca(2+) entry (ROCE) via TRPC3 and TRPC6 more strongly than STIM1. Coimmunoprecipitation experiments have revealed that when compared with STIM1, STIM1L binds more abundantly to Orai1 and also to TRPC3 and TRPC6. These results suggest that the higher binding capacity of STIM1L to SOCCs and ROCCs plays an important role in the regulation of Ca(2+) signaling such as the augmentation of SOCE via Orai1 and the inhibition of ROCE via TRPC3 and TRPC6.

Adenylate cyclase/cAMP/protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca²âº entry mediated via transient receptor potential canonical 6 channels.

Receptor-operated Ca²âº entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca²âº concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca²âº measurements with thapsigargin-induced Ca²âº-depletion/Ca²âº-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

An essential role for Rax in retina and neuroendocrine system development.

In vertebrates, the central nervous system (CNS) develops as a highly hierarchical, patterned organ with a vast diversity of neuronal and glial cell types. The vertebrate retina is developmentally a part of the CNS. Establishment of the vertebrate retina requires a series of developmental steps including specification of the anterior neural plate, evagination of the optic vesicles from the ventral forebrain, and differentiation of cells. The transcription factor RAX is a paired-type homeoprotein that plays a critical role in the eye and forebrain development of vertebrate species. Rax is initially expressed in the anterior neural region of developing mouse embryos, and later in the retina, pituitary gland, hypothalamus, and pineal gland. The targeted deletion of Rax in the mouse results in no eye formation and abnormal forebrain formation. In humans, mutations in the RAX gene lead to anophthalmia and microphthalmia. These observations indicate that RAX plays a pivotal role in the establishment of the retina. In addition, recent studies have reported that retina and pituitary gland tissues can be induced in a culture system from embryonic stem cells, using RAX expression as an indicator of neuronal progenitor cells in the induced tissue, and suggesting that the Rax gene is a key factor in neuronal regeneration. This review highlights the biological functions and molecular mechanisms of RAX in retina, pituitary, hypothalamus, and pineal gland development.

OBIF, an osteoblast induction factor, plays an essential role in bone formation in association with osteoblastogenesis.

In vertebrate bone formation, the functional mechanisms of transcription factors in osteoblastic differentiation have been relatively well elucidated; however, the exact roles of cell-extrinsic molecules are less clear. We previously identified human and mouse Obif, an osteoblast induction factor, also known as Tmem119, which encodes a single transmembrane protein. OBIF is predominantly expressed in osteoblasts in mouse. While exogenous Obif expression stimulated osteoblastic differentiation, knockdown of Obif inhibits the osteoblastic differentiation of pre-osteoblastic MC3T3-E1 cells. In order to investigate an in vivo role of OBIF in bone formation, we generated Obif-deficient mice by targeted gene disruption. Analyses of micro-computed tomography (mCT) revealed that Obif(-/-) mice exhibit significantly reduced cortical thickness in the mid-shaft of the femur at postnatal day 14 (P14). Furthermore, progressive bone hypoplasia is observed after 8 weeks. The expression levels of osteoblast marker genes, Collagen 1a1, Osteopontin, Runx2, and Osterix, in the calvaria were decreased in Obif(-/-) mice at P4. These data indicate that Obif plays an essential role in bone formation through regulating osteoblastogenesis.

Nicotine- and tar-free cigarette smoke extract induces cell injury via intracellular Ca2+-dependent subtype-specific protein kinase C activation.

Nicotine- and tar-free cigarette smoke extract (CSE) is reported to induce cell damage via activation of protein kinase C (PKC) and NADPH oxidase (NOX) in rat C6 glioma cells. Here we determined PKC isozyme(s) activated by CSE and their activation mechanism. In C6 glioma cells, mRNAs for PKCα, PKCδ, PKCε, and PKCι were expressed. CSE triggered translocation of PKCα and PKCε to plasma membrane. CSE-induced cell damage and PKC translocation were inhibited by chelating intracellular Ca(2+) but not extracellular Ca(2+). These results suggest that CSE induces cell damage through intracellular Ca(2+)-dependent activation of PKCα and PKCε and subsequent NOX activation.

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase.

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-L-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of (•)OH) but not by superoxide dismutase, indicating involvement of (•)OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H(2)O(2) or O(2)(-) generated by the xanthine/xanthine oxidase system, indicating involvement of H(2)O(2) or O(2)(-) in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Sumoylation controls retinal progenitor proliferation by repressing cell cycle exit in Xenopus laevis.

Precisely controlled progenitor proliferation is essential for normal development. However, molecular mechanisms, which control the correct timing of cell cycle withdrawal during development, have been poorly understood. We show here that ubc9, a sumo-conjugating enzyme, controls the cell cycle exit of retinal progenitors. We found that ubc9 is highly expressed in retinal progenitors and stem cells in Xenopus embryos. Ubc9 physically and functionally associates with Xenopus hmgb3, which is required for retinal cell proliferation, and prolonged expression of ubc9 and hmgb3 results in suppression of the cell cycle exit of retinal progenitors in a sumoylation-dependent manner. Overexpression of ubc9 and hmgb3 decreased expression of the cell-cycle inhibitor p27(Xic1). Furthermore, progenitor proliferation is regulated, at least in part, by sumoylation of transcription factor Sp1. These results suggest a significant role of sumoylation for cell cycle regulation in retinal progenitors.

Function and regulation of endothelin type A receptor-operated transient receptor potential canonical channels.

The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.