Human chromosome 1 localization of the gene for a prostaglandin F2α, receptor negative regulatory protein

A protein that copurifies with the bovine prostaglandin F_2α receptor (FP) has been isolated and the corresponding rat cDNA has been cloned. Transfection experiments suggest that this protein inhibits binding of [³H]prostaglandin F_2α ([³H]PGF_2α) to FP. Histologically, this protein (FP regulatory protein or FPRP) shows a distribution coinciding well with those cells and tissues that respond to PGF_2α. A portion of the 3′ untranslated region of the human homolog to fprp was subcloned, sequenced, and oligonucleotide primers chosen that allow polymerase chain reaction (PCR) amplification specifically of the human fprp sequence. These primers were then used in a PCR-based mapping protocol. The human fprp gene was first localized through human/rodent somatic cell hybrids to human chromosome 1 (100% concordance), and further through yeast artificial chromosome (YAC) pools to region 1p13.1-q21.3 (level 1 mapping). In view of the specific histologic localization of this negative regulator, possible pathological conditions are mentioned that may cosegregate with this chromosomal region. Received: 25 May 1995 / Revised: 2 October 1995     

Delayed internalization and lack of recycling in a beta<Subscript>2</Subscript>-adrenergic receptor fused to the G protein alpha-subunit

Background  

Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β₂-adrenergic receptor (β₂AR) and Gα_s indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα_s-fused β₂AR.     

Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice

We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL). These studies tested if prostaglandin F_2α (PGF_2α) or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT) or caspase-3 deficient female mice, 25–26 days old, were given 10 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP) followed by 10 IU human chorionic gonadotropin (hCG) IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v.), the FAS-activating antibody Jo2 (2 micrograms, i.v.), or PGF_2α (10 micrograms, i.p.) at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay) in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF_2α or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 ± 1.8% and 13.7 ± 2.2 % of the cells, respectively and resulted in 16.35 ± 0.7% (PGF_2α) and 14.3 ± 2.5% TUNEL-positive cells when compared to 1.48 ± 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF_2α , Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF_2α at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact that the receptor for PGF_2α has not been shown to be directly coupled to caspase-8 recruitment and activation. We hypothesize that PGF_2α initiates luteolysis in vivo, at least in part, by increasing the bioactivity or bioavailability of cytokines, such as FasL and that multiple endocrine factors work in concert to activate caspase-3-driven apoptosis during luteolysis.     

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF(2α) in engineered Escherichia coli

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l⁻¹ PGF_2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF_2α. Furthermore, these E. coli cells produced PGF_2α in a medium supplemented with AA and secreted the PGF_2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF_2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF_2α at significantly reduced prices and greater purity.     

Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

Background  

Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α_1A-adrenergic receptor (α_1A-AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α_1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.     

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells

Background  

Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCα) with caveolin in CHO cells. PKCα is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca²⁺ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult     

Co-localization of prostaglandin F synthase,cyclooxygenase-1 and prostaglandin F receptor in mouse Leydig cells

In order to promote better understanding of the physiological roles of prostaglandin F_2α in the mouse testis, we investigated the protein expression and the cellular localization of the enzymes cyclooxygenase and prostaglandin F synthase that are essential for the production of prostaglandin F_2α, and the binding site, which is the prostaglandin F_2α receptor (FP). Western blot exhibited the expression of FP protein in wild type mouse testis, and that of prostaglandin F synthase and cyclooxygenase-1 proteins in the both of wild type mouse and FP-deficient mouse testes. The expression of prostaglandin F synthase and cyclooxygenase-1 were detected intensely in Leydig cell-rich fraction, and that of FP was detected equally in Leydig cell-rich fraction and the other fraction. Immunohistochemistry for cyclooxygenase-1 and prostaglandin F synthase demonstrated their co-localization in mouse Leydig cells. Histochemistry for FP demonstrated the localization in Leydig cells and in spermatids of seminiferous tubules. Double histochemical staining confirmed the co-localization of cyclooxygenase-1, prostaglandin F synthase and FP in the Leydig cells. These findings indicate that prostaglandin F_2α may have an effect on the functions of Leyding cells in an autocrine fashion. It implies that prostaglandin F synthase and FP are involved in the control of testosterone release from Leydig cells and in spermatogenesis via the local pathway and the hypothalamo-hypophysial-testis pathway, and affect the testicular function.     

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α<Subscript>1A</Subscript> adrenergic receptor

Background  

Phenylephrine (PHE), an α₁ adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α_1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2ainhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2α. These data suggest that IGF-I may be involved in regulating CL function, and maintai     

Dysfunctional interferon-α production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus

Background  

It is well known that interferon (IFN)-α is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-α producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum.     

Interleukin-1α enhances the aggressive behavior of pancreatic cancer cells by regulating the α6β1-integrin and urokinase plasminogen activator receptor expression

Background  

In human pancreatic cancer progression, the α₆β₁-integrin is expressed on cancer cell surface during invasion and metastasis formation. In this study, we investigated whether interleukin (IL)-1α induces the alterations of integrin subunits and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) expression in pancreatic cancer cells. We hypothesize that the alterations of integrin subunits and uPA/uPAR expression make an important role in signaling pathways responsible for biological behavior of pancreatic cancer cells.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

Background  

The L-type Ca²⁺ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca²⁺ signaling but poorly understood. We characterized a single-base frame-shift mutant of α_1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca²⁺ channel fragments.     

Effect of a povidone-iodine intrauterine infusion on progesterone levels and endometrial steroid receptor expression in mares

Background  

Intrauterine infusions have been widely used for the treatment of endometritis in the mare. Nevertheless, their consequences on endocrine and endometrial molecular aspects are unknown. We studied the effect of a 1% povidone-iodine solution intrauterine infusion on progesterone levels, endometrial histology and estrogen (ERα) and progesterone (PR) receptor distribution by immunohistochemistry.     

Anandamide-derived Prostamide F2α Negatively Regulates Adipogenesis

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB₁ receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F_2α ethanolamide (PGF_2αEA), of which bimatoprost is a potent synthetic analog. PGF_2αEA/bimatoprost act via prostaglandin F_2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF_2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF_2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF_2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF_2αEA versus prostaglandin F_2α biosynthesis accelerates adipogenesis. PGF_2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.     

Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

Background  

Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.     

The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.     

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia

Purpose  

The present study evaluated mRNA expression of interferon-alpha (IFN-α), IFN-α receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2′,5′-oligoadenylate synthetase (2′5′OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III).     

Cellular and molecular effects of unoprostone as a BK channel activator

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF_2α analog; and PGF_2α were examined in HCN-1A cells, a model system for studies of large conductance Ca²⁺ activated K⁺(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca²⁺]_i changes and protection against long-term irreversible glutamate-induced [Ca²⁺]_i increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC₅₀ of 0.6 ± 0.2 nM and had no effect on Cl⁻ currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF_2α activated a Cl⁻ current sensitive to [Ca²⁺]_i chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca²⁺ signaling Cl⁻ current activation. Latanoprost and PGF_2α, but not unoprostone, increased [Ca²⁺]_i. Unoprostone, PGF_2α only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca²⁺ deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF_2α. Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.     

Characterization of subcellular localization and stability of a splice variant of G alphai2

Background  

Alternative mRNA splicing of α_i2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sα_i2. In the sα_i2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of α_i2. Whereas α_i2 is found predominantly at cellular plasma membranes, sα_i2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sα_i2 have been suggested to constitute a specific targeting signal.