Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

c-mos and cdc2 Cooperate in the Translational Activation of Fibroblast Growth Factor Receptor-1 during Xenopus Oocyte Maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3′ untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.

Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

A platelet–endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1

One crucial role of endothelium is to keep the innermost surface of a blood vessel antithrombotic. However, the endothelium also expresses prothrombotic molecules in response to various stimuli. The balance between the antithrombotic and prothrombotic nature of the endothelium is lost under certain conditions. During atherosclerosis, the attachment of platelets to the vessel surface has been suggested to promote the proliferation of smooth muscle cells and intimal thickening as well as to affect the prognosis of the disease directly through myocardial infarction and stroke. Dysfunctional endothelium, which is often a result of the action of oxidized low-density lipoprotein (OxLDL), tends to be more procoagulant and adhesive to platelets. Herein, we sought the possibility that the endothelial lectin-like OxLDL receptor-1 (LOX-1) is involved in the platelet–endothelium interaction and hence directly in endothelial dysfunction. LOX-1 indeed worked as an adhesion molecule for platelets. The binding of platelets was inhibited by a phosphatidylserine-binding protein, annexin V, and enhanced by agonists for platelets. These results suggest that negative phospholipids exposed on activation on the surface of platelets are the epitopes for LOX-1. Notably, the binding of platelets to LOX-1 enhanced the release of endothelin-1 from endothelial cells, supporting the induction of endothelial dysfunction, which would, in turn, promote the atherogenic process. LOX-1 may initiate and promote atherosclerosis, binding not only OxLDL but also platelets.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

An initiator element mediates autologous downregulation of the human type A γ-aminobutyric acid receptor β1 subunit gene

The regulated expression of type A γ-aminobutyric acid receptor (GABAAR) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABAAR subunit gene expression, including decreased β1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABAAR number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABAAR β1 subunit gene that is neuron specific and autologously down-regulated by GABA. β1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABAAR activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3′ flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of β1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of β1 subunit GABAAR gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.

Suppressor T cells regulate the nonanergic cell population that remains after peripheral tolerance is induced to the Mls-1 antigen in T cell receptor Vβ8.1 transgenic mice

We have found suppressor T cells that inhibit the proliferative response of naive CD4+ T cells in T cell receptor (TCR) Vβ8.1 transgenic mice rendered tolerant in vivo by inoculation of Mls-1a-positive cells. This suppression was mediated by CD4+ T cells but not by CD8+ T cells or double-negative (DN) cells, and splenic CD4+ T cells from tolerant mice displayed a greater suppression than lymph node CD4+ T cells. Cell contact was required for efficient suppression, and known inhibitory cytokines such as IL-4, IL-10, and transforming growth factor β were not involved. Suppressor T cells inhibited IL-2 production by naive CD4+ T cells, and the addition of exogenous IL-2 diminished the suppressed activity while having little activity on tolerant T cells. Suppression was abolished by the elimination of CD25+ T cells in the tolerant CD4+ T cell subset. CD25+CD4+ T cells suppressed the proliferative response of the residual fraction of the nonanergic population, namely, 6C10+CD4+ T cells still present in the tolerant mice. However, 6C10−CD4+ T cells still had reduced reactivity to Mls-1a even after CD25+CD4+ T cells were removed and exogenous IL-2 was added. Suppressor cells appear to affect only residual nonanergic cells in situ, thereby facilitating the maintenance of the unresponsive state in vivo. These data provide a framework for understanding suppressor T cells and explain the difficulties and variables in defining their activity in other systems, because suppressor T cells apparently control only a small population of nonanergic cells in the periphery and may be viewed as a homeostatic mechanism.

A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 μM and Hill coefficient (Hinh) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system

In the “selective” cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1

IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients.