Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (h beta c) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3R alpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type h beta c exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type h beta c, despite failing to undergo IL-3-stimulated h beta e tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and h beta c tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effecters, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence. (C) 2000 Elsevier Science Ltd. All rights reserved.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Immunophilin chaperones in steroid receptor signalling

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7, MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Effect of chronic morphine treatment on alpha(2)-adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens

1 The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha (2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2 In chronically saline treated (CST) preparations. morphine (1 muM) and the alpha (2)-adrenoceptor agonist (clonidine, 1 muM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 muM) and clonidine (1 muM) decreased mean EJP amplitude by 68+/-8% (n = 7) and 79+/-8% (n = 7) respectively. 3 When stimulating the sympathetic axons at 0.03 Hz. the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n = 7 compared with 9.9+/-0.3 mV, n = 7). 4 Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5 Results from the present study indicate that the effectiveness of alpha (2)-adrenocrptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha (2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.