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PubMed1995

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.

Animals↗
PubMed2003

Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling.

Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORalpha. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORalpha binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo.

Animals↗
PubMed1996

Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex.

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

Adenocarcinoma↗
PubMed1999

A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

Actin-Related Protein 2↗
PubMed2000

Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology.

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

Amino Acid Sequence↗
PubMed1997

A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole.

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPS gene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditional vps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition, vps18tsf cells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18tsf mutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney.

The serum- and glucocorticoid-induced kinase-1 (sgk1) increases the activity of a number of epithelial ion channels and transporters. The present study examines the distribution and subcellular localization of sgk1 protein in the rat kidney and the regulation of levels of expression induced by steroids. The results indicate that the kidney expresses predominantly the sgk1 isoform with a distribution restricted to the thick ascending limb of Henle, distal convoluted, connecting and cortical collecting tubules. Within cells, sgk1 strongly associates with the microsomal fraction of homogenates and it colocalizes with the Na+,K+-ATPase to the basolateral membrane. Analysis of the levels of expression of sgk1 by Western blotting and immunohistochemistry indicates constitutive high expression under basal conditions. Approximately half of the basal level is maintained by glucocorticoids whereas physiological fluctuations of aldosterone produce minor changes in sgk1 abundance in adrenal-intact animals. These results do not support the notion that physiological changes of aldosterone concentration turn the expression of sgk1 'on and off' in the mammalian kidney. Additionally, localization of sgk1 to the basolateral membrane indicates that the effects mediated by sgk1 do not require a direct interaction with the ion channels and transporters whose activity is modulated, since most of these proteins are located in the apical membrane of renal epithelial cells.

Adrenalectomy↗
PubMed1995

Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus.

The regulation of transcription of the hepatitis B virus core promoter is an important event in the viral life cycle. Two messages, precore and pregenomic RNAs, that are initiated 30 nucleotides apart are produced by the core promoter. Precore RNA encodes nucleocapsid protein and pregenomic RNA core and polymerase. The latter transcript also serves as a template for viral genome replication via reverse transcription. We have previously defined a basal core promoter, which contains four TA-rich sequences (TA1 through TA4) but no canonical TATA element, that can direct transcription of both messages. In this study, we demonstrated that a stretch of 15 nucleotides containing TA4 is sufficient to direct precise initiation of both precore and pregenomic transcripts. This sequence can function as both an initiator and a TATA element. Mutational analysis further revealed that sequences essential for either function are colocalized. The significance of this finding with respect to the basal transcription mechanism and regulation of viral gene expression is discussed.

Base Sequence↗
PubMed1998

Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly.

We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the alpha adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.

Amino Acid Sequence↗
PubMed2002

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells.

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

CD4-Positive T-Lymphocytes↗
PubMed2003

Stem cell antigen-1 expression in the pulmonary vascular endothelium.

Although the function of the cell surface protein stem cell antigen-1 (Sca-1) has not been identified, expression of this molecule is a characteristic of bone marrow-derived hematopoietic stem cell populations. Expression of Sca-1, however, is not restricted to hematopoietic tissue. By RT-PCR and Western analysis, we found that Sca-1 is expressed in the adult mouse lung. Sca-1 immunohistochemistry revealed a linear staining pattern on the endothelial surface of large and small pulmonary arteries and veins and alveolar capillaries. Expression of Sca-1 in the pulmonary endothelium was confirmed by dual fluorescent microscopy on lung sections and by fluorescence-activated cell sorting analysis of digested lung tissue; each of these methods showed colocalization with the endothelial marker platelet/endothelial cell adhesion molecule-1. In the kidney, Sca-1 expression was also noted in large vessels, but, in contrast to the lung, was not observed in capillaries. Overall, our data indicate that Sca-1 expression helps define the surface phenotype of endothelial cells throughout the pulmonary vasculature.

Animals↗
PubMed1996

Functional alterations in the dentate gyrus after induction of long-term potentiation, kindling, and mossy fiber sprouting.

1. The effects of long-term potentiation (LTP) and kindling on membrane currents evoked in the dentate gyrus (DG) by stimulation of the perforant path (PP) were studied by current-source density (CSD) analysis in urethan-anesthetized rats. The spatial and temporal patterns of the evoked currents were analyzed after induction of LTP, at 24 h after three afterdischarges (ADs) induced by kindling stimulation, and at 4-5 wk after 70-120 generalized tonic-clonic (class V) kindled seizures. 2. The amplitude of the ipsilateral monosynaptic population excitatory postsynaptic current (iEPSC) evoked in the middle and outer stratum moleculare (STM) of the DG by PP stimulation increased by 20-100% after induction of LTP (N = 7 of 7). There was a trend to shorter onset latency of the iEPSC after induction of LTP, but the duration and spatial distribution of the iEPSC were unchanged (N = 7 of 7). The latency of inward current generated by synchronous granule cell discharge during the population spike decreased by 0.5-1 ms after induction of LTP (N = 6 of 7). 3. There were no significant alterations in the spatial and temporal pattern of the monosynaptic iEPSC evoked by PP stimulation at 24 h after three ADs induced by kindling stimulation (N = 6 of 6), or at 4-5 wk after the last of 70-120 class V kindled seizures (N = 14 of 14). 4. The spatial and temporal distribution of low-amplitude inward currents generated after the population spike was not altered after induction of LTP (7 of 7), or at 24 h after three ADs induced by kindling stimulation (N = 6 of 6). In kindled rats studied at 4-5 wk after the last of 70-120 class V seizures, there was an alteration in evoked currents after the population spike consisting of a distinct peak of net inward current at a latency of 9-12 ms in the proximal STM (N = 10 of 14). Inward current at this latency and location was not observed in > 100 normal rats, in age-matched normal controls (N = 4 of 4), or after acute seizures induced by pentylenetetrazol (35 mg/kg ip, N = 5 of 5). 5. Timm histochemistry was combined with CSD analysis to examine the relationship of the inward current at 9-12 ms to the terminal field of the sprouted mossy fiber pathway, which forms asymmetric, putatively excitatory synapses in the inner STM of the DG in kindled rats. In six of eight kindled rats with sprouted mossy fiber terminals demonstrated by Timm histochemistry, CSD analysis revealed that the peak amplitude of the inward current colocalized with the laminar distribution of sprouted mossy fiber terminals. In the remaining two of the eight kindled rats with sprouted terminals in the inner STM, there was no alteration in the spatial and temporal distribution of inward current after the population spike. 6. In conclusion, LTP and kindling induced distinct alterations in currents evoked in the DG by stimulation of the PP. After induction of LTP, there was an acute increase in the amplitude of the monosynaptic iEPSC and reduction in the latency of currents associated with granule cell discharge, but there was no alteration in the spatial or temporal organization of multisynaptic activity. In contrast, long-lasting effects of kindling included an alteration in the spatial and temporal organization of multisynaptic currents, which was consistent with excitatory synaptic transmission by synaptic terminals of the sprouted mossy fiber pathway. The functional alterations induced by LTP and kindling may have implications for associative properties, information processing, and epileptogenesis in the DG.

Animals↗
PubMed2000

Tyrosine hydroxylase in vasopressinergic axons of the pituitary posterior lobe of rats under salt-loading as a manifestation of neurochemical plasticity.

In this study, we attempted to test whether tyrosine hydroxylase (TH), the first rate-limiting enzyme of catecholamine synthesis, is confined to the perikarya of activated magnocellular vasopressinergic (VPergic) neurons or is also present in their distal axons in the pituitary posterior lobe (PL). In addition, we evaluated the possible correlation between TH and VP turnover in the axons of rats drinking 2% NaCl for 1, 2, and 3 weeks. To this aim, we examined the large swellings of VPergic axons, the so-called Herring bodies, using the double-immunofluorescent technique and the avidinbiotin technique, combined with image analysis. Here we have demonstrated for the first time a colocalization of TH and VP in Herring bodies, which is a strong argument in favor of TH transport from the perikarya of VPergic neurons via axons toward their terminals. TH-immunoreactive (IR) and VP-IR materials were distributed in Herring bodies with seeming zonality. The number of VP-IR Herring bodies decreased by a factor of four over the first week of osmotic stimulation, remaining at almost the same low level until the end of the experiment. Conversely, the content of the VP-IR material within the individual Herring bodies fell gradually during the three weeks of salt-loading. The results suggest that VP depletion from Herring bodies prevails in its transport into these structures during the whole period of osmotic stimulation. In contrast to VP-IR Herring bodies, the number of TH-IR Herring bodies and the content of TH-IR material within the individual Herring bodies increased progressively during the entire experiment. The synchronization of the VP depletion and TH accumulation in Herring bodies during long-term osmotic stimulation raised the question about a possible functional interaction between both substances.

Administration, Oral↗
PubMed1992

Distinct sites of production and deposition of the putative cell death marker clusterin in the human thymus.

Clusterin is a multifunctional protein endowed with cell-aggregating, complement-inhibitory, and lipid-binding properties. Since several studies have demonstrated highly increased clusterin gene expression in epithelial and nervous tissues regressing as a consequence of tissue involution and apoptotic cell death, clusterin is also considered as a specific marker of dying cells. To determine whether clusterin expression is also upregulated during thymocyte death occurring during the negative selection process we analyzed the cellular distribution of clusterin mRNA and protein by in situ hybridization and immunocytochemistry in the human thymus. We observed that the expression of clusterin mRNA was confined to cells present in the thymic medulla, concentrated mainly around Hassal's bodies. Immunostaining of adjacent sections with antikeratin Ab revealed that cells containing clusterin mRNA were predominantly epithelial. By contrast no clusterin mRNA was found in thymocytes by in situ hybridization and Northern blot analysis of total RNA from purified thymocyte populations. Clusterin protein colocalized with the membrane attack complex of complement and vitronectin in the center of the largest Hassal's bodies, but was not detectable by immunocytochemistry in or at the surface of epithelial cells. Our results demonstrate that clusterin gene expression does not take place in apoptotic thymocytes, and therefore that clusterin synthesis by the dying cell is probably not a prerequisite to its death. However, synthesis of clusterin by medullary epithelial cells may be related to their terminal differentiation, and, furthermore, its presence in Hassal's bodies raises the possibility that the secreted protein is involved in the disposal of cell debris resulting from thymocyte apoptosis.

Apoptosis↗
PubMed2005

Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction.

Eps15 and its related protein Eps15R are key components of the clathrin-mediated endocytic pathway. We searched for new binding partners of Eps15 using a yeast two-hybrid screen. We report here that ubiquilin (hPLIC1), a type-2 ubiquitin-like protein containing a ubiquitin-like domain (UBL) and a ubiquitin-associated domain (UBA), interacts with both Eps15 and Eps15R. Using glutathione-S-transferase pull-down experiments, we show that the first ubiquitin-interacting motif of Eps15 (UIM1) interacts directly with the UBL domain of ubiquilin, whereas it does not bind to ubiquitinated proteins. The second UIM of Eps15 (UIM2) binds poorly to the UBL domain but does bind to ubiquitinated proteins. Two other UIM-containing endocytic proteins, Hrs and Hbp, also interact with ubiquilin in a UIM-dependent manner, whereas epsin does not. Immunofluorescence analysis showed that endogenous Eps15 and Hrs, but not epsin, colocalize with green-fluorescent-protein-fused ubiquilin in cytoplasmic aggregates that are not endocytic compartments. We have characterized these green-fluorescent-protein-fused-ubiquilin aggregates as ubiquitin-rich intracytoplasmic inclusions that are recruited to aggresomes upon proteasome inhibition. Moreover, we show that endogenous Eps15 and endogenous ubiquilin colocalize to cytoplasmic aggregates and aggresomes. Finally, we show that the recruitment of Eps15 into ubiquilin-positive aggregates is UIM dependent. Altogether, our data identify ubiquilin as the first common UIM-binding partner of a subset of UIM-containing endocytic proteins. We propose that this UIM/UBL-based interaction is responsible for the sequestration of certain UIM-containing endocytic proteins into cytoplasmic ubiquitin-rich protein aggregates.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Morphine-induced chemotaxis and brain-derived neurotrophic factor expression in microglia.

The addition of morphine at 1 mum induced morphological changes of cultured microglia such that they changed from having globular or bipolar rod-like shapes to being flat and lamellipodial, with membrane ruffling at the edge, which was stained with phalloidin. The membrane ruffling was clearly colocalized with Rac. Morphine also induced chemotaxis in Boyden chamber analysis at concentrations of 1 mum or more in microglia and the microglial cell line EOC 2. All of these changes were abolishable by naloxone, antisense oligodeoxynucleotide for mu-opioid receptor (MOR), pertussis toxin (PTx), and wortmannin, but not genistein or 1,10-phenanthroline. The addition of morphine to microglia stimulated the gene expression of brain-derived neurotrophic factor (BDNF) as early as the 1 hr point, and this lasted for >12 hr. Morphine induced BDNF gene expression and ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation, and these were abolishable by naloxone, wortmannin, PD98059, genistein, and 1,10-phenanthroline. The addition of conditioned medium derived from the culture of morphine-treated microglia also increased the phosphorylation of ERK1/2. All of these findings suggest that morphine induces significant changes in both morphology and gene expression at relatively high concentrations, but the underlying signaling pathways downstream of MOR and G(i/o) appear to be different from each other. Phosphoinositide 3-kinase gamma activation and Rac activation are involved in chemotaxis, whereas indirect pathways through ERK1/2 phosphorylation induced by unknown growth factors generated through an MOR-mediated metalloprotease activation are linked to the enhanced BDNF gene expression.

Animals↗
PubMed2004

Expression of keratinocyte growth factor and its receptor in human breast cancer: its inhibitory role in the induction of apoptosis possibly through the overexpression of Bcl-2.

Keratinocyte growth factor (KGF), a mesenchymal cell derived paracrine growth factor that regulates normal epithelial cell proliferation, appears to be an essential mediator of steroids in various reproductive organs. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human breast carcinoma tissues by immunohistochemistry. We also compared the results with the expression of estrogen receptor alpha(ERalpha), ERbeta, the proliferative activity assessed by the labeling index (LI) for the Ki-67 antigen, apoptotic frequency assessed by terminal dUTP nick end-labeling (TUNEL) index, and the expression of Bcl-2. All of KGF-positive cases were ERalpha- positive (p<0.05), but not that of ERbeta, while all of KGFR-positive cases were ERbeta-positive (p<0.05), but not that of ERalpha. The specimens with the coexpression of KGF and KGFR significantly correlated with a lower TUNEL index (p<0.05), but not with Ki-67 LI in breast cancer tissues. Further analysis at the cellular level revealed that Bcl-2 was colocalized in KGFR-positive cells, and these cells were almost negative for TUNEL staining. Bcl-2-positive cells were also associated with ERbeta, as expected. Therefore, the results indicate that ERalpha may be involved in KGF expression, and that the coexpression of KGF and KGFR may play an inhibitory role in the induction of apoptosis possibly through the up-regulation of Bcl-2 expression in human breast cancer.

Adult↗
PubMed2002

Beta-amyloid-induced synthesis of the ganglioside GD3 is a requisite for cell cycle reactivation and apoptosis in neurons.

We have shown that cortical neurons challenged with toxic concentrations of beta-amyloid peptide (betaAP) enter the S phase of the cell cycle before apoptotic death. Searching for a signaling molecule that lies at the border between cell proliferation and apoptotic death, we focused on the disialoganglioside GD3. Exposure of rat cultured cortical neurons to 25 microm betaAP(25-35) induced a substantial increase in the intracellular levels of GD3 after 4 hr, a time that precedes neuronal entry into S phase. GD3 levels decreased but still remained higher than in the control cultures after 16 hr of exposure to betaAP(25-35). Confocal microscopy analysis showed that the GD3 synthesized in response to betaAP colocalized with nuclear chromatin. The increase in GD3 was associated with a reduction of sphingomyelin (the main source of the ganglioside precursor ceramide) and with the induction of alpha-2,8-sialyltransferase (GD3 synthase), the enzyme that forms GD3 from the monosialoganglioside GM3. A causal relationship between GD3, cell-cycle activation, and apoptosis was demonstrated by treating the cultures with antisense oligonucleotides directed against GD3 synthase. This treatment, which reduced betaAP(25-35)-stimulated GD3 formation by approximately 50%, abolished the neuronal entry into the S phase and was protective against betaAP(25-35)-induced apoptosis.

Amyloid beta-Peptides↗