Increase In Nuclear Binding Research Articles

Role of protein kinase C and NF-kappaB in proteolysis-inducing factor-induced proteasome expression in C(2)C(12) myotubes.

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4α-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 μM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 μM). Proteolysis-inducing factor-induced activation of PKCα, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCα (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCα (pKS1), which showed no activation of PKCα in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-κB (NF-κB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-κBα and an increase in nuclear binding of NF-κB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-κBα, induced by PIF, necessary for the release of NF-κB from its inactive cytosolic complex.

Aluminium Lactate Treatment of DQ12 Quartz Inhibits Its Ability to Cause Inflammation, Chemokine Expression, and Nuclear Factor-κB Activation

In 1997, an IARC Working Group classified quartz (crystalline silica) as a Group 1 lung carcinogen, but only in some industries, i.e., the quartz hazard is a variable entity. The reactivity of the quartz surface may underlie its ability to cause inflammation, and treatments that ameliorate this reactivity will reduce the quartz hazard. In this study we treated quartz (Q) with aluminium lactate (AL), a procedure that is reported to decrease the quartz hazard, and explored the effect this had on the highly reactive quartz surface and on proinflammatory events in rat lungs. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance and the hemolysis assay. Eighteen hours after instillation of Q into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL). In addition, Q induced an increase in BAL macrophage inflammatory protein-2 (MIP-2) while ALQ had no significant effect compared to control. Epithelial damage, as indicated by BAL protein and gamma glutamyl transpeptidase, also increased with Q but not with ALQ. Furthermore, Q induced an increase in MIP-2 mRNA by BAL cells while ALQ had no effect compared to controls. There was an increase in nuclear binding of the transcription nuclear factor κB (NF-κB) in the Q-exposed BAL cells and again no effect on nuclear NF-κB binding in BAL cells from ALQ-exposed rats. In conclusion, treatment of the quartz surface with aluminium lactate reduced the reactivity of the particles both in terms of hydroxyl radical generation and in terms of the induction of molecular signaling events leading to inflammation.

Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts.

The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system.

Glucocorticoid agonists as well as antagonists are effective inducers of mouse mammary tumor virus RNA in mouse mammary tumor cells treated with inhibitors of ADP‐ribosylation

Glucocorticoids increase expression of specific genes by a mechanism involving binding to and "activation" of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroid-receptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoid-regulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP-ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2-fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor-treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP-ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid-sensitive genomes.

The postovulatory cat oviduct and uterus: correlation of morphological features with progesterone receptor levels.

Oviducts and uteri were obtained from cats at estrus and up to 14 days after coitus. Progesterone binding sites were estimated by Scatchard analysis, serum levels of progesterone were determined by radioimmunoassay, nd key morphological parameters of the lining of the epithelium of the oviduct and the deep uterine glands were quantitated. Progesterone cytosol binding was highest at estrus, significantly depleted by day 4, and continued a gradual decline out to day 14 in both tissues. Nuclear binding was markedly elevated at day 4; with time the binding decreased until by day 14 it was equal to or lower than that detected at estrus. The initial depletion of cytosol binding and rise in nuclear binding coincided with the initial rise in serum progesterone levels. Within the oviduct, the increase in nuclear binding coincided with the onset of regressive changes within the epithelium. These changes included atrophy, deciliation, and apoptosis. Within the uterine glands, the increase in nuclear binding coincided with the onset of the progestational response. This response included cell hypertrophy, glycogen synthesis and deposition, and with longer exposure to elevated levels of progesterone, the disappearance of these glycogen deposits. These results clearly illustrate the immediate effect of low levels of progesterone on the subcellular compartmentalization of the progesterone receptor system, and the down regulation of the total receptor system when target tissues are exposed to a continuous elevation of serum progesterone. These results also illustrate that similar changes in receptor compartmentalization induce different morphological responses within target tissues from the same animal.

Nuclear translocation of the 1,25-dihydroxycholecalciferol receptor in mouse kidney.

We have recently demonstrated that cytosol from mouse kidney contains a receptor-like binding protein for 1,25-dihydroxycholecalciferol (1,25-(OH)2D3). The present studies were undertaken to investigate whether this component undergoes nuclear translocation, thus adding support to the contention that this binding moiety represents a receptor for 1,25-(OH)2D3. Kidney slices from vitamin D-deficient mice were incubated with 1,25-(OH)2[3H]D3 and specific binding was assessed in cytosol and 0.3 M KCl extracts of isolated nuclei. The appearance of specific nuclear-bound 1,25-(OH)2[3H]D3 exhibited a saturation time course and the increase in nuclear binding was accompanied by a concomitant decrease in cytoplasmic binding such that, by 120 min, approximately 80% of specifically bound 1,25-(OH)2[3H]D3 was associated with the nuclear fraction and approximately 20% remained in the cytoplasmic compartment. The nuclear transfer was found to be temperature-dependent and specific for the dihydroxy metabolite. Both the cytoplasmic and nuclear receptors sedimented at 3.2 S in 0.3 M KCl sucrose density gradients. However, under low salt conditions, the cytoplasmic receptor was transformed to yield an approximately 5.4 S peak while the nuclear receptor sedimented unchanged t 3.2 S. In summary, the cytoplasmic 1,25-(OH)2D3 receptor in mammalian kidney undergoes temperature-dependent nuclear translocation and the cytoplasmic and nuclear forms of the receptor can be distinguished by their sedimentation properties.

Relationship between the changes in cytoplasmic progesterone receptor concentration and in nuclear progesterone binding sites in the hen hypothalamus and pituitary during the ovulatory cycle.

The amount of specific [3H]-progesterone bindings was measured in cytosols and in nuclear fractions of the hen hypothalamus and pituitary during the ovulatory cycle. In both tissues, a decrease in cytosol binding from the peak at 18 and 8 hr before ovulation was coincident with an increase in nuclear binding attained a peak 14 to 6 hr before ovulation. The results suggest that progesterone may act on the hypothalamus and pituitary 18-14 hr before ovulation and 8-6 hr before ovulation.

Estrogen-Independent Nuclear Binding of Receptor Protein of Rat Uterine Cytosol by Removal of Low Molecular Weight Inhibitor*

A cell-free system prepared from the ovariectomized rat uterus was used to study the uptake of the cytoplasmic estrogen receptor (RE) by isolated nuclei. At 0–4 C, dialysis of cytosol containing [3H]estradiol (E2)-RE complexes resulted in a 3- to 6-fold increase in nuclear binding of [3H]E2-RE in comparison with nondialyzed cytosol. Removal of the low molecular weight materials by Sephadex G-25 chromatography also enhanced binding of [3H]E22-RE complexes to nuclei. Incubation of nuclei at 0 C with cytosol dialyzed in the absence of estrogen revealed the appearance of the estrogen-binding component in nuclei which showed high affinity for EE (Kd = 6 × 10-10 M at 0 C). This nuclear estrogen-binding component was not occupied with estrogen, since a slightly higher amount of binding with [3H]EE was obtained at 0 than at 37 C. The effects of dialysis on the sedimentation constants were also studied. Dialysis of [3H]E2-RE complexes in cytosol at 0–4 C caused a change of the sedimentation constant from 4.3S to 5...

3H]-Estradiol binding by chick liver nuclear extracts: mechanism of increase in binding following estradiol injection

Specific high affinity binding of [ 3H]-estradiol by 0.5 M KCl extracts of chick liver nuclei is substantially increased by estradiol injection of the immature chick. The effect is observed shortly after estradiol injection, while the estradiol-induced production of serum phosphoproteins (vitellogenic response) is not detectable until about 24 hr.Cycloheximide given 90 min before estradiol inhibits the increase in nuclear binding for 12–15 hr. At 24–48 hr the levels of nuclear binding are similar to those in the estradiol-treated animals not given cycloheximide, but serum phosphoprotein levels are depressed by about 80% at 48 hr. By 75 hr however the serum of the cycloheximide-treated estrogenized chicks contains about twice as much phosphoprotein as does serum of chicks given estradiol alone.It is suggested that the inhibition of protein synthesis for 12–15 hr delays the vitellogenic response until sufficient levels of nuclear [ 3H]-estradiol binding protein can be synthesized. A correlation between the levels of nuclear [ 3H]-estradiol binding at 24 hr and phosphoprotein at 48 hr is shown in a dose-response experiment. In vitro , nafoxidine-HCl (Upjohn 11,100 A) inhibits binding of [ 3H]-estradiol by the chick liver nuclear extracts. In vivo , a single injection of nafoxidine with estradiol inhibits phosphoprotein production.Injection of nafoxidine alone results in a small but significant increase in [ 3H]-estradiol binding by nuclear extracts, but it is not estrogenic. A possible interpretation is that nafoxidine transfers low levels of a putative cytosol receptor to the nucleus, but is unable to induce the amplification mechanism required to give the levels of nuclear estradiol-binding protein needed for the vitellogenic response.

Progesterone-binding Components of Chick Oviduct: II. NUCLEAR COMPONENTS

Abstract Specific progesterone-binding components are shown to be present in oviduct nuclei of estrogen-treated chicks. The macromolecular complex containing 3H-progesterone can be detected by sucrose gradient centrifugation and distinguished from chick plasma transcortin by Agarose gel filtration. The nuclear binding components behave identically to the cytoplasmic components previously described in our ultracentrifugal, chromatographic, and inactivation studies. The macromolecular-progesterone complex formed in vivo or in vitro can be extracted from nuclei by 0.3 m KCl. Little or no salt-extractable progesterone-binding activity can be detected in oviduct nuclei prior to progesterone administration. The binding molecules appear to exist initially in oviduct cytoplasm, where they presumably function as specific target tissue receptors. Following injection of 3H-progesterone in vivo or incubation of oviduct slices in vitro with 3H-progesterone, a progressive increase in nuclear binding occurs. The data indicate an absolute prerequisite for an initial interaction of the steroid with the cytoplasm, and a concomitant depletion of cytoplasmic receptor as nuclear binding increases. These results, and the apparent identity of the cytoplasmic and nuclear components, support the hypothesis that the nuclear macromolecular-steroid complex arises by a hormone-dependent transfer of the cytoplasmic receptor complex into the nucleus.