Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Multiple forms of actin in Physarum polycephalum

Actin in the acellular slime mold Physarum polycephalum consists of three major forms closely spaced at isoelectric point (IP) 4.7 and a minor form at IP 5.1. Amino acid analysis has shown the IP 5.1 actin to be nearly identical to the 4.7 actins. In actin purified from acetone powder, both actin forms were present. Both forms bound to DNase I and have the same molecular weight of about 43 000 on sodium dodecyl sulfate (SDS) polyacrylamide gels. On 2-D gels of nuclear proteins, both forms of actin were present. The IP 4.7 actins account for 8.6% of total plasmodial protein, and the IP 5.1 form for about 0.7%. In the nucleus the IP 4.7 actins comprise 2.7% of total nuclear protein, and the 5.1 actin about 0.4%. No cell cycle-associated change in the concentration of actins was observed in either total plasmodial extracts or in isolated nuclei. Pulse-labelling experiments have shown that in total plasmodia actin synthesis occurs throughout the cell cycle, with no relative changes in the rate of synthesis. In isolated nuclei labelled during mitosis and early S-phase, there is about twice as much labelled actin as in nuclei labelled prior to mitosis. This result may indicate an increase in the transport of actin into the nucleus.     

Periodic accumulation of cdc15 mRNA is not necessary for septation in Schizosaccharomyces pombe

Analysis of Schizosaccharomyces pombe mutants that are defective in septum formation and cytokinesis has identified the product of the cdc15 gene as a key element in formation of a division septum. S. pombe cells lacking cdc15p function cannot assemble a functional medial ring, and do not make a division septum. cdc15 mRNA accumulates periodically during the cell cycle, peaking after entry into mitosis, and increased expression of the gene in G2-arrested cells can promote F-actin ring formation. Here, we have investigated the effects of mutations that block cell division upon the expression of cdc15 in synchronised cell populations, and analysed the expression of cdc15 when septum formation is induced by ectopic activation of the septation signalling network. We concluded the following: (i) the septation signalling network genes are not required for periodic accumulation of cdc15 mRNA; (ii) induction of septum formation in G2-arrested cells by activation of the septation signalling network does not result in accumulation of cdc15 mRNA, which is therefore not a prerequisite for septum formation; (iii) failure to turn off septum formation at the end of mitosis results in continued expression of cdc15; and (iv) periodic accumulation of cdc15 mRNA is mediated by a 97 bp region 5' to the mRNA start site.     

Temperature-shock induction of multiple flagella induces additional synthesis of flagellum specific mRNAs and tubulin

Four mRNAs (alpha- and beta-tubulin, flagellar calmodulin and Class-I), specifically expressed when Naegleria amebae differentiate into flagellates, were followed at 5-10 min intervals during the temperature-shock induction of multiple flagella in order to better understand how basal body and flagellum number are regulated. Surprisingly, tubulin synthesis continued during the 37 min temperature shock. An initial rapid decline in alpha- and beta-tubulin and flagellar calmodulin mRNAs was followed by a rapid re-accumulation of mRNAs before the temperature was lowered. mRNA levels continued to increase until they exceeded control levels by 4-21%. Temperature shock delayed flagella formation 37 min, produced twice as much tubulin protein synthesis and three fold more flagella. Labeling with an antibody against Naegleria centrin suggested that basal body formation was also delayed 30-40 min. An extended temperature shock demonstrated that lowering the temperature was not required for return of mRNAs to near control levels suggesting that induction of multiple flagella and the formation of flagella per se are affected in different ways. We suggest that temperature-shock induction of multiple flagella reflects increased mRNA accumulation combined with interference with the regulation of the recently reported microtubule-nucleating complex needed for basal body formation.     

The gravireceptor of Phycomyces. Its development following gravity exposure

The gravitropism of a mature stage IV Phycomyces sporangiophore has a shorter and more uniform latency if the sporangiophore is exposed horizontally to gravity during its earlier development (stage II and stage III). This early exposure to an altered gravitational orientation causes the sporangiophore to develop a gravireceptor as it matures to stage IV and resumes elongation. A technique has been developed to observe the spatial relationship between the vacuole and the protoplasm of a living sporangiophore and to show the reorganization caused by this exposure to altered gravity. Possible gravireceptor mechanisms are discussed.     

Regulation of Actin and Its mRNA by Thyroid Hormones in Cultures of Fetal Human Brain During Second Trimester of Gestation

Abstract: The effect of thyroid hormones (THs) on the expression of actin gene during fetal human brain development and the period of sensitivity to the hormones have been investigated. Developmental profile of actin in the cytoskeletal (CSK) and noncytoskeletal (non-CSK) fractions in the fetal cerebra showed a pronounced rise in the level of CSK actin at weeks 17–19. Northern blot analysis also revealed a sharp rise in the level of actin mRNA at weeks 16–18, temporally coinciding with the period of rise of THs and peak expression of TH receptors in the fetal brain. In organ cultures of weeks 13–23 fetal cerebra, THs elicited a general stimulation of CSK proteins at all ages studied with a preferential effect on actin at weeks 17–19. During this period, THs also stimulated the rate of synthesis of actin. Kinetics of induction of actin by TH in the non-CSK and CSK fractions in organ cultures of week 17 fetal cerebra showed an increased level of actin in both fractions within 1 h. Subsequently (at 5 and 18 h), induction was evident only in the insoluble CSK fraction, suggesting an effect of the hormone on the intracellular distribution of actin between the soluble non-CSK fraction and the insoluble CSK fraction. Correspondingly, in cultures of week 17 fetal cerebra, THs elicited an increase in actin mRNA level within 30 min of hormonal exposure. The overall results suggest that THs regulate the expression of actin gene by stimulating the rate of synthesis as well as intracellular distribution of actin during the mid phase of the second trimester of gestation.     

Angiotensin II induces MMP-2 in a p47phox-dependent manner

Activated matrix metalloproteinases (MMPs) in patients with acute coronary syndromes may contribute to plaque destabilization. Since reactive oxygen species (ROS) induce MMP-2 and angiotensin II (ANG II) enhances NADPH-oxidase-dependent ROS formation, we assessed whether ANG II induces MMP-2 in a NADPH-oxidase-dependent manner. MMP-2 mRNA expression and activity were analyzed in wildtype and p47phox-deficient (p47phox-/-) murine smooth muscle cells (SMC). To address a clinical implication, sections of human atherosclerotic arteries were stained for MMP-2, p47phox, ANG II, AT1-receptor, and alpha-smooth muscle cell actin (alpha-SMC actin). MMP-2 protein expression and activity from these arteries were compared to those without atherosclerosis. ANG II enhances mRNA synthesis and activity of MMP-2 in a p47phox-dependent manner. Immunohistochemical analyses revealed a co-localization of MMP-2 with p47phox, ANG II, AT1-receptor, and alpha-SMC actin. MMP-2 protein expression and gelatinolytic activity are increased in atherosclerotic arteries. Thus, activation of the renin-angiotensin system may contribute to plaque destabilization via ROS-dependent induction of MMP-2.     

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.     

Translational control of mRNA expression during the early mitogenic response in Swiss mouse 3T3 cells: identification of specific proteins

Addition of serum or epidermal growth factor to quiescent Swiss mouse 3T3 cells in culture leads to a number of specific changes in the pattern of protein synthesis. Earlier experiments with actinomycin D suggested that the altered expression of these proteins was controlled at either the pretranslational or translational level. Here we have identified and further characterized the regulation of mRNA expression for ten of these proteins, including protein synthesis elongation factor eEF-1 alpha, poly A binding protein, vimentin, the multiple forms of the actin protein family, and alpha- and beta-tubulin. Using an in vitro translation system, we determined the change in the level of mRNA encoding for each of these proteins after serum stimulation. The results showed that the amount of mRNA coding for eEF-1 alpha, poly A binding protein, vimentin, and alpha- and beta-tubulin remains unchanged during this time, whereas that of the actin family increases. Thus, with the exception of the actin family, the results argue that the expression of all the proteins identified is regulated at the translational level. The importance of this latter group of proteins in cell growth and the abundance of their cognate mRNAs should prove them useful tools in elucidating the mechanisms involved in the activation of translationally repressed mRNA during the mitogenic response.     

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.