Increase In Actin mRNA Research Articles

Seasonal changes in actin and Cdk5 expression in different brain regions of the Yakut ground squirrel (Spermophilus undulatus)

In this study we described the seasonal profile of the actin and Cdk5 levels in different brain structures of Yakut long-tailed ground squirrels (Spermophilus undulatus) during their annual cycle. Experiments were performed with adult Yakut ground squirrels, both male and female, weighing 600–800 g (n = 35) at different stages of their annual cycle, viz., in the summer, the period of the maximum activity of the animals; in the autumn, during preparation for hibernation; in the winter, during hibernation; in the spring, at the exit of the animals from the hibernating state. Our results indicate that actin mRNA increased by 1.9 times (p = 0.0001) in the frontal cortex, the hippocampus, and the caudal brainstem during hibernation. In the brainstem, a significant increase in actin mRNA started to develop in autumn, in normothermic animals at the stage of their preparation for hibernation (p = 0.0078). At the exit of animals from the torpid state, the level of hippocampal expression decreased significantly by 4.5 times; in the cortex and brainstem it decreased to the level of summer animals. In contrast, the dynamics in the cerebellum had opposite direction: actin mRNA level decreased significantly during the preparation for hibernation (p = 0.037), remained low in torpid animals (p = 0.051), and increased after awakening. The changes in the total protein level were observed only in the hippocampus, along with increased expression of Cdk5 mRNA during hibernation (p = 0.003) and at the exit from it (p = 0.001). Detected differences in the seasonal metabolic profile of cytoskeleton proteins in the hippocampus of Yakut long-tailed ground squirrels support a substantial structural plasticity of this brain structure during the hibernation cycle that was described previously in morphological and biochemical studies.

Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells.

Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h. Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished. In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis. These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes. Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium. Microinjection of nonpolymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of [35S]methionine into newly synthesized actin within 1 h. This decrease continued for at least 19 h. Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin. Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.

Effect of Collagen Gel Configuration on the Cytoskeleton in Cultured Rat Hepatocytes

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.

Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS-treated animals. In the ventral prostate, 5 days after EDS-treatment, a 4.0 +/- 0.3-fold up-regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 +/- 0.2-fold increase in actin mRNA. In the epididymis, a 2.0 +/- 0.5-fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS-treated rats, the AR mRNA level was not changed (1.02 +/- 0.17-fold of controls), and there was also no change in actin mRNA level at 5 days after EDS-treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis. Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H-R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS-treated rats with testosterone-implants, remained unaltered compared to control rats (9.1 +/- 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS-treated rats without testosterone-implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H-R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding). Immunoprecipitation and Western blotting of the testicular AR using specific monoclonal and polyclonal antibodies indicated that the total testicular amount of immunodetectable AR protein in long-term testosterone deprived rats was very low when compared to that in control rats or rats with testosterone-implants. This is in disagreement with results obtained in the ligand binding assay, and may point to a structural modification of the AR in the testis that possibly occurs in the prolonged absence of androgens.

G-actin pool and actin messenger RNA during development of the apical processes of the retinal pigment epithelial cells of the chick

It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15–E21) reaching lengths of 20 μm or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F-actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F-actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 ± 0.5 μg/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 μg/ml (13 μ M). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in β actin mRNA and a four- to fivefold increase in message for γ actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA.

Effect of glucocorticoids and 1,25-dihydroxyvitamin D3 on the developmental expression of the rat intestinal vitamin D receptor gene.

In this study the ontogenesis of rat intestinal vitamin D receptor (VDR) gene expression was examined. When Northern and slot blot analyses were used to examine the expression of intestinal VDR mRNA in 15-, 18-, 22-, and 28-day-old rats, induction of VDR mRNA was not observed until 22 days postpartum. Since little is known, particularly in the neonate, concerning the in vivo regulation of VDR gene expression, we examined the possibility that glucocorticoids and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] could affect the developmental expression of the intestinal VDR gene. To examine the effect of glucocorticoids, rat pups received three sequential injections (one per day) of hydrocortisone (5, 2.5, and 2.5 mg/100 g BW). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However, a significant 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17. The hydrocortisone effective period coincides with the glucocorticoid-sensitive period of rat intestinal development. It should be noted, however, that the up-regulation of VDR was accompanied by an increase in actin mRNA, suggesting that the effect is not specific for VDR. Similarly, when rats were bilaterally adrenalectomized on day 17 (killed on day 22), a 4-fold decrease in VDR mRNA was observed, accompanied by a decrease in actin mRNA. However, when rats were injected with 1,25-(OH)2D3 (25 ng/day.100 g BW) from days 15-17, levels of intestinal VDR mRNA were significantly increased by 1.5-fold, and this change was specific for VDR mRNA. In summary, our results indicate that hydrocortisone and 1,25-(OH)2D3 can precociously induce intestinal VDR mRNA, suggesting the involvement of glucocorticoids and 1,25-(OH)2D3 in the regulation of VDR gene expression in the developing rat intestine. However, our results also indicate that the effect of glucocorticoids (unlike the effect of 1,25-(OH)2D3) is not specific for VDR mRNA, but may reflect general effects of glucocorticoids on intestinal maturation.

Comparison of the effects of class 1 and class 2 heparin‐binding growth factors on protein synthesis and actin mRNA expression in BALB/c‐3T3 cells

Biological effects of class 1 or class 2 heparin-binding growth factors (HBGFs) were compared in BALB/c-3T3 cells. Changes in protein synthesis, as monitored by two-dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43-kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potentiate the changes in protein synthesis elicited by HBGF-1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.