ERK1/2 is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells

We previously reported that cyclic compressive force (CCF) induced interleukin-6 mRNA expression in osteocyte-like MLO-Y4 cells. But little is known about how the stimuli are converted into the biochemical signals in MLO-Y4 cells. The aim of this research was to study the effect of CCF on the IL-6 secretion and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in this process. The cells were exposed to CCF with different magnitudes (1000, 2000 and 4000 μstrain), frequencies (0.5, 1.0 and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) by a four-point bending system. The IL-6 secretion and ERK1/2 phosphorylation of the cells were determined by ELISA and Western blotting, respectively. The results showed that IL-6 protein secretion was significantly up-regulated in response to CCF in a magnitude-, frequency- and duration-dependent fashion. The phosphorylation of ERK1/2 also increased in all cases but not depended on the magnitude, frequency or duration of CCF. Furthermore, the inhibition of the ERK1/2 pathway by its specific inhibitor PD098059 decreased but not completely abrogated the IL-6 secretion from stressed MLO-Y4 cells. These findings demonstrate that CCF-induced IL-6 secretion occurs via a mechanism that involves ERK1/2 signaling pathway and suggest that modulation of this event contributes to the pathogenesis of osteoporosis and stress-induced pathological bone resorption as well.     

Mechanisms of cellular adhesion : II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells

cAMP/theophylline exaggerates cell shape—whether the fibroblastic morphology of controls or the epithelioid shape of colchicine-treated cells. The ultrastructural basis is that cAMP/theophylline increases the number and linearity of microtubules and microfilament bundles, although where also treated with colchicine, the cells adopt a well-spread shape maintained by microfilament bundles alone. Since interference reflection microscopy shows that colchicine promotes the marked alignment of focal contacts (which terminate microfilament bundles) it is concluded that microtubules encourage angular cell form and modify the pattern of adhesions by influencing the directionality of microfilament bundle formation although they are inessential for the maintenance of the spread form or adhesion per se.     

Corticosterone regulates the expression of neuropeptide Y and reelin in MLO-Y4 cells

Osteocytes that have a dendritic appearance are widely believed to form a complex cellular network system and play crucial roles in mechanotransduction as a principal bone mechanosensor, which is the basis of their neuronallike biology, as previously reported. Neuropeptide Y (NPY) and reelin mRNA, which are brain-specific neurogenic markers, have been identified in osteocytes. However, changes in the production of NPY and reelin in response to specific biochemical stimulation are unknown. In this study, we investigated the in vitro effect of corticosterone, one of the endogenous glucocorticoids, on the expression of NPY and reelin in the MLO-Y4 osteocyte cell line. Cells were treated with corticosterone at different concentrations (10(-9) M-10(-5) M) for 1, 3, 6, 12 and 24 h. As revealed, corticosterone reduced the MLO-Y4 cell viability and proliferation in a dose- and time-dependent manner based on an MTT assay and a Vi-CELL analyzer. The cells were then incubated with corticosterone (10(-6) μM), and the NPY and reelin expression levels were detected at 1, 3, 6, 12 and 24 h using real-time PCR and Western blot analysis. These results demonstrated that at the gene and the protein levels, corticosterone significantly upregulated the NPY and reelin expression in a time-dependent manner. The application of a glucocorticoid receptor antagonist, RU486, reversed the reduced cell viability and the increased expression of NPY and reelin that were caused by corticosterone. To the best of our knowledge, this is the first report to verify that corticosterone regulates the NPY and reelin expression in osteocytes.     

Longterm conditions of mimicked weightlessness influences the cytoskeleton in thyroid cells

Weightlessness influences the human immune and hormone system, reduces bone mass, leads to muscle atrophy and cardiac atrophy. Effects on control mechanisms for proliferation, programmed cell death and differentiation are well documented. The principal aim of this study was to investigate changes of the cytoskeleton in thyroid cells cultured in vector-averaged gravity under clinostat rotation. After 12 hours the formation of multicellular spheroids started. An increase of extracellular matrix proteins and beta 1-integrin was observed. Laser scanning confocal microscopy of ML-1 follicular thyroid carcinoma cells and normal thyroid HTU-5 cells immunostained with anti-cytokeratin to demonstrate these intermediate filaments revealed that cytokeratin filaments extended from centers, were thickened, coalesced and shortened as compared to control cells. Moreover, vimentin was highly disorganized. The vimentin network formed a coiled aggregate closely associated with the nucleus. Western blot analyses of talin, alpha- and beta-tubulin showed a clear increase of these proteins in cells cultured under simulated 0 g. Our data suggest that the effects of microgravity on cultured human thyroid cells are accompanied by noticeable functional cellular changes. Future studies to clarify the pathway that regulate the observed integrin activation and the mechanisms by which they function have to be performed.     

Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells

Blockade of mevalonate synthesis by the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL-5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL-5 cells also lose vinculin-related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non-sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability. © 1993 Wiley-Liss, Inc.     

DBC2 significantly influences cell-cycle, apoptosis, cytoskeleton and membrane-trafficking pathways

The tumor suppressor DBC2 belongs to a previously uncharacterized gene family, RHOBTB (Bric-a-brac, Tramtrack, Broad-complex). The biological roles of RHOBTB proteins, including DBC2, remain unclear. To understand the physiological functions of DBC2, a global approach was applied. Expression of DBC2 was manipulated in HeLa cells and RNA profiling of the cells was performed by microarray analyses. DBC2 was introduced into HeLa cells by a mammalian expression vector with a constitutive promoter. DBC2 knockdown was achieved by RNA interference with small interfering RNA. RNA profiles of these samples were performed by microarray analysis using Affymetrix GeneChip HG-U133A 2.0. The microarray data were analyzed by Microarray Suite 5.0 (MAS 5.0) and Robust Multichip Average (RMA). A list of genes whose expression was significantly altered (p<0.001) was generated and overlaid onto a cellular pathway map in the Ingenuity Systems' Pathway Knowledge Base (Winter'04 Release). Two networks were found to react substantially to DBC2 expression; namely, more than half of participating genes are affected. One of the networks regulates cell growth through cell-cycle control and apoptosis. The other network is related to cytoskeleton and membrane trafficking. Our findings suggest that the biological roles of DBC2 are related directly and/or indirectly to these cellular machineries.     

Involvement of the cytoskeleton in oxytocin secretion by cultured bovine luteal cells

A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8-12 of the estrous cycle) were stimulated with prostaglandin F2alpha (PGF2alpha; 1 microM), noradrenaline (NA; 10 microM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 microM) or two antimicrotubule agents (colchicine or vinblastine; 1 microM) before stimulation with PGF2alpha, NA, or GH. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.     

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.     

Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Hydrostatic pressure influences morphology and expression of VE-cadherin of vascular endothelial cells

Bovine aortic endothelial cells (BAECs) were exposed to hydrostatic pressures of 50, 100, and 150 mmHg and changes in morphology and expression of vascular endothelial (VE)-cadherin were studied. After exposure to hydrostatic pressure, BAECs exhibited elongated and tortuous shape without predominant orientation, together with the development of centrally located, thick stress fibers. Pressured BAECs also exhibited a multilayered structure unlike those under control conditions and showed a significant increase in proliferation compared with control cells. Western blot analysis demonstrated that protein level of VE-cadherin were significantly lower under pressure conditions than under control conditions. Inhibition of VE-cadherin expression, using an antibody to VE-cadherin, induced the formation of numerous randomly distributed intercellular gaps, elongated and tortuous shapes, and multilayering. These responses were similar to those of pressured BAECs. The exposure of BAECs to hydrostatic pressure may therefore downregulate the expression of VE-cadherin, resulting in loss of contact inhibition followed by increased proliferation and formation of a multilayered structure.     

Lipid, apolipoprotein, and lipoprotein synthesis and secretion during cellular differentiation in caco-2 cells

Summary Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, variable results have been reported regarding their ability to synthesize and secrete lipids and apolipoproteins. The major goal of this investigation is to examine the capacity of Caco-2 cells to elaborate and secrete lipids, lipoproteins, and apolipoproteins at different degrees of morphological and functional differentiation. Cells were cultured in medium with 5% fetal bovine serum (FBS), on permeable polycarbonate filters from 2 to 30 d in the presence of ¹⁴C-oleate or ³⁵S-methionine. Cellular differentiation, as assessed by morphology (light and electron microscopy), transepithelial resistance, free fatty acid flux, and sucrase activity, progressed steadily up to 20 d of culture. Caco-2 cells esterified oleic acid mainly into phospholipids, triglycerides (TG), and smaller amounts of cholesterol esters. Lipid synthesis began as early as 2 d, and TG secretion was enhanced with increased duration of culture. However, very low efficiency of lipid export was observed at all levels of differentiation, reaching a maximum of only 6% of intracellular lipids. VLDL and LDL were the dominant lipoproteins secreted, with HDL comprising <20% of the total. VLDL secretion increased, while LDL decreased, whereas the lipid composition of lipoproteins varied little with increasing duration of culture. Apoprotein B and A-I synthesis and secretion increased markedly from 11 to 20 d of culture. The ratio of apo B-100/B-48 decreased between 11 and 30 d, consistent with enhanced apo B editing of more mature enterocytes. Taken together, our data suggest that from 20 d of culture, Caco-2 cells are morphologically and functionally mature, capable of lipid esterification, and lipoprotein and apolipoprotein synthesis. However, despite their functional and morphological similarities to mature enterocytes, Caco-2 cells have a very limited lipid export capacity.     

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Fetal Leydig cells: cellular origin, morphology, life span, and special functional features.

The Leydig cells, responsible for testicular androgen production, have two growth phases during the life-span of mammals. The fetal population appears during fetal life and is responsible for the androgen-induced differentiation of the male genitalia. The fetal Leydig cells disappear after birth, and the other population, the adult Leydig cells, appears during puberty and persists for the whole adult life. The fetal Leydig cells, evidently due to the intrauterine endocrine milieu and their special functional requirements in genital differentiation, differ both morphologically and functionally from the adult population. The purpose of this review is to elucidate the special features of the mammalian fetal Leydig cell population, which presents an intriguing experimental model for studies of function and regulation of steroidogenic cells.     

The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.     

Effects of oriented substrates on cell morphology,the cell cycle,and the cytoskeleton in Ros 17/2.8 cells

Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts. The underlying mechanism, however, of cellular sensing and responding to the gravity vector is poorly understood. This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-, downward- or edge-on-oriented substrates. Cell morphology and nuclear translocation, cell proliferation and the cell cycle, and cytoskeletal reorganization were found to vary significantly in the three orientations. All of the responses were duration-dependent. These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.