A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation. (57/20287)

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.  (+info)

The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. (58/20287)

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.  (+info)

Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema. (59/20287)

We determined the effects of low dose radiation (<200 cGy) on the cell-cell integrity of confluent monolayers of pulmonary microvascular endothelial cells (PMEC). We observed dose- and time-dependent reversible radiation induced injuries to PMEC monolayers characterized by retraction (loss of cell-cell contact) mediated by cytoskeletal F-actin reorganization. Radiation induced reorganization of F-actin microfilament stress fibers was observed > or =30 minutes post irradiation and correlated positively with loss of cell-cell integrity. Cells of irradiated monolayers recovered to form contact inhibited monolayers > or =24 hours post irradiation; concomitantly, the depolymerized microfilaments organized to their pre-irradiated state as microfilament stress fibers arrayed parallel to the boundaries of adjacent contact-inhibited cells. Previous studies by other investigators have measured slight but significant increases in mouse lung wet weight >1 day post thoracic or whole body radiation (> or =500 cGy). Little or no data is available concerning time intervals <1 day post irradiation, possibly because of the presumption that edema is mediated, at least in part, by endothelial cell death or irreversible loss of barrier permeability functions which may only arise 1 day post irradiation. However, our in vitro data suggest that loss of endothelial barrier function may occur rapidly and at low dose levels (< or =200 cGy). Therefore, we determined radiation effects on lung wet weight and observed significant increases in wet weight (standardized per dry weight or per mouse weight) in < or =5 hours post thoracic exposure to 50 200 cGy x-radiation. We suggest that a single fraction of radiation even at low dose levels used in radiotherapy, may induce pulmonary edema by a reversible loss of endothelial cell-cell integrity and permeability barrier function.  (+info)

The role of local actin instability in axon formation. (60/20287)

The role of localized instability of the actin network in specifying axonal fate was examined with the use of rat hippocampal neurons in culture. During normal neuronal development, actin dynamics and instability polarized to a single growth cone before axon formation. Consistently, global application of actin-depolymerizing drugs and of the Rho-signaling inactivator toxin B to nonpolarized cells produced neurons with multiple axons. Moreover, disruption of the actin network in one individual growth cone induced its neurite to become the axon. Thus, local instability of the actin network restricted to a single growth cone is a physiological signal specifying neuronal polarization.  (+info)

Expression and distribution of the type 1 and type 3 inositol 1,4, 5-trisphosphate receptor in developing vascular smooth muscle. (61/20287)

The recent discoveries of inositol 1,4,5-trisphosphate (IP3) receptor subtypes with different affinities for IP3 and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP3 receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP3 receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP3 receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP3 receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP3 receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP3 receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP3 receptor during development from the type 3 with low affinity for IP3 to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle.  (+info)

Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter. (62/20287)

Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.  (+info)

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I. (63/20287)

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.  (+info)

The isoflavone genistein inhibits internalization of enteric bacteria by cultured Caco-2 and HT-29 enterocytes. (64/20287)

The dietary isoflavone genistein is the focus of much research involving its role as a potential therapeutic agent in a variety of diseases, including cancer and heart disease. However, there is recent evidence that dietary genistein may also have an inhibitory effect on extraintestinal invasion of enteric bacteria. To study the effects of genistein on bacterial adherence and internalization by confluent enterocytes, Caco-2 and HT-29 enterocytes (cultivated for 15-18 d and 21-24 d, respectively) were pretreated for 1 h with 0, 30, 100, or 300 micromol/L genistein, followed by 1-h incubation with pure cultures of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Pretreatment of Caco-2 and HT-29 enterocytes with genistein inhibited bacterial internalization in a dose-dependent manner (r = 0.60-0.79). Compared to untreated enterocytes, 1-h pretreatment with 300 micromol/L genistein was generally associated with decreased bacterial internalization (P < 0. 05) without a corresponding decrease in bacterial adherence. Using Caco-2 cell cultures, decreased bacterial internalization was associated with increased integrity of enterocyte tight junctions [measured by increased transepithelial electrical resistance (TEER)], with alterations in the distribution of enterocyte perijunctional actin filaments (visualized by fluorescein-labeled phalloidin), and with abrogation of the decreased TEER associated with S. typhimurium and E. coli incubation with the enterocytes (P < 0.01). Thus, genistein was associated with inhibition of enterocyte internalization of enteric bacteria by a mechanism that might be related to the integrity of the enterocyte tight junctions, suggesting that genistein might function as a barrier-sustaining agent, inhibiting extraintestinal invasion of enteric bacteria.  (+info)