The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.     

Intracellular acidification, guanine-nucleotide binding proteins, and cytoskeletal actin

The addition of propionic acid to rabbit neutrophils causes cell acidification and increases the amount of actin associated with the cytoskeleton. Both responses are rapid, and while the cell acidification is somewhat long-lasting, the increase in cytoskeletal actin is transient. It reaches a maximum value within 15 seconds and then returns to the basal level. Unlike fMet-Leu-Phe, however, propionic acid does not cause a rise in the intracellular concentration of free calcium. Pretreatment of the cells with pertussis toxin inhibits the propionic acid-produced increase in cytoskeletal actin but not the decrease in intracellular pH. However, the rate of return to the base line of the cell acidification produced by propionic acid is diminished in cells pretreated with pertussis toxin. On the other hand, both the decrease in intracellular pH and the increase in cytoskeletal actin produced by fMet-Leu-Phe are inhibited by pertussis toxin treatment. The results presented here suggest two important points. First, while cell acidification may trigger directly or indirectly the association of actin with the cytoskeleton, it is certainly not sufficient. Second, a functional guanine-nucleotide regulatory protein is required for stimulated cytoskeletal actin. One or more components of the G-protein and/or their effects on phosphoinositide hydrolysis may increase the number of actin monomers and the availability of preexisting actin filaments to these monomers.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

Effects of phorbol esters on cytoskeletal proteins in cultured bovine chromaffin cells: induction of neurofilament phosphorylation and reorganization of actin

Bovine chromaffin cells normally express mostly nonphosphorylated neurofilaments (NFs) in primary culture, and thus provide a unique model for examining the kinase capable of phosphorylating these proteins in situ. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C induced NF phosphorylation both in the perikaryon and in neuritic extensions of neurite-bearing cells as judged by immunofluorescence using monoclonal anti-NF antibodies which distinguish between phosphorylated and nonphosphorylated epitopes. NF phosphorylation was suppressed by pretreating the cells with sphingosine, an inhibitor of protein kinase C, and was not observed in the presence of the phorbol ester. 4 alpha-phorbol-12,13-didecanoate (PDD) which does not activate protein kinase C, arguing that protein kinase C was responsible for the observed phosphorylation. Immunochemical analysis of cytoskeletal extracts indicated that TPA induced a 3 to 6-fold increase in NF phosphorylation and showed that the 150,000 dalton NF subunit was the principal protein kinase C substrate. In addition to the TPA effect on NF phosphorylation, TPA provoked a reversible membrane ruffling, which eventually resulted in a flattening of chromaffin cells. These morphological alterations were linked with actin patching and the development of stress fibers, respectively. Sphingosine blocked the TPA-induced membrane ruffling and actin patching, and these phenomena were correlated with increased protein kinase C activity. In contrast, there was no change in the localization of microtubules and NFs. The actin reorganization and NF phosphorylation induced by TPA suggest that at least two distinct proteins of the neuronal cytoskeleton are susceptible to the influence of protein kinase C activation. It remains to be established whether protein kinase C plays a role in the regulatory mechanism controlling actin organization and neurofilament phosphorylation during neuronal differentiation.     

Stretch-induced Raf-1 activation in mesangial cells requires actin cytoskeletal integrity

Glomerular capillary hypertension is a determinant of glomerulosclerosis and is modelled in vitro by exposure of mesangial cells to cyclic mechanical strain. In response to strain, Erk is activated and mediates extracellular matrix accumulation and mesangial cell proliferation. Erk activation is dependent on an intact cytoskeleton. Since Raf-1 lies upstream of Erk in response to numerous stimuli, and since its activation is dependent on membrane recruitment, we postulated that the cytoskeleton was essential for Raf-1 membrane recruitment and Erk activation. Primary rat mesangial cells (passages 8-20) were stretched at 1 Hz and 27 kPa. Raf-1 was both phosphorylated on serine-338 (S338) and activated within 2 min of strain. The Raf-1 inhibitor, GW5074, dose-dependently blocked strain-induced Erk activation and Raf-1 phosphorylation. Although phosphatidylinositol-3-kinase (PI3-K) may mediate Raf-1 activation, PI3-K inhibition with wortmannin or LY294002 had no effect on stretch-induced Raf-1 activation. Cytoskeletal disruption with cytochalasin D and the Rho-kinase inhibitor, Y-27632, however, blocked both Raf-1 phosphorylation and activation. Furthermore, membrane localization of Raf-1 was increased by strain and prevented by cytoskeletal disruption. Thus, strain leads to rapid membrane localization, S338 phosphorylation, and activation of Raf-1. These events are independent of PI3-K, but require Rho-kinase activation and an intact actin cytoskeleton.     

The response of fenestrations, actin, and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells

To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hep1 cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep1 cells had pores; some of which appeared to be fenestrations (diameter 55 +/- 28 nm, porosity 2.0 +/- 1.4%), rudimentary sieve plates, bristle-coated micropinocytotic vesicles and expressed caveolin-1, von Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 +/- 2.6% (P < 0.01) and pore diameter to 104 +/- 59 nm (P < 0.001). GFP-actin was expressed throughout the cells, whereas GFP-caveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hep1 cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus.     

Big G,little G: G proteins and actin cytoskeletal reorganization

It has been known for some time that stimulation of heterotrimeric G protein-coupled receptors can cause cytoskeletal reorganization. A recent report in Developmental Cell demonstrates that the activation of a tyrosine kinase, C-terminal Src kinase, by heterotrimeric G protein subunits provides the trigger for Rho-dependent actin stress fiber formation.     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

Identification of anti-adipogenic proteins in adult bovine serum suppressing 3T3-L1 preadipocyte differentiation

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed. [BMB Reports 2013; 46(12): 582-587]     

Role of caveolin-1 in fibrotic diseases

Fibrosis underlies the pathogenesis of numerous diseases and leads to severe damage of vital body organs and, frequently, to death. Better understanding of the mechanisms resulting in fibrosis is essential for developing appropriate treatment solutions and is therefore of upmost importance. Recent evidence suggests a significant antifibrotic potential of an integral membrane protein, caveolin-1. While caveolin-1 has been widely studied for its role in the regulation of cell signaling and endocytosis, its possible implication in fibrosis remains largely unclear. In this review we survey involvement of caveolin-1 in various cellular processes and highlight different aspects of its antifibrotic activity. We hypothesize that caveolin-1 conveys a homeostatic function in the process of fibrosis by (a) regulating TGF-β1 and its downstream signaling; (b) regulating critical cellular processes involved in tissue repair, such as migration, adhesion and cellular response to mechanical stress; and (c) antagonizing profibrotic processes, such as proliferation. Finally, we consider this homeostatic function of caveolin-1 as a possible novel approach in treatment of fibroproliferative diseases.     

Serotonin induced actin polymerization and association with cytoskeletal elements in cultured bovine aortic endothelium

Serotonin, (5-hydroxytryptamine, 5-HT) binds to cultured endothelial cell stress fibers as identified by fluorescence microscopy and in vitro induces actin polymerization as measured by DNase 1 inhibition and differential centrifugation; the structural change in actin in the presence of 5-HT resembles actin paracrystals as visualized by negative staining under electron microscopy. These observations indicate that 5-HT, which is taken up by endothelium by a non-mediated diffusion, may interact directly with actin to affect cytoskeletal changes.     

Identification of a functional switch for actin severing by cytoskeletal proteins

Actin severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microM). Here we demonstrate that high calcium releases an autoinhibited conformation in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever actin in vivo.     

Degenerative muscle fiber accelerates adipogenesis of intramuscular cells via RhoA signaling pathway

In some pathological conditions such as Duchenne muscular dystrophy, it has been known that a fatty infiltration in skeletal muscle is often observed and that is also one of primary factors to induce marked decline of muscular strength. However, the mechanism of fatty infiltration, cellular origin of accumulated adipocytes and its significance are not fully understood. The fact that persistent degenerative muscle fibers are present on dystrophic muscle leads us to hypothesize that muscle fiber condition affects fatty infiltration in skeletal muscle. We employed a single fiber culture system to determine whether fiber condition affects an appearance of adipocytes on the fibers. Artificially hyper-contracted muscle fibers (HCF), generated from isolated intact fibers (IF) of rat extensor digitrum longus muscle, were maintained as non-adherent cultures for 5–7 days. Interestingly, there appeared to be considerable numbers of mature adipocytes on HCF, whereas no adipocytes were seen on IF, indicating that cells on HCF spontaneously differentiated into mature adipocytes. Activation of RhoA signaling by the addition of thrombin decreased the number of adipocytes on HCF in a dose-dependent manner, whereas the number of MyoD-positive myoblasts increased. In contrast, Y-27632, a specific inhibitor of Rho kinases (ROCK), induced adipogenic differentiation of cells derived from IF. In addition, administration of Y-27632 into mouse regenerating muscle resulted in fat accumulation in the muscle. Taken together, the present studies clearly demonstrated that muscle fiber condition affects fat accumulation in skeletal muscle and that is possibly mediated by the RhoA signaling pathway.     

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.     

Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells

We studied actin cytoskeletal remodeling and the role of leukotrienes and tyrosine phosphorylation in the response of endothelial cells to different types of cyclic mechanical stretching. Human aortic endothelial cells were grown on deformable silicone membranes subjected to either cyclic one-directional (strip) stretching (10%, 0.5 Hz), or biaxial stretching. After 1 min of either type of stretching, actin cytoskeletons of the stretched cells were already disrupted. After stretching for 10 and 30 min, the percentage of the stretched cells that had disrupted actin cytoskeletons were significantly increased, compared with control cells without stretching. Also, at these two time points, biaxial stretching consistently produced higher frequencies of actin cytoskeleton disruption. At 3 h, strip stretching caused the formation of stress fiber bundles, which were oriented nearly perpendicular to the stretching direction. With biaxial stretching, however, actin cytoskeletons in many stretched cells were remodeled into three-dimensional actin structures protruding outside the substrate plane, within which cyclic stretching was applied. In both stretching conditions, actin filaments were formed in the direction without substrate deformation. Moreover, substantially inhibiting either leukotriene production with nordihydroguaiaretic acid or tyrosine phosphorylation with tyrphostin A25 did not block the actin cytoskeletal remodeling. However, inhibiting both leukotriene production and tyrosine phosphorylation completely blocked the actin cytoskeletal remodeling. Thus, the study showed that the remodeling of actin cytoskeletons of the stretched endothelial cells include rapid disruption first and then re-formation. The resulting pattern of the actin cytoskeleton after remodeling depends on the type of cyclic stretching applied, but under either type of cyclic stretching, the actin filaments are formed in the direction without substrate deformation. Finally, leukotrienes and tyrosine phosphorylation are necessary for actin cytoskeletal remodeling of the endothelial cells in response to mechanical stretching.     

Lumican affects actin cytoskeletal organization in human melanoma A375 cells

AIMS: Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. MAIN METHODS: Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. KEY FINDINGS: A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. SIGNIFICANCE: Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.     

Role of actin cytoskeletal dynamics in activation of the cyclic AMP pathway and HWP1 gene expression in Candida albicans

Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.     

猪脂肪前体细胞分化过程中聚脂相关基因的表达模式

本实验采用胶原酶消化法分离猪皮下脂肪前体细胞,用含850 nmol/L 胰岛素和50 nmol/L地塞米松的诱导培养液进行诱导,采用油红O提取法测定了细胞中的甘油三酯含量,同时采用实时定量RT-PCR方法检测了细胞分化过程中聚脂相关基因的表达.结果显示:转录因子PPAR γ和C/EBP β在诱导后12 h即迅速表达,SREBP-1 mRNA表达水平在诱导后12 h出现显著下调,随后逐渐升高,96 h达到最高水平;脂肪合成相关酶基因GPDH、FAS、ACC和LPL呈现出与SREBP-1相似的表达模式;脂肪酸转运相关基因aP2、FAT、FATP1与VLDLR的表达量随着细胞分化过程的延长而不断增加,并且与细胞内甘油三酯的含量变化高度相关.本实验结果表明,PPAR γ、C/EBP β和SREBP-1可能是调控猪脂肪前体细胞分化的关键转录因子.猪皮下脂肪组织在聚脂过程中,在分化早期可能以脂肪细胞自身合成脂肪酸为主,而后期则主要依赖细胞外脂肪酸的跨膜转运.这些结果可能有助于揭示脂肪细胞的分化调控规律.