Gelsolin Expression Research Articles

A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system.

Two isoforms of the Ca2+-sensitive, actin-binding protein gelsolin have been identified thus far; one is an intracellular protein, cytoplasmic gelsolin, and the other is a secretory protein called plasma gelsolin. Gelsolin expression in the mammalian CNS appears to be localized mainly to oligodendrocytes where it is presumed that the cytoplasmic isoform predominates. Here, we show that oligodendrocytes not only contain cytoplasmic gelsolin, but they also express a novel gelsolin isoform that we have named gelsolin-3. Cytoplasmic gelsolin, plasma gelsolin, and gelsolin-3 arise by alternative splicing from the same gene. The N-terminal amino acid sequence unique to gelsolin-3 is shown to be encoded by a single exon in a region previously thought to be an intron in the human gelsolin gene. In situ hybridization analysis confirmed that gelsolin-3 mRNA is localized primarily to oligodendrocytes in rat brain. In other tissues, gelsolin-3 shows a more restricted pattern of expression than cytoplasmic gelsolin. These data support the view that the gelsolin isoforms have differential roles in the regulation of the actin cytoskeleton.

Myofibrillar myopathy. III. Abnormal expression of cyclin-dependent kinases and nuclear proteins.

The pathological process in myofibrillar myopathy (MFM) (previously also referred to as "desmin storage" or "intermediate filament myopathy") results in dissolution of myofibrils, accumulation of products of the degradative process, and abnormal ectopic expression of desmin, dystrophin, gelsolin, NCAM, and N-terminal components of beta-amyloid precursor protein. We now demonstrate that the abnormal fiber regions in MFM immunoreact strongly for (a) CDC2 kinase, the mitotic kinase that phosphorylates and disassembles intermediate filaments; (b) cyclin-dependent kinases CDK2, CDK4, and CDK7, which are involved in regulation of the cell cycle; (c) lamin B, which normally supports the inner nuclear membrane; and (d) the nuclear matrix associated protein. The normal muscle fiber lies in a terminally differentiated state and is refractory to reentry into the cell cycle. The abnormal expression of multiple cyclin-dependent kinases in the terminally differentiated muscle fiber implies inappropriate activation of positive regulators of mitosis and may signal a mitotic catastrophe. The dissolution of myofibrils may be due to hyperphosphorylation occurring during this event.

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells.

Carcinogenic progression in most epithelial systems is a multistep process and presents as numerous (un)stable intermediate stages prior to the development of a fully malignant phenotype. Recently, we reported the neoplastic transformation of an SV40 immortalized, neonatal human prostate epithelial cell line (267B1) by multiple exposures to X-rays [1, 2]. The parental 267B1 cells acquired anchorage-independence and exhibited morphological transformation following exposure to two consecutive doses of 2 Gy. Exposure of either the parental 267B1 cells or the anchorage-independent derivatives (F3-SAC) to a total dose of 30 Gy of X-rays yielded tumorigenic transformants (267B1-XR and 267B1-SXR, respectively). All of these radiation-treated derivatives (F3-SAC, 267B1-XR, and 267B1-SXR) were characterized by reduced cell size and poorly organized actin stress fibers [2, 3]. The present study examines the protein expression changes associated with cytoskeletal alterations during the different steps of neoplastic progression induced by X-rays in the in vitro human prostate cell system. This analysis was achieved by using the high resolving power of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in the 267B1, F3-SAC, 267B1-XR, and 267B1-SXR cells. We report changes in the expression of gelsolin in the partially transformed, anchorage-independent, nontumorigenic (F3-SAC) cells and a progressive loss of expression of tropomyosin isoforms (TM-1 and TM-3), and myosin light chain-2 (MLC-2) in the tumorigenic (267B1-XR; 267B1-SXR) cells, respectively. In contrast, our results demonstrate that the levels of the small GTP-binding protein Rho-A, an active participant in the actin stress fiber organization, are not altered during neoplastic progression of these 267B1 cells. Thus the changes in synthesis of gelsolin, tropomyosins, and MLC-2 provide a rationale for the alterations in the actin stress fiber formation and reduction in cell size during the exposure of prostate epithelial cells to multiple doses of X-rays.

Modulation of growth and differentiation of human colon carcinoma cells by histone deacetylase inhibitory trichostatins

Histone deacetylase inhibitors such as sodium butyrate or (R)-trichostatin A {(R)-TSA; 7- [4-(dimethylamino) phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2, 4-heptadienamide} have been reported to modulate the proliferation and differentiation of certain cell types. In this study, we analyzed the effects of these agents on KM12 human colon carcinoma (HCC) cells in culture. We found that (R)-TSA induced cell flattening, inhibited anchorage-dependent and anchorage-independent growth, increased the level of the differentiation marker carcinoembryonic antigen, and increased the expression of gelsolin, a candidate tumor suppressor, in these HCC cells. Cells treated with (R)-TSA for 3 h exhibited a high degree of histone (primarily H4) acetylation. (R)-TSA exerted these effects at concentrations in the range between 0.1 to 1 mu M that are at least 1,000 times lower than those required to achieve similar effects by n-butyrate. Trichostatin C, which exhibits some histone deacetylase inhibitory activity but not the analogs (S)-TSA or (R)- or (S)-trichostatic acid, which lack histone deacetylase inhibitory activity, also showed some growth inhibitory activity. These results indicate that histone deacetylase inhibitors may lead to suppression of the transformed phenotype and enhanced differentiation in the KM12 HCC cells.

Biphasic Pattern of Gelsolin Expression and Variations in Gelsolin-Actin Interactions during Myogenesis

During myogenesis in vitro, the amount of gelsolin in myogenic cells increased by a factor of ∼3 from about 200 ng to a maximum of 750 ng per milligram of total protein. Gelsolin increased steadily from the myoblast state to terminally differentiated myotubes containing abundant cross-striated myofibrils. At the same time, the amount of total actin varied by only about 30%, the molar ratio of gelsolin:actin increased from 1:500 to approximately 1:150. This modulation of gelsolin expression was observed both in avian and mammalian myocultures. Once the state of terminal differentiation in myocultures was attained, the amount of gelsolin decreased again. On the other hand, gelsolin decreased continuously in the postnatal mouse muscle by a factor of ∼5 between Day 1 and Day 12 after birth. When myogenic cells from various stages of differentiation were extracted with Triton X-100, the majority of gelsolin was soluble, whereas a minor fraction was tightly associated with the cytoskeleton. The actual amount of insoluble gelsolin depended on both the Ca 2+ concentration during extraction and the degree of differentiation. Whereas at [Ca 21] > 10 5 M about one-third of the total gelsolin was associated with the cytoskeleton at all stages of differentiation, the amount of insoluble gelsolin after Triton extraction in the presence of EGTA increased from 3 to 17% during differentiation. The amount of soluble actin decreased from 40 to 25% during the same period, independent of the Ca 2+ concentration. We calculated that the amount of gelsolin associated with the cytoskeletal or myofibrillar system is approximately 20-fold higher in differentiated myotubes than in early myotubes, indicating a functional role of gelsolin for myofibrillar assembly.

Trichostatin A Induces Morphological Changes and Gelsolin Expression by Inhibiting Histone Deacetylase in Human Carcinoma Cell Lines

Trichostatin A (TSA) is a Streptomyces metabolite which specifically inhibits mammalian histone deacetylase at a nanomolar concentration and causes accumulation of highly acetylated histone molecules in mammalian cells. The effects of TSA on the morphology and the cell cycle of the human carcinoma cell lines, T24 and HeLa, were investigated. The morphology of T24 and HeLa cells dramatically changed and actin stress fibers reappeared during the treatment with TSA. The morphological change was not observed with chemically synthesized (S)-TSA and trichostatic acids, which are inactive to inhibit histone deacetylase. Cell cycle progression of these cells was blocked by TSA at G1 phase (HeLa) or G1 and G2 phases (T24). An RNA synthesis inhibitor, actinomycin D, and a protein synthesis inhibitor, cycloheximide, inhibited the morphological changes by TSA, suggesting that TSA induces expression of a new gene(s) followed by de novo protein synthesis, which is required for the actin microfilament reorganization. An approximately 7-fold (T24) or 12-fold (HeLa) increase in the intracellular level of gelsolin, an actin regulatory protein, was found in the cells treated with TSA for 24 h. These results suggest that gelsolin is one of the putative proteins necessary for the morphological changes of human carcinoma cells induced by TSA.

High gelsolin content of developing oligodendrocytes

The actin-binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5-bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5-positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL-1-, GC-, and MBP-positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP-positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Localization and characterization of gelsolin in nervous tissues: gelsolin is specifically enriched in myelin-forming cells

Gelsolin is a Ca(2+)-sensitive actin filament-severing protein. To elucidate the role of gelsolin in nervous tissues, we have investigated localization and expression of gelsolin in rat CNS and PNS using biochemical and morphological methods with a polyclonal antibody against the COOH-terminal fragment of plasma gelsolin. Immunohistochemical study showed that gelsolin was specifically enriched in oligodendrocytes and Schwann cells, and was also detected in myelin sheath, especially around the Ranvier's nodes. The immunohistochemical stainings using indirect immunofluorescence, avidin-biotin-peroxidase complex, and immunogold methods were carefully confirmed by immunoblotting against the tissue homogenates. The expressional changes of gelsolin in developing brain were investigated. The protein was detectable in newborn rat brain; however, it began to increase at 8-10 d after birth and reached maximal at 20-30 d when myelinogenesis actively occurred. After this period, the protein decreased gradually, although myelin basic protein was increasing until 6 months after birth. The immunostaining of gelsolin in Schwann cells was enhanced upon regeneration of injured sciatic nerves by freezing. Immunoelectron microscopy revealed that gelsolin was present not only in the cytoplasm but also in compact myelin. Following solubilization by detergents, gelsolin in the myelin fraction could be purified using anion exchange and blue Sepharose column chromatographies. The purified protein possessed a Ca(2+)-dependent severing activity against actin filaments similar to that of cytoplasmic and plasma gelsolin. These data strongly suggest that gelsolin in nervous tissues might be involved in lamellipodial movement to wrap axons of myelin-forming cells by modulating actin polymerization.

Gelsolin expression in normal human keratinocytes is a function of induced differentiation.

The epidermis is a self-renewing tissue comprised mainly of keratinocytes that exhibit different degrees of maturation depending upon their location (i.e. basal or suprabasal) within the tissue 1,2. Under non-perturbed conditions, the turnover rate within the epidermis is relatively slow and the keratinocytes exist primarily in a non-migratory mode with cells exhibiting attachment to the basement membrane and to other surrounding cells via families of surface receptors known as integrins and cadherins3,4. Upon partial or full thickness injury, keratinocytes become activated and assume a migratory phenotype5. This activation process includes modulation of integrin expression, actin reorganization and changes within the complement of actin-associated proteins that enable the keratinocyte to migrate over and through the provisional wound matrix to re-establish a complete epithelial barrier.KeywordsTerminal DifferentiationHuman Epidermal KeratinocytesBasal KeratinocytesNormal Human KeratinocytesCornified EnvelopeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Demonstration of a Circulating 65K Gelsolin Variant Specific for Familial Amyloidosis, Finnish Type

Familial amyloidosis, Finnish type (FAF), is a dominantly inherited form of systemic amyloidosis caused by a point mutation G654 to A654 in the gelsolin gene. The mutation leads to the expression of mutant Asn-187 gelsolin and the accumulation of amyloid in tissues.Here we demonstrate that patients with FAF have an abnormal 65K gelsolin species in the circulation that cosegregates with the disease. The 65K variant is detected by immunoblotting using a monoclonal antigelsolin antibody or polyclonal antipeptide (P-gel 231-242) antibodies. The 65K gelsolin variant is lacking in normal subjects and unaffected family members. It is the putative circulating precursor of tissue amyloid in PAP.

Actin structural proteins in cell motility

The machinery for cell locomotion is based in a network of polymerized actin filaments supporting the peripheral cytoplasm. This network or 'gel' consists of actin filaments in a variety of configurations, including cables, loose bundles, and branching arrays; all formed by the interaction of actin-associated proteins with actin filaments. For cell locomotion to occur, this network must be reversibly disassembled or 'solated' to allow protrusion, then re-assembled to stabilize the resulting extension. Thus, proteins to promote both 'solation' and 'gelation' of actin are important for efficient cell locomotion. Because of their distribution, control, and in vitro effects on actin filaments, two such proteins, gelsolin and actin-binding protein (ABP) should play especially important roles in cell motility. Support for this premise is found in in vivo studies of mouse kidney fibroblasts which demonstrated increased translocational locomotion after cytoplasmic gelsolin expression was increased genetically and in melanoma cells missing actin-binding protein which behave as expected for a cell unable to achieve efficient actin gelation. Since malignant transformation is known to affect the expression and distribution of several of these actin structural proteins, including gelsolin, further investigations of the role these proteins play in cell motility will be important to the determination of tumor cell motility and hence metastatic propensity.

Elevated gelsolin and α-actin expression in a flat revertant R1 of Ha- [formula omitted] oncogene-transformed [formula omitted] cells

Expressions of gelsolin and α-actin have been investigated in a revertant cell line R1 and compared with the parental human activated Ha- ras oncogene-transformed NIH 3T3 ( EJ- NIH 3T3 ), untransformed NIH 3T3 and partially revertant R2 cells. Gelsolin mRNA expression was strongest in R1 cells, intermediate in R2 and NIH 3T3 cells, and low in EJ- NIH 3T3 cells. Southern blot analysis gave neither signs of gross rearrangements nor amplification of the gelsolin gene. α-actin mRNA expression was restored in R1 cells to the level of NIH 3T3 cells. In R2 and EJ- NIH 3T3 cell lines, no α-actin transcript was detected. High gelsolin expression and restoration of α-actin expression may be associated with the acquirement of flat morphology and ordered cell growth pattern, which imply loss of tumorigenicity of R1 cells.

Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.

Expression of gelsolin by cos cell secretion

Expression of gelsolin by cos cell secretion

Gelsolin is expressed in early erythroid progenitor cells and negatively regulated during erythropoiesis.

We have identified an approximately 85-kD protein in chicken erythrocytes which is immunologically, structurally, and functionally related to the gelsolin found in many muscle and nonmuscle cell types. Cell fractionation reveals a Ca2+-dependent partitioning of gelsolin into the soluble cytoplasm and the membrane-associated cytoskeleton of differentiating or mature erythrocytes. Depending on either the presence of Ca2+ during cell lysis or on the preincubation of the intact cells with the Ca2+-ionophore A23187, up to 40% of the total cellular gelsolin is found associated with the membrane skeleton. Expression of gelsolin shows a strong negative regulation during erythroid differentiation. From quantitations of its steady-state molar ratio to actin, gelsolin is abundant in early progenitor cells as revealed from avian erythroblastosis virus- and S13 virus-transformed cells which are arrested at the colony forming unit erythroid (CFU-e) stage of erythroid development. In these cells, which have a rudimentary and unstable membrane skeleton, gelsolin remains quantitatively cytoplasmic, irrespective of the Ca2+ concentration. During chicken embryo development and maturation, the expression of gelsolin decreases by a factor of approximately 10(3) in erythroid cells. This down regulation is independent from that of actin, which is considerably less, and is observed also when S13-transformed erythroid progenitor cells are induced to differentiate under conditions where the actin content of these cells does not change. In mature erythrocytes of the adult the amount of gelsolin is low, and significantly less than required for potentially capping of all membrane-associated actin filaments. We suggest that the gelsolin in erythroid cells is involved in the assembly of the actin filaments present in the membrane skeleton, and that it may provide for a mechanism, by means of its severing action on actin filaments, to extend the meshwork of the spectrin-actin-based membrane skeleton in erythroid cells during erythropoiesis.