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1.
Stem Cell Res ; 15(2): 281-9, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26209815

ABSTRACT

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover, depolymerizing actin reduced FAK, p38 and JNK activation during OB differentiation of hMSCs, while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation.


Subject(s)
Actin Cytoskeleton/metabolism , Actin Cytoskeleton/drug effects , Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cofilin 1/antagonists & inhibitors , Cofilin 1/genetics , Cofilin 1/metabolism , Destrin/antagonists & inhibitors , Destrin/genetics , Destrin/metabolism , Focal Adhesion Kinase 1/metabolism , Humans , Lim Kinases/metabolism , MAP Kinase Kinase 4/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteoblasts/cytology , Osteogenesis/drug effects , Phalloidine/toxicity , Phosphorylation/drug effects , RNA Interference , RNA, Small Interfering/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Biochem Biophys Res Commun ; 441(2): 301-7, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24103754

ABSTRACT

Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca(2+) release in response to inositol 1,4,5-trisphosphate (InsP3) or fertilizing sperm, raising the possibility that intracellular Ca(2+) signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca(2+) response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca(2+) signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis.


Subject(s)
Calcium Signaling , Destrin/antagonists & inhibitors , Oocytes/metabolism , Starfish/metabolism , Actin Cytoskeleton/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Cells, Cultured , Destrin/immunology , Exocytosis , Inositol 1,4,5-Trisphosphate/pharmacology , Meiosis , Microinjections , Oocytes/cytology , Oocytes/ultrastructure , Sperm-Ovum Interactions/drug effects , Sperm-Ovum Interactions/physiology , Starfish/growth & development
3.
BMC Cell Biol ; 14: 45, 2013 Oct 05.
Article in English | MEDLINE | ID: mdl-24093776

ABSTRACT

BACKGROUND: ADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently. RESULTS: Either ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates. CONCLUSION: Although ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.


Subject(s)
Actins/metabolism , Adenocarcinoma/metabolism , Cofilin 1/genetics , Destrin/genetics , Gene Expression Regulation, Neoplastic , Mammary Neoplasms, Animal/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actins/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Cell Adhesion/drug effects , Cell Movement , Cofilin 1/antagonists & inhibitors , Cofilin 1/metabolism , Destrin/antagonists & inhibitors , Destrin/metabolism , Epidermal Growth Factor/pharmacology , Female , Focal Adhesions/drug effects , Focal Adhesions/metabolism , Focal Adhesions/ultrastructure , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Neoplasm Metastasis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Signal Transduction , Tumor Cells, Cultured
4.
Cell Death Differ ; 19(6): 958-67, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22139132

ABSTRACT

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.


Subject(s)
Apoptosis , Cofilin 1/metabolism , Destrin/metabolism , Mitochondria/metabolism , Actins/metabolism , Animals , Caspase 3/metabolism , Cells, Cultured , Cofilin 1/antagonists & inhibitors , Cofilin 1/genetics , Cytochromes c/metabolism , Destrin/antagonists & inhibitors , Destrin/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Hydrogen Peroxide/toxicity , Mice , RNA Interference , RNA, Small Interfering/metabolism , Staurosporine/pharmacology
5.
J Biol Chem ; 283(10): 6013-21, 2008 Mar 07.
Article in English | MEDLINE | ID: mdl-18171679

ABSTRACT

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Movement , Focal Adhesions/metabolism , Lim Kinases/metabolism , Microfilament Proteins/metabolism , Neoplasm Proteins/metabolism , Actin Depolymerizing Factors/antagonists & inhibitors , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Animals , Ascites , Carcinoma, Hepatocellular/pathology , Cell Adhesion , Cell Line, Tumor , Cytoskeleton/metabolism , Cytoskeleton/pathology , Destrin/antagonists & inhibitors , Destrin/metabolism , Epithelium/metabolism , Epithelium/pathology , Focal Adhesions/pathology , Lim Kinases/antagonists & inhibitors , Lysophospholipids/metabolism , Microfilament Proteins/antagonists & inhibitors , Neoplasm Invasiveness , Neoplasm Proteins/antagonists & inhibitors , Phosphoric Monoester Hydrolases , Phosphorylation , RNA, Small Interfering , Rats
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