Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.
Actin Cytoskeleton/*drug effects ; Actins/metabolism ; Animals ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chickens ; Chondrogenesis/*drug effects ; Cytochalasin D/*pharmacology ; Mesoderm/cytology/drug effects ; Myosin Light Chains/metabolism ; Nucleic Acid Synthesis Inhibitors/*pharmacology ; Phosphorylation ; Staurosporine/*pharmacology ; Stress Fibers/drug effects

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates beta 1 integrin- mediated cell adhesion. However, the molecular mechanisms underlying CD98-mediated activation of beta 1 integrin are presently unclear. In this study, the effects of CD98 signaling on the expression and clustering of beta 1 integrin were investigated. Activation of CD98 augmented surface expression of beta 1 integrin on MCF-7 cells. Cross-linking CD98 induced clustering of beta 1 integrins. Inhibition of phosphorylation of focal adhesion kimase (FAK) by PP2, an inhibitor of Src family kinase, reduced cell-extracellular matrix adhesion, but not surface expression and clustering of beta1 integrin on MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, phalloidin or cytochalasin D inhibited CD98-mediated induction of cell-ECM adhesion, but not surface expression and clustering of b1 integrins. The inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated cell adhesion were diminished by pretreatment of cells with Mn2+, which is shown to induce conformational change of integrins. These results provide the first evidence that CD98 activation increases not only beta1 integrin affinity but also its surface expression and clustering and the latter is independent of FAK/Src and cytoskeleton.
Antigens, CD29/*biosynthesis/genetics ; Antigens, CD98/agonists/*metabolism ; Cell Line, Tumor ; Cytochalasin D/pharmacology ; Cytoskeleton/drug effects/enzymology ; Focal Adhesion Kinase 2/genetics/*metabolism ; Focal Adhesions/drug effects/enzymology ; Humans ; Microscopy, Confocal ; Multiprotein Complexes/*biosynthesis/genetics ; Mutant Proteins/genetics/metabolism ; Phalloidine/pharmacology ; Phosphorylation/drug effects ; Protein Binding ; Pyrimidines/pharmacology ; Signal Transduction/physiology ; Transfection

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology