Distinct Roles for CARMIL Isoforms in Cell Migration

Molecular mechanisms for cell migration, especially how signaling and cytoskeletal systems are integrated, are not understood well. Here, we examined the role of CARMIL (capping protein, Arp2/3, and Myosin-I linker) family proteins in migrating cells. Vertebrates express three conserved genes for CARMIL, and we examined the functions of the two CARMIL genes expressed in migrating human cultured cells. Both isoforms, CARMIL1 and 2, were necessary for cell migration, but for different reasons. CARMIL1 localized to lamellipodia and macropinosomes, and loss of its function caused loss of lamellipodial actin, along with defects in protrusion, ruffling, and macropinocytosis. CARMIL1-knockdown cells showed loss of activation of Rac1, and CARMIL1 was biochemically associated with the GEF Trio. CARMIL2, in contrast, colocalized with vimentin intermediate filaments, and loss of its function caused a distinctive multipolar phenotype. Loss of CARMIL2 also caused decreased levels of myosin-IIB, which may contribute to the polarity phenotype. Expression of one CARMIL isoform was not able to rescue the knockdown phenotypes of the other. Thus, the two isoforms are both important for cell migration, but they have distinct functions.     

Heat Shock Protein 27 Plays Two Distinct Roles in Controlling Human Breast Cancer Cell Migration on Laminin-5

It has recently been reported that phosphorylation of the small heat shock protein 27 (hsp27) enhances p38 MAP kinase dependent migration of bovine and human vascular endothelial cells. We have examined the role of hsp27 in controlling the constitutive migration of human breast cancer cells on the extracellular matrix molecule laminin-5. In a haptotaxis assay, anisomycin- or heat shock-induced phosphorylation of hsp27 enhances migration of MDA-MB-231 breast cancer cells constitutively overexpressing hsp27. Under these conditions, hsp27 redistributes to the nucleus. Unphosphorylated hsp27, which remains in the cytosol, induces resistance to a subset of drugs that inhibit haptotactic migration of these cells. We conclude that hsp27 plays two distinct roles in controlling migration of breast cancer cells: phosphorylated hsp27 enhances migration, while unphosphorylated hsp27 can sustain migration in the presence of inhibitory drugs.     

Protein Kinase C,Focal Adhesions and the Regulation of Cell Migration

Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.     

Fibroblast Growth Factor Receptors-1 and -3 Play Distinct Roles in the Regulation of Bladder Cancer Growth and Metastasis: Implications for Therapeutic Targeting

Fibroblast growth factor receptors (FGFRs) are activated by mutation and overexpressed in bladder cancers (BCs), and FGFR inhibitors are currently being evaluated in clinical trials in BC patients. However, BC cells display marked heterogeneity in their responses to FGFR inhibitors, and the biological mechanisms underlying this heterogeneity are not well defined. Here we used a novel inhibitor of FGFRs 1–3 and RNAi to determine the effects of inhibiting FGFR1 or FGFR3 in a panel of human BC cell lines. We observed that FGFR1 was expressed in BC cells that also expressed the “mesenchymal” markers ZEB1 and vimentin, whereas FGFR3 expression was restricted to the E-cadherin- and p63-positive “epithelial” subset. Sensitivity to the growth-inhibitory effects of BGJ-398 was also restricted to the “epithelial” BC cells and it correlated directly with FGFR3 mRNA levels but not with the presence of activating FGFR3 mutations. In contrast, BGJ-398 did not strongly inhibit proliferation but did block invasion in the “mesenchymal” BC cells in vitro. Similarly, BGJ-398 did not inhibit primary tumor growth but blocked the production of circulating tumor cells (CTCs) and the formation of lymph node and distant metastases in mice bearing orthotopically implanted “mesenchymal” UM-UC3 cells. Together, our data demonstrate that FGFR1 and FGFR3 have largely non-overlapping roles in regulating invasion/metastasis and proliferation in distinct “mesenchymal” and “epithelial” subsets of human BC cells. The results suggest that the tumor EMT phenotype will be an important determinant of the biological effects of FGFR inhibitors in patients.     

Disentangling Membrane Dynamics and Cell Migration; Differential Influences of F-actin and Cell-Matrix Adhesions

Cell migration is heavily interconnected with plasma membrane protrusion and retraction (collectively termed “membrane dynamics”). This makes it difficult to distinguish regulatory mechanisms that differentially influence migration and membrane dynamics. Yet such distinctions may be valuable given evidence that cancer cell invasion in 3D may be better predicted by 2D membrane dynamics than by 2D cell migration, implying a degree of functional independence between these processes. Here, we applied multi-scale single cell imaging and a systematic statistical approach to disentangle regulatory associations underlying either migration or membrane dynamics. This revealed preferential correlations between membrane dynamics and F-actin features, contrasting with an enrichment of links between cell migration and adhesion complex properties. These correlative linkages were often non-linear and therefore context-dependent, strengthening or weakening with spontaneous heterogeneity in cell behavior. More broadly, we observed that slow moving cells tend to increase in area, while fast moving cells tend to shrink, and that the size of dynamic membrane domains is independent of cell area. Overall, we define macromolecular features preferentially associated with either cell migration or membrane dynamics, enabling more specific interrogation and targeting of these processes in future.     

Inhibition of PKA Blocks Fibroblast Migration in Response to Growth Factors

Cell migration requires precise coordination of many signaling pathways to achieve directed motility. We report here that NIH3T3 fibroblasts expressing a dominant negative PKA subunit (dnPKA) show diminished migration in response to serum or growth factors. This effect is not a general effect on cell motility, but rather a decreased capacity to enhance migration in response to stimuli. Control (neo) and dnPKA cells show very similar haptotactic migration toward fibronectin, but dnPKA cells show reduced stimulation of migration in response to EGF/PDGF or serum. These effects were not due to alterations in cell growth or adhesion to fibronectin. Forskolin, which elevates cyclic adenosine monophosphate (cAMP) levels, dramatically inhibited neo cell motility in a scrape migration assay, although dnPKA cell migration was unaffected. The MEK selective inhibitor U0126 and the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 inhibited migrating neo cells and were able to further inhibit residual dnPKA cell migration. Our data show that intermediate or well-controlled levels of PKA activity are required for optimal growth factor-stimulated migration in fibroblasts. PKA may play an important role in the signaling processes that lead to motility.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

成纤维细胞生长因子9在抑郁症及其运动干预调节中的作用

成纤维细胞生长因子9（fibroblast growth factor 9, FGF9）是成纤维细胞生长因子（fibroblast growth factor,FGF）家族成员之一,属于一种自分泌或旁分泌生长因子。在脑组织中,FGF9主要表达于海马和皮质区,具有促进细胞增殖和维持细胞存活的功能。研究发现,FGF家族在抑郁症患者的多个脑区出现表达紊乱,FGF9在抑郁行为中扮演着负调控角色,但其介导抑郁行为的分子机制尚不清楚。本文综述了FGF9及其家族成员在抑郁中的作用; 围绕其受体（FGFR）信号在中枢神经系统中的功能特点,深入分析FGF9调节抑郁行为中的作用机制;结合运动抗抑郁的神经营养假说,提出经由FGFR/GSK3β/β-catenin通路的FGF信号,可能介导抑郁症的运动干预机制的假设。这些将为FGF9介导抑郁行为和运动抗抑郁的有关研究提供理论的基础和探索的思路。     

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.

Methodology/Principal Findings

We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate.

Conclusions/Significance

This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.     

Distinct Roles of Myosin Va in Membrane Remodeling and Exocytosis of Secretory Granules

Hormone‐ and neuropeptide‐containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans‐ Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin‐coated vesicles. The resulting mature SGs undergo Ca²⁺‐dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F‐actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP‐inhibited binding of SGs to F‐actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant‐negative myosin Va‐tail or shRNA‐based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary, our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.     

Roles for Rho/ROCK and Vinculin in Parietal Endoderm Migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.     

人FGF-1对免疫系统的影响

酸性成纤维细胞生长因子 (acidfibroblastgrowthfactor,aFGF或FGF-1 )是成纤维细胞生长因子家族成员之一 ,是一种重要的生长因子。人FGF 1 (FGF-1 )是一个 1 7～ 1 8kDa的非糖基化多肽 ,三胚层来源的细胞都可以表达。FGF-1的生物学效应非常广泛 ,在组织和器官发育、血管发生、血细胞生成、肿瘤发生、伤口愈合等方面发挥重要的作用。FGF-1对人体的免疫系统也有重要的影响 ,能提高多种刺激诱导的T细胞增殖、凋亡及细胞因子的产生。主要概述了FGF-1的生物学效应、对免疫系统的影响及其潜在的临床应用价值。