The tetraspanin CD151 regulates cell morphology and intracellular signaling on laminin-511

The tetraspanin CD151 forms a stable complex with integrin alpha3beta1, a widely expressed laminin receptor, and is implicated in the regulation of integrin alpha3beta1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin alpha3beta1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin beta1-activating mAb TS2/16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin alpha3beta1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin alpha3beta1 functions in two independent aspects: potentiation of integrin alpha3beta1-mediated cell adhesion and promotion of integrin alpha3beta1-stimulated signaling events involving tyrosine phosphorylation.     

CD44 regulates myoblast migration and differentiation

CD44 is a transmembrane protein that plays a role in cell-cell interactions and motility in a number of cell types. Cell-cell interactions are critical for myoblast differentiation and fusion but whether CD44 regulates myogenesis is unknown. Here, we show that CD44 plays a functional role in early myogenesis. Analyses of myofiber cross-sectional area, after local injury in mouse tibialis anterior (TA) muscles, revealed that growth was transiently delayed in the absence of CD44. A muscle-intrinsic role for CD44 is suggested as primary myoblasts from CD44(-/-) mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation-inducing in vitro environment. Chemotaxis of CD44(-/-) myoblasts toward hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was totally abrogated, although expression of their respective receptors did not appear to differ from wild-type. Furthermore, motility of CD44(-/-) myoblasts was decreased at early stages of differentiation as determined by time-lapse microscopy. Wild-type myoblasts contained two subpopulations of slow- and fast-migrating cells, whereas CD44(-/-) myoblasts were composed predominantly of the slower migrating subpopulation. Taken together, these data suggest that myoblast migration and differentiation are closely linked and CD44 is a key regulator.     

CCN4 regulates vascular smooth muscle cell migration and proliferation

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.     

DGCR6L,a novel PAK4 interaction protein,regulates PAK4-mediated migration of human gastric cancer cell via LIMK1

Overexpression, genetic amplification and mutations of p21-activated kinase 4 (PAK4) were found in a variety of human cancers. PAK4 regulated actin cytoskeleton reorganization by phosphorylating LIMK1 and promoted cancer cells migration. Using yeast two-hybrid screen, we identified a novel PAK4 binding protein, DGCR6L, which was associated with cancer cell metastasis. We confirmed PAK4 binding to the DGCR6L specifically by GST pull-down assay, and found an association between endogenous PAK4 and DGCR6L by immunoprecipitation in mammalian cells. Furthermore, L115 of DGCR6L was the critical amino acid to bind 466–572aa in the very C-terminus of PAK4. Importantly, DGCR6L was required for the formation of PAK4-DGCR6L-β-actin complex. Overexpressed DGCR6L promoted migration of AGS cells mediated by PAK4, whereas knock-down of DGCR6L markedly inhibited the migration of those cells. Moreover, DGCR6L (L115V), which did not bind to PAK4, lost the ability to promote AGS cells migration. DGCR6L colocalized with PAK4 or F-actin and enhanced the phosphorylation level of LIMK1 and cofilin in a dose dependent manner. Taken together, our results demonstrated that DGCR6L, a novel PAK4 interacting protein, regulated PAK4-mediated migration of human gastric cancer cells via LIMK1.     

The PCH family member proline-serine-threonine phosphatase-interacting protein 1 targets to the leukocyte uropod and regulates directed cell migration

Pombe Cdc15 homology (PCH) family members have emerged as important regulators of membrane-cytoskeletal interactions. Here we show that PSTPIP1, a PCH family member expressed in hematopoietic cells, regulates the motility of neutrophil-like cells and is a novel component of the leukocyte uropod where it colocalizes with other uropod components, such as type I PIPKIgamma. Furthermore, we show that PSTPIP1 association with the regulator of endocytosis, dynamin 2, and PSTPIP1 expression impairs transferrin uptake and endocytosis. We also show that PSTPIP1 localizes at the rear of neutrophils with a subpopulation of F-actin that is specifically detected by the binding of an F-actin probe that detects a more stable population of actin. Finally, we show that actin polymerization, but not the microtubule network, is necessary for the polarized distribution of PSTPIP1 toward the rear of the cell. Together, our findings demonstrate that PSTPIP1 is a novel component of the leukocyte uropod that regulates endocytosis and cell migration.     

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.     

Caspase 6 regulates B cell activation and differentiation into plasma cells

Caspase (Casp) family proteases regulate not only lymphocyte apoptosis but also lymphocyte activation and development. In this study, we show that Casp6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry. B cells from Casp6 knockout (Casp6 KO) mice examined ex vivo have more cells in G(1) than wild-type B cells, and mitogen-induced G(1) entry of Casp6 KO B cells is much faster than that of wild-type B cells. Even so, S phase entry and proliferation are not increased in Casp6 KO B cells. Rather than proliferating, activated Casp6 KO B cells preferentially differentiate into syndecan-1(+) plasma cells and produce Abs. In Casp6 KO mice compared with WT mice, serum levels of IgG1, IgG2a, and IgG2b are increased and Ag-specific Ab responses are also enhanced along with increased percentages of syndecan-1(+) plasma cells. Casp6 may regulate both B cell activation and differentiation by modifying requirements for G(0) B cells to enter G(1).     

ARHGEF10L expression regulates cell proliferation and migration in gastric tumorigenesis

ABSTRACT

We recently reported that Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) activated Rho GTPases as guanine nucleotide exchange factor to stimulate liver tumorigenesis. The present study continued to explore the effect of ARHGEF10L on the tumorigenic process of gastric cancer. This study detected increased expression of ARHGEF10L in GC tissues compared to peritumoral tissue samples. SGC7901 cells with ARHGEF10L overexpression showed increased cell proliferation, cell migration, and tube-like structure formation abilities, as well as increased expression of GTP-RhoA, ROCK1, and phospho-Ezrin/Radixin/Moesin. ARHGEF10L overexpression downregulated the expression of E-cadherin and upregulated the expression of N-cadherin and Slug, indicating an activation of EMT in the transfected cells. RNA-sequencing assay detected an increased expression of Heat shock 70 kDa protein 6 in the SGC7901 cells overexpressing ARHGEF10L. The above results suggest that ARHGEF10L expression can stimulate gastric tumorigenesis by prompting RhoA-ROCK1-phospho-ERM signaling, inducing EMT and increasing HSPA6 expression.     

Pax 4 and 6 regulate gastrointestinal endocrine cell development.

The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. We hereby report that deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation.     

A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.     

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration

Background

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer.

Methods and results

We found that PCI inhibits cathepsin L with an inhibition rate (k₂) of 3.0 × 10⁵ M⁻¹ s⁻¹. Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells.

Conclusions

Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L.

General significance

Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.     

The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation

Transforming growth factor-alpha (TGF-alpha) is a member of the EGF growth factor family. Both transmembrane TGF-alpha and the proteolytically released soluble TGF-alpha can bind to the EGF/TGF-alpha tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-alpha physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-alpha in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-alpha. CD9 expression strongly decreased the growth factor- and PMA- induced proteolytic conversions of transmembrane to soluble TGF-alpha and strongly enhanced the TGF- alpha-induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-alpha. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-alpha resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-alpha decreased the autocrine growth stimulatory effect of TGF-alpha in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21(CIP1). These data reveal that the association of CD9 with transmembrane TGF-alpha regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.     

Syndecan-2 cytoplasmic domain regulates colon cancer cell migration via interaction with syntenin-1

The cell surface heparan sulfate proteoglycan, syndecan-2, is crucial for the tumorigenic activity of colon cancer cells. However, the role played by the cytoplasmic domain of the protein remains unclear. Using colon cancer cells transfected with various syndecan-2-encoding genes with deletions in the cytoplasmic domain, it was shown that syndecan-2-induced migration activity requires the EFYA sequence of the C-terminal region; deletion of these residues abolished the rise in cell migration seen when the wild-type gene was transfected and syndecan-2 interaction with syntenin-1, suggesting that syntenin-1 functioned as a cytosolic signal effector downstream from syndecan-2. Colon cancer cells transfected with the syntenin-1 gene showed increased migratory activity, whereas migration was decreased in cells in which syntenin-1 was knock-down using small inhibitory RNA. In addition, syntenin-1 expression potentiated colon cancer cell migration induced by syndecan-2, and syndecan-2-mediated cell migration was reduced when syntenin-1 expression diminished. However, syntenin-1-mediated migration enhancement was not noted in colon cancer cells transfected with a gene encoding a syndecan-2 mutant lacking the cytoplasmic domain. Furthermore, in line with the increase in cell migration, syntenin-1 mediated Rac activation stimulated by syndecan-2. Together, the data suggest that the cytoplasmic domain of syndecan-2 regulates colon cancer cell migration via interaction with syntenin-1.     

BNIP3L/NIX-dependent mitophagy regulates cell differentiation via metabolic reprogramming

Macroautophagy/autophagy is the process by which cellular components are degraded and recycled within the lysosome. These components include mitochondria, the selective degradation of which is known as mitophagy. Mitochondria are dynamic organelles that constantly adapt their morphology, function, and number to accommodate the metabolic needs of the cell. Extensive metabolic reconfiguration occurs during cell differentiation, when mitochondrial activity increases in most cell types. However, our data demonstrate that during physiologic retinal ganglion cell (RGC) development, mitophagy-dependent metabolic reprogramming toward glycolysis regulates numbers of RGCs, which are the first neurons to differentiate in the retina and whose axons form the optic nerve. We show that during retinal development tissue hypoxia triggers HIF1A/HIF-1 stabilization, resulting in increased expression of the mitophagy receptor BNIP3L/NIX. BNIP3L-dependent mitophagy results in a metabolic shift toward glycolysis essential for RGC neurogenesis. Moreover, we demonstrate that BNIP3L-dependent mitophagy also regulates the polarization of proinflammatory/M1 macrophages, which undergo glycolysis-dependent differentiation during the inflammatory response. Our results uncover a new link between hypoxia, mitophagy, and metabolic reprogramming in the differentiation of several cell types in vivo. These findings may have important implications for neurodegenerative, metabolic and other diseases in which mitochondrial dysfunction and metabolic alterations play a prominent role.