pericyte; endothelial cell; VEGF; Bcl-w; PDGFRβ; integrin αvβ3
Calcium- and integrin-binding protein 1 (CIB1) has been shown to be involved in cell spreading and migration. The signaling events regulated by CIB1 during cell migration are poorly understood. Here we found that accumulation of CIB1 at the tip of the filopodia requires an intact cytoskeleton. Depletion of CIB1 using shRNA affects formation of FAK- and phosphotyrosine-rich focal adhesions without affecting stress fiber formation. Overexpression of CIB1 results in cell migration on fibronectin and Erk1/2 MAP kinase activation. CIB1-induced cell migration is dependent upon Erk1/2 activation, since it is inhibited by the MEK-specific inhibitor PD98059. Furthermore, CIB1-induced cell migration, as well as Erk1/2 activation, is dependent on PKC, Src family kinases as well as PI-3 kinase as it is inhibited by bisindolylmaleimide 1, PP2, and wortmannin respectively in a dose-dependent manner. Co-expression of dominant-negative Cdc42 completely abolished CIB1-induced cell migration. Additionally, co-expression of constitutively active, but not dominant negative PAK1, a CIB1 binding protein, inhibited CIB1-induced cell migration. These results suggest that CIB1 positively regulates cell migration and is necessary for the recruitment of FAK to the focal adhesions. Furthermore, CIB1-induced cell migration is dependent on MAP kinase signaling and its function is attenuated by PAK1.
CIB1; Cell migration; PAK-1; Cdc42; FAK
Under physiological conditions, circulating platelets are discoid in shape.1 On these platelets, the fibrinogen receptor (integrin αIIbβ3) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin αIIbβ3 through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.2 Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.
platelets; integrin αIIbβ3; Galpha13; RhoA; clot retraction; thrombin; fibrinogen
Robo4; Slit2; VEGF; tube formation; permeability; angiogenesis; endothelial cells; migration
Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.
JAM-A; integrin; αvβ3; FGF-2; breast cancer; cell migration and invasion; T47D; MDA-MB-231; siRNA
Deletion of the highly conserved gene for the major Ca2+ efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P<0.001) ATP levels, significantly (P<0.001) greater cytosolic Ca2+ concentration ([Ca2+]c) and ~10-fold higher mitochondrial sequestration, indicating Ca2+ overload. Investigating the mechanism involved, we used coimmunoprecipitation studies to show that CASK (Ca2+/calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b’s enzymatic activity, consequent Ca2+ accumulation, and a ~6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca2+ homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.
hyperactivated motility; flagellum’s principal piece; calcium efflux; electron dense mitochondria
Inflammation and angiogenesis are integral parts of wound healing. However, excessive and persistent wound-induced inflammation and angiogenesis in an avascular tissue such as the cornea may be associated with scarring and visual impairment. Junctional adhesion molecule A (Jam-A) is a tight junction protein that regulates leukocyte transmigration as well as fibroblast growth factor-2 (FGF-2)-induced angiogenesis. However its function in wound-induced inflammation and angiogenesis is still unknown. In this study, we report spontaneous corneal opacity in Jam-A deficient mice associated with inflammation, angiogenesis and the presence of myofibroblasts. Since wounds and/or corneal infections cause corneal opacities, we tested the role of Jam-A in wound-induced inflammation, angiogenesis and scarring by subjecting Jam-A deficient mice to full thickness corneal wounding. Analysis of these wounds demonstrated increased inflammation, angiogenesis, and increased number of myofibroblasts thereby indicating that Jam-A regulates the wound-healing response by controlling wound-induced inflammation, angiogenesis and scarring in the cornea. These effects were not due to inflammation alone since the inflammation-induced wound-healing response in Jam-A deficient mice was similar to wild type mice. In order to determine the molecular mechanism associated with the observed aberrant corneal wound healing in Jam-A deficient mice, we assessed the expression of the components of vascular endothelial growth factor A (VEGF-A)/vascular endothelial growth factor receptor- 2(VEGFR-2) signaling pathway. Interestingly, we observed increased levels of VEGF-A mRNA in Jam-A deficient eyes. We also observed nuclear localization of phosphorylated SMAD3 (pSMAD3) indicative of TGFβ pathway activation in the Jam-A deficient eyes. Furthermore the increased wound-induced corneal inflammation, angiogenesis, and scarring in Jam-A deficient mice was attenuated by treatment with DC101, an anti-vascular endothelial growth factor receptor-2 (VEGFR-2) antibody. Our results suggest that in the absence of Jam-A, the VEGF-A/VEGFR-2 pathway is upregulated, thereby augmenting wound induced corneal inflammation, angiogenesis, and myofibroblast accumulation leading to scarring.
Members of the polo-like kinases (Plk1, Plk2, Plk3, and Plk4) are involved in the regulation of various stages of the cell cycle and have been implicated in cancer progression. Unlike its other family members the expression of Plk3 remains steady during cell cycle progression, suggesting that its activity may be spatiotemporally regulated. However, the mechanism of regulation of Plk3 activity is not well understood. Here, we show that calcium- and integrin-binding protein 1 (CIB1), a Plk3 interacting protein, is widely expressed in various cancer cell lines. Expression of CIB1 mRNA as well as protein is increased in breast cancer tissue as compared to normal tissue. CIB1 constitutively interacts with Plk3 as determined by both in vitro and in vivo assays. This interaction of CIB1 with Plk3 is independent of intracellular Ca2+. Furthermore, binding of CIB1 results in inhibition of Plk3 kinase activity both in vitro and in vivo. Interestingly, this inhibition of the Plk3 activity by CIB1 is Ca2+-dependent. Taken together, our results suggest that CIB1 is a regulatory subunit of Plk3 and it regulates Plk3 activity in a Ca2+-dependent manner. Furthermore, upregulation of CIB1 in cancer cells could thus inhibit Plk3 activity leading to abnormal cell cycle regulation in breast cancer cells. Thus in addition to Plk3, CIB1 may be a potential biomarker and target for therapeutic intervention of breast cancer.
CIB1; calcium- and integrin-binding protein 1; polo-like kinase 3; Plk3; breast cancer
Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype.
T47D; CIB1; Plk3; multinucleation; cytokinesis; microtubules; centrosomes
Polycationic polymers have been used to condense therapeutic DNA into sub-micron particles, offering protection from shear-induced or enzymatic degradation. However, the spontaneous nature of this self-assembly process gives rise to the formation of multimolecular aggregates, resulting in significant polyplex heterogeneity. Additionally, cytotoxicity issues and serum instability have limited the in vivo efficacy of such systems. One way these issues can be addressed is through the inclusion of poly(ethylene glycol) (PEG). PEG has known steric effects that inhibit polyplex self-aggregation. A variety of PEGylated gene delivery formulations have been previously pursued in an effort to take advantage of this material’s benefits. Due to such interest, our aim was to further explore the consequences of PEG inclusion on the structure and activity of gene delivery vehicle formulations. We explored the complexation of plasmid DNA with varying ratios of a PEGylated tri-lysine peptide (PEG-K3) and 25 kDa polyethylenimine (PEI). Atomic force and scanning electron microscopy were utilized to assess the polyplex size and shape, and revealed that a critical threshold of PEG was necessary to promote the formation of homogeneous polyplexes. Flow cytometry and fluorescence microscopy analyses suggested that the presence of PEG inhibited transfection efficiency as a consequence of changes in intracellular trafficking, and promoted an increased reliance on energy-independent mechanisms of cellular uptake. These studies provide new information on the role of PEG in delivery vehicle design and lay the foundation for future work aimed at elucidating the details of the intracellular transport of PEGylated polyplexes.
Poly(ethylene glycol); Polyplex Formulation Design; Gene Delivery; Cellular Uptake; Transfection Efficiency
Regulation of integrin binding to the specific complementary sites on extra-cellular matrix (ECM) proteins plays a major role in cell adhesion and migration. In addition to regulating single integrin-ligand bonds by affinity modulation, cells regulate their adhesiveness by forming integrin clusters. Although it is clear that cells exhibit different adhesion and migration behaviors on surfaces coated with different concentrations of ECM proteins, it is not clear if this response is mediated by changes in the availability of integrin binding sites or by differential intracellular signaling that may affect integrin binding and clustering.
To quantify how the concentration of ECM affects integrin clustering, we seeded cells expressing the integrin αIIbβ3 on different concentrations of the complementary ECM protein fibrinogen (Fg) and measured the resulting integrin cluster properties. We observed heterogeneity in the properties of integrin clusters, and to characterize this population heterogeneity we use a probabilistic modeling approach to quantify changes to the distributions of integrin cluster size, shape, and location.
Our results indicate that in response to increasing ECM density cells form smaller integrin clusters that are less elongated and closer to the cell periphery. These results suggest that cells can sense the availability of ECM binding sites and consequently regulate integrin clustering as a function of ECM density.
Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within sub-micron sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes.
In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging from 30 – 50 nm were created, with minimal dependence of complex size on N:P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivitization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison with PEI-DNA complexes. Transfection experiments in Chinese Hamster Ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.
Endomitosis is a form of mitosis in which both karyokinesis and cytokinesis are interrupted and is a hallmark of megakaryocyte differentiation. Very little is known about how such a dramatic alteration of the cell cycle in a physiological setting is achieved. Thrombopoietin-induced signaling is essential for induction of endomitosis. Here we show that calcium- and integrin-binding protein 1 (CIB1), a known regulator of platelet integrin αIIbβ3 outside-in signaling, regulates endomitosis. We observed that CIB1 expression is increased in primary mouse megakaryocytes compared to mononuclear bone marrow cells as determined by Western blot analysis. Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy. Overexpression of CIB1 in Dami cells resulted in multilobated nuclei and led to increased time for a cell to complete cytokinesis as well as increased incidence of furrow regression as observed by time-lapse microscopy. Additionally, we found that surface expression of integrin αIIbβ3, an important megakaryocyte marker, was enhanced in CIB1 overexpressing cells as determined by flow cytometry. Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells. Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells. Furthermore, PMA treatment augmented the interaction between CIB1 and Plk3, which depended on the duration of treatment. These data suggest that CIB1 is involved in regulating endomitosis, perhaps through its interaction with Plk3.
Junctional Adhesion Molecules (JAMs) that are expressed in endothelial and epithelial cells and function in tight junction assembly, also perform important roles in testis where the closely-related JAM-A, JAM-B, and JAM-C are found. Disruption of murine Jam-B and Jam-C has varying effects on sperm development and function, however deletion of Jam-A has not yet been studied. Here we show for the first time that in addition to expression in the Sertoli-Sertoli tight junctions in the seminiferous tubules, the ∼32 kDa murine JAM-A is present in elongated spermatids and in the plasma membrane of the head and flagellum of sperm. Deletion of Jam-A, using the gene trap technology, results in flagellar defects at the ultrastructural level. In Jam-A-deficient mice, which have reduced litter size, both progressive and hyperactived motility are significantly affected (P<0.0001) before and, more severely, after capacitation. The findings show that JAM-A is involved in sperm tail formation and is essential for normal motility, which may occur via its signal transduction and protein phosphorylation properties. Detection of JAM-A in human sperm protein indicates that its role may be conserved in sperm motility and that JAM-A may be a candidate gene for the analysis of idiopathic sperm motility defects resulting in male subfertility in the human population.
spermiogenesis; elongated spermatid; progressive and hyperactivated motility; sperm flagellar defects; sperm membrane protein