Lumican inhibits cell migration through α2β1 integrin

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K_d ≥ 200 nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.     

PI3Kγ activation by CXCL12 regulates tumor cell adhesion and invasion

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kγ regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.     

RGS10 restricts upregulation by chemokines of T cell adhesion mediated by α4β1 and αLβ2 integrins

Chemokines rapidly and transiently upregulate α4β1 and αLβ2 integrin-mediated adhesion during T lymphocyte extravasation by activating Gα-dependent inside-out signaling. To limit and terminate Gα-mediated signaling, cells can use several mechanisms, including the action of regulator of G protein signaling (RGS) proteins, which accelerate the GTPase activity of Gα subunits. Using human T cells silenced for or overexpressing RGS10, we show in this article that RGS10 functions as an inhibitor of Gα(i)-dependent, chemokine-upregulated T cell adhesion mediated by α4β1 and αLβ2. Shear stress-dependent detachment and cell spreading analyses revealed that RGS10 action mainly targets the adhesion strengthening and spreading phases of α4β1-mediated cell attachment. Associated with these observations, chemokine-stimulated Vav1-Rac1 activation was longer sustained and of higher intensity in RGS10-silenced T cells, or inhibited in cells overexpressing RGS10. Of importance, expression of constitutively activated Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM-1 to levels similar to those in control transfectants. Instead, adhesion under flow conditions, soluble binding experiment, flow cytometry, and biochemical analyses revealed that the earlier chemokine-triggered integrin activation step was mostly independent of RGS10 actions. The data strongly suggest that RGS10 opposes activation by chemokines of the Vav1-Rac1 pathway in T cells, leading to repression of adhesion strengthening mediated by α4β1. In addition to control chemokine-upregulated T cell attachment, RGS10 also limited adhesion-independent cell chemotaxis and activation of cdc42. These results identify RGS10 as a key molecule that contributes to the termination of Gα-dependent signaling during chemokine-activated α4β1- and αLβ2-dependent T cell adhesion.     

Type I PIPK-α regulates directed cell migration by modulating Rac1 plasma membrane targeting and activation

Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) is a critical regulator of cell migration, but the roles of the type I phosphatidylinositol-4-phosphate 5-kinases (PIPKIs), which synthesize PI4,5P₂, have yet to be fully defined in this process. In this study, we report that one kinase, PIPKI-α, is a novel upstream regulator of Rac1 that links activated integrins to the regulation of cell migration. We show that PIPKI-α controls integrin-induced translocation of Rac1 to the plasma membrane and thereby regulates Rac1 activation. Strikingly, this function is not shared with other PIPKI isoforms, is independent of catalytic activity, and requires physical interaction of PIPKI-α with the Rac1 polybasic domain. Consistent with its role in Rac1 activation, depletion of PIPKI-α causes pronounced defects in membrane ruffling, actin organization, and focal adhesion formation, and ultimately affects the directional persistence of migration. Thus, our study defines the role of PIPKI-α in cell migration and describes a new mechanism for the spatial regulation of Rac1 activity that is critical for cell migration.     

Lumican Accelerates Wound Healing by Enhancing α2β1 Integrin-Mediated Fibroblast Contractility

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast’s mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.     

PIPKIγ regulates focal adhesion dynamics and colon cancer cell invasion

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ^K188,200R or PIPKIγ^D316K, two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ^K188,200R, resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ^K188,200R or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.     

Short form of α9 promotes α9β1 integrin-dependent cell adhesion by modulating the function of the full-length α9 subunit

The α9β1 integrin is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin-C, and osteopontin. A 2.3-kb truncated form of α9 integrin subunit cDNA was identified by searching the Medline database. This splice variant, which we called the short form of α9 integrin (SFα9), encodes a 632-aa isoform lacking transmembrane and cytoplasmic domains, and its authentic expression was verified by PCR and Western blotting. SFα9 is expressed on the cell surface but cannot bind ligand in the absence of the full-length α9 subunit. Over-expression of SFα9 in cells expressing full-length α9 promotes α9-dependent cell adhesion. This promoting effect of SFα9 requires the authentic cytoplasmic domain of the co-expressed full-length α9 subunit. Thus, SFα9 is a novel functional modulator of α9β1 integrin by inside-out signaling.     

Negative feedback by SNAI2 regulates TGFβ1-induced amelotin gene transcription in epithelial–mesenchymal transition

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor β1 (TGFβ1)-induced epithelial–mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFβ1-induced AMTN gene expression was attenuated by SNAI2 and TGFβ1-induced SNAI2, without inhibition of the TGFβ1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFβ1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFβ1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.     

β2-Chimaerin binds to EphA receptors and regulates cell migration

Ephrins and Eph receptors have key roles in regulation of cell migration during development. We found that the RacGAP β2-chimaerin (chimerin) bound to EphA2 and EphA4 and inactivated Rac1 in response to ephrinA1 stimulation. EphA4 bound to β2-chimaerin through its kinase domain and promoted binding of Rac1 to β2-chimaerin. In addition, knockdown of endogenous β2-chimaerin blocked ephrinA1-induced suppression of cell migration. These results suggest that β2-chimaerin is activated by EphA receptors and mediates the EphA receptor-dependent regulation of cell migration.

Structured summary

MINT-7013428: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with Chimaerin beta 2 (uniprotkb:Q80XD1-2) and EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013515: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by anti tag coimmunoprecipitation (MI:0007)MINT-7013410: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with Chimaerin beta 1 (uniprotkb:Q80XD1-1) and EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013503: Chimaerin beta 1 (uniprotkb:Q80XD1-1) physically interacts (MI:0218) with EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013472: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with EphA2 (uniprotkb:O43921) by anti tag coimmunoprecipitation (MI:0007)MINT-7013450: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with EphA2 (uniprotkb:O43921) and Chimaerin beta 2 (uniprotkb:P52757-1) by anti tag coimmunoprecipitation (MI:0007)MINT-7013491: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)     

GARP regulates the bioavailability and activation of TGFβ

Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-β (proTGFβ) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFβ activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFβ1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFβ1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFβ-binding protein for binding to proTGFβ1. Association between GARP and proTGFβ1 prevents the secretion of TGFβ1. Integrin α(V)β(6) and to a lesser extent α(V)β(8) are able to activate TGFβ from the GARP-proTGFβ1 complex. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP. Our results show that GARP is a latent TGFβ-binding protein that functions in regulating the bioavailability and activation of TGFβ.     

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell–matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82’s contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.     

miR-218 regulates focal adhesion kinase–dependent TGFβ signaling in fibroblasts

Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)–expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor β (TGFβ) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre–miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFβ to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFβ to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFβ to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.     

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G₂/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G₂/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.