Syndecan-4/PAR-3 signaling regulates focal adhesion dynamics in mesenchymal cells.

BACKGROUND: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvß3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.

Curcumin-loaded nanoemulsion: a new safe and effective formulation to prevent tumor reincidence and metastasis.

Curcumin is widely considered beneficial to human health, but insolubility and instability greatly hamper reproducible exploitation of the advantageous traits. Here we report on the development, characterization and evaluation of a curcumin-loaded nanoemulsion (CUR-NEM) that is highly effective in preventing post-surgery tumor reincidence and metastasis. The method of fabrication utilized safe excipients and generated particles of 200 nm (PDI ≤ 0.2) with negative zeta potential (-30 mV) and a high yield of curcumin (95%), which can be converted by lyophilization to a dry powder. In vitro assays showed that CUR-NEM is safe in non-cancerous human cells (HEK-293T) and preferentially cytotoxic in gastric (AGS), colon (HT29-ATCC, HT29-US), breast (MDA-MB-231) and melanoma (B16F10) cells. In addition, in melanoma cells the nanoformulation increases intracellular curcumin accumulation and reactive oxygen species (ROS) formation, while preventing cell-migration and invasion. In vivo studies in C57BL/6 mice demonstrated that a single dose, applied topically to the wounded area after surgical excision of primary tumors formed upon subcutaneous injection of syngeneic B16F10 cells, was sufficient to completely prevent reincident tumor growth and spontaneous lung metastasis, while in untreated animals 70% reincidence and metastasis were observed. In vivo experiments also showed that the fluorescence signal due to curcumin was maintained at least 15 days after topical application of CUR-NEM, while when administered in DMSO the curcumin signal disappeared within 4 days. Importantly, the administration of a dose 22 times larger than that applied topically to animals after tumor surgery did not alter biochemical parameters. Due to the safety and efficacy of the formulation, we envisage it as ideal for topical application in cancer patients following surgery, to prevent tumor reincidence and metastasis. In addition, other routes of administration/protocols could also be proposed to treat/prevent malignant tumors in patients.

Gold nanoparticles as tracking devices to shed light on the role of caveolin-1 in early stages of melanoma metastasis.

AIM: To track early events during lung metastasis, we labeled cells expressing (B16F10CAV1) or lacking CAV1 (B16F10mock) with gold nanoparticles conjugated to the peptide TAT (AuNPs-PEG-TAT). METHODS: B16F10 expressing or lacking CAV1 were labeled with AuNPs-PEG-TAT. The physicochemical properties and cytotoxicity of these nanoparticles, as well as their effects on migration and invasiveness of B16F10 cells in vitro were evaluated. Ex vivo lung distribution of the labeled cells after tail vein injection into C57BL/6 mice was examined. RESULTS: AuNPs-PEG-TAT did not affect B16F10 viability, migration and invasiveness. The metastatic and tumorigenic capability of the labeled B16F10 was also not modified in comparison to unlabeled B16F10 cells. CAV1 expression favored the retention of B16F10 cells in the lungs of mice 2 h post injection, suggesting CAV1 promoted adherence to endothelial cells and transendothelial migration. CONCLUSIONS: We developed a protocol to label B16F10 cells with AuNPs-PEG-TAT that permits subsequent tracking of cells in mice. CAV1 overexpression was found to increase retention and transendothelial migration of B16F10 cells in the lung.

αVß3 Integrin regulates astrocyte reactivity.

BACKGROUND: Neuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVß3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses. METHODS: Wild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers. RESULTS: Thy-1 induced astrocyte migration only after TNF priming. Increased expression of αVß3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of ß3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while ß3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1. CONCLUSIONS: Therefore, inflammation induces expression of αVß3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of ß3 Integrin levels modulates these responses regardless of inflammation.

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration.

Our previous reports indicate that ligand-induced αVß3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca(2+) to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVß3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVß3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca(2+), hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVß3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca(2+) entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

Up-regulation of the kinin B2 receptor pathway modulates the TGF-ß/Smad signaling cascade to reduce renal fibrosis induced by albumin.

The presence of high protein levels in the glomerular filtrate plays an important role in renal fibrosis, a disorder that justifies the use of animal models of experimental proteinuria. Such models have proved useful as tools in the study of the pathogenesis of chronic, progressive renal disease. Since bradykinin and the kinin B2 receptor (B2R) belong to a renoprotective system with mechanisms still unclarified, we investigated its anti-fibrotic role in the in vivo rat model of overload proteinuria. Upon up-regulating the kinin system by a high potassium diet we observed reduction of tubulointerstitial fibrosis, decreased renal expression of α-smooth muscle actin (α-SMA) and vimentin, reduced Smad3 phosphorylation and increase of Smad7. These cellular and molecular effects were reversed by HOE-140, a specific B2R antagonist. In vitro experiments, performed on a cell line of proximal tubular epithelial cells, showed that high concentrations of albumin induced expression of mesenchymal biomarkers, in concomitance with increases in TGF-ß1 mRNA and its functionally active peptide, TGF-ß1. Stimulation of the tubule cells by bradykinin inhibited the albumin-induced changes, namely α-SMA and vimentin were reduced, and cytokeratin recovered together with increase in Smad7 levels and decrease in type II TGF-ß1 receptor, TGF-ß1 mRNA and its active fragment. The protective changes produced by bradykinin in vitro were blocked by HOE-140. The development of stable bradykinin analogues and/or up-regulation of the B2R signaling pathway may prove value in the management of chronic renal fibrosis in progressive proteinuric renal diseases.

Pre-stimulation of the kallikrein system in cisplatin-induced acute renal injury: an approach to renoprotection.

Antineoplastic treatment with cisplatin is frequently complicated by nephrotoxicity. Although oxidative stress may be involved, the pathogenic mechanisms responsible for renal damage have not been completely clarified. In order to investigate the role of the renal kinin system in this condition, a group of rats was submitted to high potassium diet to stimulate the synthesis and excretion of tissue kallikrein 1 (rKLK1) previous to an intraperitoneal injection of 7 mg/kg cisplatin. A significant reduction in lipoperoxidation, evidenced by urinary excretion of malondialdehyde and renal immunostaining of hidroxy-nonenal, was accompanied by a decline in apoptosis. Coincident with these findings we observed a reduction in the expression of renal KIM-1 suggesting that renoprotection may be occurring. Stimulation or indemnity of the renal kinin system deserves to be evaluated as a complementary pharmacological measure to diminish cisplatin nephrotoxicity.

Thy-1-interacting molecules and cellular signaling in cis and trans.

Thy-1, discovered almost 50 years ago, was for many years a subject of great scientific interest. The putative functions attributed to this molecule could not be confirmed due, at least in part, to a ligand that took a long time to be identified. This chapter describes the properties of Thy-1 and the regulation of its expression. Also, the interactions that have been described for Thy-1 in both cis and trans, and the signaling mechanisms reported to emanate from such interactions are discussed. The consequences of Thy-1-activated signaling pathways for different cell types and organisms are also reviewed. Since the discovery of αVß3 integrin as a receptor for Thy-1 in astrocytes, many more functions have been attributed to Thy-1 interactions in trans toward other cell types. Recently, a ligand for neuronal Thy-1 was unveiled and shown to elicit signaling in cis. The ligand and the receptor for Thy-1 turned out to be the same molecule, αVß3 integrin, which upon interaction with Thy-1 yields bidirectional astrocyte-to-neuron communication. Thus, Thy-1 biology is again beginning to make progress in answering main questions surrounding this enigmatic molecule. Some of these remaining questions are highlighted in this chapter.

Antihypertensive and renoprotective effect of the kinin pathway activated by potassium in a model of salt sensitivity following overload proteinuria.

The albumin overload model induces proteinuria and tubulointersitial damage, followed by hypertension when rats are exposed to a hypersodic diet. To understand the effect of kinin system stimulation on salt-sensitive hypertension and to explore its potential renoprotective effects, the model was induced in Sprague-Dawley rats that had previously received a high-potassium diet to enhance activity of the kinin pathway, followed with/without administration of icatibant to block the kinin B2 receptor (B2R). A disease control group received albumin but not potassium or icatibant, and all groups were exposed to a hypersodic diet to induce salt-sensitive hypertension. Potassium treatment increased the synthesis and excretion of tissue kallikrein (Klk1/rKLK1) accompanied by a significant reduction in blood pressure and renal fibrosis and with downregulation of renal transforming growth factor-ß (TGF-ß) mRNA and protein compared with rats that did not receive potassium. Participation of the B2R was evidenced by the fact that all beneficial effects were lost in the presence of the B2R antagonist. In vitro experiments using the HK-2 proximal tubule cell line showed that treatment of tubular cells with 10 nM bradykinin reduced the epithelial-mesenchymal transdifferentiation and albumin-induced production of TGF-ß, and the effects produced by bradykinin were prevented by pretreatment with the B2R antagonist. These experiments support not only the pathogenic role of the kinin pathway in salt sensitivity but also sustain its role as a renoprotective, antifibrotic paracrine system that modulates renal levels of TGF-ß.

Thy-1-mediated cell-cell contact induces astrocyte migration through the engagement of αVß3 integrin and syndecan-4.

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVß3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.