Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells.

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-ß estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Type I collagen proteolysis by matrix metalloproteinase-2 contributes to focal adhesion kinase activation and vascular smooth muscle cell proliferation in the aorta in early hypertension.

INTRODUCTION: Increased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. METHODS: Male Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. RESULTS: 2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. CONCLUSIONS: Increase in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

The Sall2 transcription factor promotes cell migration regulating focal adhesion turnover and integrin ß1 expression.

SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin ß1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin ß1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin ß1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin ß1 axis could be relevant for those processes.

Focal adhesion kinase inhibitors in the treatment of solid tumors: Preclinical and clinical evidence.

Focal Adhesion Kinase (FAK) is a 125-kDa cytoplasmic protein kinase that is implicated in several cellular functions. This protein is an attractive molecular target for cancer therapy because a wide variety of studies have demonstrated associations between the activation or elevated expression of FAK and tumor progression, invasion, and drug resistance in malignant tumors. Here, we review the strategies used to inhibit FAK activity in solid tumors. We also include an overview of the preclinical (in vitro and in vivo) and clinical studies on FAK inhibitors.

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells.

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

Circulating extracellular vesicles from patients with breast cancer enhance migration and invasion via a Src­dependent pathway in MDA­MB­231 breast cancer cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype associated with high rates of metastasis, heterogeneity, drug resistance and a poor prognosis. Extracellular vesicles (EVs) are vesicles of endosomal and plasma membrane origin, and are secreted by healthy and cancer cells. In cancer, EVs contribute to tumor progression by mediating escape from the immune system surveillance, and are involved in extracellular matrix degradation, invasion, angiogenesis, migration and metastasis. Furthermore, EVs have been identified in several human fluids. However, the role of EVs from patients with breast cancer in the migration and invasion of human breast cancer cells is not fully understood. The present study investigated whether EVs isolated from Mexican patients with breast cancer can induce cellular processes related to invasion in breast cancer. Moreover, plasma fractions enriched in EVs and deprived of platelet­derived EVs obtained from blood samples of 32 Mexican patients with biopsy­diagnosed breast cancer at different clinical stages who had not received treatment were analyzed. Furthermore, one control group was included, which consisted of 20 Mexican healthy females. The present results demonstrated that EVs from women with breast cancer promote migration and invasion, and increase matrix metalloproteinase (MMP)­2 and MMP­9 secretion in TNBC MDA­MB­231 cells. In addition, it was found that EVs from patients with breast cancer induced Src and focal adhesion kinase activation, and focal adhesions assembly with an increase in focal adhesions number, while the migration and invasion was dependent on Src activity. Collectively, EVs from Mexican patients with breast cancer induce migration and invasion via a Src­dependent pathway in TNBC MDA­MB­231 cells.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Heparan sulfate proteoglycan triggers focal adhesion kinase signaling during Trypanosoma cruzi invasion

BACKGROUND Trypanosoma cruzi, the etiologic agent of Chagas disease, is capable of triggering different signaling pathways that modulate its internalisation in mammalian cells. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, has been demonstrated as a mechanism of T. cruzi invasion in cardiomyocytes. Since the involved cell surface receptors are not yet known, we evaluated whether heparan sulfate proteoglycans (HSPG), a molecule involved in T. cruzi recognition and in the regulation of multiple signaling pathways, are able to trigger the FAK signaling pathway during T. cruzi invasion. METHODS To investigate the role of HSPG in the regulation of the FAK signaling pathway during trypomastigote entry, we performed heparan sulfate (HS) depletion from the cardiomyocyte surface by treatment with heparinase I or p-nitrophenyl-β-D-xylopyranoside (p-n-xyloside), which abolishes glycosaminoglycan (GAG) attachment to the proteoglycan core protein. Wild-type (CHO-k1) and GAG-deficient Chinese hamster ovary cells (CHO-745) were also used as an approach to evaluate the participation of the HSPG-FAK signaling pathway. FAK activation (FAK Tyr397) and spatial distribution were analysed by immunoblotting and indirect immunofluorescence, respectively. FINDINGS HS depletion from the cardiomyocyte surface inhibited FAK activation by T. cruzi. Cardiomyocyte treatment with heparinase I or p-n-xyloside resulted in 34% and 28% FAK phosphorylation level decreases, respectively. The experiments with the CHO cells corroborated the role of HSPG as a FAK activation mediator. T. cruzi infection did not stimulate FAK phosphorylation in CHO-745 cells, leading to a 36% reduction in parasite invasion. FAK inhibition due to the PF573228 treatment also impaired T. cruzi entry in CHO-k1 cells. MAIN CONCLUSION Jointly, our data demonstrate that HSPG is a key molecule in the FAK signaling pathway activation, regulating T. cruzi entry.

Depletion of Host Cell Focal Adhesion Kinase Increases the Susceptibility to Invasion by Trypanosoma cruzi Metacyclic Forms.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.

Focal adhesion kinase-dependent activation of the early endocytic protein Rab5 is associated with cell migration.

Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.

Increased expression of FAK isoforms as potential cancer biomarkers in ovarian cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is expressed in most human cell types (example: Epithelial cells, fibroblasts and endothelial), it serves a key role in the control of cell survival, proliferation and motility. The abnormal expression of FAK has been associated with poor prognosis in cancer, including ovarian cancer. However, although FAK isoforms with specific molecular and functional properties have been characterized, there are a limited number of published studies that examine FAK isoforms in ovarian cancer. The aim of the present study was to analyze the expression level of FAK and its isoforms in ovarian cancer. The expression of FAK kinase and focal adhesion targeting (FAT) domains was determined with immunohistochemistry in healthy ovary, and serous and mucinous cystadenoma, borderline tumor and carcinoma samples. Additionally, the expression of FAK and its isoforms were investigated in three ovarian cancer-derived cell lines with western blotting and reverse transcription-semi-quantitative polymerase chain reaction. An increased expression of FAK kinase domain was determined in serous tumor samples and was associated with advancement of the lesion. FAK kinase domain expression was moderate-to-low in mucinous tumor samples. The expression of the FAK FAT domain in tumor samples was reduced, compared with healthy ovary samples; however, the FAT domain was localized to the cellular nucleus. Expression of alternative transcripts FAK°, FAK28,6 and FAK28 was determined in all three cell lines investigated. In conclusion, FAK kinase and FAT domains are differentially expressed among ovarian tumor types. These results indicated the presence of at least two isoforms of FAK (FAK and the putative FAK-related non-kinase) in tumor tissue, which is supported by the cells producing at least three FAK alternative transcripts. These results may support the use of FAK and its isoforms as biomarkers for ovarian cancer.

Lonomia obliqua bristle extract modulates Rac1 activation, membrane dynamics and cell adhesion properties.

Lonomia obliqua is a caterpillar of potential therapeutic interest whose venom is able to induce severe blood leakage and modulate leukocyte migration. Since both phenotypes are associated with changes in cytoskeleton dynamics and cell adhesion properties, the aim of this study was to analyze the effects of Lonomia obliqua bristle extract (LOBE) in cell adhesion and migration signaling. Proteomic analysis revealed that epithelial cells (CHO-K1) exposed to LOBE (30 µg/mL, 30 min) exhibited changes in levels of actin regulatory proteins, including RhoGTPases. These changes correlated with an increase in the activity of the RhoGTPase family member Rac as measured by Förster resonance energy transfer (FRET). When plated in migration promoting conditions, CHO-K1 cells exposed to LOBE (10 µg/mL) showed an increase in membrane ruffling after short (30 min) period of incubation that was accompanied by changes in the distribution of the adhesion markers paxillin, vinculin and an increase of focal adhesion kinase autophosphorylation levels (Y397), suggesting changes in cell-extracellular matrix (ECM) adhesion properties and signaling. These data suggest that LOBE possesses bioactive molecules that are capable to modulated cell migration signaling, cytoskeletal dynamics and cell-ECM properties of several cell types.

Focal adhesion kinase, RhoA, and p38 mitogen-activated protein kinase modulates apoptosis mediated by angiotensin II AT2 receptors.

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT2 receptors (AT 2 R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT 2 R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT 2 cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT 2 cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT 2 cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT 2 R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role.

NADPH-oxidase-derived ROS alters cell migration by modulating adhesions dynamics.

BACKGROUND INFORMATION: Cell migration requires the coordinated activation of structural and signalling molecules, such as the RhoGTPase Rac1. It is known that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex assembly, which generates reactive oxygen species (ROS) at the cell membrane, also relies on Rac1 activation, indicating a possible effect of ROS during cell migration. In this study, we evaluated the effect of NADPH-oxidase-derived ROS on the migration process. RESULTS: Using time-lapse videos of CHO.K1 cells plated on fibronectin (2 µg/ml) or collagen (5 µg/cm2 ), we observed that depletion of ROS by N-acetyl-cysteine (NAC, 10 mM), an unspecific antioxidant, or diphenyliodonium (DPI, 10 µM), a NADPH-oxidase inhibitor, induced a ∼50% decrease in migration speed and severely impacted migration directionality. Then, we analysed the effects of NADPH oxidase on three migratory events: protrusion rate, adhesion process and signalling pathways related to cell migration. DPI induced an increase of ∼3 protrusion/cell, which were 2× faster but had a ∼50% retraction when compared with control. By pull-down assay, we observed no changes on Rac1 activation, indicating that ROS-mediated effects were related to downstream molecules, such as adhesion-related molecules. A reduction of the adhesion marker FAK-Y397 levels in cells treated with NAC and DPI was observed. In order to analyse adhesion dynamics, CHO.K1 cells transfected with paxillin-GFP analysed with total internal reflectance fluorescence (TIRF) indicated that DPI (5 µM) induced larger adhesions when compared with control. CONCLUSION: These results indicate that the local generation of NADPH-oxidase-derived ROS can modulate cell migration due to changes on adhesion dynamics and signalling. SIGNIFICANCE: This study highlights the physiological requirement of ROS for cell migration and the potential use of these molecules as targets to modulate the cell migration process at different diseases.

Synergistic antitumor activity by combining trastuzumab with retinoic acid in HER2 positive human breast cancer cells.

Breast cancer can be classified into molecular subtypes. Tumors overexpressing HER2 protein are more aggressive and metastatic; hence, patients have a poor prognosis. Anti-HER2 strategies, such as the monoclonal antibody Trastuzumab (Tz), have therefore been developed. Despite this progress, not all patients respond to the treatment. Retinoic acid (RA) has been proposed as an adjuvant treatment of breast carcinoma because of its ability to inhibit cell growth. We evaluated the effect of Tz in combination with RA on the viability, adhesion, migration, invasion and expression of migration-related proteins in SKBR3 and BT-474 human breast cancer cells. MTT, pharmacological interaction analysis, immunofluorescence, adhesion/migration/invasion and Western blot assays were performed. The coadministration of both drugs synergistically decreased cell survival. Tz+RA significantly decreased adhesion/migration/invasion in both cell types. Tz+RA strongly reduced FAK and HER2 expression and induced nuclear FAK translocation. In addition, a granular distribution of HER2 receptor was observed after the combined treatment. In conclusion, the coadministration of both drugs in patients with this type of cancer could contribute to the improvement of their prognosis and reduce the adverse effects of therapy because the applied Tz doses would be lower due to the adjuvant effect of RA.

Focal adhesion kinases in head and neck squamous cell carcinoma.

BACKGROUND: Focal adhesion kinase (FAK) phosphorylation contributes to the regulation of growth factors that promote cellular adhesion, mobility, and survival, being a key factor in tumor development. The objective of this study was to evaluate the immunohistochemical expression patterns of FAK and its phosphorylated forms, FAK Tyr-576 and FAK Tyr-925, in head and neck squamous cell carcinoma and non-neoplastic adjacent epithelial tissue (AE). METHODS: The percentage of immunohistochemistry stained cells and its correlation with clinicopathological variables and prognosis were determined using samples from 54 patients. RESULTS: FAK, FAK Tyr-576, and FAK Tyr-925 overexpression was observed in tumor zones and AE. FAK Tyr-576 immunostaining showed a relationship with tumor clinicopathological parameters. Moreover, positive immunostaining of FAK Tyr-576 in AEsue was associated with patients prognoses. CONCLUSIONS: Increased expression of FAK Tyr-576 could enable identification of tumors with a more aggressive behavior and epithelial alterations before the appearance of clinical or histological manifestations.

Hypertonic Saline (NaCl 7.5%) Reduces LPS-Induced Acute Lung Injury in Rats.

Acute respiratory distress syndrome (ARDS) is the most severe lung inflammatory manifestation and has no effective therapy nowadays. Sepsis is one of the main illnesses among ARDS causes. The use of fluid resuscitation is an important treatment for sepsis, but positive fluid balance may induce pulmonary injury. As an alternative, fluid resuscitation with hypertonic saline ((HS) NaCl 7.5%) has been described as a promising therapeutical agent in sepsis-induced ARDS by the diminished amount of fluid necessary. Thus, we evaluated the effect of hypertonic saline in the treatment of LPS-induced ARDS. We found that hypertonic saline (NaCl 7.5%) treatment in rat model of LPS-induced ARDS avoided pulmonary function worsening and inhibited type I collagen deposition. In addition, hypertonic saline prevented pulmonary injury by decreasing metalloproteinase 9 (MMP-9) activity in tissue. Focal adhesion kinase (FAK) activation was reduced in HS group as well as neutrophil infiltration, NOS2 expression and NO content. Our study shows that fluid resuscitation with hypertonic saline decreases the progression of LPS-induced ARDS due to inhibition of pulmonary remodeling that is observed when regular saline is used.

TNFα contributes for attenuating both Y397FAK and Y416Src phosphorylations in osteoblasts.

OBJECTIVE: Our poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor (TNF)α-induced focal adhesion kinase (FAK) and Src phosphorylation. MATERIAL AND METHODS: MC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNFα treatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNFα content at conditioned medium. Tumor necrosis factor-α-neutralizing antibodies also were used. RESULTS: It was possible to show that TNFα provokes attenuation at Y-phosphorylation of both FAK (at Y397 ) and Src (at Y416 ) proteins (P < 0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide (LPS)-stimulated macrophages, it being significantly different than control (FAK and Src, P < 0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti-TNFα neutralizing antibody (2 µg ml(-1) ) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated (P < 0.05). Moreover, we also showed that TNFα impairs osteoblast adhesion, suggesting an interesting role on osteoblast performance. CONCLUSIONS: Altogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNFα on osteoblast performance.

Focal adhesion kinase signaling in cardiac hypertrophy and failure

Focal adhesion kinase (FAK) is a broadly expressed tyrosine kinase implicated in cellular functions such as migration, growth and survival. Emerging data support a role for FAK in cardiac development, reactive hypertrophy and failure. Data reviewed here indicate that FAK plays a critical role at the cellular level in the responses of cardiomyocytes and cardiac fibroblasts to biomechanical stress and to hypertrophic agonists such as angiotensin II and endothelin. The signaling mechanisms regulated by FAK are discussed to provide insight into its role in the pathophysiology of cardiac hypertrophy and failure.