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.

Telomerase inhibitors TMPyP4 and thymoquinone decreased cell proliferation and induced cell death in the non-small cell lung cancer cell line LC-HK2, modifying the pattern of focal adhesion.

G-quadruplexes (G4) are structures formed at the ends of telomeres rich in guanines and stabilized by molecules that bind to specific sites. TMPyP4 and thymoquinone (TQ) are small molecules that bind to G4 and have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines, LC-HK2 (non-small cell lung cancer - NSCLC) and RPE-1 (hTERT-immortalized), were treated with TMPyP4 (5 µM) and TQ (10 µM). Both inhibitors decreased telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S-phase. TMPyP4 reduced cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a concentration-dependent manner, increasing the frequency of senescent cells and inducing cell cycle arrest in G1 phase. Thus, the present results showed that TMPyP4 and TQ, although acting as telomerase inhibitors, had a broader effect on other signaling pathways and processes in cells, differing from each other. However, they act both on malignant and immortalized cells, and further studies are needed before their anti-cancer potential can be considered.

Resinas acrílicas utilizadas em placas estabilizadoras: resistência a fraturas e ensaio microbiológico / Acrylic resins used in stabilizer plates: resistance to fractures and microbiological testing

O aprimoramento da tecnologia CAD/CAM permitiu a confecção de placas estabilizadoras digitais, no entanto, há lacunas acerca do comportamento desses dispositivos frente a variáveis mecânicas e biológicas ainda desconhecidas. Dessa forma, o objetivo desse trabalho foi verificar se as resinas utilizadas na tecnologia digital das placas fresadas e impressas são capazes de influenciar na resistência à fratura e na adesão de micro-organismos em detrimento das resinas empregadas na confecção das placas termopolimerizáveis convencionais e se as propriedades testadas se relacionam com a variação da espessura desses dispositivos. Para tanto, foi realizado um estudo in vitro que contou com as fases mecânica e microbiológica, sendo cada uma composta pelos grupos Convencional (GC) (n=30), Fresado (GF) (n=30) e Impresso (GI) (n=30), de acordo com o método de confecção, possuindo cada um três subgrupos (n=10) que variaram a espessura dos espécimes de 1 a 3 mm. Na fase mecânica, as amostras, com formato de barras de 65 mm, foram testadas quanto à resistência à fratura na máquina de ensaios universal, com célula de carga de 500 kgf e velocidade de 1 mm/min; na microbiológica, a forma foi de discos com 15 mm de diâmetro, submetidos à adesão de micro-organismos à superfície com a exposição a Streptococcus mutans e mantidos por 24h a 35 °C em estufa para sucessiva contagem de UFC, com controle positivo. Os testes foram feitos em triplicata e os dados foram armazenados no SPSS 22.0 e a análise estatística contou com a ANOVA e o pósteste de Tukey. Nos resultados da fase mecânica, o ANOVA identificou diferenças estatisticamente significativas entre os grupos (p≤0,005), mostrando que GC e GF apresentaram melhores resultados. O pós-teste de Tukey considerou diferença estatisticamente significativa entre GF e GI (p=0,031), elencando GF como superior. Para a fase microbiológica, não foram verificadas diferenças estatísticas entre os grupos (p>0,005) em nenhuma das análises. Observou-se que para a resistência a fraturas, as resinas do modo fresado descreveram resultados superiores e/ou próximos aos da técnica convencional; e que as resinas da técnica digital e convencional, quanto à adesão superficial de micro-organismos, não diferiram entre si (AU).

CAD/CAM technology allowed the stabilizing splints manufacture, however there are gaps about these devices' behavior in face of mechanical and biological variables that are still unknown. Thus, the objective of this project was to verify if the resins used in digital technology of milled and printed stabilizer splints can influence fracture resistance and microorganism adhesion, in detriment of the resin applied in manufacture of conventional thermopolymerizable splints and if the tested properties are related to thickness variation of these devices. The method included mechanical and microbiological steps, each one composed by Conventional (CG) (n=30), Milled (MG) (n=30) and Printed (PG) (n=30) groups, according to the method of preparation, each one having three subgroups (n=10) that varied the specimen thickness from 1 to 3 mm. In the mechanical step, the samples, in the form of 65 mm bars, were tested for fracture resistance in a universal testing machine, with load cell of 500 kgf and speed of 1 mm/min; in the microbiological step, the shape was in form of 15 mm diameter discs, subjected to microorganism adhesion to the surface with exposure to Streptococcus mutans and kept for 24 hours at 35 °C in oven for successive CFU counting, with positive control. The tests were performed in triplicate and data were stored in SPSS 22.0 and statistical analysis used ANOVA and Tukey's post hoc. In the results of mechanical step ANOVA had identified statistically significant differences between the groups (p≤0,005), showing CG and MG had presented better results. Tukey´s post hoc had considered statistically significant difference between MG and PG (p=0,031), listing MG as superior. For the microbiological step, they did not show statistical differences between the groups (p>0.005) in any analysis. It was observed milled resins described superior results and/or close to the conventional technique one, for fracture resistance; and resins of digital and conventional technique, regarding the microorganism adhesion to the surface, did not differ from each other (AU).

Actin Stress Fibers Response and Adaptation under Stretch.

One of the many effects of soft tissues under mechanical solicitation in the cellular damage produced by highly localized strain. Here, we study the response of peripheral stress fibers (SFs) to external stretch in mammalian cells, plated onto deformable micropatterned substrates. A local fluorescence analysis reveals that an adaptation response is observed at the vicinity of the focal adhesion sites (FAs) due to its mechanosensor function. The response depends on the type of mechanical stress, from a Maxwell-type material in compression to a complex scenario in extension, where a mechanotransduction and a self-healing process takes place in order to prevent the induced severing of the SF. A model is proposed to take into account the effect of the applied stretch on the mechanics of the SF, from which relevant parameters of the healing process are obtained. In contrast, the repair of the actin bundle occurs at the weak point of the SF and depends on the amount of applied strain. As a result, the SFs display strain-softening features due to the incorporation of new actin material into the bundle. In contrast, the response under compression shows a reorganization with a constant actin material suggesting a gliding process of the SFs by the myosin II motors.

Protein tyrosine phosphatase 1B targets focal adhesion kinase and paxillin in cell-matrix adhesions.

Protein tyrosine phosphatase 1B (PTP1B, also known as PTPN1) is an established regulator of cell-matrix adhesion and motility. However, the nature of substrate targets at adhesion sites remains to be validated. Here, we used bimolecular fluorescence complementation assays, in combination with a substrate trapping mutant of PTP1B, to directly examine whether relevant phosphotyrosines on paxillin and focal adhesion kinase (FAK, also known as PTK2) are substrates of the phosphatase in the context of cell-matrix adhesion sites. We found that the formation of catalytic complexes at cell-matrix adhesions requires intact tyrosine residues Y31 and Y118 on paxillin, and the localization of FAK at adhesion sites. Additionally, we found that PTP1B specifically targets Y925 on the focal adhesion targeting (FAT) domain of FAK at adhesion sites. Electrostatic analysis indicated that dephosphorylation of this residue promotes the closed conformation of the FAT 4-helix bundle and its interaction with paxillin at adhesion sites.

Focal adhesion signaling: vascular smooth muscle cell contractility beyond calcium mechanisms.

Smooth muscle cell (SMC) contractility is essential to vessel tone maintenance and blood pressure regulation. In response to vasoconstrictors, calcium-dependent mechanisms promote the activation of the regulatory myosin light chain, leading to increased cytoskeleton tension that favors cell shortening. In contrast, SMC maintain an intrinsic level of a contractile force independent of vasoconstrictor stimulation and sustained SMC contraction beyond the timescale of calcium-dependent mechanisms suggesting the involvement of additional players in the contractile response. Focal adhesions (FAs) are conceivable candidates that may influence SMC contraction. They are required for actin-based traction employed by cells to sense and respond to environmental cues in a process termed mechanotransduction. Depletion of FA proteins impairs SMC contractility, producing arteries that are prone to dissection because of a lack of mechanical stability. Here, we discuss the role of calcium-independent FA signaling mechanisms in SMC contractility. We speculate that FA signaling contributes to the genesis of a variety of SMC phenotypes and discuss the potential implications for mechanical homeostasis in normal and diseased states.

Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy.

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

Rab40-Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration.

Rab40b is a SOCS box-containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b-Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b-Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b-Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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.

Role of Src/FAK in migration and invasion mediated by extracellular vesicles from MDA-MB-231 cells stimulated with linoleic acid.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in occidental diets, which mediate a variety of processes in human breast cancer cells, including migration and invasion. Extracellular vesicles (EVs) are vesicles released from endosomes and plasma membrane that are composed of a variety of molecules, including proteins, nucleic acids and lipids. EVs from cancer cells promote processes related with cancer progression. In the present study, we demonstrate that treatment of MDA-MB-231 cells with EVs from MDA-MB-231 cells stimulated with LA (LA EVs) promote migration and invasion via Src activity. LA EVs induce activation of FAK via Src activity and of Src and Akt2. LA EVs also induce the assembly of focal adhesions and MMP-9 secretion. These findings demonstrate that LA EVs mediate an autocrine and/or paracrine Src/FAK signaling pathway to promote migration and invasion.

K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity.

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.

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.

RHBDD2 overexpression promotes a chemoresistant and invasive phenotype to rectal cancer tumors via modulating UPR and focal adhesion genes.

The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.

Bisphenol A induces focal adhesions assembly and activation of FAK, Src and ERK2 via GPER in MDA-MB-231 breast cancer cells.

Bisphenol A (BPA) is an industrial synthetic chemical used in the production of polycarbonate plastics and epoxy resins. Human exposition to BPA is primarily through eating food, and drinking liquids, because BPA can leach from polycarbonate plastic containers, beverage cans and epoxy resins. BPA induces proliferation and migration in human breast cancer cells. The G protein-coupled estrogen receptor (GPER) is a G protein-coupled receptor coupled with Gs proteins that is activated by estrogen and estrogenic compounds and it is the receptor for BPA. However, the signal transduction pathways that mediate migration via BPA/GPER in triple negative breast cancer (TNBC) cells has not been studied in detail. Here, we demonstrate that BPA induces an increase of GPER expression and activation of FAK, Src and ERK2, and an increase of focal adhesion assembly via GPER in TNBC MDA-MB-231 cells. Moreover, BPA induces FAK and ERK2 activation, focal adhesion assembly and migration via epidermal growth factor receptor (EGFR) transactivation. Collectively our data showed that BPA via GPER and/or EGFR transactivation induces activation of signal transduction pathways that mediate migration in TNBC MDA-MB-231 cells.

Anastrozole promotes implantation by altering the expression of paxillin and fak in rat luminal uterine epithelium / El anastrozol promueve la implantación alterando la expresión de paxilina y fak en el epitelio uterino luminal de rata

An alternative hyper-ovulator inducer to replace clomiphene citrate (CC) is needed as it is unsuitable for women with polycystic ovarian syndrome and is associated with low pregnancy rates. Anastrozole is an effective hyper-ovulator inducer, but has not been well researched. In order to determine the effectiveness of anastrozole as a hyper-ovulator inducer and to an extent compare it with CC in similar situations, this study ascertained the effects of these drugs on the expression of the focal adhesion proteins, paxillin and FAK, which are uterine receptivity markers in the surface luminal uterine epithelial cells of day 1 and day 6 pregnant Wistar rats. The results show that paxillin is localized in focal adhesions at the base of the uterine epithelial cells at day 1 of pregnancy whereas at day 6, paxillin disassembles from the basal focal adhesions and localizes and increases its expression apically. FAK is faintly expressed at the basal aspect of the uterine epithelial cells while moderately expressed at the cell-to-cell contact at day 1 in all groups from where it disassembles and relocates apically and becomes more intensely expressed at day 6 of pregnancy in untreated and anastrozole treated rats. Although paxillin is localized apically at day 6, its expression is significantly down-regulated with CC treatment suggesting its interference with the implantation process. These findings seem to suggest that anastrozole could favor implantation.

Para reemplazar el citrato de clomifeno (CC) es necesario un inductor de hiperovulación alternativo, ya que no es adecuado para mujeres con síndrome de ovario poliquístico y está asociado con tasas bajas de embarazo. El anastrozol es un inductor eficaz del hiper-ovulador, pero no se ha investigado adecuadamente. Con el fin de determinar la efectividad del anastrozol como inductor del hiper-ovulador y, en cierta medida, compararlo con CC en situaciones similares, este estudio determinó los efectos de estos fármacos en la expresión de las proteínas de adhesión focal, paxillin y FAK, uterinas marcadores de receptividad en la superficie luminal de células uterinas epiteliales, del día 1 y día 6 en ratas Wistar preñadas. Los resultados muestran que la paxilina se localiza en adherencias focales en la base de las células epiteliales uterinas en el día 1 del embarazo, mientras que en el día 6, la paxilina se desmonta de las adherencias focales basales y localiza y aumenta su expresión apicalmente. FAK se expresa débilmente en el aspecto basal de las células epiteliales uterinas, mientras que se expresa moderadamente en el contacto de célula a célula en el día 1 en todos los grupos, donde se separa y se reubica apicalmente y se expresa con mayor intensidad el día 6 de la preñez, en pacientes no tratados y tratados. ratas tratadas con anastrozol. Aunque la paxillina se localiza apicalmente en el día 6, su expresión está significativamente disminuida con el tratamiento con CC, lo que sugiere su interferencia con el proceso de implantación. Estos hallazgos sugieren que el anastrozol podría favorecer el proceso de implantación.

Endothelial cell sprouting driven by RhoJ directly activated by a membrane-anchored Intersectin 1 (ITSN1) RhoGEF module.

Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.

Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice.

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.

Aquaporin-2 and Na+ /H+ exchanger isoform 1 modulate the efficiency of renal cell migration.

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin ß1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na+ /H+ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.

Losartan through Hsp70 Avoids Angiotensin II Induced Mesenchymal Epithelial Transition in Proximal Tubule Cells from Spontaneously Hypertensive Rats.

BACKGROUND/AIMS: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR). METHODS: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy. RESULTS: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc). CONCLUSION: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR.

Early transcriptomic responses associated with the membrane-initiated action of cortisol in the skeletal muscle of rainbow trout (Oncorhynchus mykiss).

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.