Progesterone receptor-Src kinase signaling pathway mediates neuroprogesterone induction of the luteinizing hormone surge in female rats.

Neural circuits in female rats are exposed to sequential estradiol and progesterone to regulate the release of luteinizing hormone (LH) and ultimately ovulation. Estradiol induces progesterone receptors (PGRs) in anteroventral periventricular nucleus (AVPV) kisspeptin neurons, and as estradiol reaches peak concentrations, neuroprogesterone (neuroP) synthesis is induced in hypothalamic astrocytes. This local neuroP signals to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signaling through Src family kinase (Src) underlies the LH surge. As observed in vitro, PGR and Src are co-expressed in AVPV neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Furthermore, estradiol treatment increased the number of AVPV cells that had extranuclear PGR and Src in close proximity (< 40 nm). Infusion of the Src inhibitor (PP2) into the AVPV region of ovariectomized/adrenalectomized (ovx/adx) rats attenuated the LH surge in trunk blood collected 53 h post-estradiol (50 µg) injection that induced neuroP synthesis. Although PP2 reduced the LH surge in estradiol benzoate treated ovx/adx rats, activation of either AVPV PGR or Src in 2 µg estradiol-primed animals significantly elevated LH concentrations compared to dimethyl sulfoxide infused rats. Finally, antagonism of either AVPV PGR or Src blocked the ability of PGR or Src activation to induce an LH surge in estradiol-primed ovx/adx rats. These results indicate that neuroP, which triggers the LH surge, signals through an extranuclear PGR-Src signaling pathway.

First person - Sepideh Fallah.

First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Sepideh Fallah is first author on ' Src family kinases inhibit differentiation of intestinal epithelial cells through the Hippo effector YAP1', published in BiO. Sepideh is a postdoctoral researcher in the lab of Prof. Jean-François Beaulieu at Université de Sherbrooke, Quebec, Canada, investigating how SFKs negatively regulate the differentiation of absorptive and goblet cells through upregulating of YAP1 activity.

Src family kinases inhibit differentiation of intestinal epithelial cells through the Hippo effector YAP1.

Intestinal cell lineage differentiation is a tightly regulated mechanism that involves several intracellular signaling pathways affecting the expression of a variety of transcription factors, which ultimately regulate cell specific gene expression. Absorptive and goblet cells are the two main epithelial cell types of the intestine. Previous studies from our group using an shRNA knockdown approach have shown that YAP1, one of the main Hippo pathway effectors, inhibits the differentiation of these two cell types. In the present study, we show that YAP1 activity is regulated by Src family kinases (SFKs) in these cells. Inhibition of SFKs led to a sharp reduction in YAP1 expression at the protein level, an increase in CDX2 and the P1 forms of HNF4α and of absorptive and goblet cell differentiation specific markers. Interestingly, in Caco-2/15 cells which express both YAP1 and its paralog TAZ, TAZ was not reduced by the inhibition of SFKs and its specific knockdown rather impaired absorptive cell differentiation indicating that YAP1 and TAZ are not always interchangeable for regulating cell functions. This article has an associated First Person interview with the first author of the paper.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action.

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine's antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine's antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor-mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine's antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine's antidepressant action.

Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway.

Oxidized low-density lipoprotein (oxLDL) induced a foam-cell-like phenotype of the vascular smooth muscle cells (VSMCs), leading to the inflammatory responses incorporating Toll-like receptor- (Tlr-) mediated cellular alterations. However, the role of Tlr4 in foam cell formation and underlying molecular pathways has not been comprehensively elucidated. To further investigate the mechanism, VSMCs were incubated with different doses of oxLDL, and then, the lipid, reactive oxygen species (ROS) accumulation, Tlr family genes, and the foam cell phenotype were explored. We observed that oxLDL induced foam cell-like phenotype in VSMCs and led to lipid and ROS accumulation in a dose-dependent manner. Furthermore, in the Tlr family, Tlr4 demonstrated the strongest upregulation under oxLDL stimulation. Simultaneously, oxLDL induced activation of Src, higher expression of Nox2, and lower expression of Mnsod, Sirt1, and Sirt3. By interfering the TLR4 expression, the phenotype alteration, lipid accumulation in VSMCs, and Src kinase activation induced by oxLDL were abolished. After interfering Src activation, the oxLDL-induced lipid accumulation and foam cell phenotype in VSMCs were also alleviated. Furthermore, the ROS accumulation, upregulated Nox2 expression, downregulated Sirt1, Sirt3, and Mnsod expression in VSMCs under oxLDL stimulation were also relieved after the knockdown of Tlr4. Additionally, overexpression of Sirt1 and Sirt3 ameliorated the ROS accumulation and foam cell-like marker expression in VSMCs. These results demonstrated that beyond its familiar role in regulating inflammation response, Tlr4 is a critical regulator in oxLDL-induced foam cell formation in VSMCs via regulating Src kinase activation as well as Sirt1 and Sirt3 expression.

Caveolin-1 promotes radioresistance in rhabdomyosarcoma through increased oxidative stress protection and DNA repair.

The aim of this work was to investigate whether Caveolin-1 (Cav-1), a membrane scaffolding protein widely implicated in cancer, may play a role in radiation response in rhabdomyosarcoma (RMS), a pediatric soft tissue tumor. For this purpose, we employed human RD cells in which Cav-1 expression was stably increased via gene transfection. After radiation treatment, we observed that Cav-1 limited cell cycle arrest in the G2/M phase and enhanced resistance to cell senescence and apoptosis via reduction of p21Cip1/Waf1, p16INK4a and Caspase-3 cleavage. After radiotherapy, Cav-1-mediated cell radioresistance was characterized by low accumulation of H2AX foci, as confirmed by Comet assay, marked neutralization of reactive oxygen species (ROS) and enhanced DNA repair via activation of ATM, Ku70/80 complex and DNA-PK. We found that Cav-1-overexpressing RD cells, already under basal conditions, had higher glutathione (GSH) content and greater catalase expression, which conferred protection against acute treatment with hydrogen peroxide. Furthermore, pre-treatment of Cav-1-overexpressing cells with PP2 or LY294002 compounds restored the sensitivity to radiation treatment, indicating a role for Src-kinases and Akt pathways in Cav-1-mediated radioresistance. These findings were confirmed using radioresistant RD and RH30 lines generated by hypofractionated radiotherapy protocol, which showed marked increase of Cav-1, catalase and Akt, and sensitivity to PP2 and LY294002 treatment. In conclusion, these data suggest that concerted activity of Cav-1 and catalase, in cooperation with activation of Src-kinase and Akt pathways, may represent a network of vital mechanisms that allow irradiated RMS cells to evade cell death induced by oxidative stress and DNA damage.

Concomitant use of a dual Src/ABL kinase inhibitor eliminates the in vitro efficacy of blinatumomab against Ph+ ALL.

Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome-positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Src-mediated Tyr353 phosphorylation of IP3R1 promotes its stability and causes apoptosis in palmitic acid-treated hepatocytes.

Palmitic acid (PA)-induced hepatocyte apoptosis is critical for the progression of nonalcoholic fatty liver disease (NAFLD). Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular Ca2+-release channel and is involved in PA-induced hepatocyte apoptosis. While the expression of IP3R1 is elevated in patients with NAFLD and in hepatocytes treated with PA, it remains unclear how PA promotes the expression of IP3R1. In present study, our results showed that PA induced mitochondrial dysfunction and apoptosis, which is accompanied with the increase of the IP3R1 expression in hepatic cells. The inhibition of IP3R1 expression using siRNA ameliorated the PA-induced mitochondrial dysfunction. Furthermore, PA enhanced the stability of the IP3R1 protein instead of an increase in its mRNA levels. PA also promoted the phosphorylation of IP3R1 at the Tyr353 site and increased the phosphorylation of src in hepatic cells. Moreover, an inhibitor of src kinase (SU6656) significantly reduced the Tyr353 phosphorylation of IP3R1 and decreased its stability. In addition, SU6656 improved mitochondrial function and reduced apoptosis in hepatocytes. Conclusion: PA promotes the Tyr353 phosphorylation of IP3R1 by activating the src pathway and increasing the protein stability of IP3R1, which consequently results in mitochondrial Ca2+ overload and mitochondrial dysfunction in hepatic cells. Our results also suggested that inhibition of the src/IP3R1 pathway, such as by SU6656, may be a novel potential therapeutic approach for the treatment of NAFLD.

Inhibitory effects of Paris saponin I, II, ⠥ and ⠦ on HUVEC cells through regulation of VEGFR2, PI3K/AKT/mTOR, Src/eNOS, PLCγ/ERK/MERK, and JAK2-STAT3 pathways.

Rhizoma Paris is a popular Chinese medicine in clinics. It contains four main saponins which are its major bioactive compounds. These saponins are Paris saponin I, II, VI and VII (PSI, PSII, PSVI and PSVII, respectively). Up to now, the research using HUVEC cells to evaluate the anti-angiogenic activity of four saponins is blank. The purpose of this study was to evaluate the anti-angiogenic properties (also known as angiotoxicity) of the four saponins in Rhizoma Paris on vascular endothelial cells-HUVEC cells, and to investigate the underlying mechanism, which has not been studied before. In this study, MTT assay, Lactate dehydrogenase (LDH) assay, wound healing experiments, transwell cell invasion assay, tubule formation experiment, DAPI staining, AV-PI double staining, and cell cycle analysis were used to determine the effects of Paris saponins. The results showed that, with increases in concentrations of PSI, PSII, PSVI and PSVII, the viability of HUVEC cells decreased significantly. In addition, four saponins dose-dependent enhanced LDH release and inhibited HUVEC cell migration, invasion, and angiogenesis. In terms of mechanism, PSI significantly inhibited protein expression in multiple signaling pathways. In particular, with the VEGF2 as the target, it activate the downstream PI3K / AKT / mTOR, SRC / eNOS, P38, PLCγ / ERK / MERK and JAK2/STAT3 signaling pathways. In conclusion, PSI, PSII, PSVI and PSVII can inhibit endothelial cell proliferation, migration and invasion, block endothelial cell cycle, induce endothelial cell apoptosis, act on protein expression in several anti-angiogenic signaling pathways, and finally inhibit angiogenesis in vitro. This study provides further data support for the clinical application of Paris saponins as antiangiogenic drugs.

Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion.

Epithelia can eliminate apoptotic cells by apical extrusion. This is a complex morphogenetic event where expulsion of the apoptotic cell is accompanied by rearrangement of its immediate neighbors to form a rosette. A key mechanism for extrusion is constriction of an actomyosin network that neighbor cells form at their interface with the apoptotic cell. Here we report a complementary process of cytoskeletal relaxation that occurs when cortical contractility is down-regulated at the junctions between those neighbor cells themselves. This reflects a mechanosensitive Src family kinase (SFK) signaling pathway that is activated in neighbor cells when the apoptotic cell relaxes shortly after injury. Inhibiting SFK signaling blocks both the expulsion of apoptotic cells and the rosette formation among their neighbor cells. This reveals the complex pattern of spatially distinct contraction and relaxation that must be established in the neighboring epithelium for apoptotic cells to be extruded.

EPS8 phosphorylation by Src modulates its oncogenic functions.

BACKGROUND: EPS8 is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking. Its expression is increased in cancer, enhancing mitogenesis, migration and tumorigenesis. Src phosphorylates EPS8 at four tyrosine residues, although the function is unknown. Here we investigated the pro-oncogenic role of EPS8 tyrosine phosphorylation at Src target sites in HNSCC. METHODS: Plasmids expressing EPS8 Src-mediated phosphorylation site mutants (Y485F, Y525F, Y602F, Y774F and all four combined [FFFF]) were expressed in cells containing a normal endogenous level of EPS8. In addition, cells were treated with dasatinib to inhibit Src activity. EPS8 downstream targets were evaluated by western blotting. Wound closure, proliferation, immunofluorescence and tumorgenicity assays were used to investigate the impact of phenylalanine mutations on EPS8 biological functions. RESULTS: FOXM1, AURKA, and AURKB were decreased in cells expressing FFFF- and Y602F-EPS8 mutants, while cells harbouring the Y485F-, Y525F- and Y774F-EPS8 mutants showed no differences compared to controls. Consistent with this, dasatinib decreased the expression of EPS8 targets. Moreover, Y602F- and FFFF-EPS8 mutants reduced mitogenesis and motility. Strikingly though, FFFF- or Y602F-EPS8 mutants actually promoted tumorigenicity compared with control cells. CONCLUSIONS: Phosphorylation of EPS8 at Y602 is crucial for signalling to the cell cycle and may provide insight to explain reduced efficacy of dasatinib treatment.

IQGAP1 promotes anoikis resistance and metastasis through Rac1-dependent ROS accumulation and activation of Src/FAK signalling in hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) has a crucial role in the progression of hepatocellular carcinoma (HCC). Tumour cells must develop anoikis resistance in order to survive before metastasis. This study aimed to investigate the mechanism of IQGAP1 in HBV-mediated anoikis evasion and metastasis in HCC cells. METHODS: IQGAP1 expression was detected by immunohistochemistry, real-time PCR and immunoblot analysis. Lentiviral-mediated stable upregulation or knockdown of IGAQP1, immunoprecipitation, etc. were used in function and mechanism study. RESULTS: IQGAP1 was markedly upregulated in HBV-positive compared with HBV-negative HCC cells and tissues. IQGAP1 was positively correlated to poor prognosis of HBV-associated HCC patients. IQGAP1 overexpression significantly enhanced the anchorage-independent growth and metastasis, whereas IQGAP1-deficient HCC cells are more sensitive to anoikis. Mechanistically, we found that HBV-induced ROS enhanced the association of IQGAP1 and Rac1 that activated Rac1, leading to phosphorylation of Src/FAK pathway. Antioxidants efficiently inhibited IQGAP1-mediated anoikis resistance and metastasis. CONCLUSIONS: Our study indicated an important mechanism by which upregulated IQGAP1 by HBV promoted anoikis resistance, migration and invasion of HCC cells through Rac1-dependent ROS accumulation and activation of Src/FAK signalling, suggesting IQGAP1 as a prognostic indicator and a novel therapeutic target in HCC patients with HBV infection.

PAG1 directs SRC-family kinase intracellular localization to mediate receptor tyrosine kinase-induced differentiation.

All receptor tyrosine kinases (RTKs) activate similar downstream signaling pathways through a common set of effectors, yet it is not fully understood how different receptors elicit distinct cellular responses to cause cell proliferation, differentiation, or other cell fates. We tested the hypothesis that regulation of SRC family kinase (SFK) signaling by the scaffold protein, PAG1, influences cell fate decisions following RTK activation. We generated a neuroblastoma cell line expressing a PAG1 fragment that lacks the membrane-spanning domain (PAG1TM-) and localized to the cytoplasm. PAG1TM- cells exhibited higher amounts of active SFKs and increased growth rate. PAG1TM- cells were unresponsive to TRKA and RET signaling, two RTKs that induce neuronal differentiation, but retained responses to EGFR and KIT. Under differentiation conditions, PAG1TM- cells continued to proliferate and did not extend neurites or increase ß-III tubulin expression. FYN and LYN were sequestered in multivesicular bodies (MVBs), and dramatically more FYN and LYN were in the lumen of MVBs in PAG1TM- cells. In particular, activated FYN was sequestered in PAG1TM- cells, suggesting that disruption of FYN localization led to the observed defects in differentiation. The results demonstrate that PAG1 directs SFK intracellular localization to control activity and to mediate signaling by RTKs that induce neuronal differentiation.

YES1 amplification confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive cancer.

BACKGROUND: Trastuzumab-emtansine (T-DM1), one of the most potent HER2-targeted drugs, shows impressive efficacy in patients with HER2-positive breast cancers. However, resistance inevitably occurs and becomes a critical clinical problem. METHODS: We modelled the development of acquired resistance by exposing HER2-positive cells to escalating concentrations of T-DM1. Signalling pathways activation was detected by western blotting, gene expression was analysed by qRT-PCR and gene copy numbers were determined by qPCR. The role of Yes on resistance was confirmed by siRNA-mediated knockdown and stable transfection-mediated overexpression. The in vivo effects were tested in xenograft model. RESULTS: We found that Yes is overexpressed in T-DM1-resistant cells owing to amplification of chromosome region 18p11.32, where the YES1 gene resides. Yes activated multiple proliferation-related signalling pathways, including EGFR, PI3K and MAPK, and led to cross-resistance to all types of HER2-targeted drugs, including antibody-drug conjugate, antibody and small molecule inhibitor. The outcome of this cross-resistance may be a clinically incurable condition. Importantly, we found that inhibiting Yes with dasatinib sensitised resistant cells in vitro and in vivo. CONCLUSIONS: Our study revealed that YES1 amplification conferred resistance to HER2-targeted drugs and suggested the potential application of the strategy of combining HER2 and Yes inhibition in the clinic.

The Leptin induced Hic-5 expression and actin puncta formation by the FAK/Src-dependent pathway in MCF10A mammary epithelial cells / La leptina promueve la expresión de Hic-5 y la formación de puntos de actina por la vía dependiente de FAK-Src en células epiteliales mamarias MCF10A

Introduction: Leptin is a hormone secreted by adipocytes that has been associated with the epithelial-mesenchymal transition (EMT). Additionally, leptin promotes the migration and invasion of mammary epithelial cells through the activation of FAK and Src kinases, which are part of a regulatory complex of signaling pathways that promotes the expression of proteins related to the formation of proteolytic structures involved in the invasion and progression of cancer. Recently, overexpression and activation of Hic-5 during the EMT have been shown to induce the formation of actin puncta; these structures are indicative of the formation and functionality of invadopodia, which promote the local degradation of extracellular matrix components and cancer metastasis. Objective: To evaluate the role of FAK and Src kinases in the expression of Hic-5 during the epithelial-mesenchymal transition induced by leptin in MCF10A cells. Materials and methods: We used specific inhibitors of FAK (PF-573228) and Src (PP2) to evaluate Hic-5 expression and subcellular localization by Western blot and immunofluorescence assays and to investigate the formation of actin puncta by epifluorescence in MCF10A cells stimulated with leptin. Results: Leptin induced an increase in Hic-5 expression and the formation of actin puncta. Pretreatment with inhibitors of FAK (PF-573228) and Src (PP2) promoted a decrease in Hic-5 expression and actin puncta formation in the non-tumorigenic mammary epithelial cell line MCF10A. Conclusion: In MCF10A cells, leptin-induced Hic-5 expression and perinuclear localization, as well as the formation of actin puncta through a mechanism dependent on the kinase activity of FAK and Src.

Introducción. La leptina es una hormona secretada por los adipocitos que se ha relacionado con el proceso de la transición de epitelio a mesénquima (Epithelial-Mesenchymal Transition, EMT). Promueve la migración e invasión de las células del epitelio mamario mediante la activación de las cinasas FAK y Src, un complejo regulador de vías de señalización que favorecen la expresión de las proteínas relacionadas con la formación de estructuras proteolíticas implicadas en la invasión y progresión del cáncer. Recientemente, se ha descrito que la sobreexpresión y activación de la proteína Hic-5 durante el mencionado proceso de transición, favorece la formación de los puntos de actina (indicativa de la formación y funcionalidad de los invadopodios), lo cual promueve la degradación local de los componentes de la matriz extracelular y la metástasis del cáncer. Objetivos. Evaluar el papel de las cinasas FAK y Src sobre la expresión y localización subcelular de Hic-5 y la formación de puntos de actina inducida por la leptina en la línea celular MCF10A de epitelio mamario no tumoral. Materiales y métodos. Se utilizaron los inhibidores específicos de la FAK (PF-573228) y la Src (PP2) para evaluar el papel de ambas cinasas en los niveles de expresión y localización subcelular de la proteína Hic-5 mediante Western blot e inmunofluorescencia, así como la formación de puntos de actina mediante la tinción con faloidina-TRITC en células MCF10A estimuladas con leptina. Resultados. La leptina indujo el incremento en la expresión de Hic-5 y la formación de puntos de actina. El tratamiento previo con los inhibidores de las cinasas FAK (PF-573228) y Src (PP2), promovió la disminución en la expresión de Hic-5 y de los puntos de actina en la línea celular MCF10A de epitelio mamario no tumoral. Conclusión. La leptina indujo la expresión y la localización perinuclear de Hic-5 y la formación de puntos de actina mediante un mecanismo dependiente de la actividad de las cinasas FAK y Src en las células MCF10A.

La leptina promueve la expresión de Hic-5 y la formación de puntos de actina por la vía dependiente de FAK-Src en células epiteliales mamarias MCF10A / Leptin induced Hic-5 expression and actin puncta formation by the FAK/Src-dependent pathway in MCF10A mammary epithelial cells

Resumen Introducción. La leptina es una hormona secretada por los adipocitos que se ha relacionado con el proceso de la transición de epitelio a mesénquima (Epithelial- Mesenchymal Transition, EMT). Promueve la migración e invasión de las células del epitelio mamario mediante la activación de las cinasas FAK y Src, un complejo regulador de vías de señalización que favorecen la expresión de las proteínas relacionadas con la formación de estructuras proteolíticas implicadas en la invasión y progresión del cáncer. Recientemente, se ha descrito que la sobreexpresión y activación de la proteína Hic-5 durante el mencionado proceso de transición, favorece la formación de los puntos de actina (indicativa de la formación y funcionalidad de los invadopodios), lo cual promueve la degradación local de los componentes de la matriz extracelular y la metástasis del cáncer. Objetivos. Evaluar el papel de las cinasas FAK y Src sobre la expresión y localización subcelular de Hic-5 y la formación de puntos de actina inducida por la leptina en la línea celular MCF10A de epitelio mamario no tumoral. Materiales y métodos. Se utilizaron los inhibidores específicos de la FAK (PF-573228) y la Src (PP2) para evaluar el papel de ambas cinasas en los niveles de expresión y localización subcelular de la proteína Hic-5 mediante Western blot e inmunofluorescencia, así como la formación de puntos de actina mediante la tinción con faloidina-TRITC en células MCF10A estimuladas con leptina. Resultados. La leptina indujo el incremento en la expresión de Hic-5 y la formación de puntos de actina. El tratamiento previo con los inhibidores de las cinasas FAK (PF-573228) y Src (PP2), promovió la disminución en la expresión de Hic-5 y de los puntos de actina en la línea celular MCF10A de epitelio mamario no tumoral. Conclusión. La leptina indujo la expresión y la localización perinuclear de Hic-5 y la formación de puntos de actina mediante un mecanismo dependiente de la actividad de las cinasas FAK y Src en las células MCF10A.

Abstract Introduction: Leptin is a hormone secreted by adipocytes that has been associated with the epithelial-mesenchymal transition (EMT). Additionally, leptin promotes the migration and invasion of mammary epithelial cells through the activation of FAK and Src kinases, which are part of a regulatory complex of signaling pathways that promotes the expression of proteins related to the formation of proteolytic structures involved in the invasion and progression of cancer. Recently, overexpression and activation of Hic-5 during the EMT have been shown to induce the formation of actin puncta; these structures are indicative of the formation and functionality of invadopodia, which promote the local degradation of extracellular matrix components and cancer metastasis. Objective: To evaluate the role of FAK and Src kinases in the expression of Hic-5 during the epithelial-mesenchymal transition induced by leptin in MCF10A cells. Materials and methods: We used specific inhibitors of FAK (PF-573228) and Src (PP2) to evaluate Hic-5 expression and subcellular localization by Western blot and immunofluorescence assays and to investigate the formation of actin puncta by epifluorescence in MCF10A cells stimulated with leptin. Results: Leptin induced an increase in Hic-5 expression and the formation of actin puncta. Pretreatment with inhibitors of FAK (PF-573228) and Src (PP2) promoted a decrease in Hic-5 expression and actin puncta formation in the non-tumorigenic mammary epithelial cell line MCF10A. Conclusion: In MCF10A cells, leptin-induced Hic-5 expression and perinuclear localization, as well as the formation of actin puncta through a mechanism dependent on the kinase activity of FAK and Src.

[The Involvement of Src in Airway Inflammation Induced by Repeated Exposure to Lipopolysaccharide in Mice].

Corticosteroid insensitive airway inflammation is one of major barrier to effective managements of chronic airway diseases, such as chronic obstructive pulmonary disease (COPD) and severe asthma. The role of nonreceptor tyrosine kinase Src is important in airway inflammation in mice models of atopic asthma and COPD. Thus, in this study, we determined the effects of Src inhibitor, dasatinib, on airway inflammation induced by repeated intranasal exposure to lipopolysaccharide (LPS). Male mice (A/J strain, 5 weeks old) were intranasally exposed to LPS twice daily for 3 d, and dasatinib was intranasally treated 2 h prior to each LPS exposure. A day after the last stimulation, lungs and bronchoalveolar lavage fluid (BALF) were collected. Dasatinib attenuated the accumulation of inflammatory cells in lungs, and the increase in the numbers of inflammatory cells and the accumulation of cytokines/chemokines in BALF in a dose dependent manner. Therefore, this study suggested that targeting the Src can provide a new therapeutic approach for corticosteroid insensitive pulmonary diseases.

Nonphosphorylatable Src Ser75 Mutation Increases Ethanol Preference and Consumption in Mice.

Src is highly expressed in CNS neurons and contributes not only to developmental proliferation and differentiation but also to high-order brain functions, such as those contributing to alcohol consumption. Src knock-out mice exhibit no CNS abnormalities, presumably due to compensation by other Src family kinases (SFKs), but have a shortened lifespan and osteopetrosis-associated defects, impeding investigations of the role of Src on behavior in adult mice. However, the Unique domain of Src differs from those in other SFKs and is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75, which influences its postmitotic function in neurons. Therefore, ethanol consumption in mice harboring nonphosphorylatable (Ser75Ala) or phosphomimetic (Ser75Asp) Src mutants was investigated. Mice harboring the Ser75Ala Src mutant, but not the Ser75Asp mutant, had a higher preference for and consumption of solutions containing 5% and 10% ethanol than wild-type mice. However, plasma ethanol concentrations and sensitivities to the sedative effects of ethanol were not different among the groups. In mice harboring the Ser75Ala Src mutant, the activity of Rho-associated kinase (ROCK) in the striatum was significantly lower and Akt Ser473 phosphorylation was significantly higher than in wild-type mice. These results suggest that Src regulates voluntary ethanol drinking in a manner that depends on Ser75 phosphorylation.

Src deficient mice demonstrate behavioral and electrophysiological alterations relevant to psychiatric and developmental disease.

Much evidence suggests that hypofunction of the N-methyl-d-aspartate glutamate receptor (NMDAR) may contribute broadly towards a subset of molecular, cognitive and behavioral abnormalities common among psychiatric and developmental diseases. However, little is known about the specific molecular changes that lead to NMDAR dysfunction. As such, personalized approaches to remediating NMDAR dysfunction based on a specific etiology remains a challenge. Sarcoma tyrosine kinase (Src) serves as a hub for multiple signaling mechanisms affecting GluN2 phosphorylation and can be disrupted by convergent alterations of various signaling pathways. We recently showed reduced Src signaling in post mortem tissue from schizophrenia patients, despite increased MK-801 binding and NMDA receptor complex expression in the postsynaptic density (PSD). These data suggest that Src dysregulation may be an important underlying mechanism responsible for reduced glutamate signaling. Despite this evidence for a central role of Src in NMDAR signaling, little is known about how reductions in Src activity might regulate phenotypic changes in cognition and behavior. As such, the current study sought to characterize behavioral and electrophysiological phenotypes in mice heterozygous for the Src Acl gene (Src+/- mice). Src+/- mice demonstrated decreased sociability and working memory relative to Src+/+ (WT) mice while no significant differences were seen on locomotive activity and anxiety-related behavior. In relation to WT mice, Src+/- mice showed decreased mid-latency P20 auditory event related potential (aERP) amplitudes, decreased mismatch negativity (MMN) and decreased evoked gamma power, which was only present in males. These data indicate that Src+/- mice are a promising new model to help understand the pathophysiology of these electrophysiological, behavioral and cognitive changes. As such, we propose that Src+/- mice can be used in the future to evaluate potential therapeutic approaches by targeting increased Src activity as a common final pathway for multiple etiologies of SCZ and other diseases characterized by reduced glutamate function.