A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease

Elevated c-Src is linked to altered cell–matrix adhesion rather than proliferation in KM12C human colorectal cancer cells

Elevated expression and/or activity of c-Src, the prototype of the Src family of protein tyrosine kinases, is associated with the development of human colon cancer. However, despite the known pleiotropic effects of these kinases in promoting (a) cell growth downstream of growth factor receptors, and (b) the dynamic regulation of integrin adhesions in fibroblast model systems, their precise role in epithelial cancer cells is unknown. Here we addressed whether elevated expression and activity of cellular Src alters cell proliferation and/or cell–matrix adhesion in cancer cells from the Fidler model of colorectal metastasis. Although elevated Src correlates with ability to metastasise to the liver after intrasplenic injection, we found that this was not linked to enhanced growth, either in vitro or in vivo as sub-cutaneous tumours. However, elevated Src was associated with enhanced attachment to extracellular matrix. In addition, adhesion to fibronectin, was suppressed by agents that inhibited Src activity, while enforced elevation of Src in non-metastatic cells was sufficient to stimulate adhesion to fibronectin and enhanced assembly of adhesion complexes, without influencing cell growth. Thus, we conclude that one role of elevated Src in human colon cancer cells is to modulate integrin-dependent cell–matrix attachment and formation of adhesion structures, which may, in turn, influence cell motility and integrin-dependent cellular responses

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

<p>Abstract</p> <p>Background</p> <p>Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.</p> <p>Results</p> <p>We show here that SYF cells (mouse fibroblasts lacking the NTKs Src, Yes and Fyn) exhibit a low level of basal tyrosine phosphorylation at focal adhesions. FIT-mediated tyrosine phosphorylation of NTK substrates p130Cas, paxillin and FAK and cortactin was observed at focal adhesions, while FIT-mediated phosphorylation of cortactin was also seen at the cell periphery. Phosphorylation of p130Cas in SYF cells led to activation of Rac1 and increased membrane ruffling and lamellipodium formation, events associated with cell migration. We also found that the kinase activity of Src and not FAK is essential for phosphorylation of p130Cas when the three proteins exist as a complex in focal adhesions.</p> <p>Conclusion</p> <p>These results demonstrate that tyrosine phosphorylation of p130Cas is sufficient for its localization to focal adhesions and for activation of downstream signaling events associated with cell migration. FIT provides a valuable tool to evaluate the contribution of individual components of the response to signals with multiple outputs, such as activation of NTKs.</p

Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains Differentially Modulates Src Kinase Activity in Brain Maturation

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1-/- mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (−20–30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1-/- mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain

SHP-2 Promotes the Maturation of Oligodendrocyte Precursor Cells Through Akt and ERK1/2 Signaling In Vitro

Background: Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive. Methods and Findings: In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, overexpression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes

PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

<p>Abstract</p> <p>Background</p> <p>Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because <it>pdgfa</it>-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).</p> <p>Methods</p> <p>Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.</p> <p>Results</p> <p>The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.</p> <p>Conclusion</p> <p>During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.</p

Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

<p>Abstract</p> <p>Background</p> <p>Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD). One hypothesis is that amyloid beta (Aβ) peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD.</p> <p>Methods</p> <p>Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis.</p> <p>Results</p> <p>Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals.</p> <p>Conclusions</p> <p>These data suggest that amyloid dependent microgliosis may be Src kinase dependent <it>in vitro</it> and <it>in vivo.</it> This study defines a role for Src kinase in the microgliosis characteristic of diseased brains and suggests that particular tyrosine kinase inhibition may be a valid anti-inflammatory approach to disease. Dasatinib is an FDA-approved drug for treating chronic myeloid leukemia cancer with a reported ability to cross the blood-brain barrier. Therefore, this suggests a novel use for this drug as well as similar acting molecules.</p

Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis

Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS