Regulation of Rac1 activation by the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.     

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.     

Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.     

Mitogen-activated protein kinase activity may not be necessary for the neuropathology of Niemann-Pick type C mice

Hyperphosphorylation of neurofilament and tau, and formation of cytoskeletal lesions, are notable features of several human neurodegenerative diseases, including Niemann-Pick Disease Type C (NPC). Previous studies suggested that the MAPKs, extracellular signal regulated kinase 1 and 2 (ERK1/2) may play a significant role in this aspect of NPC. To test this idea, we treated npc mice with PD98059, a specific and potent inhibitor of MAPK activation. Although activity of ERK1/2 was inhibited by 40%, a 2-week intracerebroventricular infusion of PD98059 just prior to onset of cytoskeletal pathology and symptoms in npc mice did not delay or inhibit prominent hallmarks of NPC. Unexpectedly, ERK1/2 inhibition led to aggravation of tau hyperphosphorylation, particularly in oligodendroctyes, in a manner similar to that of certain human tauopathies. Our results suggest that ERK1/2 does not play a major role in NPC neuropathology, and therefore, that MAPK inhibition is unlikely to be a useful strategy for managing the disease.     

Requirement of the enzymatic and signaling activities of plasmin for phorbol-ester-induced scattering of colon cancer cells

Colon cancer progression is associated with the activation of protein kinase C (PKC), the downregulation of functional E-cadherin and an increased expression of the serine protease urokinase (u-PA) and its receptor (u-PAR). HT29-M6 intestinal epithelial cells represent an in vitro model to study colon cancer progression. These cells are induced to scatter and to invade by phorbol esters. Using proteolytic and cell signaling inhibitors, we show that HT29-M6 cells require plasminogen for the acquisition of the scattering response to PMA. Our results indicate that, prior to inducing a state of competency for plasminogen-dependent scattering, PMA triggers an ordered succession of events where upregulation of the activity of u-PA precedes proteolysis of u-PAR and active degradation of the extracellular matrix (ECM). These events poise HT29-M6 cells to a scatter-competent state that allows the subsequent localized proteolytic activation of plasminogen to plasmin, required for the execution of scattering. Finally, we show that, in addition to its enzymatic activity directed at the degradation of ECM, plasmin generates an intracellular signal resulting in the phosphorylation of ERK 1/2. For a full motogenic activity, plasmin requires this signal since the use of a MEK inhibitor (PD98059) specifically blocks the plasmin-dependent phase of cell scattering. Our observations suggest that plasmin exerts a dual role in PMA-induced scattering of HT29-M6 cells, one directed extracellularly to promote proteolysis of the ECM and one directed to generate intracellular signaling.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

Flavanone and 2'-OH flavanone inhibit metastasis of lung cancer cells via down-regulation of proteinases activities and MAPK pathway

Flavanones richly exist in citrus and have been well characterized to have various bioactive properties. However, the anti-metastasis properties of flavanones remain unclear. The anti-metastatic effects of six flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, naringin, and naringenin were investigated in lung cancer cells. Despite little influence on cell viability, flavanone and 2'-OH flavanone markedly inhibited the invasion, motility, and cell-matrix adhesion of A549 cells. This was associated with a reduced expression of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) in treated cells. Treatment with flavanone and 2'-OH flavanone also potently attenuated the phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38(MAPK), as well as the activations of NF-kappaB and AP-1. The reduced expressions of MMP-2 and u-PA, as well as inhibition of cell invasion were obtained in the cultures treated with U0126 (ERK 1/2 inhibitor) and SB203580 (p38(MAPK) inhibitor). Thus, the inhibitory effects of flavanone and 2'-OH flavanone on the expression of MMP-2 and u-PA may be at least partly through inactivation of ERK 1/2 and p38(MAPK) signaling pathways. Finally, oral administration of flavanone and 2'-OH flavanone were evidenced by its inhibition on the metastasis of A549 cells and Lewis lung carcinoma (LLC) cells in vivo. In conclusion, flavanone and 2'-OH flavanone perturb the invasion and metastasis of lung cancer cells, thereby constituting an adjuvant treatment for metastasis control.     

Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA)

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 μM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.     

Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.     

Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs up-regulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.     

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.     

Hic-5, an adaptor protein expressed in vascular smooth muscle cells, modulates the arterial response to injury in vivo

Focal adhesion components are targets for biochemical and mechanical stimuli that evoke crucial injury. Hic-5 (hydrogen peroxide-inducible clone 5) is a multidomain adaptor protein which is implicated in the regulation of integrin signaling in focal adhesion. The aim of this research was to test the hypothesis that Hic-5, a focal adhesion LIM protein expressed in smooth muscle cells, is involved in dynamic processes by pathological stimuli in the vessel wall. Here, we describe the analysis of the function of Hic-5 using a mouse model of vascular injury that may mimic balloon angioplasty. At 4 days after vascular injury, marked down-regulation of the Hic-5 expression was observed in the smooth muscle layer, and local delivery of the Hic-5 using adenovirus vectors repressed injury-induced neointimal expansion. In addition, Hic-5 reduced cells migration into three-dimensional collagen gels, and the forced expression of Hic-5 in cells embedded in the collagen gel matrix repressed the expression of uPA that participates in smooth muscle cell migration. These results suggest that Hic-5 modulates cellular responses to pathological stimuli in the vessel wall.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Live-cell imaging of tumor proteolysis: Impact of cellular and non-cellular microenvironment

Our laboratory has had a longstanding interest in how the interactions between tumors and their microenvironment affect malignant progression. Recently, we have focused on defining the proteolytic pathways that function in the transition of breast cancer from the pre-invasive lesions of ductal carcinoma in situ (DCIS) to invasive ductal carcinomas (IDCs). We use live-cell imaging to visualize, localize and quantify proteolysis as it occurs in real-time and thereby have established roles for lysosomal cysteine proteases both pericellularly and intracellularly in tumor proteolysis. To facilitate these studies, we have developed and optimized 3D organotypic co-culture models that recapitulate the in vivo interactions of mammary epithelial cells or tumor cells with stromal and inflammatory cells. Here we will discuss the background that led to our present studies as well as the techniques and models that we employ. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Involvement of phosphatidylcholine-selective phospholipase C in activation of mitogen-activated protein kinase pathways in imidazoline receptor antisera-selected protein

Imidazoline receptor antisera-selected protein (IRAS) is considered as a candidate for the I1-imidazoline receptor (I1R), but the signaling pathway mediated by IRAS remains unknown. In our study, the signal transduction pathways of IRAS were investigated in CHO cells stably expressing IRAS (CHO-IRAS), and compared to the native I1R signaling pathways. Rilmenidine or moxonidine (10 nM-100 microM), I1R agonists, failed to stimulate [35S]-GTPgammaS binding in CHO-IRAS cell membrane preparations, suggesting that G protein may not be involved in IRAS signaling pathway. However, incubation of CHO-IRAS with rilmenidine or moxonidine for 5 min could induce an upregulation of phosphatidylcholine-selective phospholipase C (PC-PLC) activity, and an increase in the accumulation of diacylglycerol (DAG), the hydrolysate of PC-PLC, in a concentration-dependent manner. The elevated activation of PC-PLC by rilmenidine or moxonidine (100 nM) could be blocked by efaroxan, a selective I1R antagonist. Cells treated with rilmenidine or moxonidine showed an increased level of extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, which could be reversed by efaroxan or D609, a selective PC-PLC inhibitor. These results suggest that the signaling pathway of IRAS in response to I1R agonists coupled with the activation of PC-PLC and its downstream signal transduction molecule, ERK. These findings are similar to those in the signaling pathways of native I1R, providing some new evidence for the relationship between I1R and IRAS.     

SUMO-1 modification of centrosomal protein hNinein promotes hNinein nuclear localization

A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.     

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.