MMP‐2 and MMP‐13 affect vasculogenic mimicry formation in large cell lung cancer

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP‐13 and MMP‐2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln‐5) fragments cleaved by MMP‐2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three‐dimensional (3D) cultures. Transient up‐regulation of MMP‐13 or treatment with recombinant MMP‐13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln‐5 fragments cleaved by MMP‐2 stimulated EGFR and F‐actin expression. Ln‐5 fragments cleaved by MMP‐13 decreased EGFR/F‐actin expression and disrupted VM formation. MMP‐13 expression was negatively correlated with VM, Ln‐5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium‐dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP‐13 expression was progressively increased. In conclusion, MMP‐2 promoted and MMP‐13 disrupted VM formation in LCLC by cleaving Ln‐5 to influence EGFR signal activation. MMP‐13 may regulate VM and EDV formation.     

Targeting the ROS/PI3K/AKT/HIF‐1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2‐Deoxy‐d‐glucose

It is well established that cancer cells depend upon aerobic glycolysis to provide the energy they need to survive and proliferate. However, anti‐glycolytic agents have yielded few positive results in human patients, in part due to dose‐limiting side effects. Here, we discovered the unexpected anti‐cancer efficacy of Polydatin (PD) combined with 2‐deoxy‐D‐glucose (2‐DG), which is a compound that inhibits glycolysis. We demonstrated in two breast cell lines (MCF‐7 and 4T1) that combination treatment with PD and 2‐DG induced cell apoptosis and inhibited cell proliferation, migration and invasion. Furthermore, we determined the mechanism of PD in synergy with 2‐DG, which decreased the intracellular reactive oxygen (ROS) levels and suppressed the PI3K/AKT pathway. In addition, the combined treatment inhibited the glycolytic phenotype through reducing the expression of HK2. HK2 deletion in breast cancer cells thus improved the anti‐cancer activity of 2‐DG. The combination treatment also resulted in significant tumour regression in the absence of significant morphologic changes in the heart, liver or kidney in vivo. In summary, our study demonstrates that PD synergised with 2‐DG to enhance its anti‐cancer efficacy by inhibiting the ROS/PI3K/AKT/HIF‐1α/HK2 signalling axis, providing a potential anti‐cancer strategy.     

Targeted inhibition of the EGFR pathways enhances Zn‐BC‐AM PDT‐induced apoptosis in well‐differentiated nasopharyngeal carcinoma cells

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn‐BC‐AM PDT with an EGFR inhibitor AG1478 were investigated. Well‐differentiated NPC HK‐1 cells were subjected to PDT with 1 µM of Zn‐BC‐AM and were irradiated at a light dose of 1 J/cm² in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK‐1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub‐lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn‐BC‐AM PDT in HK‐1 cells. Pre‐incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn‐BC‐AM PDT‐induced formation of apoptotic cells. The efficacy of Zn‐BC‐AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn‐BC‐AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356–1363, 2009. © 2009 Wiley‐Liss, Inc.     

Beta‐elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.     

Sodium butyrate inhibits aerobic glycolysis of hepatocellular carcinoma cells via the c‐myc/hexokinase 2 pathway

Aerobic glycolysis is a well‐known hallmark of hepatocellular carcinoma (HCC). Hence, targeting the key enzymes of this pathway is considered a novel approach to HCC treatment. The effects of sodium butyrate (NaBu), a sodium salt of the short‐chain fatty acid butyrate, on aerobic glycolysis in HCC cells and the underlying mechanism are unknown. In the present study, data obtained from cell lines with mouse xenograft model revealed that NaBu inhibited aerobic glycolysis in the HCC cells in vivo and in vitro. NaBu induced apoptosis while inhibiting the proliferation of the HCC cells in vivo and in vitro. Furthermore, the compound inhibited the release of lactate and glucose consumption in the HCC cells in vitro and inhibited the production of lactate in vivo. The modulatory effects of NaBu on glycolysis, proliferation and apoptosis were related to its modulation of hexokinase 2 (HK2). NaBu downregulated HK2 expression via c‐myc signalling. The upregulation of glycolysis in the HCC cells induced by sorafenib was impeded by NaBu, thereby enhancing the anti‐HCC effect of sorafenib in vitro and in vivo. Thus, NaBu inhibits the expression of HK2 to downregulate aerobic glycolysis and the proliferation of HCC cells and induces their apoptosis via the c‐myc pathway.     

Novel mitochondrion-targeting copper(II) complex induces HK2 malfunction and inhibits glycolysis via Drp1-mediating mitophagy in HCC

[Cu(ttpy-tpp)Br₂]Br (abbreviated as CTB) is a novel mitochondrion-targeting copper(II) complex synthesized by our research group, which contains tri-phenyl-phosphonium (TPP) groups as its lipophilic property. In this study, we explored how CTB affects mitochondrial functions and exerts its anti-tumour activity. Multiple functional and molecular analyses including Seahorse XF Bioanalyzer Platform, Western blot, immunofluorescence analysis, co-immunoprecipitation and transmission electron microscopy were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo. We discovered that CTB inhibited aerobic glycolysis and cell acidification by impairing the activity of HK2 in hepatoma cells, accompanied by dissociation of HK2 from mitochondria. The modification of HK2 not only led to the complete dissipation of mitochondrial membrane potential (MMP) but also promoted the opening of mitochondrial permeability transition pore (mPTP), contributing to the activation of mitophagy. In addition, CTB co-ordinately promoted dynamin-related protein 1 (Drp1) recruitment in mitochondria to induce mitochondrial fission. Our findings established a previously unrecognized role for copper complex in aerobic glycolysis of tumour cells, revealing the interaction between mitochondrial HK2-mediated mitophagy and Drp1-regulated mitochondrial fission.     

Non‐muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)

Non‐muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF‐alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365–1375, 2010. © 2010 Wiley‐Liss, Inc.     

INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.     

miR‐134 inhibits non‐small cell lung cancer growth by targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti‐EGFR therapies. However, more EGFR‐targeting miRNAs need to be explored. In this study, we identified a novel EGFR‐targeting miRNA, miRNA‐134 (miR‐134), in non‐small‐cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR‐134. In addition, the overexpression of miR‐134 inhibited EGFR‐related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR‐134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down‐regulation of EGFR by miR‐134 partially contributes to the antiproliferative role of miR‐134. Last, in vivo experiments demonstrated that miR‐134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR‐134 inhibits non‐small cell lung cancer growth by targeting the EGFR.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.     

Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy.     

Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells

Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA‐MB‐231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4‐induced increased glucose uptake. J. Cell. Physiol. 228: 1943–1948, 2013. © 2013 Wiley Periodicals, Inc.     

ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT/β-catenin/HK2 axis

Abnormal energy metabolism is one of the characteristics of tumours. In the last few years, more and more attention is being paid to the role and regulation of tumour aerobic glycolysis. Cancer cells display enhanced aerobic glycolysis, also known as the Warburg effect, whereby tumour cells absorb glucose to produce a large amount of lactic acid and energy under aerobic conditions to favour tumour proliferation and metastasis. In this study, we report that the haploinsufficient tumour suppressor ASPP2, can inhibit HCC growth and stemness characteristics by regulating the Warburg effect through the WNT/β-catenin pathway. we performed glucose uptake, lactate production, pyruvate production, ECAR and OCR assays to verify ASPP2 can inhibit glycolysis in HCC cells. The expression of ASPP2 and HK2 was significantly inversely correlated in 80 HCC tissues. Our study reveals downregulation of ASPP2 can promote the aerobic glycolysis metabolism pathway, increasing HCC proliferation, glycolysis metabolism, stemness and drug resistance. This ASPP2-induced inhibition of glycolysis metabolism depends on the WNT/β-catenin pathway. ASPP2-regulated Warburg effect is associated with tumour progression and provides prognostic value. and suggest that may be promising as a new therapeutic strategy in HCC.     

Targeting HLA-F suppresses the proliferation of glioma cells via a reduction in hexokinase 2-dependent glycolysis

HLA-F, a nonclassical HLA class I molecule, is required for regulating immune tolerance. In recent years, HLA-F has been found to play a role in a variety of cancers, including glioma (GM). Additionally, high expression of HLA-F predicts the poor overall survival of individuals with GM. However, the functions of HLA-F in GM remain to be further elucidated. In this study, we found that HLA-F expression was elevated in GM tissues. High levels of HLA-F resulted in a high cell proliferation index and predicted GM recurrence. Forced expression of HLA-F promoted the growth of murine C8-D1A cells transplanted in immunodeficient Rag2^-/- mice. In contrast, silencing HLA-F inhibited cell growth in vitro. Furthermore, targeting HLA-F with an anti-HLA-F antibody suppressed the growth of C8-D1A cells stably expressing HLA-F transplanted in immunodeficient Rag2^-/- mice. In further experiments, we found that forced expression of HLA-F contributed to the aerobic glycolysis phenotype in C8-D1A cells along with an increase in HK2 protein stabilization. Conversely, silencing HK2 by shRNA reduced HLA-F-mediated glycolysis and cell proliferation. Our data indicated that HLA-F promoted cell proliferation via HK2-dependent glycolysis. HLA-F could be a potential therapeutic target for the treatment of GM.     

Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

Positive feedback in Cav‐1‐ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells

Caveolin‐1 (Cav‐1) is the principal structural component of caveolae, and its dysregulation occurs in cancer. However, the role of Cav‐1 in pancreatic cancer (PDAC) tumorigenesis and metabolism is largely unknown. In this study, we aimed to investigate the effect of pancreatic stellate cell (PSC) Cav‐1 on PDAC metabolism and aggression. We found that Cav‐1 is expressed at low levels in PDAC stroma and that the loss of stromal Cav‐1 is associated with poor survival. In PSCs, knockdown of Cav‐1 promoted the production of reactive oxygen species (ROS), while ROS production further reduced the expression of Cav‐1. Positive feedback occurs in Cav‐1‐ROS signalling in PSCs, which promotes PDAC growth and induces stroma‐tumour metabolic coupling in PDAC. In PSCs, positive feedback in Cav‐1‐ROS signalling induced a shift in energy metabolism to glycolysis, with up‐regulated expression of glycolytic enzymes (hexokinase 2 (HK‐2), 6‐phosphofructokinase (PFKP) and pyruvate kinase isozyme type M2 (PKM2)) and transporter (Glut1) expression and down‐regulated expression of oxidative phosphorylation (OXPHOS) enzymes (translocase of outer mitochondrial membrane 20 (TOMM20) and NAD(P)H dehydrogenase [quinone] 1 (NQO1)). These events resulted in high levels of glycolysis products such as lactate, which was secreted by up‐regulated monocarboxylate transporter 4 (MCT4) in PSCs. Simultaneously, PDAC cells took up these glycolysis products (lactate) through up‐regulated MCT1 to undergo OXPHOS, with down‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1). Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.     

Low‐concentration heparin suppresses ionomycin‐activated CAMK‐II/EGF receptor‐ and ERK‐mediated signaling in mesangial cells

Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK‐II. Many studies have used heparin at concentrations of 100 µg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low‐concentration (1 µg/ml) heparin on activation of several kinases and subsequent induction of the c‐fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK‐II (by 30 s), and subsequently of the EGF receptor (EGFR), c‐Src, and Erk 1/2. Low‐dose heparin suppresses the ionomycin‐dependent phosphorylation of EGFR, c‐Src, and Erk 1/2, but not of CaMK‐II, whereas inhibition of activated CaMK‐II reduces phosphorylation of EGFR, c‐Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK‐II, including EGFR, leading in turn to a decrease in Erk‐ (but not c‐Src‐) dependent induction of c‐fos. J. Cell. Physiol. 224: 484–490, 2010. © 2010 Wiley‐Liss, Inc.     

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways.