Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions

The core fucosylation (alpha1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha1,6-fucosylation has been further reported to be very crucial by the study of alpha1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha3beta1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha3beta1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha3beta1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha3beta1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha3beta1 integrin.     

Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in Fut8-/- mice

alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine residue of N-glycans. Here we report that the loss of core fucosylation impairs the function of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), a multifunctional scavenger and signaling receptor, resulting in a reduction in the endocytosis of insulin like growth factor (IGF)-binding protein-3 (IGFBP-3) in the cells derived from Fut8-null (Fut8-/-) mice. The reduced endocytosis was restored by the re-introduction of Fut8. Serum levels of IGFBP-3 were markedly upregulated in Fut8-/- mice. These data clearly indicate that core fucosylation is crucial for the scavenging activity of LRP-1 in vivo.     

Reduced alpha4beta1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in alpha 1,6-fucosyltransferase deficient mice

Mice with a targeted gene disruption of Fut8 (Fut8(-/-)) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4, alpha4beta1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8(-/-) mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8(-/-) pre-B cells. Indeed, the frequency of preBCR(+)CD79b(low) cells in bone marrow pre-B cells in Fut8(-/-) was much lower than that in Fut8(+/+) cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.     

Blocking core fucosylation of TGF-β1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells

Posttranslational modification of proteins could regulate their multiple biological functions. Transforming growth factor-β receptor I and II (ALK5 and TGF-βRII), which are glycoproteins, play important roles in the renal tubular epithelial-mesenchymal transition (EMT). In the present study, we examined the role of core fucosylation of TGF-βRII and ALK5, which is regulated by α-1,6 fucosyltransferase (Fut8), in the process of EMT of cultured human renal proximal tubular epithelial (HK-2) cells. The typical cell model of EMT induced by TGF-β1 was constructed to address the role of core fucosylation in EMT. Core fucosylation was found to be essential for both TGF-βRII and ALK5 to fulfill their functions, and blocking it with Fut8 small interfering RNA greatly reduced the phosphorylation of Smad2/3 protein, caused the inactivation of TGF-β/Smad2/3 signaling, and resulted in remission of EMT. More importantly, even with high levels of expressions of TGF-β1, TGF-βRII, and ALK5, blocking core fucosylation also could attenuate the EMT of HK-2 cells. Thus blocking core fucosylation of TGF-βRII and ALK5 may attenuate EMT independently of the expression of these proteins. This study may provide new insight into the role of glycosylation in renal interstitial fibrosis. Furthermore, core fucosylation may be a novel potential therapeutic target for treatment of renal tubular EMT.     

Core fucosylation of μ heavy chains regulates assembly and intracellular signaling of precursor B cell receptors

α1,6-Fucosyltransferase (Fut8) knock-out (Fut8(-/-)) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint for pre-B cell differentiation and proliferation in both humans and mice. The assembly of pre-BCR on the cell surface was substantially blocked in the Fut8-knockdown pre-B cell line, 70Z/3-KD cells, and then completely restored by re-introduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re) cells. Moreover, loss of α1,6-fucosylation (also called core fucosylation) of μHC was associated with the suppression of the interaction between μHC and λ5. In contrast to Fut8(+/+) CD19(+)CD43(-) cells, the subpopulation expressing the μHC·λ5 complex in the Fut8(-/-) CD19(+)CD43(-) cell fraction was decreased. The pre-BCR-mediated tyrosine phosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells. The frequency of CD19(low)CD43(-) cells (pre-B cell enriched fraction) was also reduced in Fut8(-/-) bone marrow cells, and then the levels of IgM, IgG, and IgA of 12-week-old Fut8(-/-) mice sera were significantly lower than those of Fut8(+/+) mice. Our results suggest that the core fucosylation of μHC mediates the assembly of pre-BCR to regulate pre-BCR intracellular signaling and pre-B cell proliferation.     

Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression

We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.     

Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems

Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8(-/-)) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-β1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8(-/-) mice using a combination of neurological and behavioral tests. Fut8(-/-) mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8(-/-) mice displayed increased locomotion compared with wild-type (Fut8(+/+)) and heterozygous (Fut8(+/-)) mice. In particular, Fut8(-/-) mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8(-/-) mice as evidenced by the Y-maze tests. Furthermore, Fut8(-/-) mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8(+/+) and Fut8(+/-) mice in the PPI test under normal conditions, Fut8(+/-) mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8(-/-) mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders.     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Oncostatin M suppresses EGF-mediated protein tyrosine phosphorylation in breast cancer cells

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.     

Overexpression of phospho mutant forms of transglutaminase 2 downregulates epidermal growth factor receptor

Simultaneous upregulation of transglutaminase 2 (TG2) and epidermal growth factor receptor (EGFR) have been reported in a number of systems. Moreover, TG2 has been identified as a downstream target gene for EGF/EGFR. However, it is not known whether the relationship between EGFR and TG2 is only one-way or collaborative. Using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)), co-overexpressing native TG2 and EGFR, here we report that TG2 differentially regulates EGFR protein in the presence and absence of EGF. In the absence of EGF, TG2 facilitates EGFR downregulation whereas in the presence of EGF, TG2 has opposite effect on EGFR and facilitates Akt phosphorylation. TG2 mediated ligand-independent downregulation of EGFR was not observed in MEF(tg2-/-) cells overexpressing Ser212Ala phospho mutant form of TG2 suggesting a role of TG2 phosphorylation in this process. However, similar to native TG2, Ser212Ala-TG2 mutant was also able to attenuate ligand-dependent down regulation of EGFR in MEF(tg2-/-) cells. Interestingly, overexpression of Ser216Ala-TG2 mutant led to downregulation of EGFR in MEF(tg2-/-) cells irrespective of the ligand. These results were further confirmed in breast cancer cells expressing high levels of EGFR. Collectively, data presented here suggests that the relationship between EGFR and TG2 is collaborative and phosphorylation of TG2 play a key role in it. Phospho mutant forms of TG2 reported in this study may be utilized as a part of a novel strategy to downregulate EGFR in cancers with enhanced EGFR signaling.     

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.     

Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.