Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.
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Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. (+info)
The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling. (+info)
EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake.
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Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling. (+info)
The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.
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Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor. The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N-terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post-translational beta-hydroxylation of Asp/Asn residues. We identified the presence of erythro-beta-hydroxy-aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 M.M protein-1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro-beta-hydroxyasparagine (Hyn). The Hyn-containing modules have a consensus calcium-binding motif immediately N-terminal of the first Cys residue. The synthetic EGF module 2 (residues 356-395) of the EGF precursor was found to bind calcium with low affinity, Kd approximately 3.5 mM, i.e. similar to the affinity of other isolated calcium-binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd approximately 0.2 microM, i.e. approximately 10(4)-fold higher than that of isolated EGF modules presumably due to the influence of neighboring modules. We have detected EGF precursor in platelet-rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30-160 EGF molecules each. (+info)
The N-terminal CUB-epidermal growth factor module pair of human complement protease C1r binds Ca2+ with high affinity and mediates Ca2+-dependent interaction with C1s.
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The Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their alpha regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yielding KD values of 10.9-29.7 nM. The C1r CUB-EGF pair bound immobilized C1s with a higher KD (1.5-1.8 microM), which decreased to 31.4 nM when CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+ concentrations ranging from 5 microM with intact C1r to 10-16 microM for C1ralpha and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1-175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair. (+info)
Selective perturbation of early endosome and/or trans-Golgi network pH but not lysosome pH by dose-dependent expression of influenza M2 protein.
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Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic. However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects. To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments. We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport. At low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from the trans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin. At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes. Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases. Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell. The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport. (+info)
Extracellular matrix-dependent activation of syndecan-1 expression in keratinocyte growth factor-treated keratinocytes.
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Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Maatta, A., and Jalkanen, M. (1998) FASEB J. 12, 959-969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiRE-dependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix. (+info)
Comparisons of genomic structures and chromosomal locations of the mouse aldose reductase and aldose reductase-like genes.
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Aldose reductase (AR), best known as the first enzyme in the polyol pathway of sugar metabolism, has been implicated in a wide variety of physiological functions and in the etiology of diabetic complications. We have determined the structures and chromosomal locations of the mouse AR gene (Aldor1) and of two genes highly homologous to Aldor1: the fibroblast growth factor regulated protein gene (Fgfrp) and the androgen regulated vas deferens protein gene (Avdp). The number of introns and their locations in the mouse Aldor1 gene are identical to those of rat and human AR genes and also to those of Fgfrp and Avdp. Mouse Aldor1 gene was found to be located near the Cald1 (Caldesmon) and Ptn (Pleiotropin) loci at the proximal end of chromosome 6. The closely related genes Fgfrp and Avdp were also mapped in this region of the chromosome, suggesting that these three genes may have arisen by a gene duplication event. (+info)