RESUMO
Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)-1, -2, -3, and -4 on FSAP-mediated signaling in HEK293T and A549 cells. Cleavage of PAR-reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR-2 and to a lesser degree PAR-1, but not PAR-3 or PAR-4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR-2, but not PAR-1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N-terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR-1, but not PAR-2, prevented the apoptosis-inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR-1 and -2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.
Assuntos
Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Apoptose , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Peptídeos/química , Fosforilação , Isoformas de Proteínas , Transdução de Sinais , TromboseRESUMO
A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.
Assuntos
Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Linfócitos T Reguladores/imunologia , Trifosfato de Adenosina/imunologia , Animais , Diferenciação Celular , Movimento Celular , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Imunidade Celular , Imunofenotipagem , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2X7/genéticaRESUMO
BACKGROUND: Matriptase is a membrane serine protease essential for epithelial development, homeostasis, and regeneration, as well as a central orchestrator of pathogenic pericellular signaling in the context of inflammatory and proliferative diseases. Matriptase is an unusual protease in that its zymogen displays measurable enzymatic activity. RESULTS: Here, we used gain and loss of function genetics to investigate the possible biological functions of zymogen matriptase. Unexpectedly, transgenic mice mis-expressing a zymogen-locked version of matriptase in the epidermis displayed pathologies previously reported for transgenic mice mis-expressing wildtype epidermal matriptase. Equally surprising, mice engineered to express only zymogen-locked endogenous matriptase, unlike matriptase null mice, were viable, developed epithelial barrier function, and regenerated the injured epithelium. Compatible with these observations, wildtype and zymogen-locked matriptase were equipotent activators of PAR-2 inflammatory signaling. CONCLUSION: The study demonstrates that the matriptase zymogen is biologically active and is capable of executing developmental and homeostatic functions of the protease.
Assuntos
Precursores Enzimáticos/metabolismo , Epitélio/crescimento & desenvolvimento , Homeostase/genética , Regeneração/genética , Serina Endopeptidases/metabolismo , Animais , Precursores Enzimáticos/genética , Epitélio/metabolismo , Feminino , Mutação com Ganho de Função , Expressão Gênica , Mutação com Perda de Função , Masculino , Camundongos Transgênicos , Serina Endopeptidases/genéticaRESUMO
RATIONALE: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. OBJECTIVE: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. METHODS AND RESULTS: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. CONCLUSIONS: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.
Assuntos
Anafilaxia/metabolismo , Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Homeostase/fisiologia , Lisofosfolipídeos/deficiência , Esfingosina/análogos & derivados , Anafilaxia/patologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esfingosina/deficiênciaRESUMO
The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.
Assuntos
Coagulação Sanguínea/fisiologia , Células Epiteliais/metabolismo , Proteólise , Serina Endopeptidases/metabolismo , Tromboplastina/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Fator VIIa/metabolismo , Fator Xa/metabolismo , Células HeLa , Humanos , Células MCF-7 , Proteínas Mutantes/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transdução de SinaisRESUMO
The obligate intracellular bacterium Chlamydia exists as two distinct forms. Elementary bodies (EBs) are infectious and extra-cellular, whereas reticulate bodies (RBs) replicate within a specialized intracellular compartment termed an 'inclusion'. Alternative persistent intra-cellular forms can be induced in culture by diverse stimuli such as IFNγ or adenosine/EHNA. They do not grow or divide but revive upon withdrawal of the stimulus and are implicated in several widespread human diseases through ill-defined in vivo mechanisms. ß-Lactam antibiotics have also been claimed to induce persistence in vitro. The present report shows that upon penicillin G (pG) treatment, inclusions grow as fast as those in infected control cells. After removal of pG, Chlamydia do not revert to RBs. These effects are independent of host cell type, serovar, biovar and species of Chlamydia. Time-course experiments demonstrated that only RBs were susceptible to pG. pG-treated bacteria lost their control over host cell apoptotic pathways and no longer expressed pre-16S rRNA, in contrast to persistent bacteria induced with adenosine/EHNA. Confocal and live-video microscopy showed that bacteria within the inclusion fused with lysosomal compartments in pG-treated cells. That leads to recruitment of cathepsin D as early as 3 h post pG treatment, an event preceding bacterial death by several hours. These data demonstrate that pG treatment of cultured cells infected with Chlamydia results in the degradation of the bacteria. In addition we show that pG is significantly more efficient than doxycycline at preventing genital inflammatory lesions in C. muridarum-C57Bl/6 infected mice. These in vivo results support the physiological relevance of our findings and their potential therapeutic applications.
Assuntos
Infecções por Chlamydia/tratamento farmacológico , Chlamydia/efeitos dos fármacos , Chlamydia/fisiologia , Lisossomos/microbiologia , Penicilina G/farmacologia , Vagina/efeitos dos fármacos , Vagina/microbiologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Feminino , Inflamação/prevenção & controle , Lisossomos/efeitos dos fármacos , Camundongos , Penicilina G/uso terapêuticoRESUMO
Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220(+) CD4(-)CD8(-) (DN) T lymphocytes. The precise ontogeny of B220(+) DN T cells is unknown. B220(+) DN T lymphocytes could be derived from effector CD4(+) and CD8(+) T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220(+) DN T-cell numbers in lymphoid organs. Importantly, we found that this B220(+) DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220(+) T cells observed in normal MRL(+/+) and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220(+) T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.
Assuntos
Membrana Celular/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Western Blotting , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/metabolismo , Antígenos CD8/efeitos dos fármacos , Antígenos CD8/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Mutantes , NAD/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P2X7/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLRâMyD88âAREG/EREGâEGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.
Assuntos
Colite/prevenção & controle , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Sistema Hematopoético/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Anfirregulina , Animais , Colite/induzido quimicamente , Colite/metabolismo , Sulfato de Dextrana , Família de Proteínas EGF , Epirregulina , Ligantes , Lipopolissacarídeos/farmacologia , Metagenoma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fator 88 de Diferenciação Mieloide/deficiência , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismoRESUMO
Protein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factor-α (TNFα) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside-out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site.
Assuntos
Proteínas ADAM/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Proteína ADAM17 , Animais , Células COS , Domínio Catalítico , Células Cultivadas , Chlorocebus aethiops , Regulação para Baixo , Humanos , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Camundongos , Fosforilação , Transdução de Sinais , TransfecçãoRESUMO
ADAMs (a disintegrin and metalloproteinase) have important roles in development and diseases such as cancer. Previously, an ADAM15 splice variant (ADAM15B), which contains an inserted cytoplasmic Src-binding site, was linked to clinical aggressiveness in breast cancer, yet little was known about how this splice variant affects the function of ADAM15. Here, we show that ADAM15B has enhanced catalytic activity in cell-based assays compared with ADAM15A, which lacks a Src-binding site, using shedding of fibroblast growth factor receptor 2iiib variant as an assay for catalytic activity. Moreover, the enhanced activity of ADAM15B compared with ADAM15A depends on Src because it is abolished by Src-kinase inhibitors and in Src(-/-) cells, but not in Src(-/-) cells rescued with Src. These findings provide insights into the mechanism of how a splice variant linked to clinical agressiveness in breast cancer causes increased activity of ADAM15B, and suggest that inhibitors of the ADAM15 protease activity or of the interaction of ADAM15B with Src could be useful to treat breast cancer in patients with dysregulated ADAM15B.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Genes src/fisiologia , Proteínas de Membrana/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/fisiologia , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células COS , Domínio Catalítico/fisiologia , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Invasividade Neoplásica , Proteína Oncogênica pp60(v-src)/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Homologia de Sequência de AminoácidosRESUMO
Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrosis factor (TNF)-alpha, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growth factor (TGF)-alpha and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17-/- cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17-/- fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17-/- cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17-/- cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Selectina L/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Inibidores de Proteases/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Epidermal growth factor (EGF) is present in kidney membranes as an integral type I precursor protein, enzymatically processed to release immunoreactive materials in urine or incubation medium. The aim of this work was the elucidation of both the anchor of the serine protease activity that processes pro-EGF, and the determination of the steps of the enzymatic processing. Quantification of EGF containing molecules by RIA following gel filtration analysis demonstrated that the membrane precursor is first shed from the kidney membrane principally into a 170-kDa soluble precursor. This entire ectodomain is further processed into a 70-kDa precursor and finally into the mature 5.9 kDa urinary EGF. These species correspond to the ones found in urines. Both shedding and maturation events are clearly realized by membrane anchored serine protease activity, which remains active in detergent. By use of wild-type and knockout mice urines, we found that tissue kallikrein (TK) was not involved in the regulation of this processing.
Assuntos
Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Calicreínas/metabolismo , Rim/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Cromatografia em Gel , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/imunologia , Calicreínas/deficiência , Calicreínas/genética , Rim/enzimologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/imunologia , RatosRESUMO
Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.