Inhibition Of Protein Secretion Research Articles

Preventing protein secretion with chemical glue

A small-molecule inhibitor of protein secretion has been found that targets Cdc42, a regulator of the secretory pathway. This compound acts through a previously undescribed mechanism—by recruiting a protein inhibitor.

Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii relies on calcium-mediated exocytosis to secrete adhesins on to its surface where they can engage host cell receptors. Increases in intracellular calcium occur in response to Ins(1,4,5)P3 and caffeine, an agonist of ryanodine-responsive calcium-release channels. We examined lysates and microsomes of T. gondii and detected evidence of cADPR (cyclic ADP ribose) cyclase and hydrolase activities, the two enzymes that control the second messenger cADPR, which causes calcium release from RyR (ryanodine receptor). We also detected endogenous levels of cADPR in extracts of T. gondii. Furthermore, T. gondii microsomes that were loaded with 45Ca2+ released calcium when treated with cADPR, and the RyR antagonists 8-bromo-cADPR and Ruthenium Red blocked this response. Although T. gondii microsomes also responded to Ins(1,4,5)P3, the inhibition profiles of these calcium-release channels were mutually exclusive. The RyR antagonists 8-bromo-cADPR and dantrolene inhibited protein secretion and motility in live parasites. These results indicate that RyR calcium-release channels that respond to the second-messenger cADPR play an important role in regulating intracellular Ca2+, and hence host cell invasion, in protozoan parasites.

Don't Distract Me

T cells receive both activating "stop" signals through ligation of the T cell receptor (TCR) and chemotactic or chemokinetic "go" signals from secreted chemokines. Molon et al . suggest that soluble chemokines released by the antigen-presenting cell (APC) may also contribute to the "stop" signal that stabilizes the immunological synapse formed between the T cell and the APC. Using green fluorescent protein (GFP)-tagged CCR5, a G protein-coupled chemokine receptor, they observed that CCR5 accumulated at the immunological synapse through a mechanism that did not require G i (it was not inhibited by pertussis toxin) but instead may involve coupling to G q or G 11 . This recruitment of CCR5 was inhibited if the cells were exposed to a CCR5 antagonist or if the APCs were treated with monensin, a nonspecific inhibitor of protein secretion. Furthermore, T cells that were prebound to APCs that were exposed to antigen and that had not been treated with monensin (conditions in which the CCR5 was incorporated into the immunological synapse) were less responsive to a chemotactic signal than were T cells that were either prebound to APCs that had not been exposed to antigen or antigen-exposed APCs that had been treated with monensin. In addition, stimulation of T cells with beads coated with either an antibody against CD3 (a protein in the TCR complex) or anti-CD3 plus CCR5 and CXCR4 agonists resulted in strong stimulation of T cell proliferation and interferon γ secretion comparable to that observed for T cells stimulated with antigen and a costimulatory signal. The authors propose that recruitment of selective chemokine receptors to the immunological synapse may serve to sequester these receptors, thus making the T cells less "distracted" by chemotactic signals. This may further serve as a costimulatory signal. In commentary on this article, Trautmann suggests that chemokines may be "immunotransmitters" analogous to the neurotransmitters released at neuronal synapses. B. Molon, G. Gri, M. Bettella, C. Gómez-Moutón, A. Lanzavecchia, C. Martínez-A, S. Mañes, A. Viola, T cell costimulation by chemokine receptors. Nat. Immunol. 6 , 465-471 (2005). [PubMed] A. Trautmann, Chemokines as immunotransmitters? Nat. Immunol. 6 , 427-428 (2005). [PubMed]

A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

The molecular mechanisms of congenital hypofibrinogenaemia.

Congenital hypofibrinogenaemia is characterized by abnormally low levels of fibrinogen and is usually caused by heterozygous mutations in the fibrinogen chain genes (alpha, beta and gamma). However, it does not usually result in a clinically significant condition unless inherited in a homozygous or compound heterozygous state, where it results in a severe bleeding disorder, afibrinogenaemia. Various protein and expression studies have improved our understanding of how mutations causing hypo- and afibrinogenaemia affect secretion of the mature fibrinogen molecule from the hepatocyte. Some mutations can perturb chain assembly as in the gamma153 Cys-->Arg case, while others such as the Bbeta Leu-->Arg and the Bbeta414 Gly-->Ser mutations allow intracellular hexamer assembly but inhibit protein secretion. An interesting group of mutations, such as gamma284 Gly-->Arg and gamma375 Arg-->Trp, not only cause hypofibrinogenaemia but are also associated with liver disease. The nonexpression of these variant chains in plasma fibrinogen is due to retention in the endoplasmic reticulum, which in turn leads to hypofibrinogenaemia.

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

BackgroundThis study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells.MethodsWe optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 51Cr-release assays were made using human LAK cells.ResultsTitration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay.ConclusionsBecause of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.

Analysis of phenomena involved in the apical growth of Phycomyces blakesleeanus.

The effects of the Ca(2+)/H(+) exchanger A23187 and the K(+)/H(+) exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca(2+) channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.

Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus.

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

Development of antibodies to feline IFN-γ as tools to elucidate the cellular immune responses to FeLV

An understanding of the nature of immune protection and the role of immune effector products such as interferon-γ (IFN-γ) in the control of infectious disease is fundamental to the rational design of effective vaccines and immunotherapeutic reagents. Murine monoclonal and sheep polyclonal antibodies (mAbs and pAbs) to feline IFN-γ (fIFN-γ) were generated firstly to facilitate further research into the role of cellular immune responses in the control of feline infectious disease, and secondly to enable evaluation of the efficacy of novel immunotherapeutic approaches. A hybridoma clone, D9, secreting IgG1 antibodies was selected for expansion and the mAbs affinity purified in vitro. Polyclonal antibodies were raised in a sheep against recombinant fIFN-γ and affinity purified. The sensitivity of the D9 mAb and the sheep anti-fIFN-γ pAb was determined using an indirect fIFN-γ enzyme-linked immunosorbent assay (ELISA) and immunoblots. These antibodies were assessed for their ability to detect the production of fIFN-γ by specific feline T cell populations ex vivo following coculture with mitogen or feline leukaemia virus (FeLV) antigens for 4 h in the presence of the protein secretion inhibitor brefeldin A (BFA). Production of fIFN-γ was evaluated using flow cytometry to simultaneously detect PE-labelled surface molecules and fluorescein isothiocyanate (FITC)-labelled intracellular fIFN-γ. Using this approach, our initial studies revealed an upregulation in virus-specific fIFN-γ-secreting CD4 +T cells in the lymph nodes of FeLV latently infected cats.

Heterologous cells cooperate to augment stem cell migration, homing, and engraftment

AbstractT-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+but not CD4+T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/−NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+cells but not CD4+cells. This correlated with CD8+augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+cytoskeletal rearrangements abrogated CD34+transendothelial migration and impaired CD34+cell homing in vivo. CD8+cells did not migrate in tandem with CD34+cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

Estudio del patrón de citocinas (Th1/Th2) producido por linfocitos T periféricos y del existente en tejido tumoral de pacientes con melanoma en diferentes estadios

ObjectivesThe Th1 or Th2 diferentiation of T cells according to their profile of cytokine expression is of great relevance in the regulation of immune responses.We have determined by flow cytometry the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with melanoma in different clinical stages. Additionally, we have studied the mRNA expression of several cytokines in melanoma tissue and compared it with normal skin. MethodsWB samples from 6 normal donors and 19 patients with melanoma were activated during 4 hours with PMA+ ionomycin in the presence or absence of an inhibitor of protein secretion (GolgiStop). Following surface staining (CD3-Cy5+CD8- FITC), fixation and permeabilization (CytoFix-CytoPerm) cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-γ, TNF-α) and Th2 citokines (IL-4, IL-10). In the second part of the study, we investigated the presence of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10 messenger RNA (mRNA) in 6 biopsy specimens of melanoma and compared it with 1 nevus and 1 normal skin by a real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) tecnique. ResultsThe most clear and relevant results of the flow cytometry study were related to IFN-γ and IL-10 production. The percentage of IFN-γ producing cells was significantively lower in melanoma patients, independently of the stage, than in controls. IL-10 production was significantly increased in the melanoma patients with respect to normal donors. There is also a tendency to increase IL-10 production in the more advanced stages of the diseae. The quantitative RT-PCR technique performed in melanoma specimens (primary or metastatic) showed cytokine transcripts of Th2 o Th0 subtypes. ConclusionsOur data sugget that activated lymphocytes from normal donors tend to exhibit a Th1 phenotype, while melanoma patients have a higher proportion of IL-10 producing lym-phocytes, a cytokine with recognized immunosuppresive properties and characteristic of the Th2 phenotype. The results obtained from the melanoma tumor support the concept of a predominance of the Th2 response in melanoma patients.

Spontaneous cytokine production and its effect on induced production.

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) by peripheral-blood B lymphocytes, T cells, CD8(-) T cells, CD8(+) T cells, CD3(-) CD16/56(+) lymphocytes (natural killer [NK] cells), CD3(+) CD16/56(+) lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-alpha production occurred most frequently, followed in descending order by IFN-gamma, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-alpha; for 12 of these patients, TNF-alpha was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-alpha and IFN-gamma, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.

Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways.

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-beta, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-kappaB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G(0) to G(1). Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.

Retinol mobilization from cultured rat hepatic stellate cells does not require retinol binding protein synthesis and secretion

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

Characterization of monoclonal antibody fragments produced by plant cells.

Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.

A Study of the Protein Secretory Pathway of Aspergillus niger Using a Glucoamylase–GFP Fusion Protein

The effect of various treatments that block protein secretion was visualized in Aspergillus niger using a strain expressing a glucoamylase–GFP fusion protein. Cold shock caused the retention of the fusion protein in a reticulate network (ER) with brighter nodes that may represent Golgi bodies. Treatment of germlings with brefeldin A (BFA) also initially caused accumulation within the ER but prolonged exposure led to the formation and targeting of the fusion protein to vacuoles from the ER. Disruption of actin with cytochalasin A initially led to a faint diffuse accumulation and ultimately to the formation of aggregated bodies which were not vacuoles, suggesting that the actin cytoskeleton is important in secretory vesicle transport. Disruption of microtubules with nocodazole led to hyperbranching but did not cause intracellular accumulation, suggesting that microtubules play a role in directing vesicle transport rather than vesicle movement per se. Treatment of regenerating protoplasts confirmed that BFA and cytochalasin but not nocodazole inhibited protein secretion. When germlings were subjected to carbon starvation, vacuolation was rapidly initiated throughout the hyphae and GFP fluorescence was visible in some of the vacuoles, indicating retargeting of the fusion protein from the secretory pathway to the vacuoles.

MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A.

The effects of poliovirus 3A protein expression and poliovirus infection on the presentation of hepatitis C virus antigens in cultured chimpanzee cells were examined. Expression of poliovirus 3A protein inhibits protein secretion when expressed in isolation and was sufficient to protect chimpanzee cells from lysis by hepatitis C virus-specific cytotoxic T cells in standard (51)Cr-release assays. Poliovirus infection also inhibited antigen presentation, as determined by decreased cytotoxic T cell activation. A mutation in 3A that abrogates the inhibition of protein secretion also abolished the effects of poliovirus on antigen presentation. These results demonstrate that the inhibition of secretion observed in poliovirus-infected cells substantially reduces the presentation of new antigens on the cell surface. These observations may reflect a general mechanism by which nonenveloped viruses such as poliovirus and other viruses that do not require a functional protein secretory apparatus can evade detection by the cellular immune response.

Fibroblast growth factor-2 inhibits the maturation of pro-insulin-like growth factor-II (Pro-IGF-II) and the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in the human adrenocortical tumor cell line NCI-H295R.

The IGF system is thought to play a major role in adrenocortical tumorigenesis. In this study, we used the NCI H295R cell line as a model to investigate the effects of fibroblast growth factor-2 (FGF-2), a potent mitogen for normal adrenal cells, on the proliferation and on the expression of the IGF system in cultured adrenocortical tumor cells. Three immunoreactive FGF-2 isoforms of molecular masses 18, 22, and 24 kDa were detected in H295R cell extracts. Recombinant human FGF-2 stimulated the proliferation of adrenocortical tumor cells in a dose- and time-dependent manner, with a maximal effect at a concentration of about 1 ng/ml. Treatment of H295R cells with 10 ng/ml FGF-2 for 7 days had no significant effect on IGF-II messenger RNA levels. However, a marked increase in levels of intracellular IGF-II protein was detected by immunoblotting. In contrast, FGF-2 induced a marked decrease in the amount of IGF-II protein secreted, with the disappearance of mature IGF-II and secretion of higher molecular forms of the growth factor, suggesting modifications of IGF-II processing. Cell cultures in the presence of brefeldin A (1 microg/ml), a specific inhibitor of protein secretion, suggested that FGF-2 did not increase IGF-II synthesis but instead inhibited the secretion of pro-IGF-II from H295R cells, thereby impairing the final steps of IGF-II processing to the mature 7.5-kDa peptide. At the same concentrations, FGF-2 also decreased both IGFBP-2 messenger RNA and secreted protein, which might increase IGF-II bioavailability. No proteolysis of IGFBP-2 was detected in FGF-2-conditioned medium. Altogether, these results indicate that FGF-2 is mitogenic for NCI H295R tumor cells and regulates the expression of both IGF-II and IGFBP-2 in this tumor model. Moreover, this study shows a novel effect of FGF-2, by which this growth factor modulates the processing of pro-IGF-II.

Posttranscriptional Stimulation of Endothelial Cell Matrix Metalloproteinases 2 and 1 by Endothelioma Cells

Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24.24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis.

Brefeldin A revealing the fundamental principles governing membrane dynamics and protein transport.

Brefeldin A (BFA) has enjoyed widespread use as an inhibitor of protein secretion in eukaryotic cells. BFA, first described in 1958 (Singleton et al., 1958), is a heterocyclic lactone produced by certain fungi. In the mid-1980’s, BFA was demonstrated to have dramatic effects on the structure and functioning of intracellular organelles, particularly the Golgi apparatus, in eukaryotic cells (Doms et al., 1989; Hunziker et al., 1992; LippincottSchwartz et al., 1991; Lippincott-Schwartz et al., 1989; Misumi et al., 1986). This discovery led to intense interest in the drug both for understanding the mechanisms underlying the organization of intracellular compartments and as a tool to specifically inhibit the functioning of the secretory pathway