The Precursor but Not the Mature Form of IL1alpha Blocks the Release of FGF1 in Response to Heat Shock*

Francesca TarantiniDagger, Isabella MicucciDagger, Stephen Bellum, Matteo Landriscina§, Susan Garfinkel, Igor Prudovsky, and Thomas Maciag||

From the Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074

Received for publication, October 20, 2000, and in revised form, November 20, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Interleukin (IL)1alpha mediates proinflammatory events through its extracellular interaction with the IL1 type I receptor. However, IL1alpha does not contain a conventional signal peptide sequence that provides access to the endoplasmic reticulum-Golgi apparatus for secretion. Thus, we have studied the release of the precursor (p) and mature (m) forms of IL1alpha from NIH 3T3 cells. We have demonstrated that mIL1alpha but not pIL1alpha was released in response to heat shock with biochemical and pharmacological properties similar to those reported for the stress-mediated release pathway utilized by fibroblast growth factor (FGF)1. However, unlike the FGF1 release pathway, the IL1alpha release pathway appears to function independently of synaptotagmin (Syt)1 because the expression of a dominant-negative form of Syt1, which represses the release of FGF1, did not inhibit the release of mIL1alpha in response to temperature stress. Interestingly, whereas the expression of both mIL1alpha and FGF1 in NIH 3T3 cells did not impair the stress-induced release of either polypeptide, the expression of both pIL1alpha and FGF1 repressed the release of FGF1 in response to temperature stress. These data suggest that the release of mIL1alpha requires proteolytic processing of its precursor form and that mIL1alpha and FGF1 may utilize similar but distinct mechanisms for export.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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The interleukin(IL)1-1 gene family is currently comprised of eight members, and these include the IL1 prototypes, IL1alpha and IL1beta , as well as the IL1 receptor antagonist RA (1), IL-18, and the recently identified members, FIL1delta , FIL1epsilon , FIL1zeta , and FIL1eta (2). The IL1 prototypes are translated as 31-34-kDa precursor proteins that are cleaved by two distinct specific proteases to produce mature 17-kDa forms of the IL1 prototypes from the C-terminal end of the precursor (3). Whereas precursor (p) IL1beta is biologically inactive until it is processed into the mature (m) form by the IL1beta -converting enzyme (ICE) (4, 5), pIL1alpha is biologically active (5). Precursor IL1alpha is recognized by a calcium-dependent protease of the calpain family, and this cleavage results in the formation of the mature counterpart (6). Interestingly, the N-terminal fragment derived from pIL1alpha proteolytic processing contains a functional nuclear localization signal (7) and can be translocated to the nucleus (8). The ability of pIL1alpha to bind the IL1 type I receptors with high affinity, with subsequent activation of the signal transduction pathway, and the presence of the nuclear localization sequence anticipates the existence of a biological role for pIL1alpha , independent from the activity of mIL1alpha . Indeed, comparative studies using the pIL1alpha and mIL1alpha forms have suggested that pIL1alpha , but not mIL1alpha , is a negative regulator of cell migration (9).

Amino acid sequence (10) and x-ray crystallographic analysis (11) of the IL1 and FGF prototypes have revealed rather striking structural similarities between members of the two gene families. Like the majority of the IL1 gene family members (1, 2), the FGF gene family prototypes, FGF1 and FGF2 also lack a classical signal peptide sequence to direct secretion through the ER-Golgi apparatus (12). Whereas the release of FGF1 is regulated by a variety of stress conditions (13-18), the release of FGF2 is not regulated by stress (19), and FGF2 contains structural features that repress the stress-induced release of FGF1 (19). Because (i) the release pathways utilized by the FGF prototypes have diverged, and (ii) it is unlikely that the pathway responsible for the regulation of FGF1 would have evolved independent of the mechanisms utilized by other signal peptide-deficient gene products to gain access to the extracellular compartment, we sought to determine whether other signal peptide-deficient cytokines are also able to utilize the FGF1 release pathway for export. We focused our effort on IL1alpha because its precursor form is biologically functional (5, 20), and extracellular IL1alpha is well described as an antagonist of FGF-dependent biological activities (21-24). We report that the release pathway utilized by IL1alpha exhibits similar biochemical, pharmacological, and biological properties to the FGF1 release pathway. In contrast, unlike the FGF1 release pathway (16, 17), IL1alpha does not require the function of synaptotagmin (Syt)1 but does require the function of an intracellular protease to convert pIL1alpha to mIL1alpha , because only mIL1alpha is released in response to heat shock. Lastly, the stress-induced IL1alpha and FGF1 release pathways may be convergent, because the expression of pIL1alpha acts as a dominant-negative repressor of FGF1 release.


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ABSTRACT
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Plasmids, Transfections, and Cell Culture-- Human pIL1alpha -(1-271) and mIL1alpha -(113-271) cDNAs (7), as well as pIL1alpha ·beta -gal and mIL1alpha ·beta -gal fusion constructs, were inserted into the expression vector, pMEXneo (25), and stable NIH 3T3 cell transfectants were obtained as previously described (7). The transfectants were grown on cell culture dishes coated with 10-µg/cm2 fibronectin in Dulbecco's modified Eagle's medium (DMEM, Cellgro) supplemented with 10% (v/v) bovine calf serum (BCS, HyClone), 1× antibiotic/antimycotic (Life Technologies, Inc.), and 400 µg/ml geneticin (G418, Life Technologies, Inc.). The Syt1 constructs, full-length rat p65 Syt1 and its deletion mutant Syt1Delta (120-214) in the expression vector pMEXneo/hygro, were obtained as described (17). The Syt1 constructs were cotransfected into mIL1alpha ·beta -gal NIH 3T3 cell transfectants using 5 µg of DNA mixed with the multicomponent lipid-based transfection reagent FuGENE6 (Roche Molecular Biochemicals) following the manufacturer's instructions. Stable cotransfectants were grown on fibronectin-coated (10 µg/cm2) cell culture dishes in DMEM, supplemented with 10% (v/v) bovine calf serum, 1× antibiotic/antimycotic, 400 µg/ml G418, and 200 µg/ml hygromycin (Roche Molecular Biochemicals). The human FGF1 construct in the expression vector, pcDNA3.1/Hygro (Invitrogen) was obtained by digesting the pXZ38 plasmid (12) with EcoRV and HpaI (New England BioLabs Inc.), isolating the 836-base pair fragment containing FGF1 by electroelution, ligating into the pcDNA3.1/Hygro expression vector previously digested with EcoRV, and purifying by electroelution. The FGF1 pcDNA3.1/Hygro construct was cotransfected into mIL1alpha ·beta -gal and pIL1alpha ·beta -gal NIH 3T3 cell transfectants using 5 µg of DNA mixed with the multicomponent lipid-based transfection reagent, FuGENE6, following the manufacturer's instructions. Stable cotranfectants were selected and grown as described above. Whereas multiple clones were obtained in the mIL1alpha , FGF1, and mIL1alpha ·FGF1 backgrounds, we were limited to studying a single clone in the pIL1alpha and pIL1alpha ·FGF1 backgrounds.

Transfectants were grown to 70-80% confluency and prior to the temperature stress, the cells were washed with DMEM. The heat shock was performed in DMEM at 42 °C for 2 h, as previously described (12). When FGF1 transfectants were subjected to temperature stress, the heat shock medium was supplemented with 4 units/ml heparin (Upjohn Co.). The effects of Brefeldin A (Epicenter Technologies), 2-deoxyglucose (Sigma), and amlexanox (Takeda) on mIL1alpha release were evaluated as previously described (17-18).

Processing of Cell Lysates and Conditioned Medium and Immunoblot Analysis-- Prior to and after temperature stress, total cell lysates were obtained by sonication of cell pellets collected in Germino buffer DT (10 mM Tris, pH 7.6, containing 250 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 0.1% (v/v) Triton X-100) as described previously (26). Conditioned medium was collected, filtered through a 0.22-µm filter, and then treated with either dithiothreitol (DTT, Sigma) and (NH4)2SO4 (Sigma) or were left untreated. DTT treatment was performed at a concentration of 0.1% (w/v) DTT at 37 °C for 2 h, and (NH4)2SO4 fractionation was performed at 95% (w/v) saturation as described previously (18). Cell lysates and DTT- or (NH4)2SO4-treated medium from mIl1alpha ·beta -gal and pIL1alpha ·beta gal NIH 3T3 cell transfectants were further processed by affinity chromatography using a 0.5-ml p-aminobenzyl 1-thio-beta -D-galactopyranoside column (Sigma) previously equilibrated with Germino buffer DT. The column was washed with Germino buffer DT, followed by a wash with Germino buffer DT. The proteins were eluted with 2 ml of 0.1 M sodium borate, pH 10 and were concentrated by centrifugation using Centricon 30 concentrators (Amicon, Inc.).

Prior to immunoprecipitation, the cells were washed and scraped in cold phosphate-buffered saline, and cell pellets were obtained by centrifugation as described previously (26). The pellets were resuspended in 1 ml of cold NP lysis buffer (20 mM Tris, pH 7.5, containing 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 5% (v/v) glycerol, 2 mM EDTA, 1% (v/v) Triton X-100, 2 µg/ml aprotinin, 2 µg/ml leupeptin). Conditioned medium with or without DTT treatment were filtered through a 0.22-µm filter and concentrated using a centrifugal filter device (Ultrafree-15, Millipore) to a volume of ~500 µl. 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 5 µl of goat anti-human IL1alpha polyclonal antibodies (1.21 mg/ml, a generous gift from Dr. R. Chizzonite, Hoffmann-La Roche Inc.) were added to either cell lysates or conditioned medium, and the samples were rotated at 4 °C for 18 h. Protein A-Sepharose (Amersham Pharmacia Biotech) was added, and the samples were rotated at 4 °C for an additional 2 h. All IL1alpha samples either eluted from the p-aminobenzyl 1-thio-beta -D-galactopyranoside column or purified by immunoprecipitation were resolved by either 15% (w/v) SDS-PAGE (native forms of pIL1alpha and mIL1alpha ) or 8% (w/v) SDS-PAGE (pIL1alpha ·beta -gal and IL1alpha ·beta -gal fusion proteins) and subjected to immunoblot analysis as previously described (9). Briefly, proteins were transferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech) and probed with goat anti-human IL-1alpha polyclonal antibody at a 1:1200 dilution. IL-1alpha specific bands were visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech) following the manufacturer's instructions.

Analysis of the pIL1alpha ·beta -gal/FGF1 and mIL1alpha ·beta -gal/FGF1 NIH 3T3 cell cotransfectants was accomplished by dividing individual cell lysates and conditioned medium in half for the detection of FGF1 and IL1alpha immunoreactive bands. For FGF1 analysis, total cell lysates were obtained by sonication in cold lysis buffer containing 1% (v/v) Triton X-100 (Sigma) and adsorbed to a 1-ml heparin-Sepharose CL-6B column (Amersham Pharmacia Biotech), previously equilibrated with 50 mM Tris pH 7.4 containing 10 mM EDTA (TEB). The column was washed with TEB, and the proteins were eluted with 2 ml of TEB containing 1.5 M NaCl. The volume of the eluates was reduced using Centricon 10 concentrators (Amicon), and the eluates were analyzed by 15% (w/v) SDS-PAGE followed by immunoblot analysis using anti-human FGF1 polyclonal antibodies (27) as described previously (16). IL1alpha was analyzed by immunoprecipitation using anti-IL1alpha antibody and resolved by 8% (w/v) SDS-PAGE followed by immunoblot analysis, as described above. Cell lysates from mIL1alpha ·beta -gal, mIL1alpha ·beta -gal/p65 Syt1, and mIL1alpha ·beta -gal/Syt1Delta (120-214) mutant NIH 3T3 cell cotransfectants were obtained by sonication in NP lysis buffer and processed as described below. Conditioned medium from these cotransfectants were treated with 0.1% (w/v) DTT and divided into two equal samples. The first sample was adsorbed to heparin-Sepharose CL-6B (1 ml) and washed with TEB, and samples were eluted with 1.5 M NaCl, as reported above. Cell lysates and conditioned medium purified by heparin affinity were resolved by 10% (w/v) SDS-PAGE and subjected to immunoblot analysis using a rabbit anti-rat Syt1 polyclonal antibody as described previously (17). The second sample was processed by IL1alpha immunoprecipitation as described above. As a negative control, cell lysates were also immunoprecipitated with a control antibody (goat IgG purified immunoglobulin from pooled normal goat serum, Sigma). Cell lysates and conditioned medium purified by IL1alpha immunoprecipitation were resolved by 8% (w/v) SDS-PAGE, followed by IL1alpha immunoblot analysis, as described above. IL1alpha and Syt1 specific bands were visualized by chemiluminescence (ECL) following the manufacturer's instructions. The activity of lactate dehydrogenase in conditioned medium was utilized as an assessment of cell lysis in all experiments and was measured by a colorimetric assay using pyruvate as a substrate (Sigma). Each experiment reported was repeated at least three times with similar results in all cases, and representative data are shown in each figure.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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We evaluated the ability of human IL1alpha to enter the extracellular compartment using NIH 3T3 cells because these cells (i) are refractory to the activity of endogenous and exogenous IL1alpha (data not shown) and (ii) have proven to be valuable for the study of the FGF1 release pathway (16-18). Thus, stable NIH 3T3 cell transfectants expressing either pIL1alpha or mIL1alpha with or without the beta -gal reporter gene were obtained and were subjected to temperature stress at 42 °C for 2 h. Cell lysates and conditioned medium treated with 0.1% (w/v) DTT were processed for IL1alpha immunoblot analysis. As shown in Fig. 1, A and B, mIL1alpha and mIL1alpha ·beta -gal were readily visible in medium conditioned by heat shock. In contrast, neither pIL1alpha nor pIL1alpha ·beta -gal was detected in heat shock-conditioned medium, but both forms of the polypeptide were present in cell lysates (Fig. 1, A and B). These data suggest the following: (i) the mature (residues 113 to 271) but not the precursor (residues 1 to 271) form of IL1alpha was able to enter the extracellular compartment in response to temperature stress, (ii) the release of IL1alpha does not restrict export of the reporter gene product, beta -gal, and (iii) cell lysis does not account for the release of mIL1alpha because the absence of pIL1alpha in medium conditioned by heat shock served as a negative control.



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Fig. 1.   Mature IL1alpha but not precursor IL1alpha is released in response to temperature stress. The mIL1alpha and pIL1alpha (A) and mIL1alpha ·beta -gal and pIL1alpha ·beta -gal (B) NIH 3T3 cell transfectants were subject to heat shock (42 °C, 2 h), and CM was treated with DTT, immunoprecipitated with anti-IL1alpha , and analyzed by IL1alpha immunoblot analysis. A, lane 1, total CL from mIL1alpha transfectants; lanes 2 and 3, 37 and 42 °C CM from mIL1alpha transfectants, respectively; lane 4, total CL from pIL1alpha transfectants; lanes 5 and 6, 37 and 42 °C CM from pIL1alpha transfectants, respectively. B, lane 1, total CL from mIL1alpha ·beta -gal transfectants; lanes 2 and 3, 37 and 42 °C CM from mIL1alpha ·beta -gal transfectants, respectively; lane 4, total CL from pIL1alpha ·beta -gal transfectants; lanes 5 and 6, 37 and 42 °C CM from pIL1alpha ·beta -gal transfectants, respectively. C and D, mIL1alpha ·beta -gal and pIL1alpha ·beta -gal NIH 3T3 cell transfectants were subject to heat shock (42 °C, 2 h), CM was treated with DTT (0.1%, w/v), (NH4)2SO4 (95%, w/v) or left untreated. The IL1alpha ·beta -gal fusion proteins were isolated by PATG affinity chromatography (C) or by IL1 IP (D) followed by IL1 immunoblot analysis. C, lanes 1 and 2, total CL from mIL1alpha ·beta -gal transfectants at 37 and 42 °C, respectively, purified by PATG affinity; lanes 3-5, 37 °C CM from mIL1alpha ·beta -gal transfectants, after no treatment, (NH4)2SO4, or DTT treatment, respectively; lanes 6-8, 42 °C CM from mIL1alpha ·beta -gal transfectants after no treatment, (NH4)2SO4, or DTT treatment, respectively. D, lanes 1 and 2, total CL from mIL1alpha ·beta -gal transfectants, purified by IL1alpha IP with anti-IL1alpha (lane 1) or control (lane 2) antibody; lanes 3 and 4, 37 °C CM from mIL1alpha ·beta -gal transfectants after no treatment or DTT treatment, respectively; lanes 5 and 6, 42 °C CM from mIL1alpha ·beta -gal transfectants, after no treatment or DTT treatment, respectively.

Because FGF1 (14) but not FGF2 (19) was released from NIH 3T3 cells in response to heat shock as a DTT-sensitive latent homodimer (14), and both reducing agents and (NH4)2SO4 were able to activate the heparin affinity of latent FGF1(16), we questioned whether these reagents would also affect the ability of IL1alpha to be recognized by affinity reagents. Because mIL1alpha ·beta -gal does not exhibit heparin-binding affinity (data not shown), we utilized beta -gal affinity and IL1alpha immunoprecipitation to assess this issue. As shown in Fig. 1C, IL1alpha immunoblot analysis of medium conditioned by temperature stress was performed using p-aminobenzyl-1-thio-beta -D-galactopyranoside (PATG) affinity and failed to detect the presence of the IL1alpha ·beta -gal fusion product. However, treatment of heat shocked-conditioned medium with either 0.1% (w/v) DTT or 95% (w/v) (NH4)2SO4 did resolve the presence of mIL1alpha ·beta -gal in medium conditioned by heat shock from mIL1alpha ·beta -gal NIH 3T3 cell transfectants. In contrast, the use of IL1alpha immunoprecipitation detected the presence of IL1alpha ·beta -gal in medium conditioned by heat shock, and the addition of either DTT (Fig. 1D) or (NH4)2SO4 (data not shown) to the medium did not alter the ability of the anti-IL1alpha antibody to recognize the fusion protein (Fig. 1D). These data suggest that whereas mIL1alpha ·beta -gal is present in the extracellular compartment in a conformation that does not efficiently recognize PATG (unlike FGF1 that utilizes Cys oxidation for export, Ref. 15), this conformation may not represent mIL1alpha but rather the conformation of beta -gal. Thus, it is unlikely that mIL1alpha utilizes Cys oxidation for export.

We also examined the kinetics and pharmacologic properties of mIL1alpha ·beta -gal release in response to temperature stress, because the FGF1 release pathway exhibits relatively slow export kinetics (14). Moreover, FGF1 is sensitive to agents that interfere with translation (12), transcription (12), ATP biosynthesis (17), and assembly of the F-actin cytoskeleton (28) but is not sensitive to Brefeldin A, an agent that interferes with the function of the ER-Golgi apparatus (14). As previously observed with FGF1 release (14), the release of mIL1alpha ·beta -gal also required at least 90 min of temperature stress before IL1alpha immunoblot analysis was able to resolve the presence of the fusion protein in medium conditioned by heat shock (Fig. 2A). In addition, IL1alpha immunoblot analysis revealed that the release of mIL1alpha ·beta -gal was not sensitive to treatment of the mIL1alpha ·beta -gal NIH 3T3 cell transfectants with Brefeldin A (Fig. 2B) but was sensitive to the presence of 2-deoxyglucose (Fig. 2C) as well as cycloheximide and actinomycin D (data not shown). Further, the release of mIL1alpha ·beta -gal was also sensitive to treatment with amlexanox (Fig. 2D), an agent which induces the Src-dependent disassembly of F-actin stress fibers (28) and exhibits a dose-response to amlexanox similar to that reported for the inhibition of FGF1 release in vitro (18). These data suggest that the pathway utilized by mIL1alpha ·beta -gal for entering the extracellular compartment is dependent upon transcription, translation, ATP biosynthesis, and the actin cytoskeleton but is independent of the function of the ER-Golgi apparatus.



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Fig. 2.   Kinetics and pharmacological properties of mIL1alpha ·beta -gal release from NIH 3T3 cells in response to temperature stress. The mIL1alpha ·beta -gal NIH 3T3 cell transfectants were subjected to heat shock (42 °C, 2 h) and CM was treated with (NH4)2SO4 (95%, w/v) and purified by PATG affinity. The eluted fraction was resolved by IL1alpha immunoblot analysis. A, lane 1, CL; lane 2, CM from cells maintained at 37 °C for 120 min; lanes 3-7, CM from cells maintained at 42 °C for 45 min (lane 3), 60 min (lane 4), 75 min (lane 5), 90 min (lane 6), and 120 min (lane 7). B, mIL1alpha ·beta -gal NIH3T3 cell transfectants were subjected to heat shock (42 °C, 2 h) without pretreatment or after treatment with Brefeldin A (5 µg/ml, 30 min at 37 °C). Lanes 1 and 2, total CL from cells maintained at 37 °C without or with Brefeldin A, respectively; lanes 3 and 4, 37 °C CM without or with Brefeldin A, respectively; lanes 5 and 6, 42 °C CM without pretreatment or after treatment with Brefeldin A, respectively. C, mIL1alpha ·beta -gal NIH 3T3 cell transfectants were subjected to heat shock (42 °C, 2 h) without or with 2-DG (50 mM, 1 h at 37 °C). Lanes 1 and 2, total cell lysates from cells maintained at 37 °C with or without 2-DG, respectively; lanes 3 and 4, 37 °C CM without or with 2-DG, respectively; lanes 5 and 6, 42 °C CM without or with 2-DG, respectively. D, cells were subjected to heat shock (42 °C, 2 h) in the absence or presence of amlexanox in the medium. Total CL from cells maintained at 37 °C with (lane 1) or without (lane 2) 0.3 mM amlexanox; lanes 3 and 4, 37 °C CM with or without 0.3 mM amlexanox, respectively; lanes 5-9, 42 °C CM without 0.3 mM amlexanox (lane 5) or with 1 µM (lane 6), 37 µM (lane 7), 0.1 mM (lane 8), 0.3 mM (lane 9) amlexanox.

Because the kinetic and pharmacologic properties of mIL1alpha ·beta -gal release were remarkably similar to that previously reported for FGF1, we examined the potential role of p65 Syt1 in the IL1alpha release pathway. Syt1 is the prototype member of a gene family of vesicular transmembrane Ca2+- and acidic phospholipid-binding proteins (29) involved in the regulation of conventional exocytosis (30) and endocytosis (31). Because the extravesicular p40 domain of Syt1 is released as an aggregate with FGF1 in response to heat shock (16) and is associated with FGF1 in vivo (18), we utilized a p65 Syt1 mutant (p65 Syt1Delta (120-214)) lacking the C2A domain, which acts as a dominant negative effector of FGF1 release in response to heat shock (17). To evaluate the role of p65 Syt1 in the release of mIL1alpha ·beta -gal, we obtained stable NIH 3T3 cell cotransfectants expressing either p65 Syt1/mIL1alpha ·beta -gal or p65 Syt1Delta (120-214)/mIL1alpha ·beta -gal and examined medium conditioned by heat shock for the presence of p40 Syt1 and IL1alpha ·beta -gal using immunoblot analysis. As shown in Fig. 3A, Syt1 immunoblot analysis revealed the presence of p40 Syt1 in medium conditioned by heat shock from the p65 Syt1/mIL1alpha ·beta -gal NIH 3T3 cell cotransfectants but did not report the presence of p40 Syt1 in heat shocked-conditioned medium from either mIL1alpha ·beta -gal NIH 3T3 cell transfectants or p65 Syt1Delta (120-214)/mIL1alpha ·beta -gal NIH 3T3 cell cotransfectants. In contrast, however, IL1alpha immunoblot analysis did reveal the presence of mIL1alpha ·beta -gal in medium conditioned by temperature stress independent of the expression of either p65 Syt1 or p65 Syt1Delta (120-214) (Fig. 3B). These data suggest that unlike the stress-induced FGF1 release pathway (17), the IL1alpha release pathway may not utilize the function of p65 Syt1. However, it is not possible to eliminate the function of another member of the Syt gene family in the regulation of mIL1alpha release.



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Fig. 3.   Expression of p65 Syt1Delta (120-214) does not alter the release of mIL1alpha ·beta -gal in response to heat shock. The mIL1alpha ·beta -gal NIH 3T3 cell transfectants, mIL1alpha ·beta -gal/p65 Syt1 NIH 3T3 cell cotransfectants, and mIL1alpha ·beta -gal/p65 Syt1Delta (120-214) mutant NIH 3T3 cell cotransfectants were subjected to heat shock (42 °C, 2 h), and the level of expression of the transfected proteins in CL or CM was analyzed by heparin-Sepharose affinity followed by Western blot analysis with a rabbit anti-human Syt1 antibody (A) or IL1alpha IP followed by IL1alpha immunoblot analysis (B). A, Syt1 immunoblot: lanes 1-3, total CL from mIL1alpha ·beta -gal transfectants (lane 1), mIL1alpha ·beta -gal/p65 Syt1 cotransfectants (lane 2), and mIL1alpha ·beta -gal/p65 Syt1Delta (120-214) cotransfectants (lane 3). Lanes 4-6, 37 °C CM from mIL1alpha ·beta -gal transfectants, mIL1alpha ·beta -gal/p65 Syt1, and mIL1alpha ·beta -gal/p65 Syt1Delta (120-214) mutant cotransfectants, respectively; lanes 7-9, 42 °C CM from mIL-1alpha ·beta -gal single transfectants, mIL1alpha ·beta -gal/p65 Syt1, and mIL1alpha ·beta -gal/Syt1Delta (120-214) cotransfectants, respectively. B, IL1alpha immunoblot: total CL from mIL1alpha ·beta -gal transfectants (lanes 1 and 2), mIL1alpha ·beta -gal/p65-Syt1 cotransfectants (lanes 3 and 4), and mIL1alpha ·beta -gal/Syt1Delta (120-214) cotransfectants (lanes 5 and 6) after IP with a goat anti-human IL-1alpha antibody (lanes 1, 3, and 5) or a control antibody (lanes 2, 4, and 6); lanes 7-9, 37 °C CM from mIL1alpha ·beta -gal transfectants (lane 7), mIL1alpha ·beta -gal/p65- Syt1 cotransfectants (lane 8), and mIL1alpha ·beta -gal/Syt1Delta (120-214) cotransfectants (lane 9). Lanes 10-12, 42 °C CM from mIL1alpha ·beta -gal transfectants (lane 10), mIL1alpha ·beta -gal/p65-Syt1 cotransfectants (lane 11), and mIL1alpha ·beta -gal/Syt1Delta (120-214) cotransfectants (lane 12).

Although the data with p65 Syt1Delta (120-214) expression suggest that the IL1alpha and FGF1 release pathways may have diverged, we questioned whether the coexpression of IL1alpha and FGF1 in the NIH 3T3 cell could confirm this premise. Thus we obtained stable NIH 3T3 cell cotransfectants in which pIL1alpha ·beta -gal and mIL1alpha ·beta -gal NIH 3T3 transfectants were cotransfected with FGF1 and examined their ability to release mIL1alpha ·beta -gal, pIL1alpha ·beta -gal, and FGF1 in response to heat shock. As shown in Fig. 4, IL1alpha and FGF1 immunoblot analysis revealed the presence of both mIL1alpha ·beta -gal and FGF1 in medium conditioned by heat shock from the FGF1/mIL1alpha ·beta -gal NIH 3T3 cell cotransfectants. However, IL1alpha and FGF1 immunoblot analysis failed to detect the presence of either pIL1alpha or FGF1 from the FGF1/pIL1alpha ·beta -gal NIH 3T3 cell cotransfectants in response to temperature stress (Fig. 4). Because the precursor form but not the mature form of IL1alpha was able to repress the release of FGF1 in response to heat shock, the precursor domain of IL1alpha may contain a structural feature that may function as a dominant-negative effector of FGF1 release. Although we do not know the element within the precursor domain of IL1alpha responsible for this event, these data do suggest that the IL1alpha and FGF1 release pathways may indeed be convergent.



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Fig. 4.   Expression of pIL1alpha but not mIL1alpha represses the release of FGF1 in response to heat shock. The mIL1alpha ·beta -gal and pIL1alpha ·beta -gal NIH 3T3 cell transfectants, mIL1alpha ·beta -gal/FGF1, and pIL1alpha ·beta -gal/FGF1 NIH 3T3 cell cotransfectants were subjected to heat shock (42 °C, 2 h), and the level of expression of transfected proteins in CL or CM was analyzed by IP followed by IL1alpha immunoblot analysis with antibodies (A) or by heparin-Sepharose affinity followed by FGF1 immunoblot analysis (B). A, IL1alpha immunoblot. Lanes 1-4, total CL from mIL1alpha ·beta -gal transfectants (lane 1), mIL1alpha ·beta -gal/FGF1 cotransfectants (lane 2), pIL1alpha ·beta -gal transfectants (lane 3), and pIL1alpha ·beta -gal/FGF1 cotransfectants (lane 4). Lanes 5-6, 37 and 42 °CM from mIL1alpha ·beta -gal/FGF1 cotransfectants, respectively; lanes 7-8, 37 and 42 °CM from mIL1alpha ·beta -gal transfectants, respectively; lanes 9-10, 37 and 42 °CM from pIL1alpha ·beta -gal/FGF1 cotransfectants, respectively; lanes 11-12, 37 and 42 °CM from pIL1alpha ·beta -gal transfectants, respectively. B, FGF1 immunoblot. Lanes 1 and 2, total cell lysates from mIL1alpha ·beta -gal/FGF1 and pIL1alpha ·beta -gal/FGF1 cotransfectants, respectively; lanes 3-4, 37 and 42 °C CM from mIL1alpha ·beta -gal/FGF1 cotransfectants, respectively; lanes 5-6, 37 and 42 °C CM from pIL1alpha ·beta -gal/FGF1 cotransfectants, respectively.

It is well established that the precursor forms of the IL1 prototypes are cell-associated polypeptides whereas the mature forms of the IL1 prototypes are present in either biological fluids or cell culture medium (32-35); an observation which suggests that the mature forms of the IL1 prototypes may be preferred for release. Indeed, in transgenic mice manipulated to overexpress mIL1alpha in basal keratinocytes, high levels of mIL1alpha were released into the circulation (36). In contrast, however, mechanical deformation of human keratinocytes was required to induce a rapid release of pIL1alpha (37). Whereas both precursor and mature forms of the IL1 prototypes have also been detected in cell culture medium conditioned by mononuclear cells treated with lipopolysaccharides (34, 38), the appearance of the precursor forms correlated with the release of LDH, a cytosolic protein used to monitor the release of cytosol because of compromised plasma membrane integrity (39). It should be noted that our experimental conditions also employed LDH release, and we did not observe any significant levels of extracellular LDH in response to temperature stress. However, because the pIL1alpha ·beta -gal release studies were limited to a single clone, we cannot eliminate the possibility that a critical ratio of pIL1alpha ·beta -gal expression may enable pIL1alpha ·beta -gal to be released in response to heat shock.

Our data are consistent with the observation that human bladder carcinoma cells selectively release mIL1alpha but not pIL1alpha in vitro (40). Moreover, Siders, et al. (41) using a variety of different cell types transfected with either the precursor or mature forms of the IL1 prototypes observed that the mature forms of IL1alpha and IL1beta were also preferred for release and that the release of mIL1alpha from pIL1alpha was dependent upon the presence of a calpain-like protease activity (41). Interestingly, whereas macrophages from the ICE-null mouse were able to release pIL1beta but not mIL1beta in response to lipopolysaccharide stimulation, these cells were also deficient in IL1alpha release suggesting a possible role for ICE in the release of IL1alpha (42). Because we were unable to observe the presence of mIL1alpha in cytosol derived from the pIL1alpha NIH 3T3 cell transfectants, even under conditions of apoptosis where ICE is functional (data not shown), it is unlikely that ICE expression is involved in the formation of mIL1alpha in the NIH 3T3 cell. Rather, the NIH 3T3 cells may be deficient in the expression of the appropriate calpain and as a result are not capable of processing intracellular pIL1alpha . Alternatively, this may also be because of the presence of a calpain inhibitor (43) or because of the absence of calpain activation in response to heat shock. However, our data do suggest that the proteolytic conversion of pIL1alpha to mIL1alpha may be prerequisite for the appearance of IL1alpha in the extracellular compartment.

Although the ability of pIL1alpha to repress the release of FGF1 in response to heat shock suggests a connection between the IL1alpha and FGF1 release pathways, we do not know whether mIL1alpha requires the function of helper genes to gain access to the extracellular compartment. However, it is interesting to note that like FGF1 (18, 44), early studies with IL1alpha using gel exclusion chromatography also noted the presence of IL1alpha in high molecular weight complexes (34, 45). Whether these represent the IL1alpha equivalent of the multiprotein aggregate of FGF1(18) is not known. It is intriguing to speculate that like the FGF1 release pathway, the release of mIL1alpha in response to temperature stress may require the function of an unknown set of helper genes.


    ACKNOWLEDGEMENTS

We thank the officers of Takeda Pharmaceuticals, Ltd. and Hoffman-LaRoche, Inc, for their generous supply of amlexanox and IL1alpha antibody, respectively and Julia Frothingham and Norma Albrecht for expert administrative assistance.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL32348 and AG98503 (to T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Geriatric Medicine, University of Florence, School of Medicine, Florence, Italy 50100.

§ Supported by a fellowship from the Catholic University of Rome.

Present address: Div. of Lung Diseases, NHLBI, National Institutes of Health, Bethesda, MD 20892.

|| To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Inst., 81 Research Drive, Scarborough, ME 04074. Tel.: 207-885-8200; Fax: 207-885-8179; E-mail: maciat@mail.mmc.org.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.C000714200


    ABBREVIATIONS

The abbreviations used are: IL, interleukin; CL, cell lysate; CM, conditioned medium; ER, endoplasmic reticulum; FGF, fibroblast growth factor; beta -gal, beta -galactosidase; ICE, IL1beta converting enzyme; PATG, p-aminobenzyl-1-thio-beta -D-galactopyranoside; Syt, Synaptotagmin; p, precursor; m, mature; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; 2DG, 2-deoxyglucose; IP, immunoprecipitation, LDH, lactate dehydrogenase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


1. Dinarello, C. (1998) Int. Rev. Immunol. 16, 457-499[Medline] [Order article via Infotrieve]
2. Smith, D. E., Renshaw, B., Ketcham, R., Kubin, M., Garka, K., and Sims, J. (2000) J. Biol. Chem. 275, 1169-1175[Abstract/Free Full Text]
3. Giri, J., Lomedico, P., and Mizel, S. (1985) J. Immunol. 134, 343-349[Abstract/Free Full Text]
4. Cerretti, D., Koslosky, C., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T., March, C., Kronheim, S., Druck, T., Cannizzaro, L., Huebner, K., and Black, R. (1992) Science 256, 97-100[Medline] [Order article via Infotrieve]
5. Mosley, B., Urdal, D., Prickett, K., Larsen, A., Cosman, D., Conlon, P., Gillis, S., and Dower, S. (1987) J. Biol. Chem. 262, 2941-2944[Abstract/Free Full Text]
6. Carruth, L., Demczuk, S., and Mizel, S. (1991) J. Biol. Chem. 266, 12162-12167[Abstract/Free Full Text]
7. Wessendorf, J., Garfinkel, S., Zhan, X., Brown, S., and Maciag, T. (1993) J. Biol. Chem. 268, 22100-22104[Abstract/Free Full Text]
8. Stevenson, F., Turck, J., Locksley, R., and Lovett, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 508-513[Abstract/Free Full Text]
9. McMahon, G., Garfinkel, S., Prudovsky, I., Hu, X., and Maciag, T. (1997) J. Biol. Chem. 272, 28202-28205[Abstract/Free Full Text]
10. Thomas, K., Rios-Candelore, M., Gimenez-Gallego, G., DiSalvo, J., Bennett, C., Rodkey, J., and Fitzpatrick, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6409-6413[Abstract]
11. Zhang, J., Cousens, L., Barr, P., and Sprang, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3446-3450[Abstract]
12. Jackson, A., Friedman, S., Zhan, X., Engleka, K., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10691-10695[Abstract]
13. Shin, J., Opalenik, S., Wehby, J., Mahesh, V., Jackson, A., Tarantini, F., Maciag, T., and Thompson, J. (1996) Biochim. Biophys. Acta 1312, 27-38[Medline] [Order article via Infotrieve]
14. Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag, T. (1995) J. Biol. Chem. 270, 33-36[Abstract/Free Full Text]
15. Tarantini, F., Gamble, S., Jackson, A., and Maciag, T. (1995) J. Biol. Chem. 270, 29039-29042[Abstract/Free Full Text]
16. Tarantini, F., LaVallee, T., Jackson, A., Gamble, S., Carreira, C., Garfinkel, S., Burgess, W., and Maciag, T. (1998) J. Biol. Chem. 273, 22209-22216[Abstract/Free Full Text]
17. LaVallee, T., Tarantini, F., Gamble, S., Carreira, C., Jackson, A., and Maciag, T. (1998) J. Biol. Chem. 35, 22217-22223[CrossRef]
18. Carreira, C., LaVallee, T., Tarantini, F., Jackson, A., Lathrop, J., Hampton, B., Burgess, W., and Maciag, T. (1998) J. Biol. Chem. 35, 22224-22231[CrossRef]
19. Shi, J., Friedman, S., and Maciag, T. (1997) J. Biol. Chem. 272, 1142-1147[Abstract/Free Full Text]
20. Maier, J., Voulalas, P., Roeder, D., and Maciag, T. (1990) Science 249, 1570-1574[Medline] [Order article via Infotrieve]
21. Minter, A., Keoshkerian, E., Chesterman, C., and Dawes, J. (1996) J. Cell. Physiol. 167, 229-237[CrossRef][Medline] [Order article via Infotrieve]
22. Garfinkel, S., Haines, D., Brown, S., Wessendorf, J., Gillespie, D., and Maciag, T. (1992) J. Biol. Chem. 267, 24375-24378[Abstract/Free Full Text]
23. Norioka, K., Mitaka, T., Mochizuki, Y., Hara, M., Kawagoe, M., and Nakamura, H. (1985) Jpn. J. Cancer Res. 85, 522-529
24. Sawada, H., Kan, M., and McKeehan, W. (1990) In Vitro Cell Dev. Biol. 26, 213-216[Medline] [Order article via Infotrieve]
25. Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T., and Barbacid, M. (1989) Mol. Cell. Biol. 9, 24-33[Medline] [Order article via Infotrieve]
26. Germino, J., Gray, J., Charbonneau, H., Vanaman, T., and Bastia, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6848-6852[Abstract]
27. Sano, H., Forough, R., Maier, J., Case, J., Jackson, A., Engleka, K., Maciag, T., and Wilder, R. (1990) J. Cell Biol. 110, 1417-1426[Abstract]
28. Landriscina, M., Prudovsky, I., Carreira, C., Soldi, R., Tarantini, F., and Maciag, T. (2000) J. Biol. Chem. 275, 32753-32762[Abstract/Free Full Text]
29. Perin, M., Fried, V., Mignery, G., Jahn, R., and Sudhof, T. (1990) Nature 345, 260-263[CrossRef][Medline] [Order article via Infotrieve]
30. Perin, M., Brose, N., Jahn, R., and Sudhof, T. (1991) J. Biol. Chem. 266, 623-629[Abstract/Free Full Text]
31. Zhang, J., Davletov, B., Sudhof, T., and Anderson, R. (1994) Cell 78, 751-760[Medline] [Order article via Infotrieve]
32. Dinarello, C. (1991) Blood 77, 1627-1652[Abstract]
33. Dinarello, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 98485-98489
34. Suttles, J., Giri, J., and Mizel, S. (1990) J. Immunol. 144, 175-182[Abstract/Free Full Text]
35. Hogquist, K., Nett, M., Unanue, E., and Chaplin, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8485-8489[Abstract]
36. Groves, R., Mizutani, H., Kieffer, J., and Kupper, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11874-11878[Abstract]
37. Lee, R., Briggs, W., Cheng, G., Rossiter, H., Libby, P., and Kupper, T. (1997) J. Immunol. 159, 5084-5088[Abstract]
38. Carinci, V., Guida, S., Fontana, M., Palla, E., Rossini, M., and Melli, M. (1992) Eur. J. Biochem. 205, 295-301[Abstract]
39. Hogquist, K., Unanue, E., and Chaplin, D. (1991) J. Immunol. 147, 2181-2186[Abstract/Free Full Text]
40. Watanabe, N., and Kobayashi, Y. (1994) Cytokine 6, 597-601[Medline] [Order article via Infotrieve]
41. Siders, W., Klimovitz, J., and Mizel, S. (1993) J. Biol. Chem. 268, 22170-22174[Abstract/Free Full Text]
42. Kuida, K., Lippke, J., Ku, G., Harding, M., Livingston, D., Su, M., and Flavell, R. (1995) Science 267, 2000-2002[Medline] [Order article via Infotrieve]
43. Croall, D., and DeMartino, G. (1991) Physiol. Rev. 71, 813-847[Free Full Text]
44. Maciag, T., Hoover, G., and Weinstein, R. (1982) J. Biol. Chem. 257, 5333-5336[Abstract/Free Full Text]
45. Mizel, S., and Rosenstreich, D. (1979) J. Immunol. 122, 2173-2179[Medline] [Order article via Infotrieve]


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