Vol. 10, No. 9
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4596-4602 0270-7306/90/094596-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Transforming Growth Factor (x in Arterioles: Cell Surface Processing of Its Precursor by Elastases SUSAN G. MUELLER, ANDREW J. PATERSON,t AND JEFFREY E. KUDLOWt* Department of Clinical Biochemistry, University of Toronto, Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4 Received 10 May 1990/Accepted 15 June 1990
Analysis of the transforming growth factor a (TGFa) cDNA predicts that the mature TGFa polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGFot to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGFa, NR6 cells were transfected with the rat TGFoa cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGFa precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGFot polypeptide. The released TGFa was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGFea but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic x.chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGFot precursor from the fixed cells. The location of TGFa in the arterioles and the ability of PMN leukocytic elastase to process the membrane-bound TGFa precursor suggests a novel role for this elastase at the wound site. Injury is followed by a rapid increase in cellular proliferation at the wound site. However, the signals initiating and perpetuating this proliferative response are not fully understood. Much of the initial response to injury involves the coagulation process and removal of foreign material. Since proteolytic enzymes are involved in these initial processes, it would be attractive to consider that some of these proteases are also signals for the growth response. One means by which proteases might signal the growth response would be by involvement in processing and/or activation of growth factors at the wound site. This concept has two requirements: (i) that the wound site contain a preformed store of unprocessed growth factor, and (ii) that this pool of growth factor be accessible to extracellular proteases capable of correct cleavage of the growth factor precursor to its active form. In concert with the first requirement, we now report that we have systematically localized transforming growth factor a (TGFox) immunoreactivity in the smooth muscle cells of small arterioles during the course of our immunohistochemical studies of TGFoa in the pituitary (16), brain (20), and ovary (19, 22). The vascular smooth muscle cell could then be an immediate source of this growth factor at an injury site. The precursor for TGFoa could fulfill the second requirement in that it is initially synthesized as a membraneanchored molecule (4, 8, 10, 21, 35). The extracellular domain of the TGFa precursor contains the sequence corresponding to the mature TGFa, suggesting that the precursor could be processed by proteolysis in the extracellular com-
partment. Thus, processing and release of TGFa from the
vascular smooth muscle cell could be accomplished by one of the proteases involved in the initial injury response. Analysis of the proteolytic cleavage sites, as predicted from the TGFo cDNA sequence, indicates the participation of an elastaselike enzyme in its processing (8, 21). Since elastases are produced by a variety of blood cells involved in the injury response, including polymorphonuclear (PMN) leukocytes (15), platelets (14), and macrophages (7), these cells could release an elastase capable of processing the TGFa precursor from its membrane-bound form to its mature, soluble form. Indeed, a previous study demonstrated that porcine pancreatic elastase could release the mature TGFot from an intermediately processed, soluble form of the TGFa precursor (13). Whether pancreatic elastase or, in this context, the more physiologically relevant elastases derived from blood elements, such as PMN leukocytic elastase, could release the fully processed mature TGFa from the intact membrane-bound TGFao precursor has not been determined. To investigate whether the TGFot precursor can be processed extracellularly such that the mature growth factor is released from the plasma membrane, we created a model cell with which to test processing by extracellularly applied enzymes. We stably transfected the NR6 strain of Swiss 3T3 cells (29) with the rat TGFa cDNA encoding the entire open reading frame for the TGFot precursor. A cloned transfectant, termed 1B2, expressed the TGFot precursor molecule, which was localized to the cell surface. In this study, we show that incubation of these cells in the presence of either PMN leukocytic or pancreatic elastase resulted in the release of the mature, active TGFot into the culture medium, indicating that the TGFot precursor is in a conformation on
* Corresponding author. t Present address: Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, AL 35294.
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the cell surface allowing its processing to the mature growth factor. If the TGFa immunohistochemically localized to the vascular smooth muscle cells is in a conformation similar to that assumed in the 1B2 cells, then the vascular smooth muscle cell may serve as an immediate source of TGFa at the wound site, with release controlled by PMN leukocytic elastase. MATERIALS AND METHODS Materials, Culture media, serum, G418, and trypsin were from GIBCO Laboratories (Grand Island, N.Y.). L-[2,3,4,53H]leucine was from ICN Radiochemicals (Irvine, Calif.). Omnifluor and [methyl-3H]thymidine were from Dupont, NEN Research Products (Boston, Mass.). 1251 was obtained from Amersham Corp. (Arlington Heights, Ill.). Protein A was labeled with 1251 by the chloramine T method. Mouse epidermal growth factor (EGF) was purified from male Swiss Webster mouse submaxillary glands (28) and was iodinated by using Iodogen (Pierce Chemical Co., Rockford, Ill.). Human PMN leukocytic elastase was from Elastin Products Co., Inc. (Pacific, Mo.). Porcine pancreatic elastase was from Boehringer Mannheim Canada, Ltd. (Dorval, Quebec, Canada). Collagenase was from Worthington Diagnostic Systems, Inc. (Freehold, N.J.). Bovine pancreatic a1-chymotrypsin and subtilisin were from Sigma Chemical Co. (St. Louis, Mo.). Immunohistochemical staining. Tissue was fixed immediately upon removal from the animal in 10%o buffered Formalin and subsequently embedded in paraffin. Tissue sections (5 ,um thick) were stained by the immunoperoxidase technique as previously described (16, 19, 20, 22). The specificity of the immunostaining was verified by replacing the MF9 monoclonal antibody with buffered saline or with MF9 preincubated with synthetic carboxy-terminal TGFa peptide (10 jig/ml) to which MF9 was raised. Cell culture. The NR6 strain of mouse Swiss 3T3 fibroblasts was cultured in Dulbecco modified Eagle medium (DMEM) containing 10% calf serum (CS), 100 mg of penicillin per liter, and 10 mg of gentamicin per liter. Cells were incubated at 37°C in 5% C02-95% air. Cel transfection, NR6 cells (106/10-cm plate) were cotransfected by the calcium phosphate precipitation technique (37) with 10 jig of pEV-2 and 1 jig of pSV2-neo expression vectors. The pEV-2 vector contained the rat TGFa cDNA which encoded the entire precursor sequence. The TGFa cDNA was placed under the control of the inducible mouse metallothionein I promoter as previously described (10). The pSV2-neo vector provided transfected cells with resistance to the antibiotic G418. Stable transfectants were established in the presence of G418. One cloned transfectant, referred to as 1B2, was chosen for all the experiments. Cell induction and labelin. Cells were routinely subcultured at a density of 5 x 10 cells per 10-cm tissue culture plate in l1o CS-DMEM. After an overnight incubation, the culture medium was replaced with 100 jiM ZnCl2-109o CS-DMEM containing 5 jiCi of L-[2,3,4,5-3H]leucine per ml. The cells were incubated for a further 2 days before experiments were conducted. Elastase treatment. Induced and labeled cells were rinsed initially with 10 mM acetic acid-150 mM NaCl, followed by 0.1% bovine serum albumin-phosphate-buffered saline (PBS) and then three times with PBS. After the final wash, 4 ml of either a pancreatic or a PMN leukocytic elastase solution was added to the cells. The elastase was diluted in
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4597
DMEM; concentrations varied from 0.1 to 10.0 U/ml. Cells were incubated at 37°C in the presence of elastase for 1 h. After the elastase treatment, the medium was removed from the cells and the cells were rinsed with 1 ml of 10 mM acetic acid; this wash was pooled with the medium. Phenylmethylsulfonyl fluoride (PMSF; 10 ,ug/ml) was added to the medium, which was clarified by low-speed centrifugation. The medium was desalted and concentrated, using a C-18 Seppak column (Waters Associates, Inc., Milford, Mass.) that had been prewashed with methanol and then equilibrated with H20. The medium was passed through the column, collected, and reloaded onto the column. This procedure was repeated twice. The column was then washed with H20, and the protein was eluted with 70o acetonitrile0.1% trifluoroacetic acid. The eluate was lyophilized, reconstituted in a urea sample buffer (7 M urea, 10% glycerol, 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 62.5 mM Tris hydrochloride, pH 6.8), boiled for 5 min, and then electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane (Schleicher and Schuell, Inc., Keene, N.H.) and analyzed by Western blot (immunoblot) analysis. Whole-cell lysates were prepared from the elastasetreated cells after the medium had been removed and the cells had been rinsed with 10 mM acetic acid. The cells were scraped from the tissue culture plates in PBS and then pelleted by low-speed centrifugation. The cells were then lysed in 300 jil of urea sample buffer, sheared by being drawn through a 23-gauge needle three times, and then boiled for 5 min. Samples (30 jil) of the cell lysates were trichloroacetic acid precipitated, and the incorporated radioactivity was determined. Normalized volumes of the cell lysates, containing the same number of trichloroacetic acid-precipitable counts, were then electrophoresed through a 15% SDSpolyacrylamide gel. The proteins were then transblotted onto a nitrocellulose membrane and analyzed by Western blot analysis. Western blot analysis. The nitrocellulose membranes were routinely blocked with 10% skim milk powder-0.3% TweenPBS for 2 h at room temperature, followed by an overnight incubation at 4°C with MF9 (10 jg/ml), a monoclonal antibody directed against mature TGFa (17), in fresh blocking solution. The blots were then washed with 0.1% Tween-PBS three times for 10 min each time. The blots were then incubated with rabbit anti-mouse antibodies (1 jig/ml) for 1 h at room temperature, followed by another three 10-min washes as described above. The blots were finally incubated with '251-labeled protein A (5 x 105 cpm/ml) for 30 min and then rewashed. After the final wash, the blots were exposed to Kodak X-MAT AR film with a Dupont Cronex intensifying screen at -70°C. EGF radioreceptor assay. EGF competing activity was measured by using formaldehyde-fixed A431 cells in 24-well plates as previously described (18). Mitogenic assay. Subconfluent normal rabbit kidney (NRK) cells grown in 5% CS-DMEM were trypsinized and plated into 24-well plates (Limbro) at a density of 4 x 104 cells per well in 500 jIl of 0.1% CS-3 ,uM thymidine-DMEM. The 24-well plates had been previously incubated with 500 jl of 5% CS-DMEM at 37°C for 1 h. This medium was removed, and the wells were rinsed twice with PBS before the seeding with NRK cells. The cells were incubated at 37°C for 3 days, after which 50 jil of culture medium from elastase-treated 1B2 cells were added to the NRK cells. The elastase treatment of the 1B2 cells was carried out as described above. The elastase in the culture medium was
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neutralized with PMSF. The NRK cells were allowed to incubate for a further 18 h, and then 2 ,uCi of [methyl3Hlthymidine was added to each well. After 5 h, the medium was aspirated, and cells were washed, trypsinized, and filtered. The filters were then assayed for radioactivity by scintillation counting, using Omnifluor in toluene scintillation fluid. Protease treatment of formaldehyde-fixed 1B2 cells. 1B2 cells were subcultured in 10-cm tissue culture plates and zinc induced as described above. Cells were rinsed twice with PBS and then fixed with 10% formaldehyde for 30 min. After the 30-min incubation, the cells were rinsed five times with PBS. The formaldehyde-fixed 1B2 cells were either stored in PBS at 4°C or used immediately. Before protease treatment, the 1B2 cells were rinsed with 10 mM acetic acid-150 mM NaCl, followed by two rinses with PBS. The formaldehydefixed 1B2 cells were then treated with 4 ml of the following protease solutions: (i) DMEM alone, (ii) 10 U of human PMN leukocytic elastase per ml, (iii) 10 U of porcine pancreatic elastase per ml, (iv) 1 U of human cathepsin G per ml, (v) 5 U of bovine pancreatic a1-chymotrypsin per ml, (vi) 10 U of collagenase per ml, (vii) 10 ,ug of trypsin per ml, or (viii) 1 U of subtilisin per ml. The 1B2 cells were incubated at 37°C for 1 h. After the protease treatment, the medium was removed from the cells and the cells were rinsed with 1 ml of 10 mM acetic acid; this wash was pooled with the medium. The medium was clarified by low-speed centrifugation, desalted, and concentrated by using a C-18 Seppak column as described above. The samples were then electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane and analyzed by Western blotting.
RESULTS Immunohistochemical localization of TGFa in arterioles. During our immunohistochemical studies on the localization of TGFa in the pituitary (16), brain (20), and ovary (19, 22), we have systematically detected TGFa immunoreactivity in the small arterioles of the tissues under examination. Figure 1 shows a typical arteriole observed in sections of the anterior pituitary gland when stained with the anti-TGFa monoclonal antibody MF9. There is strong immunoperoxidase staining of the vascular smooth muscle cells, which could be blocked by preadsorption of the MF9 antibody with the synthetic peptide to which this antibody was raised. Expression of the rat TGFa precursor in transfected NR6 cells. NR6 cells were transfected with a mammalian expression vector containing the 700-base-pair SmaI fragment of the rat TGFa cDNA (21). This fragment of the cDNA contains the entire coding sequence of the TGFa precursor. The placement of the rat TGFa cDNA in the pEV2 expression vector (10) downstream of the inducible mouse metallothionein I promoter resulted in zinc inducibility of the TGFa transcript (data not shown). One of the cloned transfectants, termed 1B2, showed about a 10-fold zinc-dependent (100 p,M ZnCl2) induction of the 1-kilobase-pair TGFa mRNA. The parental NR6 cell line did not express this TGFa mRNA. Examination of total cell lysates from zincinduced 1B2 cells by Western blot analysis using the antiTGFa monoclonal antibody MF9 demonstrated that the 1B2 cells expressed immunoreactive TGFa appearing as a doublet centered at approximately 20 kilodaltons (kDa). This species was not detectable in either the uninduced 1B2 cells or the NR6 parental cell line (Fig. 2a). On the basis of studies in other systems, this 20-kDa material represents the vari-
MOL. CELL. BIOL.
FIG. 1. Immunohistochemical localization of TGFa to arterioles in normal adult tissue. Bovine anterior pituitary glands and adherent tissue were fixed immediately upon removal from the animal in 10% buffered Formalin and subsequently embedded in paraffin. Tissue sections (5 p.m thick) were stained with the anti-TGFa monoclonal antibody MF9 by the immunoperoxidase technique (see text). Shown is a typical immunopositive arteriole found in the section. Magnification, x60. ably glycosylated TGFa precursor (4, 10, 35). We localized the TGFa precursor to the plasma membrane of the 1B2 cells by using differential centrifugation (data not shown). This plasma membrane localization is in agreement with previous reports (4, 10, 35). Western blot analysis of the serum-free culture medium conditioned by the 1B2 cells for 12 h demonstrated the presence of the fully processed 5 kDa TGFax in a zinc-dependent manner (Fig. 2b). The medium from the zinc-induced cells but not the uninduced or parental cells contained mitogenic and corresponding EGF receptor binding activity (data not shown). Processing of the cell surface rat TGFa precursor by elastases. 1B2 cells were zinc induced and treated for 1 h with various concentrations of either PMN leukocytic or pancreatic elastase. After the elastase treatment, whole-cell lysates and the culture medium were analyzed for a change in the level of TGFa immunoreactivity by Western blot analysis. We analyzed the effect of human PMN leukocytic elastase on the amount of cell-associated TGFa precursor (Fig. 3) and on the release of mature, 5-kDa TGFa into the culture medium (Fig. 4). A concentration-dependent decrease was observed in the amount of cell-associated TGFa precursor. Correspondingly, the medium from the elastase-treated cells contained a concentration-dependent increase in the amount of the mature TGFa species. The culture medium from the elastase-treated 1B2 cells was also analyzed for biological activity by the EGF radioreceptor assay and for mitogenic
VOL. 10, 1990
TGFa PROCESSING
a
NR6
1B2
EZnCI2]
0
0
100
100
ztM
b
NR6
1B2
[ZnCI2]
0 100
0
4599
;1M
100
97.4 66.2 42.7
-
3,.0
-
97.4 66.2
-
42.7
-
31.0 -
21.5
21.5 -
I, 1 4.4 -
-
14.4 -
6.2 3.0 -
FIG. 2. (a) Detection and characterization of the expressed TGFa precursor in total cellular lysates. The transfected cell line 1B2 and the parental cell line NR6 were grown in the presence or absence of 100 ,uM ZnCl2 in 10% CS-DMEM for 2 days. Total cellular lysates were prepared as described in Materials and Methods. Samples were electrophoresed through a 15% SDS-polyacrylamide gel, transblotted onto nitrocellulose, and analyzed by Western blot analysis using the anti-TGFa monoclonal antibody MF9. (b) Detection of the released TGFa in the culture medium of the 1B2 cells. The transfected cell line 1B2 and the parental cell line NR6 were grown in the presence or absence of 100 p.M ZnCl2 in 10% CS-DMEM for 36 h. The medium was then replaced by a serum-free medium, and the cells were incubated overnight. The serum-free medium was then desalted and concentrated, using a C-18 Seppak column as described in Materials and Methods. Samples were electrophoresed through a 12 to 25% SDS-polyacrylamide gradient gel, transblotted onto a nitrocellulose membrane, and then analyzed by Western blot analysis using MF9. Molecular size standards in kilodaltons are shown on the left.
activity, as measured by the stimulation of [3H]thymidine incorporation. The level of EGF-displacing activity in the medium was increased approximately sevenfold upon treatment of the induced 1B2 cells with 10 U of PMN leukocytic elastase per ml (Fig. 5). This increase in EGF-displacing activity corresponded to an increase in mitogenic activity in the culture medium (Fig. 6). Similar effects were observed for the porcine pancreatic elastase; when values were corrected for specific activity, both enzymes appeared equally active. Treatment of formaldehyde-fixed 1B2 cells with various 0
.1
1
5
10
U/mi
proteases. 1B2 cells were zinc induced and then fixed with formaldehyde. The formaldehyde-fixed 1B2 cells were then treated for 1 h at 37°C with the following proteases: PMN leukocytic elastase, pancreatic elastase, cathepsin G, alchymotrypsin, collagenase, trypsin, and subtilisin. The culture medium was analyzed by Western blot analysis for the appearance of either the mature TGFa species or intermediately processed forms of the TGFa precursor. There was no basal release of TGFa from the formaldehyde-fixed 1B2 cells (Fig. 7, lane 1). Treatment with either PMN leukocytic or pancreatic elastase resulted in the accumulation of a 5-kDa TGFa species corresponding in size to the mature TGFa (Fig. 7, lanes 2 and 3). Treatment of the formaldehyde-fixed 1B2 cells with cathepsin G, ac-chymotrypsin, collagenase, trypsin, or subtilisin failed to release any
25.7 0 6.2
-
3.0
-
.1
5
10
U/mi
aSW
184 12.0 FIG. 3. Processing of the cell surface TGFa precursor by PMN leukocytic elastase. Subconfluent 1B2 cells were induced with 100 ,uM ZnC12 in 10% CS-DMEM for 48 h. Cells were then rinsed thoroughly with PBS and incubated with various concentrations of PMN leukocytic elastase (0, 0.1, 1.0, 5.0, and 10.0 U/ml) in DMEM. After a 1-h incubation at 37°C, whole-cell lysates were prepared from the elastase-treated cells (see Materials and Methods). Normalized volumes of cell lysates, based on trichloroacetic acidprecipitable counts, were electrophoresed through a 15% SDSpolyacrylamide gel, transblotted onto a nitrocellulose membrane, and analyzed by Western blot analysis. Molecular size standards in kilodaltons are shown on the left.
FIG. 4. Release of mature TGFa into the culture medium from the cell surface TGFa precursor by PMN leukocytic elastase. 1B2 cells were induced with ZnCl2 and elastase treated as described for Fig. 3. After the 1-h incubation at 37°C, the culture medium was removed from the cells, the cells were rinsed with 10 mM acetic acid, and the wash was pooled with the culture medium. PMSF (10 p.g/ml) was added to the medium, which was clarified by low-speed centrifugation, desalted, and concentrated as described above. Samples were electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane and analyzed by Western blot analysis using MF9. Molecular size standards in kilodaltons are shown on the left.
4600
MOL. CELL. BIOL.
MUELLER ET AL.
800
I
~
1
600
3
4
5
6
7
8
6.2 -
2b400
(
2
3.0 -
200
0
0
.1
1
Elastase Concentration (U/ml) FIG. 5. Release of EGF-displacing activity into the culture medium by PMN leukocytic elastase treatment of induced 1B2 cells. 1B2 cells were induced with ZnCl2 and elastase treated as described for Fig. 3. After the 1-h incubation at 37°C, the culture medium was removed from the cells. PMSF was added to the medium to neutralize the elastase. The medium was then assayed for EGFdisplacing activity, using the EGF radioreceptor assay. Each point is the mean of triplicate determinations (coefficient of variation,
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4596-4602 0270-7306/90/094596-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Transforming Growth Factor (x in Arterioles: Cell Surface Processing of Its Precursor by Elastases SUSAN G. MUELLER, ANDREW J. PATERSON,t AND JEFFREY E. KUDLOWt* Department of Clinical Biochemistry, University of Toronto, Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4 Received 10 May 1990/Accepted 15 June 1990
Analysis of the transforming growth factor a (TGFa) cDNA predicts that the mature TGFa polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGFot to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGFa, NR6 cells were transfected with the rat TGFoa cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGFa precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGFot polypeptide. The released TGFa was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGFea but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic x.chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGFot precursor from the fixed cells. The location of TGFa in the arterioles and the ability of PMN leukocytic elastase to process the membrane-bound TGFa precursor suggests a novel role for this elastase at the wound site. Injury is followed by a rapid increase in cellular proliferation at the wound site. However, the signals initiating and perpetuating this proliferative response are not fully understood. Much of the initial response to injury involves the coagulation process and removal of foreign material. Since proteolytic enzymes are involved in these initial processes, it would be attractive to consider that some of these proteases are also signals for the growth response. One means by which proteases might signal the growth response would be by involvement in processing and/or activation of growth factors at the wound site. This concept has two requirements: (i) that the wound site contain a preformed store of unprocessed growth factor, and (ii) that this pool of growth factor be accessible to extracellular proteases capable of correct cleavage of the growth factor precursor to its active form. In concert with the first requirement, we now report that we have systematically localized transforming growth factor a (TGFox) immunoreactivity in the smooth muscle cells of small arterioles during the course of our immunohistochemical studies of TGFoa in the pituitary (16), brain (20), and ovary (19, 22). The vascular smooth muscle cell could then be an immediate source of this growth factor at an injury site. The precursor for TGFoa could fulfill the second requirement in that it is initially synthesized as a membraneanchored molecule (4, 8, 10, 21, 35). The extracellular domain of the TGFa precursor contains the sequence corresponding to the mature TGFa, suggesting that the precursor could be processed by proteolysis in the extracellular com-
partment. Thus, processing and release of TGFa from the
vascular smooth muscle cell could be accomplished by one of the proteases involved in the initial injury response. Analysis of the proteolytic cleavage sites, as predicted from the TGFo cDNA sequence, indicates the participation of an elastaselike enzyme in its processing (8, 21). Since elastases are produced by a variety of blood cells involved in the injury response, including polymorphonuclear (PMN) leukocytes (15), platelets (14), and macrophages (7), these cells could release an elastase capable of processing the TGFa precursor from its membrane-bound form to its mature, soluble form. Indeed, a previous study demonstrated that porcine pancreatic elastase could release the mature TGFot from an intermediately processed, soluble form of the TGFa precursor (13). Whether pancreatic elastase or, in this context, the more physiologically relevant elastases derived from blood elements, such as PMN leukocytic elastase, could release the fully processed mature TGFa from the intact membrane-bound TGFao precursor has not been determined. To investigate whether the TGFot precursor can be processed extracellularly such that the mature growth factor is released from the plasma membrane, we created a model cell with which to test processing by extracellularly applied enzymes. We stably transfected the NR6 strain of Swiss 3T3 cells (29) with the rat TGFa cDNA encoding the entire open reading frame for the TGFot precursor. A cloned transfectant, termed 1B2, expressed the TGFot precursor molecule, which was localized to the cell surface. In this study, we show that incubation of these cells in the presence of either PMN leukocytic or pancreatic elastase resulted in the release of the mature, active TGFot into the culture medium, indicating that the TGFot precursor is in a conformation on
* Corresponding author. t Present address: Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, AL 35294.
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the cell surface allowing its processing to the mature growth factor. If the TGFa immunohistochemically localized to the vascular smooth muscle cells is in a conformation similar to that assumed in the 1B2 cells, then the vascular smooth muscle cell may serve as an immediate source of TGFa at the wound site, with release controlled by PMN leukocytic elastase. MATERIALS AND METHODS Materials, Culture media, serum, G418, and trypsin were from GIBCO Laboratories (Grand Island, N.Y.). L-[2,3,4,53H]leucine was from ICN Radiochemicals (Irvine, Calif.). Omnifluor and [methyl-3H]thymidine were from Dupont, NEN Research Products (Boston, Mass.). 1251 was obtained from Amersham Corp. (Arlington Heights, Ill.). Protein A was labeled with 1251 by the chloramine T method. Mouse epidermal growth factor (EGF) was purified from male Swiss Webster mouse submaxillary glands (28) and was iodinated by using Iodogen (Pierce Chemical Co., Rockford, Ill.). Human PMN leukocytic elastase was from Elastin Products Co., Inc. (Pacific, Mo.). Porcine pancreatic elastase was from Boehringer Mannheim Canada, Ltd. (Dorval, Quebec, Canada). Collagenase was from Worthington Diagnostic Systems, Inc. (Freehold, N.J.). Bovine pancreatic a1-chymotrypsin and subtilisin were from Sigma Chemical Co. (St. Louis, Mo.). Immunohistochemical staining. Tissue was fixed immediately upon removal from the animal in 10%o buffered Formalin and subsequently embedded in paraffin. Tissue sections (5 ,um thick) were stained by the immunoperoxidase technique as previously described (16, 19, 20, 22). The specificity of the immunostaining was verified by replacing the MF9 monoclonal antibody with buffered saline or with MF9 preincubated with synthetic carboxy-terminal TGFa peptide (10 jig/ml) to which MF9 was raised. Cell culture. The NR6 strain of mouse Swiss 3T3 fibroblasts was cultured in Dulbecco modified Eagle medium (DMEM) containing 10% calf serum (CS), 100 mg of penicillin per liter, and 10 mg of gentamicin per liter. Cells were incubated at 37°C in 5% C02-95% air. Cel transfection, NR6 cells (106/10-cm plate) were cotransfected by the calcium phosphate precipitation technique (37) with 10 jig of pEV-2 and 1 jig of pSV2-neo expression vectors. The pEV-2 vector contained the rat TGFa cDNA which encoded the entire precursor sequence. The TGFa cDNA was placed under the control of the inducible mouse metallothionein I promoter as previously described (10). The pSV2-neo vector provided transfected cells with resistance to the antibiotic G418. Stable transfectants were established in the presence of G418. One cloned transfectant, referred to as 1B2, was chosen for all the experiments. Cell induction and labelin. Cells were routinely subcultured at a density of 5 x 10 cells per 10-cm tissue culture plate in l1o CS-DMEM. After an overnight incubation, the culture medium was replaced with 100 jiM ZnCl2-109o CS-DMEM containing 5 jiCi of L-[2,3,4,5-3H]leucine per ml. The cells were incubated for a further 2 days before experiments were conducted. Elastase treatment. Induced and labeled cells were rinsed initially with 10 mM acetic acid-150 mM NaCl, followed by 0.1% bovine serum albumin-phosphate-buffered saline (PBS) and then three times with PBS. After the final wash, 4 ml of either a pancreatic or a PMN leukocytic elastase solution was added to the cells. The elastase was diluted in
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DMEM; concentrations varied from 0.1 to 10.0 U/ml. Cells were incubated at 37°C in the presence of elastase for 1 h. After the elastase treatment, the medium was removed from the cells and the cells were rinsed with 1 ml of 10 mM acetic acid; this wash was pooled with the medium. Phenylmethylsulfonyl fluoride (PMSF; 10 ,ug/ml) was added to the medium, which was clarified by low-speed centrifugation. The medium was desalted and concentrated, using a C-18 Seppak column (Waters Associates, Inc., Milford, Mass.) that had been prewashed with methanol and then equilibrated with H20. The medium was passed through the column, collected, and reloaded onto the column. This procedure was repeated twice. The column was then washed with H20, and the protein was eluted with 70o acetonitrile0.1% trifluoroacetic acid. The eluate was lyophilized, reconstituted in a urea sample buffer (7 M urea, 10% glycerol, 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 62.5 mM Tris hydrochloride, pH 6.8), boiled for 5 min, and then electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane (Schleicher and Schuell, Inc., Keene, N.H.) and analyzed by Western blot (immunoblot) analysis. Whole-cell lysates were prepared from the elastasetreated cells after the medium had been removed and the cells had been rinsed with 10 mM acetic acid. The cells were scraped from the tissue culture plates in PBS and then pelleted by low-speed centrifugation. The cells were then lysed in 300 jil of urea sample buffer, sheared by being drawn through a 23-gauge needle three times, and then boiled for 5 min. Samples (30 jil) of the cell lysates were trichloroacetic acid precipitated, and the incorporated radioactivity was determined. Normalized volumes of the cell lysates, containing the same number of trichloroacetic acid-precipitable counts, were then electrophoresed through a 15% SDSpolyacrylamide gel. The proteins were then transblotted onto a nitrocellulose membrane and analyzed by Western blot analysis. Western blot analysis. The nitrocellulose membranes were routinely blocked with 10% skim milk powder-0.3% TweenPBS for 2 h at room temperature, followed by an overnight incubation at 4°C with MF9 (10 jg/ml), a monoclonal antibody directed against mature TGFa (17), in fresh blocking solution. The blots were then washed with 0.1% Tween-PBS three times for 10 min each time. The blots were then incubated with rabbit anti-mouse antibodies (1 jig/ml) for 1 h at room temperature, followed by another three 10-min washes as described above. The blots were finally incubated with '251-labeled protein A (5 x 105 cpm/ml) for 30 min and then rewashed. After the final wash, the blots were exposed to Kodak X-MAT AR film with a Dupont Cronex intensifying screen at -70°C. EGF radioreceptor assay. EGF competing activity was measured by using formaldehyde-fixed A431 cells in 24-well plates as previously described (18). Mitogenic assay. Subconfluent normal rabbit kidney (NRK) cells grown in 5% CS-DMEM were trypsinized and plated into 24-well plates (Limbro) at a density of 4 x 104 cells per well in 500 jIl of 0.1% CS-3 ,uM thymidine-DMEM. The 24-well plates had been previously incubated with 500 jl of 5% CS-DMEM at 37°C for 1 h. This medium was removed, and the wells were rinsed twice with PBS before the seeding with NRK cells. The cells were incubated at 37°C for 3 days, after which 50 jil of culture medium from elastase-treated 1B2 cells were added to the NRK cells. The elastase treatment of the 1B2 cells was carried out as described above. The elastase in the culture medium was
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neutralized with PMSF. The NRK cells were allowed to incubate for a further 18 h, and then 2 ,uCi of [methyl3Hlthymidine was added to each well. After 5 h, the medium was aspirated, and cells were washed, trypsinized, and filtered. The filters were then assayed for radioactivity by scintillation counting, using Omnifluor in toluene scintillation fluid. Protease treatment of formaldehyde-fixed 1B2 cells. 1B2 cells were subcultured in 10-cm tissue culture plates and zinc induced as described above. Cells were rinsed twice with PBS and then fixed with 10% formaldehyde for 30 min. After the 30-min incubation, the cells were rinsed five times with PBS. The formaldehyde-fixed 1B2 cells were either stored in PBS at 4°C or used immediately. Before protease treatment, the 1B2 cells were rinsed with 10 mM acetic acid-150 mM NaCl, followed by two rinses with PBS. The formaldehydefixed 1B2 cells were then treated with 4 ml of the following protease solutions: (i) DMEM alone, (ii) 10 U of human PMN leukocytic elastase per ml, (iii) 10 U of porcine pancreatic elastase per ml, (iv) 1 U of human cathepsin G per ml, (v) 5 U of bovine pancreatic a1-chymotrypsin per ml, (vi) 10 U of collagenase per ml, (vii) 10 ,ug of trypsin per ml, or (viii) 1 U of subtilisin per ml. The 1B2 cells were incubated at 37°C for 1 h. After the protease treatment, the medium was removed from the cells and the cells were rinsed with 1 ml of 10 mM acetic acid; this wash was pooled with the medium. The medium was clarified by low-speed centrifugation, desalted, and concentrated by using a C-18 Seppak column as described above. The samples were then electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane and analyzed by Western blotting.
RESULTS Immunohistochemical localization of TGFa in arterioles. During our immunohistochemical studies on the localization of TGFa in the pituitary (16), brain (20), and ovary (19, 22), we have systematically detected TGFa immunoreactivity in the small arterioles of the tissues under examination. Figure 1 shows a typical arteriole observed in sections of the anterior pituitary gland when stained with the anti-TGFa monoclonal antibody MF9. There is strong immunoperoxidase staining of the vascular smooth muscle cells, which could be blocked by preadsorption of the MF9 antibody with the synthetic peptide to which this antibody was raised. Expression of the rat TGFa precursor in transfected NR6 cells. NR6 cells were transfected with a mammalian expression vector containing the 700-base-pair SmaI fragment of the rat TGFa cDNA (21). This fragment of the cDNA contains the entire coding sequence of the TGFa precursor. The placement of the rat TGFa cDNA in the pEV2 expression vector (10) downstream of the inducible mouse metallothionein I promoter resulted in zinc inducibility of the TGFa transcript (data not shown). One of the cloned transfectants, termed 1B2, showed about a 10-fold zinc-dependent (100 p,M ZnCl2) induction of the 1-kilobase-pair TGFa mRNA. The parental NR6 cell line did not express this TGFa mRNA. Examination of total cell lysates from zincinduced 1B2 cells by Western blot analysis using the antiTGFa monoclonal antibody MF9 demonstrated that the 1B2 cells expressed immunoreactive TGFa appearing as a doublet centered at approximately 20 kilodaltons (kDa). This species was not detectable in either the uninduced 1B2 cells or the NR6 parental cell line (Fig. 2a). On the basis of studies in other systems, this 20-kDa material represents the vari-
MOL. CELL. BIOL.
FIG. 1. Immunohistochemical localization of TGFa to arterioles in normal adult tissue. Bovine anterior pituitary glands and adherent tissue were fixed immediately upon removal from the animal in 10% buffered Formalin and subsequently embedded in paraffin. Tissue sections (5 p.m thick) were stained with the anti-TGFa monoclonal antibody MF9 by the immunoperoxidase technique (see text). Shown is a typical immunopositive arteriole found in the section. Magnification, x60. ably glycosylated TGFa precursor (4, 10, 35). We localized the TGFa precursor to the plasma membrane of the 1B2 cells by using differential centrifugation (data not shown). This plasma membrane localization is in agreement with previous reports (4, 10, 35). Western blot analysis of the serum-free culture medium conditioned by the 1B2 cells for 12 h demonstrated the presence of the fully processed 5 kDa TGFax in a zinc-dependent manner (Fig. 2b). The medium from the zinc-induced cells but not the uninduced or parental cells contained mitogenic and corresponding EGF receptor binding activity (data not shown). Processing of the cell surface rat TGFa precursor by elastases. 1B2 cells were zinc induced and treated for 1 h with various concentrations of either PMN leukocytic or pancreatic elastase. After the elastase treatment, whole-cell lysates and the culture medium were analyzed for a change in the level of TGFa immunoreactivity by Western blot analysis. We analyzed the effect of human PMN leukocytic elastase on the amount of cell-associated TGFa precursor (Fig. 3) and on the release of mature, 5-kDa TGFa into the culture medium (Fig. 4). A concentration-dependent decrease was observed in the amount of cell-associated TGFa precursor. Correspondingly, the medium from the elastase-treated cells contained a concentration-dependent increase in the amount of the mature TGFa species. The culture medium from the elastase-treated 1B2 cells was also analyzed for biological activity by the EGF radioreceptor assay and for mitogenic
VOL. 10, 1990
TGFa PROCESSING
a
NR6
1B2
EZnCI2]
0
0
100
100
ztM
b
NR6
1B2
[ZnCI2]
0 100
0
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;1M
100
97.4 66.2 42.7
-
3,.0
-
97.4 66.2
-
42.7
-
31.0 -
21.5
21.5 -
I, 1 4.4 -
-
14.4 -
6.2 3.0 -
FIG. 2. (a) Detection and characterization of the expressed TGFa precursor in total cellular lysates. The transfected cell line 1B2 and the parental cell line NR6 were grown in the presence or absence of 100 ,uM ZnCl2 in 10% CS-DMEM for 2 days. Total cellular lysates were prepared as described in Materials and Methods. Samples were electrophoresed through a 15% SDS-polyacrylamide gel, transblotted onto nitrocellulose, and analyzed by Western blot analysis using the anti-TGFa monoclonal antibody MF9. (b) Detection of the released TGFa in the culture medium of the 1B2 cells. The transfected cell line 1B2 and the parental cell line NR6 were grown in the presence or absence of 100 p.M ZnCl2 in 10% CS-DMEM for 36 h. The medium was then replaced by a serum-free medium, and the cells were incubated overnight. The serum-free medium was then desalted and concentrated, using a C-18 Seppak column as described in Materials and Methods. Samples were electrophoresed through a 12 to 25% SDS-polyacrylamide gradient gel, transblotted onto a nitrocellulose membrane, and then analyzed by Western blot analysis using MF9. Molecular size standards in kilodaltons are shown on the left.
activity, as measured by the stimulation of [3H]thymidine incorporation. The level of EGF-displacing activity in the medium was increased approximately sevenfold upon treatment of the induced 1B2 cells with 10 U of PMN leukocytic elastase per ml (Fig. 5). This increase in EGF-displacing activity corresponded to an increase in mitogenic activity in the culture medium (Fig. 6). Similar effects were observed for the porcine pancreatic elastase; when values were corrected for specific activity, both enzymes appeared equally active. Treatment of formaldehyde-fixed 1B2 cells with various 0
.1
1
5
10
U/mi
proteases. 1B2 cells were zinc induced and then fixed with formaldehyde. The formaldehyde-fixed 1B2 cells were then treated for 1 h at 37°C with the following proteases: PMN leukocytic elastase, pancreatic elastase, cathepsin G, alchymotrypsin, collagenase, trypsin, and subtilisin. The culture medium was analyzed by Western blot analysis for the appearance of either the mature TGFa species or intermediately processed forms of the TGFa precursor. There was no basal release of TGFa from the formaldehyde-fixed 1B2 cells (Fig. 7, lane 1). Treatment with either PMN leukocytic or pancreatic elastase resulted in the accumulation of a 5-kDa TGFa species corresponding in size to the mature TGFa (Fig. 7, lanes 2 and 3). Treatment of the formaldehyde-fixed 1B2 cells with cathepsin G, ac-chymotrypsin, collagenase, trypsin, or subtilisin failed to release any
25.7 0 6.2
-
3.0
-
.1
5
10
U/mi
aSW
184 12.0 FIG. 3. Processing of the cell surface TGFa precursor by PMN leukocytic elastase. Subconfluent 1B2 cells were induced with 100 ,uM ZnC12 in 10% CS-DMEM for 48 h. Cells were then rinsed thoroughly with PBS and incubated with various concentrations of PMN leukocytic elastase (0, 0.1, 1.0, 5.0, and 10.0 U/ml) in DMEM. After a 1-h incubation at 37°C, whole-cell lysates were prepared from the elastase-treated cells (see Materials and Methods). Normalized volumes of cell lysates, based on trichloroacetic acidprecipitable counts, were electrophoresed through a 15% SDSpolyacrylamide gel, transblotted onto a nitrocellulose membrane, and analyzed by Western blot analysis. Molecular size standards in kilodaltons are shown on the left.
FIG. 4. Release of mature TGFa into the culture medium from the cell surface TGFa precursor by PMN leukocytic elastase. 1B2 cells were induced with ZnCl2 and elastase treated as described for Fig. 3. After the 1-h incubation at 37°C, the culture medium was removed from the cells, the cells were rinsed with 10 mM acetic acid, and the wash was pooled with the culture medium. PMSF (10 p.g/ml) was added to the medium, which was clarified by low-speed centrifugation, desalted, and concentrated as described above. Samples were electrophoresed through a 15% SDS-polyacrylamide gel. The protein was then transblotted onto a nitrocellulose membrane and analyzed by Western blot analysis using MF9. Molecular size standards in kilodaltons are shown on the left.
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800
I
~
1
600
3
4
5
6
7
8
6.2 -
2b400
(
2
3.0 -
200
0
0
.1
1
Elastase Concentration (U/ml) FIG. 5. Release of EGF-displacing activity into the culture medium by PMN leukocytic elastase treatment of induced 1B2 cells. 1B2 cells were induced with ZnCl2 and elastase treated as described for Fig. 3. After the 1-h incubation at 37°C, the culture medium was removed from the cells. PMSF was added to the medium to neutralize the elastase. The medium was then assayed for EGFdisplacing activity, using the EGF radioreceptor assay. Each point is the mean of triplicate determinations (coefficient of variation,
