Abstract:
A method of inhibiting, ameliorating or reversing the effects of TGF-β in biological systems by exposing the bioligical systems with an agent which substantially represents active site peptide of the TGF-β molecules.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims priority from an earlier filed provisional patent application Ser. No. 60/050,202, filed Jun. 19, 1997. 
    
    
     ACKNOWLEDGMENT 
     This invention was made with the US Government support awarded by the National Institutes of Health. The US Government may have certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Transforming growth factor β (TGF-β) is a family of 25-kDa structurally homologous dimeric proteins containing one interchain disulfide bond and four intrachain disulfide bonds. The TGF-β family is composed of three-known members (TGF-β 1 , TGF-β 2 , and TGF-β 3 ) in mammalian species. TGF-β is a bifunctional growth regulator: it is a growth inhibitor for epithelial cells, endothelial cells, T-cells, and other cell types and a mitogen for mesenchymal cells. TGF-β also has other biological activities, including stimulation of collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) synthesis, stimulation of angiogenesis, and induction of differentiation in several cell lineages. 
     TGF-β has been implicated in the pathogenesis of various diseases such as intimal hyperplasia following angioplasty, tissue fibrosis, and glomerulonephritis. Neutralizing antibodies to TGF-β have been used experimentally to reduce scarring of wounds, to prevent lung injury in adult respiratory distress syndrome (ARDS), and to block restenosis following angioplasty in animal models. These promising results warrant the development of TGF-β antagonists (inhibitor) that might be useful in inhibiting, ameliorating or reversing the effects of TGF-β and treating diseases. 
     SUMMARY OF THE INVENTION 
     A method of inhibiting, ameliorating or reversing the effects of TGF-β in biological systems, comprising the step of exposing said biological systems with a binding agent, said binding agent is a peptide which substantially resembles a segment of the TGF-β molecules. 
     The binding agent is a peptide which substantially resembles an active site of the TGF-β molecules, hereinafter referred to as active site peptide. 
     The binding agent may be in the form of a peptide or other chemical agents which substantially resembles an active site of the TGF-β molecule, the binding agents occupy the TGF-β cellular receptors making the TGF-β cellular receptors unavailable for the binding of TGF-β molecules. 
     The biological systems may be either in-vitro or in-vivo. 
     Most preferably, the binding agent of TGF-β substantially corresponds to a peptide having an amino acid sequence substantially extending from residue 41 to residue 65 of TGF-β amino acid sequence. Most preferably, said binding agents correspond to an amino acid sequence motif preferably represented by WSXD (SEQ ID NO:10) and/or RSXD (SEQ ID NO:11), wherein X represents any amino acid. 
     Yet another object of the present invention is to provide an active site of TGF-β molecules. And to provide an amino acid sequence which substantially represents an active site of TGF-β molecules. 
     Yet another object of the present invention is to provide an agonist of TGF-β molecules, comprising a carrier molecule have a plurality of said binding agent. Furthermore, a method of producing an agonist of TGF-β molecules, comprising the step of conjugating a plurality of said binding agents to a carrier molecule. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 . Effect of various concentrations of pentacosapeptides, decapeptides, and their structural variants on  125 I-TGF-β 1 , (Panels A and D),  125 I-TGF-β 2  (Panel B), and  125 I-TGF-β 3  (Panel C) binding to TGF-β receptors in mink lung epithelial cells. 
     Cells were incubated with  125 I-TGF-β 1  (Panels A and D),  125 I-TGF-β 2  (Panel B), and  125 I-TGF-β 3  (Panel C) both with and without 100-fold excess of unlabeled TGF-β isoforms and various concentrations of β 1   25  (41-65)(SEQ ID NO:4), β 2   25  (41-65)(SEQ ID NO:5), and β 3   25  (41-65)(SEQ ID NO:6)(Panels A, B, and C) or of β 1   10  (49-58)(SEQ ID NO:7), β 2   10  (49-58)(SEQ ID NO:8), β 3   10  (49-58)(SEQ ID NO:9), β 1   10  (49-58) W52A, β 2   10  (49-58) S53A, β 2   10  (49-58) D55A, β 1   25  (41-65) W52A/D55A and β 3   25  (41-65) R52A/D55A (Panel D). The specific binding of  125 I-labeled TGF-β isoforms was then determined. The specific binding obtained in the absence of peptide antagonists was taken as 0% inhibition. The specific binding (0% inhibition) of  125 I-TGF-β 1 ,  125 I-TGF-β 2 , and  125 I-TGF-β 3  were 3930+540 cpm/well, 4512±135 cpm/well and 4219±125 cpm/well, respectively. The error bars are means ± of triplicate cultures. FIG. 2.  125 I-TGF-β 1 -affinity labeling of cell-surface TGF-β receptors after incubation of mink lung epithelial cells with  125 I-TGF-β 1  in the presence of various concentrations of β 1   25  (41-65) and β 3   25  (41-65). 
     Cells were incubated with  125 I-TGF-β 1  in the presence of 100-fold excess of unlabeled TGF-β 1  (lane 1) and of various concentrations of β 1   25  (41-65) (lanes 8-13) and β 3   25  (41-65) (lanes 2-7). The  125 I-TGF-β 1 -affinity labeling was carried out in the presence of DSS. The  125 I-TGF-β 1  affinity-labeled TGF-β receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. The arrow indicates the location of the  125 I-TGF-β 1  affinity-labeled type V TGF-β receptor (TβR-V). The brackets indicate the locations of the  125 I-TGF-β 1  affinity-labeled type I, type II, and type III TGF-β receptors (TβR-I, TβR-II, and TβR-III). 
     FIG.  3 . Effect of β 1   25  (41-65) on TGF-β 1 -induced growth inhibition as measured by DNA synthesis (Panels A and B) and TGF-β 1 -induced PAI-1 expression (Panel C) in mink lung epithelial cells. 
     (Panel A) Cells were incubated with various concentrations of TGF-β 1  in the presence of 18 μM β 1   25  (41-65). [Methyl- 3 H]thymidine incorporation into cellular DNA was then determined. The [methyl- 3 H]thymidine incorporation into cellular DNA in cells treated with and without 10 pM TGF-β 1  were taken as 100 and 0% inhibition. The error bars are means± S.D. of triplicate cultures. (Panel B) Cells were incubated with 0.25 pM TGF-β, in the presence of various concentrations of β 1   25  (41-65). The [methyl- 3 H]thymidine incorporation into cellular DNA in cells treated with and without 10 pM TGF-β 1  were taken as 100 and 0% inhibition, respectively. The error bars are means± S.D. of triplicate cultures. (Panel C) Cells were treated with 0.25 and 2.5 pM TGF-β 1  and various concentrations of β 1   25  (41-65) for 3 hr. The transcriptional expressions of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were determined by Northern blot analysis. 
     FIG.  4 . Effect of β 1   25  (41-65)-CA and β 1   25  (41-65)-BSA conjugates on  125 I-TGF-β 1  binding to TGF-β receptors in mink lung epithelial cells (Panel A) and on mink lung epithelial cell growth as measured by DNA synthesis (Panel B). 
     (Panel A) Cells were incubated with  125 I-TGF-β 1  in the presence and absence of 100-fold excess of unlabeled TGF-β 1  and various concentrations of β 1   25  (41-65)-CA conjugate. The specific binding of  125 I-TGF-β 1  was then determined. The specific binding of  125 I-TGF-β 1  obtained in the absence of the conjugates was taken as 0% inhibition. The error bars are means± S.D. of triplicate cultures. (Panel B) Cells were treated with various concentrations of β 1   25  (41-65)-CA or β 1   25  (41-65)-BSA conjugate. [Methyl- 3 H]thymidine incorporation into cellular DNA was determined. The [methyl- 3 H]thymidine incorporation into cellular DNA in cell treated with and without 10 pM TGF-β 1  were taken as 100 and 0% inhibition, respectively. The error bars are means± S.D. of triplicate cultures. 
     FIG. 5A shows the amino acid sequence of human TGF-β 1  (SEQ ID NO:1), human TGF-β 2  (SEQ ID NO:2), and human TGF-β 3  (SEQ ID NO:3); FIG. 5B shows the amino acid sequence of β 1    25 (41-65)(SEQ ID NO:4), β 2   25 (41-65)(SEQ ID NO:5), and β 3   25 (41-25)(SEQ ID NO:6), which consist of amino acids 41-65 of TGF-β 1 , TGF-β 2 , and TGF-β 3 , respectively. 
    
    
     DESCRIPTION OF THE INVENTION 
     The object of the invention is to develop TGF-β antagonists or inhibitors with specificities toward the type V TGF-β receptor and other TGF-β receptor types (type I, type II, and type III receptors). It was discovered that three chemically synthesized pentacosapeptides (i.e. binding agents), β 1   25  (41-65), β 2   25  (41-65), and β 3   25  (41-65), whose amino acid sequences were derived from and correspond to the 41st to 65th amino acid residues of TGF-β 1 , TGF-β 2 , and TGF-β 3 , inhibit the binding of radiolabeled TGF-β 1 , TGF-β 2 , and TGF-β 3  to TGF-β receptors in mink lung epithelial cells (i.e. cellular receptors). It was also discovered that the W/RXXD motif in the sequences determines their potencies and that they block TGF-β-induced growth inhibition and TGF-β-induced expression of PAI-1 in mink lung epithelial cells. It was also discovered that these TGF-β peptide antagonists can be converted to partial agonists (i.e. agent which mimics the effects of TGF-β) by conjugation to carriers such as proteins or synthetic polymers. 
     To develop synthetic peptide antagonists of TGF-β, seven pentacosapeptides were synthesized β 1   25  (21-45), β 1   25  (41-65), β 1   25  (51-75), β 1   25  (61-85), β 1   25  (71-95), and β 1   25  (81-105), whose amino acid sequences overlap one another and cover most of the human TGF-β 1  molecule, the monomer of which has 112 amino acid residues (1). The antagonist activities of these peptides were first tested for their abilities to inhibit  125 I-labeled TGF-β 1  ( 125 I-TGF-β 1 ) binding to cell-surface TGF-β receptors in mink lung epithelial cells, a standard model system for investigating TGF-β receptor types and TGF-β-induced cellular responses (2). β 1   25  (41-65), whose amino acid sequence corresponds to the 41st-65th amino acid residues of TGF-β 1 , completely inhibited the  125 I-TGF-β 1  binding (specific binding without peptides=3672±524 cpm/well) to TGF-β receptors in mink lung epithelial cells at 34 μM. The other six pentacosapeptides did not show any effect on  125 I-TGF-β 1  binding to TGF-β receptors in these epithelial cells even at a concentration of 136 μM. This suggests that β 1   25  (41-65) is a TGF-β inhibitor or antagonist and contain an active-site amino acid sequence of TGF-β 1 . 
     TGF-β isoforms (TGF-β 1 , TGF-β 2 , and TGF-β 3 ) have been shown to exhibit different potencies in inducing cellular responses in certain cell types or systems. There is 70% amino acid sequence homology at the 41st to 65th amino acid residues among these three TGF-β isoforms (1-3). To determine the potencies of β 1   25  (41-65), β 2   25  (41-65), and β 3   25  (41-65) in terms of TGF-β antagonist activity, applicant measured the effects of these peptides on the binding of  125 I-labeled TGF-β 1 , TGF-β 2 , and TGF-β 3  to TGF-β receptors in mink lung epithelial cells. As shown in FIG. 1, both β 1   25  (41-65) and β 2   25  (41-65) inhibited  125 I-TGF-β 1  and  125 I-TGF-β 2  binding to TGF-β receptors in a concentration-dependent manner with an IC 50  of ˜1-2 μM (FIG. 1, A and B). β 3   25  (41-65) was weaker with an IC 50  of ˜20 μM for inhibiting  125 I-TGF-β 1  and  125 I-TGF-β 2  binding to TGF-β receptors (FIG.  1 A and B). In contrast, β 1   25  (41-65) and β 3   25  (41-65) showed equal potency (IC 50 =˜0.06-0.08 μM) when  125 I-TGF-β 3  was used as ligand for testing the inhibitory activity (FIG.  1 C). β 2   25  (41-65) also had an IC 50  of ˜0.08 μM for inhibiting  125 I-TGF-β 3  binding to TGF-β receptors in these epithelial cells (data not shown). These results show that both β 1   25  (41-65) and β 2   25  (41-65) are more potent antagonists than β 3   25  (41-65) for  125 I-TGF-β 1  and  125 I-TGF-β 2 , and that all three pentacosapeptides are potent antagonists for  125 I-TGF-β 3  with equal IC 50 . 
     The region spanning residues 41-65 includes a loop in the three-dimensional structures of TGF-β 1  and TGF-β 2  (4,5). This loop is accessible to solvent according to X-ray and NMR analyses (4,5). There are two reasons why a WSXD (for TGF-β 1  and TGF-β 2 ) or RSXD (for TGF-β 3 ) motif in the loop is a good candidate site whereby these pentacosapeptides and their parent molecules could interact with TGF-β receptors. The W/RSXD (52nd-55th amino acid residues) motif is located on the exposed surface of the loop, and the side chains of the amino acid residues in the motif orient toward the solvent (4,5). Also, this motif may determine the affinities of β 1   25  (41-65), β 2   25  (41-65), and β 3   25  (41-65), and their parent molecules for binding to TGF-β receptors. Both β 1   25  (41-65) and β 2   25  (41-65) share the same motif (WSXD) and have equal potencies (IC 50 =1-2 μM) for the inhibition of  125 I-TGF-β 1  binding to TGF-β receptors. β 3   25  (41-65) possesses a distinct motif of RSXD and is a weaker inhibitor (IC 50  of 20 EM). The Kds for TGF-β 1  and TGF-β 2  binding to the type V TGF-β receptor are identical (˜0.4 nM), whereas the Kd of TGF-β 3  binding to the type V receptor is higher (˜5 nM) (6). To test the possibility that the W/RSXD motif is the active site of these peptides, applicant synthesized three decapeptides designated β 1   10  (49-58), β 2   10  (49-58), and β 1   10  (49-58) whose amino acid sequences correspond to the 49th-58th amino acid residues of TGF-β 1 , TGF-β 2 , and TGF-β 3 , respectively. The W/RSXD variants of these decapeptides in which the W-52, S-53, or D-55 residue was replaced by an alanine residue were also synthesized and designated β 2   10  (49-58) W52A, β 2   10  (49-58) S53A, and β 2   10  (49-58) D55A, respectively. The abilities of these decapeptides to inhibit  125 I-TGF-β 1  binding to TGF-β receptors in mink lung epithelial cells were then examined. As shown in FIG. 1D, both β 1   10  (49-58) and β 2   10  (49-58) inhibited the  125 I-TGF-β 1  binding to TGF-β receptors in a concentration-dependent manner with an IC 50  of ˜40-70 μM. β 3   31  (49-58) did not show any inhibitory activity at concentrations up to 300 μM. β 2   10  (49-58) S53A was equipotent with an IC 50  of 40 μM. The other variants, β 2   10  (49-58) W52A and β 2   10  (49-58) D55A, failed to inhibit  125 I-TGF-β 1  binding to TGF-β receptors in these epithelial cells. Identical experiments with β 1   10  (49-58) W52A, β 1   10  (49-58) S53A, and β 1   10  (49-58) D55A were also carried out, and the results were similar to those shown in FIG. 2D with the β 2   10  (49-58) variants (data not shown). These results suggest that the WXXD motif is important for the inhibitory activity ofthe decapeptides β 1   10  (49-58) and β 2   10  (49-58). To prove that the W/RXXD motif is also important for the inhibitory activities of the pentacosapeptides β 1   25  (41-65) and B 3   25  (41-65), applicant prepared variants of β 1   25  (41-65) and β 3   25  (41-65), in which both W- or R-52 and D-55 were replaced by alanine residues. These were designated β 1   25  (41-65) W52A/D55A and β 3   25  (41-65) R52A/D55A, respectively, and tested for their inhibitory activities. FIG. 1D shows that β 1   25  (41-65) W52A/D55A and β 3   25  (41-65) R52A/D55A did not inhibit  125 I-TGF-β 1  binding to TGF-β receptors. These results support that the motif W/RXXD is involved in the interactions of the peptide antagonists eith TGF-β receptors. 
     Mink lung epithelial cells express all known and well-characterized TGF-β receptors (type I, type II, type III, and type V receptors) (6). To determine the relative sensitivities of TGF-β receptor types to inhibition by β 1   25  (41-65) and β 3   25  (41-65) with respect to ligand binding, applicant performed  125 I-TGF-β 1 -affinity labeling of cell-surface TGF-β receptors after incubation of mink lung epithelial cells with  125 I-TGF-β 1  in the presence of various concentrations of β 1   25  (41-65) and β 3   25  (41-65). As shown in FIG. 2, all cell-surface TGF-β receptors (type I, type II, type III, and type V receptors) were affinity-labeled with  125 I-TGF-β 1  in the absence of the antagonists (lanes 7 and 13). β 1   25  (41-65) appeared to inhibit the  125 I-TGF-β 1 -affinity labeling of all TGF-β receptor types in a concentration-dependent manner (lanes 8-12). However, B 1   25  (41-65) inhibition of the  125 I-TGF-β 1 -affinity labeling of the type V TGF-β receptor was greater than its inhibition of other TGF-β receptor types. The  125 I-TGF-β 1 -affinity labeling of the type V TGF-β receptor was almost completely abolished by β 1   25  (41-65) at 2.3 μM , whereas the  125 I-TGF-β 1 -affinity labeling of other TGF-β receptor types was only partially inhibited (30-40%) (FIG. 2, lane 10). This result is consistent with applicant&#39;s observation that the affinity for TGF-β 1  binding to the type V TGF-β receptor is ˜20-40-fold lower than those for TGF-β 1  binding to other TGF-β receptor types (6). β 3   25  (41-65) showed a weak activity in blocking the  125 I-TGF-β 1 -affinity labeling of the type V TGF-β receptor (FIG. 2, lanes 2-5). 
     It has been demonstrated that β 1   25  (41-65), β 2   25  (41-65), and β 3   25  (41-65) are potent inhibitors for  125 I-TGF-β 1  binding to TGF-β receptors. To fulfill the criteria for TGF-β antagonists or inhibitor, these pentacosapeptides have to be shown to block TGF-β-induced cellular responses. One prominent biological activity of TGF-β is growth inhibition. Applicant investigated the effect of β 1   25  (41-65) on TGF-β 1 -induced growth inhibition by exposing mink lung epithelial cells to various concentrations of TGF-β 1  in the presence of 18 μM β 1   25  (41-65) and measuring cellular DNA synthesis. As shown in FIG. 3A, DNA synthesis inhibition induced by 0.025 and 0.25 pM TGF-β 1  was completely blocked by β 1   25  (41-55). In the presence of β 1   25  (41-65), the dose-response curve of TGF-β 1  shifted to the right. β 1   25  (41-65) blocked TGF-β 1 -induced growth inhibition in a concentration-dependent manner (FIG.  3 B). It is important to note that β 1   25  (41-65) (0.1 to 36 μM) did not have an effect on DNA synthesis in the absence of TGF-β 1 . These results suggest that β 1   25  (41-65) is a TGF-β antagonist which blocks TGF-β-induced growth inhibition. The other prominent biological activity of TGF-β is transcriptional activation of collagen, fibronectin, and PAI- 1. To see if β 1   25  (41-65) is able to block this activity, applicant investigated the effect of β 1   25  (41-65) on PAI-1 expression in mink lung epithelial cells stimulated by 0.25 and 2.5 pM TGF-β 1 . As shown in FIG. 3C, β 1   25  (41-65) completely blocked the PAI-1 expression stimulated by TGF-β 1  (lane 7 versus lanes 3 and 5). These results further support that β 1   25  (41-65) is a potent TGF-β antagonist. 
     The dimeric structure of TGF-β has been shown to be required for its biological activities. The hetero-oligomerization of TGF-β receptors induced by the TGF-β dimer appears to trigger signaling. If β 1   25  (41-65) contains the active site sequence involved in the interaction of TGF-β 1  with TGF-β receptors, one may be able to convert its antagonist activity to agonist activity by conjugating β 1   25  (41-65) to carrier proteins, such that the β 1   25  (41-65)-protein conjugates would carry multiple valences of the putative active site. To test this possibility, β 1   25  (41-65) was conjugated to carrier proteins CA (carbonic anhydrase) and BSA (bovine serum albumin) using the cross-linking agent DSS. DSS mainly cross-links the α-amino group of β 1   25  (41-65) to the ε-amino groups of the carrier proteins. The β 1   25  (41-65)-BSA and β 1   25  (41-65)-CA conjugates contained ˜5-10 molecules of β 1   25  (41-65) per molecule of carrier protein. As shown in FIG. 4A, the β 1   25  (41-65)-CA conjugate inhibited  125 I-TGF-β 1  binding to TGF-β receptors in mink lung epithelial cells with an IC 50  of ˜0.05 μM. The β 1   25  (41-65)-BSA conjugate had a similar IC 50  of ˜0.06 μM (data not shown). These IC 50  are ˜20-fold lower than that of β 1   25  (41-65) prior to conjugation. In the control experiments, both BSA and CA conjugated without peptides did not have inhibitory activity. These results suggest that the multiple valences of the active site in the protein conjugates enhance its affinity for binding to TGF-β receptors. Potential agonist activities of the β 1   25  (41-65)-protein conjugates was also examined. As shown in FIG. 4B, both β 1   25  (41-65)-CA and β 1   25  (41-65)-BSA conjugates induced a small but significant growth inhibition as measured by DNA synthesis with an ED 50  of 0.1 μM, although neither showed significant effects on the expression of PAI-1 in mink lung epithelial cells (data not shown). The growth inhibition (30-40%) induced by 0.2 μM β 1   25  (41-65)-CA could be abolished in the presence of 10 μM β 1   25  (41-65) (data not shown). These results suggest that these β 1   25  (41-65)-protein conjugates are partial TGF-β agonists. 
     Applicant shows that the W/RXXD motif is a primary site involved in the interaction with TGF-β receptors. This is supported by several lines of evidence including: 1) among seven pentacosapeptides whose amino acid sequences cover most of the TGF-β 1  molecule, only β 1   25  (41-65), which contains the W/RXXD motif in the middle of the peptide amino acid sequence, has TGF-β antagonist activity; 2) pentacosapeptides and decapeptides containing this W/RXXD motif are potent TGF-β antagonists; 3) replacement of W-52/R-52 and D-55 by alanine residues abolishes the antagonist activities of these decapeptides and pentacosapeptides; 4) conjugation of the β 1   25  (41-65) antagonist to carrier proteins creates a partial TGF-β agonist; 5) several proteins that possess W/RXXD motifs have TGF-β agonist and antagonist activities. 
     Most of the experiments in this study were performed using mink lung epithelial cells. However, the antagonist activities of the pentacosapeptides (i.e. binding agents or active site peptides) have been substantially reproduced using a human line i.e. human lung fibroblasts (data not shown). Since TGF-β is highly conserved across the species, this invention disclosed herein is substantially valid across species. 
     Experimental Procedures 
     Material 
     Na 125 I (17 Ci/mg) and [methyl- 3 H]thymidine (67 Ci/mmole) were purchased from ICN Radiochemicals (Irvine, Calif.). High molecular-weight protein standards (myosin, 205 kDa; β-galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serum albumin, 66 kDa), chloramine T, bovine serum albumin (BSA), and human carbonic anhydrase I (CA) were purchased from Sigma Company (St. Louis, Mo.). Disuccinimidyl suberate (DSS) was obtained from Pierce (Rockford, IL). TGF-β 1  was purchased from Austral Biologicals (San Ramon, Calif.). TGF-β 2  and TGF-β 3  were purchased from R&amp;D Systems (Minneapolis, Minn.). 
     Preparation of Pentacosapeptides and Decapeptides. 
     The amino acid sequences of all pentacosapeptides and decapeptides were derived from those of TGF-β 1 , TGF-β 2 , and TGF-β 3 . For pentacosapeptides β 1   25  (41-65), β 2   25  (41-65), and β 3   25  (41-65), other versions in which cysteine-44 and cysteine-48 were replaced by serine residues were also synthesized. These C44S/C48S versions of β 1   25  (41-65) and β 2   25  (41-65), and β 3   25  (41-65) had the same TGF-β antagonist activity. The C44S/C48S versions had better stability in solution during storage, so they were used in most of the experiments. The peptides were synthesized using tert-butoxycarbonyl chemistry on an Applied Biosystems Model 431A peptide synthesizer and purified using Sephadex G-25 column chromatography and reverse-phase HPLC (C-8 column). The purity of the synthesized peptides were verified by automated Edman degradation on an Applied Biosystems Model 477A gas/liquid phase protein sequenator with an on-line Applied Biosystems Model 120A phenylthiohydantoin amino acid analyzer. The purity of all peptides was estimated to be ≧95%. 
     Preparation of β 1   25  (41-65)-Carbonic Anhydrase (CA) and β 1   25  (41-65)-Bovine Serum Albumin (BSA) Conjugates 
     One hundred fifty μl of 3 mM β 1   25  (41-65) in phosphate buffer saline (pH adjusted to 9.0) was mixed with 300 μl of 0.1 M NaHCO 3  (pH ˜9.0) containing BSA or CA (0.5 mg) and 10 μl of 27 mM DSS in dimethyl sulfoxide. After 18 hr at 4° C., the reaction mixture was mixed with 50 μl of 1 M ethanolamine HCl in 0.1 M NaHCO 3  (˜pH 9.0). After 2 hr at room temperature, the reaction mixture was dialyzed against 2 liters of 0.1 M NaHCO 3  (pH 9.0). After four changes of the dialysis solution, the sample was stored at 4° C. prior to use. The molar ratio of β 1   25  (41-65)/carrier protein in the conjugate was determined by amino acid composition analysis. 
     Specific Binding of  125 I-labeled TGF-β 1 , TGF-β 2 , and TGF-β 3  ( 125 I-TGF-β 1 ,  125 I-TGF-β 2 , and  125 I-TGF-β 3 ) to TGF-β Receptors in Mink Lung Epithelial Cells 
       125 I-TGF-β 1 ,  125 I-TGF-β 2 , and  125 I-TGF-β 3  were prepared by iodination of TGF-β 1 , TGF-β 2 , and TGF-β 3  with Na 125 I as described previously (7). The specific radioactivities of  125 I-TGF-β 1 ,  125 I-TGF-β 2 , and  125 I-TGF-β 3  were 1-3×10 5  cpm/ng. Mink lung epithelial cells were grown on 24-well clustered dishes to near confluence in Dulbecco&#39;s modified Eagle medium (DMEM) containing 10% fetal calf serum. The epithelial cells were incubated with 0.1 nM  125 I-TGF-β 1 ,  125 I-TGF-β 2 , or  125 I-TGF-β 3  both with and without 100-fold excess of unlabeled TGF-β 1 , TGF-β 2 , or TGF-β 3  in binding buffer (7). After 2.5 hr at 0° C., the cells were washed two times with binding buffer, and the cell-associated radioactivity was determined. The specific binding of  125 I-labeled TGF-β isoforms to TGF-β receptors in the cells was calculated by subtracting non-specific binding (in the presence of 100-fold excess of the unlabeled TGF-β isoforms) from total binding. All experiments were carried out in triplicate cell cultures. 
       125 I-TGF-β 1 -affinity Labeling of Cell-surface TGF-β Receptors in Mink Lung Epithelial Cells 
     Mink lung epithelial cells grown on 60-mm Petri dishes were incubated with 0.1 InM  125 I-TGF-β 1  in the presence of various concentrations of P 25  (41-65) or β 3   25  (41-65) in binding buffer. After 2.5 hr at 0° C.,  125 I-TGF-β 1 -affinity labeling was carried out in the presence of DSS as described previously. The  125 I-TGF-β 1  affinity-labeled TGF-β receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. 
     [Methyl- 3 H]thymidine Incorporation 
     Mink lung epithelial cells grown on 24-well clustered dishes were incubated with various concentrations of TGF-β 1  in the presence and absence of β 1   25  (41-65) or with various concentrations of β 1   25  (41-65)-CA, and β 1   25  (41-65)-BSA in DMEM containing 0.1% fetal calf serum. After 16 hr at 37° C., the cells were pulsed with 1 μCi/ml of [methyl- 3 H]thymidine for 4 hr. The cells were then washed twice with 1 ml of 10% trichloroacetic acid and once with 0.5 ml of ethanol:ether (2:1, v/v). The cells were then dissolved in 0.4 ml of 0.2N NaOH and counted with a liquid scintillation counter. 
     RNA Analysis 
     Mink lung epithelial cells were grown overnight in 12-well clustered dishes in DMEM containing 10% fetal calf serum. The medium was then changed to DMEM containing 0.1% fetal calf serum and the cells were incubated with 0.25 and 2.5 pM TGF-β 1  in the presence of various concentrations of β 1   25  (41-65) for 2.5 hr. Total cellular RNA was extracted using RNAzol B (Tel-Test Inc.) according to the manufacturer&#39;s protocol. RNA was electrophoresed in 1.2% agarose-formaldehyde gel and transferred to Duralon-UV membranes using 10×SCC. The membranes were probed at 42° C. with a random-primed, radiolabeled one kb fragment from the Hind III and NeoI digests of PAI-1 cDNA and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA. The blots were washed with 0.1×SCC containing 0.1% SDS at room temperature. 
     References 
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