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a Departments of Surgery
b and Medical Biochemistry,
c Molecular, Cellular and Developmental Biology Program,
d and Arthur G. James Cancer Hospital and Research Institute, The Ohio State University, Columbus, Ohio 43215
e Department of Surgery, Children's Hospital, Columbus, Ohio 43205
f Department of Animal, Dairy and Veterinary Sciences, Clemson University, Clemson, South Carolina 29634
g Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Complementary DNAs have been reported for human, mouse, and porcine CTGF (hCTGF, mCTGF/fisp-12, pCTGF) [1–3, 5, 14]. After cleavage of their respective signal peptides, the secreted forms of all three proteins are predicted to comprise 323 residues (of which 38 are conserved cysteine residues) and to exhibit > 95% homology to each other [14]. It has been suggested that the CTGF protein may be organized into four structurally distinct modules and that CTGF is thus a multifunctional mosaic protein [15]. In hCTGF or pCTGF, residues 27–97, 101–167, 199–243, and 256–330 resemble, respectively, an insulin-like growth factor-binding motif (module I), a von Willebrand type C repeat (module II), a glycoconjugate-binding domain (module III), and a dimerization and/or receptor-binding motif that may contain a cysteine knot (module IV) [5, 14, 15]. Forms of CTGF of 36 to 38 kDa have been detected in human endothelial cell-conditioned medium [1], by in vitro translation of hCTGF RNA [1], and by radioimmunoprecipitation of metabolically labeled mouse or human fibroblasts or pig endometrial explants using CTGF antisera [3, 6, 14]. While the size of these CTGF proteins is consistent with predictions regarding the size of the primary translational product from cDNA analysis, we have recently shown that pig uterine luminal flushings (ULF) do not contain readily detectable levels of 38-kDa CTGF but instead contain 10-, 16-, and 20-kDa immunoreactive forms of the protein [5]. A detailed analysis of 10-kDa CTGF revealed that it corresponded to two microheterogeneous forms of pCTGF that represented the C-terminal 102 or 103 residues of the primary translational product and thus comprised module IV flanked by 8 or 9 additional residues at its N-terminus plus 19 presumptive residues at its C-terminus [5]. Since the 10-kDa proteins were mitogenic for cultured Balb/c 3T3 cells, these data suggested that modules I–III of CTGF are not essential for CTGF biological activity. Similar low-mass forms of CTGF are present in conditioned medium from cultures of mouse or human fibroblasts [6].
In view of the importance of uterine secretory proteins, especially growth factors, in uterine and placental growth [16–20], as well as the need to more fully understand CTGF processing, these studies were focused on the characterization of 16- to 20-kDa CTGF in ULF and on identifying a putative mechanism by which low-mass forms of CTGF might be generated in utero.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For purification of CTGF, uteri were collected randomly from gilts at a local slaughterhouse. Uterine horns were flushed with 20–50 ml PBS to obtain ULF, which were subsequently clarified by centrifugation at 13 500 x g for 30 min at 4°C and passage of the supernatant through glass wool. CTGF levels and CTGF-degrading properties were studied in ULF obtained by flushing each uterine horn of 45 mixed-breed gilts on Days 0–18 of the estrous cycle or pregnancy (Day 0 = 1st day of estrus) with 10 ml PBS.
For Northern blot analysis, uteri were obtained at hysterectomy from pigs on Days 10–16 of the estrous cycle or of pregnancy [21]. Endometrium was dissected from underlying myometrium, immediately frozen in liquid nitrogen, and stored at -86°C. Northern blotting was performed using a 32P-labeled 596-base pair (bp) pCTGF cDNA (nucleotides 294–889) as previously described [14].
Purification and Isolation of 16–20-kDa CTGF Proteins from ULF
Heparin-affinity chromatography CTGF proteins were purified from ULF using successive steps of cation-exchange chromatography, heparin-affinity fast protein liquid chromatography (FPLC), and reverse-phase HPLC that have previously been used for isolation of 10-kDa CTGF [5]. Briefly, Balb/c 3T3 cell mitogens in 2.0-, 1.6-, 1.5-, 2.2-, and 1.8-liter samples of ULF from, respectively, 52, 28, 42, 43, and 37 uteri (202 total) were individually eluted from a BioRex 70 cation-exchange column (Bio-Rad Laboratories, Richmond, CA) by 0.3–0.6 M NaCl in PBS. Fractions from each run were combined into two separate pools of mitogenic material, each of which was subjected to two cycles of heparin-affinity chromatography using an EconoPac heparin column (0.7 x 3.6 cm; Bio-Rad) in the first step and a TSK heparin 5PW column (0.8 x 7.5 cm; TosoHaas, Philadelphia, PA) in the second step [5]. TSK heparin column fractions containing proteins that were eluted by 0.8 M NaCl and that demonstrated mitogenic activity for 3T3 cells were divided into two pools (regions I and II). Each pool (3 ml) was subjected to C8 reverse-phase HPLC as described previously [5]. In view of the lability of CTGF mitogenic activity under the acidic conditions employed in HPLC [5], CTGF was subsequently detected in fractions containing the column eluate by Western blotting.
Characterization of CTGF
DNA synthesis assays Fractions from heparin-affinity FPLC of ULF were tested for their ability to stimulate DNA synthesis as measured by [3H&; incorporation into the DNA of confluent quiescent Balb/c 3T3 cells grown for 7 days in 200 µl of Dulbecco's Modified Eagle's medium (DMEM)/10% bovine calf serum in 96-well culture plates [5].
SDS-PAGE and Western blotting Aliquots (150 µl) of selected HPLC fractions were evaporated to dryness in a SpeedVac concentrator (Savant Instruments, Farmingdale, NY) and reconstituted in 35 µl 10 mM Tris-HCl (pH 7.4). Twenty microliters of this concentrate was mixed with 20 µl of double-strength sample buffer, boiled, and used for analytical SDS-PAGE in which samples were electrophoresed at 20 µl/lane under reducing conditions on duplicate 18% polyacrylamide mini gels for approximately 1 h at 200 mA. SDS-PAGE was also performed directly on unfractionated ULF from 45 animals. To permit quantitative comparisons between different animals, the amount of ULF applied to each lane (i.e., 10–35 µl) was normalized to 0.1% of the total volume of ULF recovered.
Proteins in the gels were transferred to nitrocellulose membranes in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid (pH 11.0) for 90 min at 300 V. Blots were blocked for 30 min with 10 mM Tris-HCl/0.15 M NaCl/0.25% BSA and incubated overnight with 1:1000 dilution of rabbit preimmune serum or 1:1000 dilution of rabbit anti-pCTGF[247–260] peptide antiserum (rabbit A; see [5]). Immunoreactive bands were visualized using alkaline phosphatase-conjugated goat anti-rabbit IgG followed by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate chromogenic substrates. Intensities of individual CTGF bands were calculated using a Storm 860 scanner and ImageQuanNT software (Molecular Dynamics, Sunnyvale, CA). Data were expressed as mean ± SEM pixels per band for each CTGF isoform at each reproductive stage examined.
N-Terminal microsequencing Fractions containing HPLC-purified immunoreactive CTGF proteins were pooled, dried, and subjected to preparative SDS-PAGE and Western blotting to polvinyldifluoride (PVDF) as described previously [5]. Proteins of interest were located by staining the PVDF with 0.1% Coomassie R250 in 50% methanol for 2 min, excised, and submitted for N-terminal analysis by the BioSciences Sequencing Facility at The Ohio State University, Columbus, OH, and the Molecular Biology Core Laboratory at Case Western Reserve University, Cleveland, OH.
Degradation of 38-kDa CTGF by ULF
Radioimmunoprecipitation (RIPA) of 35S-labeled 38-kDa CTGF Pig aortic smooth muscle cells were isolated as described previously [5] and used between passages 3 and 5. Cells were grown to approximately 80% confluency in T-75 culture flasks in 15 ml DMEM containing 10% bovine calf serum. They were then transferred to serum-free cysteine/methionine-deficient DMEM for 1 h and labeled for 4 h at 37°C with 4–5 ml of the same medium containing 100 µCi/ml [35S]cysteine/methionine (ICN Biomedical, Costa Mesa, CA). Labeling medium was aspirated, and the cell monolayer was lysed in 3 ml cold RIPA buffer (50 mM Tris-HCl [pH 8.0] containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS). Cell lysate from two flasks (6 ml) was then precleared by addition of 160 µl rabbit B preimmune serum (see below) for 30 min at room temperature followed by shaking with 400 µl protein A agarose beads (Pierce Chemical Co., Rockford, IL) for 30 min at 4°C. The supernatant was then divided into two aliquots of approximately 2.5 ml, to which was added either 80 µl of CTGF[247–260] peptide antiserum or 80 µl of preimmune serum (rabbit B; see [5, 6, 14]). After incubation for 1 h at room temperature, immune complexes were allowed to precipitate for 1 h at 4°C by addition of 200 µl of protein A beads. Beads were washed four times with 15 ml of RIPA buffer, resuspended in 7.5 ml PBS, and aliquoted into microfuge tubes (0.5 ml/tube). Tubes were centrifuged to pellet the beads, and the supernatant was removed by aspiration.
Incubation of 35S-labeled 38-kDa CTGF with ULF CTGF of 38 kDa, complexed on protein A beads, was mixed with either PBS alone or various dilutions (1:8–1:4096 in PBS) of ULF from a Day 14 nonpregnant gilt for 0, 10, 20, or 180 min at 37°C in a final volume of 20 µl. The effect of heat treatment on CTGF-degrading activity was determined by boiling a 1:8 dilution of ULF for 1 min prior to its addition to 38-kDa CTGF for 180 min at 37°C. All reactions were terminated by addition of 20 µl of double-strength SDS-PAGE sample buffer followed by boiling. Aliquots (18 µl) were subjected to SDS-PAGE under reducing conditions on 18% polyacrylamide gels, after which the gels were soaked in En3Hance (NEN DuPont, Boston, MA), dried, and exposed to x-ray film for 5–8 days.
Degradation of 38-kDa recombinant human CTGF (rhCTGF)
Baculovirus-derived 38-kDa rhCTGF was produced and purified essentially as described previously [4] and was kindly provided by Dr. G. Martin and colleagues (FibroGen Inc, South San Francisco, CA). One microgram of rhCTGF was incubated overnight at 37°C in a total volume of 10 µl PBS containing aliquots of ULF that were all normalized to 0.0007% of the total volume of ULF recovered. Negative control reactions were performed with PBS in the absence of ULF. Since it was not possible to distinguish rhCTGF breakdown products from endogenous low-mass pCTGF proteins in many of the ULF samples, degradation was assessed by the disappearance of the 38-kDa rhCTGF band and was scored semiquantitatively as follows: -, no breakdown (i.e., same 38-kDa rhCTGF signal as in negative control); +, moderate proteolysis (
50% breakdown of 38-kDa rhCTGF); ++, high proteolysis (> 50% breakdown of 38-kDa rhCTGF).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For structural analysis, peak fractions containing each of the CTGF proteins were dried, subjected to SDS-PAGE, and transferred to PVDF membranes. For each protein, N-terminal sequence data were obtained for up to 16 cycles and showed that relative to the previously described pCTGF primary translational product [14], the 16-kDa, 18-kDa, and 20-kDa forms of CTGF from region I commenced at Ala197, Asp186, and Asp186, respectively, and that the two microheterogeneous 16-kDa proteins from region II commenced at Ala197 and Cys199. These data are summarized in Figure 3, which shows the relationship of the CTGF proteins to each other and to the junction of modules II and III within the pCTGF primary translational product.
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Northern blots of endometrial RNA demonstrated a single 2.4-kilobase pCTGF transcript at all stages examined (Days 10, 12, and 14 of the cycle and Days 10, 12, 14, and 16 of pregnancy; data not shown). This transcript was the same size as that previously reported in Day 16 cyclic pig endometrium [14] as well as cultured endothelial cells and fibroblasts of human or mouse origin [1, 3, 6, 7]. These data suggested that the occurrence of 16- to 20-kDa CTGF proteins was not likely a result of alternative splicing of pCTGF RNA.
To investigate whether 38-kDa CTGF was posttranslationally processed by proteases in ULF, metabolically labeled native 38-kDa pCTGF was immunoprecipitated from 35S-labeled pig aortic smooth muscle cells and incubated with ULF from a Day 14 cycling pig. As shown in Figure 4A, 38-kDa CTGF was stable when incubated for 3 h at 37°C in ULF at dilutions greater than 1:256 or in PBS alone. However, there was a dose-dependent decrease in the amount of 38-kDa CTGF after its exposure to ULF dilutions of 1:256–1:8. Moreover, samples exposed to these higher concentrations of ULF contained a ~10-kDa protein that was absent from samples treated with lower concentrations of ULF in which 38-kDa CTGF was stable (Fig. 4A). Processing of 38-kDa CTGF was extremely rapid, with a substantial decrease in its levels within 10 min of exposure to a 1:8 dilution of ULF at 37°C (Fig. 4B). CTGF-degrading activity was destroyed by exposure of ULF to 100°C for 1 min (Fig. 4C). When the same ULF used in these studies was analyzed by Western blotting, endogenous CTGF proteins of 10 and 16–20 kDa were readily detected, whereas 38-kDa CTGF was absent (Fig. 4D). In contrast to 38-kDa CTGF, the 10- to 20-kDa forms of CTGF were all completely stable in ULF for 2 days at 37°C (Fig. 4D).
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When aliquots of unpurified ULF were subjected to Western blotting, 10-, 16-, and 20-kDa CTGF were detected by anti-CTGF[247–260] (Fig. 5) but not by preimmune serum (data not shown), as we have previously demonstrated [5]. Two proteins of 12 kDa and ~40 kDa were detected nonspecifically, since they reacted equally with the CTGF antiserum and the preimmune serum (data not shown). The 10-, 16-, and 20-kDa isoforms of CTGF were detected during the estrous cycle at moderate to high levels in 4 of 5 pigs on Day 14, 5 of 5 pigs on Day 16, and 2 of 5 pigs on Day 18 (Fig. 5A). The highest levels of immunoreactive CTGF were present on Days 16 (n = 3) or 18 (n = 2). CTGF levels were very low or nondetectable on Days 0, 10, or 12 of the cycle (Fig. 5A). During pregnancy, CTGF signals were weak or absent in 5 of 5 pigs on Day 10 but were then present at moderate to high levels in 5 of 6 pigs on Day 12, 5 of 5 pigs on Day 14, 5 of 5 pigs on Day 16, and 1 of 4 pigs on Day 18 (Fig. 5B). Overall, ULF from pregnant animals demonstrated the highest level of CTGF on Day 12. Although the three mass forms of CTGF were usually present, ULF from Days 16 and 18 of pregnancy contained predominantly the 10-kDa CTGF protein (Fig. 5B). These changes were verified by densitometric scanning (Table 1), which also showed that the intensity of the total CTGF signal in cycling animals was elevated between 4- and 13-fold on Days 14, 16, or 18 as compared to Days 0, 10, or 12. Moreover, the intensity of the total CTGF signal on Day 12 of pregnancy was approximately 7-fold greater than that on Day 10 of pregnancy and about 4-fold greater than that on Day 12 of the cycle (Table 1).
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Finally, the same individual ULF samples were incubated at normalized dilutions with 38-kDa rhCTGF, and the level of proteolytic activity for CTGF was determined by Western blotting. CTGF proteolysis did not occur using ULF from cycling animals in 1 of 1 pig on Day 0, 1 of 1 pig on Day 10, 3 of 3 pigs on Day 12, 1 of 5 pigs on Day 14, and 1 of 4 pigs on Day 18 (Fig. 5A). Moderate to high levels of proteolytic activity for 38-kDa CTGF were present in 4 of 5 pigs on Day 14, 5 of 5 pigs on Day 16, and 3 of 4 pigs on Day 18 of the cycle (Fig. 5A). In pregnant animals, proteolytic activity for CTGF was absent from 5 of 5 pigs on Day 10 but present at moderate-high levels in 6 of 6 pigs on Day 12 and 4 of 4 pigs on Day 18 and at high levels in 5 of 5 pigs on Day 14 and 5 of 5 pigs on Day 16 (Fig. 5B). Hence, overall there was a high correlation between the presence and intensity of endogenous 10- to 20-kDa CTGF proteins and proteolytic activity for 38-kDa CTGF.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of the truncated forms of CTGF so far identified, most have N-termini that commence between modules II and III (16-, 18-, and 20-kDa CTGF) or between modules III and IV (10-kDa CTGF). An exception is the 16-kDa protein commencing at Cys199, the first residue in module III. These data suggest that the modules may be relatively protease-insensitive as a result of their folding and intrachain disulfide bridging whereas the intervening cysteine-free regions may be more susceptible to proteolytic cleavage. The presence in ULF of proteases that rapidly degrade 38-kDa CTGF provides a likely explanation for the very low or nondetectable levels of the native 38-kDa protein in ULF (these results and [5]) despite its synthesis by pig endometrium [14]. Protease mapping of CTGF (not shown) demonstrates predicted trypsin-sensitive sites between Arg196 and Ala197 and a chymotrypsin-sensitive site between Leu245 and Glu246 that would generate, respectively, one of the two microheterogeneous forms of 16-kDa CTGF identified here and of 10-kDa CTGF identified earlier [5]. However, these and the other low-mass CTGF proteins may arise through cleavage by other as yet undefined mechanisms that may involve several sequential proteolytic steps. While protease activity in ULF appears to be responsible for the production of 10-kDa CTGF from 38-kDa CTGF, the mechanism of production of 16- to 20-kDa CTGF requires further investigation. However, a proteolytic process is the most likely explanation, since there was a high correlation between the levels of 16- to 20-kDa CTGF and proteolytic activity for 38-kDa CTGF in individual ULF samples. It is possible that the experimental conditions used may not have been optimal for production of 16- to 20-kDa CTGF in vitro. For example, important cofactors may have been absent or the proteases responsible for production of 16- to 20-kDa CTGFs may be associated with uterine cell surfaces, unlike those that generate 10-kDa CTGF, which are evidently soluble. In this respect it is of interest that 38-kDa CTGF is frequently cell-associated and appears to bind to heparin-like molecules in the ECM [6, 14, 22]. Alternatively, 38-kDa CTGF may be degraded by intracellular proteases within the endoplasmic reticulum or Golgi apparatus, resulting in direct export of proteolyzed products from the cell. Thus it is possible that 38-kDa CTGF is proteolytically processed by cell-associated or secreted enzymes that result in its liberation from intracellular or extracellular locations and appearance in ULF as soluble, stable, bioactive low-mass forms.
It is well recognized that uterine fluids of several mammalian species, including the pig, contain a variety of protease activities. ULF from ovariectomized gilts contains leucine aminopeptidase, lysozyme, and cathepsin B1, D, and E, which are estrogen- and/or progesterone-dependent [23,24]. Pig ULF also contains plasminogen activator and its substrate plasminogen [25], thus providing the potential for production of plasmin, a broadly specific protease. In addition, several protease inhibitors have been identified in pig ULF, including a 14-kDa Kunitz-type plasmin/trypsin inhibitor [26, 27] and antileukoproteinase [28]. These inhibitors may control the protease activities of the conceptus, especially in species such as the pig that exhibit noninvasive placentation [26]. However, an alternative role may be to protect components of uterine secretory fluids from proteolytic degradation [26], highlighting the possibility that the level of each CTGF protein in ULF likely reflects a highly controlled balance between CTGF proteases and protease inhibitors.
Studies of CTGF isoforms and CTGF protease activity during the cycle and pregnancy revealed a number of interesting features. First, the presence of low-mass forms of CTGF was correlated with the presence of proteolytic activity for 38-kDa CTGF, suggesting a likely causative relationship and possibly coexpression of their respective genes. Secondly, CTGF protein levels in pregnant animals were higher on Day 12 of pregnancy than on Day 12 of the cycle. A similar result was obtained in a previous study showing that CTGF bioactivity in ULF (assessed by comparison of heparin-purified mitogenic activity) was higher on Day 11.5 of pregnancy as compared to the cycle [29]. Although the underlying mechanism requires further studies, it is possible that blastocyst estrogens, which peak around Days 11–12 [30], are involved in enhanced production, secretion, and/or solubilization of CTGF. Alternatively, the pig blastocyst, which undergoes rapid morphological changes at this time [31], may produce CTGF itself or alternatively may induce uterine CTGF through its production of TGFß, which occurs as early as Day 10 [32]. TGFß is a potent and rapid inducer of CTGF gene expression [2, 7–9] and has been implicated as an initiator of CTGF production in growth factor cascades during processes such as wound healing [7], fibroplasia [4], and fibrotic diseases [10, 11]. Thirdly, the peak levels of CTGF in ULF on Day 16 of the cycle contrast with the diminishing levels at the same stage of pregnancy. Again, while the underlying mechanisms remain unclear, the decrease in uterine luminal CTGF may reflect its utilization by uterine or conceptus tissues during this period, possibly for tissue remodeling and growth during the attachment and placentation phases. Several biological properties of CTGF are consistent with its potential role in these processes, including its ability to stimulate cell growth, chemotaxis, cell adhesion, and synthesis of ECM components such as type I collagen, fibronectin, and 
5 integrin [1, 4–6, 22], as well as potentiation of the activity of basic fibroblast growth factor [22], which is produced by pig uterine and conceptus tissues on Days 10–14 [33].
It is well recognized that the molecular mechanisms regulating uterine protein secretion are complex and that they involve steroid hormonal control (both embryonic and maternal) of gene transcription and translation as well as of the timing and localization of protein export and processing [20]. Although in the case of CTGF, many of these parameters require further study, our data suggest that CTGF may play a potentially important role in regulating cell function in the uterine tract in both pregnant and nonpregnant pigs. Moreover, the identification in uterine secretions of potent proteolytic activity for 38-kDa CTGF and multiple low-mass forms of CTGF suggests that limited posttranslational proteolytic processing of CTGF is an important component of its mode of action in utero.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D.R. Brigstock, Department of Surgery, Wexner Institute for Pediatric Research, Children's Hospital, 700 Children's Drive, Columbus, OH 43205. FAX: (614) 722–2716; brigstod{at}pediatrics.ohio-state.edu
Accepted: May 20, 1998.
Received: December 29, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 G. A. Surveyor, A. K. Wilson, and D. R. Brigstock Localization of Connective Tissue Growth Factor during the Period of Embryo Implantation in the Mouse Biol Reprod, November 1, 1998; 59(5): 1207 - 1213. [Abstract] [Full Text]
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T. M. Grzeszkiewicz, D. J. Kirschling, N. Chen, and L. F. Lau CYR61 Stimulates Human Skin Fibroblast Migration through Integrin alpha vbeta 5 and Enhances Mitogenesis through Integrin alpha vbeta 3, Independent of Its Carboxyl-terminal Domain J. Biol. Chem., June 8, 2001; 276(24): 21943 - 21950. [Abstract] [Full Text] [PDF]
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