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a Departments of Surgery and
b Medical Biochemistry, The Ohio State University and Children's Hospital, Columbus, Ohio 43205
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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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and ß (TGF, TGFß), [15–17] and insulin-like growth factors [18, 19] have been strongly implicated in regulating uterine remodeling, implantation, and placentation. The collective and coordinate action of these molecules on uterine and extraembryonic cells is likely to be a major mechanism whereby pregnancy is successfully established and maintained. Connective tissue growth factor (CTGF) is one of the most recently described growth factors [20]. CTGF belongs to a gene family that also includes cyr61, nov, elm1, and HICP, all of which exhibit a quite varied range of biological properties [21–25]. CTGF is encoded by a TGFß-inducible immediate early gene, although other growth factors such as basic FGF, EGF, and platelet-derived growth factor also induce CTGF expression, albeit to a lesser extent [26–29]. To date, most studies of the role of CTGF in vivo have focused on the function of CTGF in wound healing and fibrotic disorders, especially those in which TGFß appears to be important [22, 23, 28, 30, 31]. However, a role for CTGF in normal uterine physiology has been inferred from the presence of its mRNA in cycling and early-pregnant pig endometrium [32, 33]. In addition, immunoprecipitation experiments demonstrated synthesis by pig endometrial explants of the 38-kDa CTGF protein [32], which appears to undergo limited proteolysis yielding bioactive 10- to 20-kDa moieties that are readily detectable as the principal isoforms of CTGF in pig uterine secretory fluids [33, 34]. In pigs, the levels of CTGF in the uterine lumen appear to be elevated at the time of blastocyst expansion [33, 35], and the 10-kDa form of porcine CTGF has been shown to stimulate mitosis in cultured pig endometrial stromal cells [35]. Recently, CTGF was shown to be present in mouse embryonic and placental tissues on Days 14.5–18.5 of pregnancy [36]. Mouse CTGF (mCTGF), also termed fisp-12 or ßIG-M2, is a 348-residue protein (prior to signal peptide cleavage) that is 95% identical to human CTGF [26, 27]. Since a systematic study of CTGF during the periimplantation period of the mouse has not been reported, the purpose of these studies was to determine the spatial and temporal localization of CTGF on Days 1.5–6.5 of pregnancy, as well as throughout the estrous cycle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histochoice was obtained from Amresco (Solon, OH). Superfrost Plus Slides and Permount were purchased from Fisher Scientific (Pittsburgh, PA). Hydrogen peroxide was from Sigma Chemical Company (St. Louis, MO). PBS was obtained from Gibco BRL (Gaithersburg, MD). Normal goat serum (NGS) was from Vector Labs. (Burlingame, CA). Biotin-conjugated goat anti-rabbit IgG (preadsorbed to rat and mouse tissue), peroxidase-conjugated streptavidin and liquid diaminobenzidine-concentrated substrate pack were purchased from BioGenex (San Ramon, CA). Hematoxylin was obtained from Newcomer Supply (Middleton, WI). A TSK heparin 5PW column (0.8 x 7.5 cm) was obtained from TosoHaas (Philadelphia, PA). Heparin Sepharose was from Amersham Pharmacia Biotech (Piscataway, NJ). CTGF antisera raised against residues 80–93 or 246–259 of mouse CTGF were produced as previously described [34, 37]. Anti-mCTGF(80–93) was affinity purified [37] and used for immunohistochemistry and Western blotting. Anti-mCTGF(246–259) was used for Western blotting.
Animals
Swiss Webster mice (Harlan Sprague Dawley, Indianapolis, IN) were used for all experiments, which were approved by the Institutional Animal Use and Care Committee of Children's Hospital Research Foundation. To determine the stage of cycling females, a vaginal smear was performed and different cell types of the particular stage were microscopically identified. To generate pregnant mice, the females were housed with male mice of the same species at around 2000 h. Females were checked for vaginal plugs the next morning, and only those that showed a plug were considered pregnant. This time point was defined as Day 0.5 of pregnancy. Nonpregnant females or mice pregnant at the indicated day were killed and the uteri removed and fixed. A total of 42 animals were used in this study: 21 staged mice for immunohistochemistry (diestrous, n = 2; proestrous, n = 3; estrous n = 2; and pregnant Days 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5, n = 2, 2, 2, 3, 1, and 2, respectively) and 21 nonstaged cycling mice for analysis of CTGF in endometrial extracts (n = 5) or uterine luminal flushings (ULF) (n = 16). ULF were obtained by sequential passage of 1 ml PBS through each uterine horn of groups of either 6 or 10 mice. Aliquots of each ULF sample were tested for their stimulation of DNA synthesis in quiescent cultures of Balb/c 3T3 cells and also subjected directly to SDS-PAGE and Western blotting [34]. The ULF remaining from both groups of animals were pooled and subjected to heparin-affinity chromatography. Endometrial extracts were prepared by sonicating 120 mg of endometrium (scraped and pooled from 5 mice) in 200 µl of 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS followed by centrifugation at 13 000 x g for 10 min. Aliquots of the supernatant were then subjected to SDS-PAGE and Western blotting.
Immunohistochemistry
Uteri were fixed in Histochoice for 5–6 h and then left in 70% ethanol overnight. They were processed in a TP 1050 pressure-vacuum processor (Leica, Deerfield, IL) and embedded in paraffin. Sections (5 µm) were cut using a microtome and placed on Superfrost Plus slides. The slides were heated at 60°C for 1 h and deparaffinized in xylene for 5 min. Sections were then hydrated by being passed through a series of graded ethanol washes of 95%, 90%, 70%, and 50% ethanol and finally being placed in distilled water for a few minutes. The sections were then treated with 3% hydrogen peroxide to destroy any endogenous peroxidase activity, and then washed in PBS. Sections were then incubated with 10% NGS in PBS (NGS-PBS) for 30 min at room temperature, washed in PBS, and then incubated with 10 µg/ml affinity-purified anti-mCTGF IgG [37] or preimmune IgG diluted in NGS-PBS for 30–60 min at room temperature. Slides were washed in PBS and incubated for 20 min at room temperature with a 1:50 dilution of biotin-conjugated goat anti-rabbit IgG. Slides were washed in PBS and incubated with 1:20 diluted peroxidase-conjugated streptavidin for 20 min at room temperature. Slides were washed in PBS, after which the chromogenic substrate diaminobenzidine and hydrogen peroxide were added to the sections until color developed (2–5 min). Sections were counterstained with hematoxylin, mounted, and photographed using an Axioscope microscope equipped with a 35-mm camera (Carl Zeiss Inc., Thornwood, NY).
Heparin-Affinity Chromatography
Mouse ULF collected from 16 animals were pooled and subjected to heparin-affinity fast protein liquid chromatography (FPLC) using a TSK heparin 5PW column [34]. Briefly, the column was developed with a 40-ml gradient of 0.2–2 M NaCl in 10 mM Tris-HCl (pH 7.4), and the eluate was collected into forty 1-ml fractions. Fractions were assayed at 50 µl/ml for their ability to stimulate Balb/c 3T3 cell DNA synthesis as measured by [3H&; incorporation [34]. CTGF was subsequently detected in the fractions by Western blotting.
SDS-PAGE and Western Blotting
SDS-PAGE was performed under reducing conditions on endometrial extract (0.5 µl per lane), unpurified ULF (7.5 µl per lane), or fractions from heparin-affinity FPLC of ULF (equivalent of 25% of each fraction per lane) that had been desalted and concentrated using 15-µl beds of heparin Sepharose as described previously [34, 37].
Western blots were performed using either 2 µg/ml anti-mCTGF(80–93) IgG with the same concentration of normal rabbit IgG as a negative control or 1:1000 dilution of anti-mCTGF(246–259) with the same concentration of preimmune serum as a negative control [33, 34]. Immunoreactive bands were visualized using alkaline phosphatase-conjugated goat anti-rabbit IgG as described [33, 34].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously demonstrated the use of affinity-purified anti-mCTGF(80–93) for the specific immunohistochemical detection of CTGF in mouse and human fibroblasts [37]. Using this antibody, CTGF was localized in the nonpregnant mouse uterus to the luminal and glandular epithelial cells (Fig. 1, A–C). Staining was distributed throughout the entire cell and was not preferentially localized to either the apical or basal surface. In contrast, stromal cells and the myometrium showed low or undetectable levels of CTGF. Epithelial staining for CTGF was more intense during the early proestrous (Fig. 1A) and diestrous (Fig. 1B) stages than at estrus (Fig. 1C). Sections stained with preimmune IgG were negative for CTGF at all stages, for which a typical example is shown in Figure 1D. Staining with the CTGF antibody was reduced to negative control levels by prior incubation of the antibody with a 1000-fold excess of mCTGF(80–93) peptide antigen (data not shown).
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Localization of CTGF in the Mouse Uterus during Early Pregnancy
On Days 1.5–3.5 of pregnancy, CTGF was localized to the luminal and glandular epithelial cells in a manner similar to that observed in the nonpregnant mouse (Fig. 2, A–C), except that CTGF was preferentially localized to the apical surfaces on Days 2.5 and 3.5. Staining of stromal and myometrial cells was much lower and was comparable to that observed in cycling animals. However, on Day 4.5 of pregnancy, the time of implantation, the level of CTGF was substantially reduced in both epithelial cell types and was only slightly higher than that in stromal cells (Fig. 2, D and E). By Day 5.5, the stromal cells that had differentiated into decidual cells stained strongly for CTGF, whereas undifferentiated stromal cells beyond the zone of decidualization continued to stain weakly for CTGF (Fig. 3A). By Day 6.5, most of the stromal cells had undergone the decidual cell reaction and were strongly positive for CTGF (Fig. 3B).
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Localization of CTGF in the Periimplantation Mouse Embryo
On the Day of implantation, Day 4.5, mouse blastocysts showed overall weak staining for CTGF in both the inner cell mass and trophectoderm (Fig. 2D) as compared to preimmune controls (Fig. 2E). However, over the next 1–2 days, the intensity of CTGF staining increased and became differentially distributed throughout the various embryonic structures. In the Day 5.5 embryo, CTGF was present at highest levels in embryonic ectoderm cells in which it was preferentially localized toward the proamniotic cavity (Fig. 3A). Moderate staining was present in the proximal endoderm and ectoplacental cone while relatively weak signals were present in the extraembryonic ectoderm and trophectoderm (Fig. 3A). On Day 6.5, abundant CTGF staining was present in the embryonic ectoderm and mesoderm, with substantially weaker staining in the endoderm and extraembryonic ectoderm (Fig. 3B).
CTGF Isoforms in Endometrial Extracts and ULF
We have previously demonstrated that pig ULF contains multiple forms of bioactive CTGF that range in size from 10 to 20 kDa and compose between one third and one half of the C-terminal region of the full-length (i.e., 38 kDa) CTGF molecule [33, 34]. It was therefore of interest to determine whether similar low-mass forms of CTGF were also present in the mouse uterine tract. Western blotting of mouse endometrial extracts resulted in the detection of ~36- to 38-kDa CTGF proteins with anti-mCTGF(80–93) or anti-mCTGF(246–259) (Fig. 4). This size heterogeneity of "full-length" CTGF is consistent with previous findings [20, 38]. In addition, endometrial extracts contained 20- to 28-kDa "N-terminal" forms of CTGF that reacted with anti-mCTGF(80–93) but not with anti-mCTGF(246–259), as well as a 16-kDa "C-terminal" form that reacted with anti-mCTGF(246–259) but not with anti-mCTGF(80–93) (Fig. 4). Western blotting of unfractionated ULF resulted in the detection of the same proteins with anti-mCTGF(80–93) as were present in endometrium (Fig. 4). Although the presence of low-mass N-terminal CTGF isoforms in ULF suggested that C-terminal isoforms were also likely to be present, the latter could not be detected because of the low sensitivity of detection of anti-mCTGF(246–259) (Fig. 4). To substantiate the presence of low-mass C-terminal CTGF proteins in ULF, we took advantage of the fact that these particular isoforms are mitogenic and heparin-binding [33, 34]. Unfractionated ULF was shown to stimulate dose-dependent stimulation of Balb/c 3T3 cell DNA synthesis (data not shown). Heparin-affinity FPLC of ULF resulted in the detection of a broad profile of mitogenic activity for Balb/c 3T3 cells that was eluted from the heparin column by 0.4–1.2 M NaCl (Fig. 5). Although the presence of other heparin-binding growth factors such as platelet-derived growth factor and HB-EGF—which elute from heparin columns by 0.5 M and 1 M NaCl, respectively, and are present in uterine luminal fluids [39]—likely contributed to the overall mitogenic profile, the presence of 12- to 14-kDa C-terminal CTGF proteins in the eluate was demonstrated by their reactivity with anti-mCTGF(246–259) but not with anti-mCTGF(80–93) (Fig. 5). Similar low-mass forms of mCTGF have previously been described in fibroblast-conditioned medium [37]. In this experiment, the 12-kDa component eluted earlier from the heparin column (~0.6 M NaCl) than the 14-kDa component (~0.8 M NaCl), suggesting that the two components exhibited differential heparin affinities. A similar phenomenon has been observed for microheterogenous forms of 10-kDa pig CTGF and was attributed to variations in the N-termini of the proteins [34]. Although very low levels of a ~40-kDa protein were also detected by anti-mCTGF(246–259) in the column fractions (Fig. 5), this appears not to be authentic full-length CTGF since it was not detected by anti-mCTGF(80–93) (data not shown) and a similar protein was previously detected nonspecifically in pig ULF [33, 34].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the cycling uterus, CTGF was localized primarily to surface and glandular epithelial cells, where it was present at high levels during proestrus and at lower levels at estrus. During pregnancy, epithelial staining was reduced on Day 4.5 as compared to Days 1.5–3.5. It remains to be established whether the weaker CTGF signal in epithelial cells at estrus and on Day 4.5 of pregnancy reflects a decreased rate of CTGF production as opposed to increased rate(s) of CTGF release, utilization, and/or degradation, and how (if at all) steroid hormones are involved in these effects.
Decidualization is a highly regulated process and involves increases in DNA synthesis, as well as synthesis and deposition of desmin and extracellular matrix (ECM) molecules such as laminin and fibronectin [40, 41]. Fibronectin and laminin are deposited in the endometrial ECM by decidual cells at the time of implantation [42, 43] and, in vitro, have been shown to directly promote blastocyst attachment [44].Thus, the appearance of CTGF in differentiating endometrium, coupled with its ability to stimulate DNA synthesis, chemotaxis, cell proliferation, and production of ECM components such as fibronectin, type IV collagen, and 5 integrin [20, 34, 36, 38], is consistent with a direct contribution of CTGF to the differentiation process itself. The recognition that CTGF gene expression is regulated by TGFß suggests that TGFß may be a principal molecular cue for CTGF production in the uterine tract. Moreover, it is well recognized that TGFß plays a central role in ECM production since it stimulates synthesis of ECM proteins such as collagen, fibronectin, laminin, elastin, glycosaminoglycans, and other glycoproteins, as well as synthesis of inhibitors of ECM degradation such as plasminogen activator inhibitor and tissue inhibitors of metalloproteases [45]. In addition, TGFß has been postulated as a stimulus for fibronectin production at the time of implantation [42]. In previous studies, TGFß1 accumulated in the stroma after its initial synthesis by luminal and glandular epithelial cells on Days 1–4 of pregnancy (Day 1 = day of vaginal plug). Stromal staining was present on Days 1 and 3, absent on Day 2, markedly enhanced on Day 4, and intense in the decidua on Day 5. It was thus proposed that epithelial-derived TGFß acts to stimulate stromal differentiation at the time of implantation [17]. Expression of the type I TGFß receptor occurred mainly in the luminal and glandular epithelium on Days 1–5 and spread throughout the decidua on Days 6–8 [17]. In contrast, the type II and III receptor mRNAs were absent on Days 1–3 but appeared in stromal (II, III) and epithelial (II) cells on Day 4. Expression of the type II and III receptors was predominant in the stroma and decidua on Days 5–8. Transgenic mice that had down-regulated levels of TGFß receptors on Days 3–5 demonstrated a delayed implantation reaction [17]. Collectively, these data suggested that TGFß and its receptor function to remodel and prepare the uterus for implantation [17]. Our data show that CTGF localization on Days 2.5–5.5 of pregnancy is well correlated with the production of TGFß1 and TGFß type I receptor by luminal/glandular epithelial cells during the preimplantation period and with the accumulation of TGFß1 and expression of all three TGFß receptors in the stroma/decidua during the peri- and postimplantation period. Thus the scenario in the immediate postimplantation uterus may be very similar to growth factor cascades that occur during wound healing and fibrosis in which CTGF is produced downstream of the production of TGFß [22, 28]. However, since CTGF was present at only low levels in the uterine stroma on Days 1.5–3.5, when both the type I TGFß receptor and epithelial-derived TGFß1 are present, it is possible that CTGF biosynthesis may be TGFß independent under some circumstances or that TGFß is present only in its latent form. Whatever the precise relationship between CTGF, TGFß, and steroid hormones, our data suggest that CTGF is involved in the establishment of the decidual reaction or is produced as a result of it.
The presence of CTGF in surface and glandular uterine epithelial cells in cycling mice is consistent with its deposition into the uterine lumen and detection in ULF. A recent study showed that other secretory structures such as kidney tubules and salivary, mucus, or sebaceous glands are frequently positive for CTGF [36]. Although the role of uterine epithelial CTGF has yet to be determined, it may serve as a mitogenic stimulus for its target cells, among which are fibroblasts (including endometrial stromal cells) and smooth muscle cells, but not epithelial or endothelial cells [20, 22, 34, 35, 37]. Alternatively, it may fulfil other roles such as promoting cell adhesion [36] or binding insulin-like growth factors [46], the latter of which are produced in the rodent uterus under steroidal control [47–49].
Our observations regarding the presence of CTGF in mouse ULF is consistent with our previous demonstration of CTGF in ULF of pigs [33, 34]. In both species, CTGF biological activity is attributable to heparin-binding 10- to 20-kDa C-terminal isoforms of the protein. Thus despite the dramatic differences in uterine physiology of these two species, certain aspects of CTGF biochemistry appear to have been conserved. This may be indicative of fundamentally similar mechanisms of CTGF processing, which, in the case of the pig, appears to involve rapid but limited degradation of 38-kDa CTGF by proteases in the uterine tract [33, 34]. Since a prominent N-terminal isoform of CTGF was present in endometrial extracts, it is possible that this protein contributed to the overall CTGF signal detected immunohistochemically. These results highlight the importance of determining the biological functions of the individual CTGF isoforms, identifying the specific proteases involved, and defining the mechanisms by which their production and action are regulated.
These data establish a likely role for CTGF in uterine tissue of both pregnant and nonpregnant animals. Although the physiological roles of CTGF in utero have yet to be determined, the diverse biological properties of this growth factor suggest that it may regulate multiple cellular processes in both uterine and embryonic tissues. These aspects of CTGF biology, together with the mechanisms of regulation of uterine CTGF production and the distribution of its presumptive cell surface receptor, will be exciting areas for future studies.
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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 Dr., Columbus, OH 43205. FAX: 614 722 2716;brigstod{at}pediatrics.ohio-state.edu
Accepted: July 10, 1998.
Received: April 27, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and a functional epidermal growth factor receptor (EGFR) to the inner cell mass and preferential localization of the EGFR on the basolateral surface of the trophectoderm in the mouse blastocyst. Dev Biol 1992; 154:396–409.[CrossRef][Medline] precursors in the mouse uterus during the preimplantation period and after steroid hormone treatments. Biol Reprod 1994; 50:481–491.[Abstract] in the uterus of transgenic mouse causes downregulation of TGF-ß receptors and delays the blastocyst-attachment reaction. J Mol Endocrinol 1997; 18:243–257.This article has been cited by other articles:
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