Hum. Reprod. Advance Access originally published online on May 4, 2006
Human Reproduction 2006 21(8):1990-1999; doi:10.1093/humrep/del108
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Differential effects of interleukin-1
and transforming growth factor-1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro
1 Department of Obstetrics and Gynecology and 2 Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Room I3091-950, West 28th Avenue, Vancouver, BC, Canada V5Z 4H4. E-mail: cdmaccalman{at}hotmail.com
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Abstract |
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BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1
) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1 and TGF-1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1 increased whereas TGF-1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1 and TGF-1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.
Key words:
ADAMTS-1/decidua/interleukin-1/pregnancy/transforming growth factor-1
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Introduction |
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Human pregnancy is dependent upon a regulated series of inflammatory-type responses occurring at the maternal-fetal interface (Salamonsen et al., 2000
). The reciprocal expression of growth factors, cytokines and their receptors in the embryo and/or endometrium is believed to orchestrate this complex interplay of developmental events, the molecular mechanisms of which are still not fully understood (Graham and Lala, 1992; Salamonsen et al., 2003; Fazleabas et al., 2004).
Interleukin-1 beta (IL-1) is a pro-inflammatory cytokine that plays a major regulatory role in the establishment of pregnancy (Graham and Lala, 1992; Salamonsen et al., 2000, 2003; Fazleabas et al., 2004). In particular, the spatiotemporal expression of this cytokine in the human endometrium promotes decidualization of stromal cells in both an autocrine and a paracrine manner and contributes to the ability of this dynamic cellular compartment to control the invasion of embryonic trophoblasts into the maternal vasculature and underlying myometrium (Graham and Lala, 1992; Simon et al., 1995; Wolff et al., 2000; Bigonesse et al., 2001). There is increasing evidence to suggest that the cellular mechanisms underlying the biological actions of IL-1 on the endometrium are similar to those described for this cytokine in the development/progression of inflammation and cancer, and involve the regulated proteolytic degradation/activation of specific extracellular matrix (ECM) components (Stamenkovic, 2003; Mott and Werb, 2004). Furthermore, IL-1 has also been shown to increase the proteolytic activities of the matrix metalloproteinase/tissue inhibitors of metalloproteinases (MMP/TIMP) and/or urokinase plasminogen activator/plasminogen activator inhibitor-1 (uPA/PA-1) systems in primary cultures of human endometrial stromal cells (Huang et al., 1998; Chung et al., 2001).
The anti-inflammatory cytokine, transforming growth factor-1 (TGF-1), is also a potent regulator of the proteolytic activities of the MMP/TIMP and uPA/PAI-1 systems in mammalian tissues and cells under normal and pathological conditions (Stamenkovic, 2003; Mott and Werb, 2004). Similarly, TGF-1 has been assigned key regulatory roles in decidualization of the endometrial stroma and in placental development and function (Godkin and Dore, 1998; Karmakar and Das, 2002), particularly during the first trimester of pregnancy when maximum levels of this growth factor are expressed in both maternal and fetal cellular compartments (Lala and Graham, 1990; Graham and Lala, 1992; Godkin and Dore, 1998). In addition, TGF-1 has been shown to decrease MMP and uPA activity in human endometrial stromal cells and extravillous cytotrophoblasts in vitro (Graham, 1997; Huang et al., 1998; Chung et al., 2001; Karmakar and Das, 2002). Taken together, these observations suggest that the coordinated expression of IL-1 and TGF-1 counterbalance the activities of the proteolytic mechanisms operating at the fetal–maternal interface.
The ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) are a novel family of metalloproteinases (Kaushal and Shah, 2000; Tang, 2001; Apte, 2004; Porter et al., 2005). One of the best characterized members of this gene family is ADAMTS-1, initially identified as an inflammation-associated gene in an experimental colon carcinoma model system (Kuno et al., 1997b). The proteolytic activity of ADAMTS-1 has subsequently been associated with local tissue invasion in cancer (Masui et al., 2001), in the degradation of cartilage in osteoarthritis (Nagase and Kashiwagi, 2003), and in the development of inflammation associated with these two diseases (Kuno et al., 1997b; Nagase and Kashiwagi, 2003) or in response to trauma (Sasaki et al., 2001). Gene knockout studies in mice have also highlighted the integral roles that ADAMTS-1 plays in the pre- and post-natal development of tissues, including the cyclic remodelling events that occur in the adult ovary and uterus (Shindo et al., 2000; Mittaz et al., 2004; Shozu et al., 2005). In view of these observations, it would seem likely that ADAMTS-1 also contributes to the cytokine-mediated degradation of the decidual ECM during pregnancy in humans. In these studies, we have determined whether ADAMTS-1 expression is associated with decidualization of the human endometrium and examined the ability of IL-1 and TGF-1 to regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidual tissues.
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Tissues
For the isolation of enriched cultures of stromal cells, tissue samples of first-trimester decidua parietalis were obtained from women undergoing elective termination of pregnancy. The use of these tissues was approved by the committee for ethical review of research involving human subjects, University of British Columbia, Vancouver, Canada. All patients provided informed written consent.
Immunohistochemical analysis
Tissue sections were prepared from archived permanent paraffin blocks containing mid-secretory endometrium (n = 3) obtained from women of reproductive age with proven fertility (Getsios et al., 1998), or decidual tissues (n = 3) obtained during the first trimester of pregnancy (Chou et al., 2004). These endometrial tissue sections were immunostained using a commercially available polyclonal antibody directed against human ADAMTS-1 (Biodesign International, Saco, ME, USA). A nonspecific isotype-matched antibody served as a negative control for these experiments.
Sequential incubations were performed according to the methods of MacCalman et al. (1996) and included 10% normal horse serum for 30 min, primary antibody at 37°C for 1 h, secondary biotinylated antibody at 37°C for 45 min, streptavidin-biotinylated horse-radish peroxidase (HRP) complex reagent at 37°C for 30 min and three 5 min washes in phosphate-buffered saline (PBS). The sections were then exposed to chromagen reaction solution (0.035% diaminobenzidine and 0.03% H2O2) for 10 min, washed in tap water for 5 min, counterstained in haematoxylin, dehydrated, cleared and mounted.
Cell isolation and culture
Stromal cells were isolated from the decidual tissue samples by enzymatic digestion and mechanical dissociation using a protocol previously described (Chou et al., 2003). Briefly, the decidual tissue samples were minced and subjected to 0.1% collagenase (type IV; Sigma Aldrich, St Lois, MO, USA) and 0.1% hyaluronidase (type I-S; Sigma Aldrich) digestion in a shaking water bath at 37°C for 60 min. The cell digest was then passed through a nylon sieve (38 µm). The isolated glands and any undigested tissue fragments were retained on the sieve, and the eluate containing the stromal cells collected in a 50 ml tube. The stromal cells were then pelleted by centrifugation at 800 x g for 10 min at room temperature. The cell pellet was washed once, re-suspended, and plated in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose, L-glutamine, antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin) and supplemented with 10% fetal bovine serum, 17-estradiol (E2; 30 nM) and progesterone (P4; 1 µM). All of the decidual stromal cell cultures were subsequently maintained in this culture medium unless otherwise stated.
The purity of the decidual stromal cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin and factor VIII (data not shown). These cellular markers have been used to determine the purity of human endometrial cell cultures (Irwin et al., 1989). As defined by these criteria, the stromal cell cultures used in these studies contained <1% epithelial or vascular cells. Decidualization of these cells in culture was confirmed by the immunodetection of insulin-like growth factor binding protein -1 (data not shown), a biochemical marker specific for decidualized endometrial stromal cells in vivo and in vitro (Tseng et al., 1992; Giudice, 1997; Chen et al., 1999).
Experimental culture conditions
Decidual stromal cells (passages 4–6) were plated in 60 mm2 tissue culture dishes (Becton Dickinson and Co, Franklin Lakes, NJ, USA) at a density of 5 x 106 cells/dish and grown to 80% confluency. The cells were then washed with PBS and cultured under serum-free conditions in DMEM supplemented with antibiotics, E2 (30 nM) and P4 (1 µM) for the duration of these studies.
Twenty four hours after removal of serum from the culture medium, the decidual stromal cells were again washed with PBS and cultured in the presence of TGF-1 (0.1, 1, 5 or 10 ng/ml) or IL-1 (1, 10, 100 or 1000 IU/ml) for 24 h or TGF-1 (5 ng/ml) or IL-1(100 IU/ml) for 0, 6, 12, 24 or 48 h. Decidual stromal cells cultured in the presence of vehicle (0.1% ethanol) served as controls for these studies.
To inhibit the regulatory effects of TGF-1 and IL-1 on ADAMTS-1 mRNA and protein expression levels in these primary cell cultures, decidual stromal cells were cultured in the presence of either TGF-1 (5 ng/ml) alone or in combination with a function-perturbing monoclonal antibody directed against human TGF-1 (10 µg/ml; Sigma Aldrich), or IL-1 (100 IU/ml) alone or in combination with a function-perturbing monoclonal antibody directed against this cytokine (1 or 2 µg/ml; Sigma Aldrich) for 24 h.
The time points and the concentrations of cytokines and corresponding function-perturbing antibodies examined in these studies are based upon previous reports (Huang et al., 1998; Chung et al., 2001). All of the decidual stromal cell cultures were harvested for either total RNA or protein extraction.
Generation of first strand complementary DNA (cDNA)
Total RNA was prepared from the decidual cell cultures using a RNeasy Mini Kit (Qiagen, Mississuaga, ON, Canada). The total RNA extracts were then treated with Deoxyribonuclease-1 (Sigma Aldrich) to eliminate possible contamination with genomic DNA. To verify the integrity of the RNA, aliquots of the total RNA were electrophoresed in a 1% (w/v) denaturing agarose gel containing 3.7% (v/v) formaldehyde and the 28 S and 18 S ribosomal RNA subunits visualized by ethidium bromide staining. The purity and concentration of total RNA present in each of the extracts was quantified by optical densitometry (260/280 nm) using a Du-64 UV-spectrophotometer (Beckman Coulter, Mississuaga, ON, Canada).
Aliquots (approximately 1 µg) of the total RNA extracts prepared from each of the decidual stromal cell cultures were then reverse-transcribed into cDNA using a First Strand cDNA Synthesis Kit, according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Oakville, ON, Canada).
Primer design
Nucleotide sequences specific for human ADAMTS-1 were identified in the mRNA sequence deposited in GenBank (Accession No. NM_006988
[GenBank]
; National Center for Biotechnology Information, Bethesda, MD, USA). Primers corresponding to these nucleotide sequences were synthesized at the Nucleic Acid and Protein Synthesis Unit, University of British Columbia, Vancouver, Canada. To generate a competitive cDNA fragment, a stretch of nucleotides corresponding to a sequence present within the target ADAMTS-1 PCR product was incorporated into the 3'-end of a second forward primer (Figure 1). A similar approach has been used to examine the effects of IL-1 and TGF-1 on u-PA/PAI-1 and MMP/TIMP mRNA levels in primary cultures of human endometrial stromal cells (Huang et al., 1998; Chung et al., 2001).
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Quantitative competitive-PCR (QC-PCR)
The QC-PCR strategy employed in these studies is based upon the competitive co-amplification of a known amount of competitive ADAMTS-1 PCR product added to aliquots of first strand cDNA prepared from our primary cultures of decidual stromal cells. PCR was performed using these cDNA mixtures as templates and the sets of primers specific for ADAMTS-1 under the following optimized conditions: 1 min at 94°C, 1 min at 58.5°C and 1.5 min at 72°C. This cycle was repeated 28 times followed by a final extension at 72°C for 15 min. The resultant target (620 bp) and competitive (321 bp) ADAMTS-1 PCR products were separated using gel electrophoresis and visualized by ethidium bromide staining. An aliquot of these PCR products was subcloned into the PCR II vector (Invitrogen, Carlsbad, CA, USA) and subjected to DNA sequence analysis to confirm the specificity of the primers (data not shown).
To determine the optimal amount of template cDNA to be added to each reaction mixture, PCR was performed using a fixed amount of either the competitive or target cDNA combined with decreasing concentrations of the target or competitive cDNA, respectively. An aliquot (10 µl) of the resultant ADAMTS-1 PCR products was subjected to electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining (Figure 2). The intensity of ethidium bromide staining of the PCR products was analysed by UV densitometry (Biometra, Whiteman Co., Gottigen, Germany). Volume counts (mm3) of the PCR products were then determined using Scion Image computer software (Scion Image Co., Frederick, MD, USA).
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On the basis of these results, an aliquot (0.0625 pg/µl) of the competitive ADAMTS-1 cDNA was subsequently added to each of the aliquots of the first strand cDNA generated from our human decidual stromal cell cultures. QC-PCR was performed under the optimized conditions for the ADAMTS-1 primers described above using 1 µl of first strand cDNA synthesized from an aliquot (1 µg) of total RNA extracted from the decidual stromal cell cultures. The ratio of target:competitive ADAMTS-1 PCR products in each reaction mixture was determined by UV densitometry as described above.
Western blot analysis
The decidual stromal cell cultures were washed three times in PBS and incubated in 100 µl of cell extraction buffer (Biosource International, Camarillo, CA, USA) supplemented with 1.0 mM phenylmethylsulphonyl fluoride and protease-inhibitor cocktail for 30 min on a rocking platform. The cell lysates were centrifuged at 10 000 x g for 30 min at 4°C and the supernatants used for Western blot analysis. The concentrations of protein in the cell lysates were determined using a BCA kit (Pierce Chemicals, Rockford, IL, USA). Western blots containing aliquots (approximately 30 µg) of the cell lysates were prepared and immunoblotted as previously described (MacCalman et al., 1996) using a polyclonal antibody directed against human ADAMTS-1 (Biodesign Intl). To standardize the amounts of protein loaded into each lane, the blots were reprobed with a monoclonal antibody directed against human -actin (Sigma Aldrich). The Amersham enhanced chemiluminescence system was used to detect the amount of each antibody bound to antigen and the resultant autoradiograms analysed by UV densitometry. The absorbance values obtained for ADAMTS-1 were then normalized relative to the corresponding -actin absorbance value.
Statistical analysis
The absorbance values obtained from the ethidium bromide stained gels containing the ADAMTS-1 QC-PCR products and the autoradiograms generated by Western blotting were subjected to statistical analysis using GraphPad Prism 4 computer software (GraphPad, San Diego, CA, USA). Statistical differences between the absorbance values were assessed by the analysis of variance. Differences were considered significant for P 
0.05. Significant differences between the means were determined using Dunnett’s test. The results are presented as the mean relative absorbance (±SEM) obtained using tissue samples obtained from 
4 different patients.
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Immunolocalization of ADAMTS-1 in the human endometrium during the menstrual cycle and pregnancy
ADAMTS-1 expression was readily detectable in the glandular epithelium, throughout the menstrual cycle. In contrast ADAMTS-1 was restricted to stromal cells surrounding the spiral arterioles of the secretory endometrium (Figure 3A), which corresponds to areas of early decidualization (Gellersen and Brosens, 2003
). Extensive ADAMTS-1 immunostaining was subsequently detected in the stromal cells of first-trimester decidua (Figure 3B), large polyhedral cells that are characteristic of this dynamic tissue (Gellersen and Brosens, 2003).
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Time-dependent effects of TGF-
1 on ADAMTS-1 mRNA and protein levels in human decidual stromal cellsADAMTS-1 mRNA transcripts were detected in all of the decidual stromal cell cultures (Figure 4A). The addition of vehicle to the culture medium had no significant effect on ADAMTS-1 mRNA levels in these cells at any of the time points examined in these studies (data not shown). A significant decrease (P < 0.05) in ADAMTS-1 mRNA levels was first detected in decidual stromal cells cultured in the presence of a fixed concentration of TGF-
1 (5 ng/ml) for 24 h (Figure 4A). Levels of the ADAMTS-1 mRNA transcript present in these primary cell cultures levels continued to decrease until the termination of these studies at 48 h.
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Western blot analysis revealed the presence of a single ADAMTS-1 protein species of 110 kDa in all of the decidual stromal cell cultures (Figure 4B). This protein species corresponds to the ADAMTS-1 zymogen (Rodriguez-Manzaneque et al., 2000
; Wachsmuth et al., 2004). ADAMTS-1 protein expression levels remained relatively constant in decidual stromal cells cultured in the presence of vehicle alone, at least at the time points examined in the present studies (data not shown). In agreement with our QC-PCR analysis, there was a significant decrease (P < 0.05) in ADAMTS-1 expression in decidual stromal cells cultured in the presence of TGF-1 (5 ng/ml) for 24 h with the levels of this protein species continuing to decline until the termination of these studies at 48 h (Figure 4B).
Time-dependent effects of IL-1 on ADAMTS-1 mRNA and protein levels in human decidual stromal cells
Significant increases in ADAMTS-1 mRNA and protein expression levels (P < 0.05) were detected in decidual stromal cells cultured in the presence of a fixed concentration of IL-1 (100 IU) for 24 h (Figure 5A and B). This was followed by a continuous increase in the levels of both the ADAMTS-1 mRNA transcript and protein species present in these primary cell cultures until the termination of our studies at 48 h.
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Concentration-dependent effects of TGF-
1 on ADAMTS-1 mRNA and protein levels in human decidual stromal cellsIn agreement with our preceding findings (Figure 4A and B), ADAMTS-1 mRNA and protein expression levels were significantly decreased (P < 0.05) in decidual stromal cells cultured in the presence of TGF-
1 (5 ng/ml) for 24 h (Figure 6A and B). In contrast, there was no significant change in the levels of the ADAMTS-1 mRNA transcript or protein species present in cells cultured with lower concentrations of this cytokine. Lower ADAMTS-1 mRNA and protein expression levels were however detected in decidual stromal cells cultured in the presence of the highest concentration of TGF-1 (10 ng/ml) examined in these studies.
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Concentration-dependent effects of IL-1
on ADAMTS-1 mRNA and protein levels in human decidual stromal cellsA significant increase (P < 0.05) in ADAMTS-1 mRNA and protein expression levels was only detected in decidual stromal cells cultured in the presence of the higher concentrations of IL-1
(100 and 1000 IU) examined in these studies (Figure 7A and B) with 1000 IU of this cytokine having the greatest regulatory effect.
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Attenuation of cytokine-modulated ADAMTS-1 mRNA and protein levels in human decidual stromal cells using monoclonal antibodies directed against TGF
1 or IL-1Function-perturbing monoclonal antibodies directed against either TGF-
1 or IL-1 had no significant effect on ADAMTS-1 mRNA or protein expression levels in our human decidual stromal cells after 24 h of culture (data not shown). However, the monoclonal antibody directed against TGF-1 abolished the decrease in ADAMTS-1 mRNA and protein levels observed in decidual stromal cells cultured in the presence of this cytokine (Figure 8A and B). Similarly, the IL-1-mediated increase in the levels of the ADAMTS-1 mRNA transcript and protein species present in these primary cell cultures was inhibited by the addition of an anti-IL-1 antibody to the culture medium (Figure 9A and B).
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In these studies, we have determined that regulated expression of the inflammation-associated metalloproteinase, ADAMTS-1, in the stroma of the human endometrium correlates with decidualization of this cellular compartment. In addition, IL-1
and TGF-1 were found to have differential effects on ADAMTS-1 mRNA and protein expression levels in primary cultures of human decidual stromal cells. Collectively, these observations provide further evidence that the cytokine-mediated proteolytic mechanisms underlying inflammation and cancer progression are present at the human fetal–maternal interface.
ADAMTS-1 mRNA transcripts have been detected in a wide array of adult human tissues including term placenta and non-pregnant uterus (Abbaszade et al., 1999; Vazquez et al., 1999). ADAMTS-1 expression has also been detected in the uterine tissues of pregnant mice (Shindo et al., 2000; Mittaz et al., 2004), but the role of this novel metalloproteinase in the development of a uterine environment that is capable of supporting pregnancy remains unclear. Although there is a significant increase in ADAMTS-1 mRNA levels in the mouse endometrium during the peri-implantation period (Kim et al., 2005), endometrial tissues of mice null-mutant for this gene have been shown to either develop large cysts (Shindo et al., 2000) or be capable of undergoing normal morphological decidualization (Mittaz et al., 2004). However, all ADAMTS-1 gene knockout female mice are reported to have reduced pregnancy rates (Shindo et al., 2000; Mittaz et al., 2004). Taken together, these observations suggest that ADAMTS-1 is neither necessary nor sufficient to mediate decidualization at least in the mouse, but may play an important role in the later stages of implantation and placentation. Alternatively other ADAMTS subtypes expressed in the endometrium may have overlapping and, thus, redundant functions in this multi-step reproductive process. A potential candidate for the partial rescue of the reproductive capacity of the ADAMTS-1 gene knockout mouse is ADAMTS-5, also known as aggrecanase-2, ADAMTS-11 or by its trivial name ‘implantin’ (Hurskainen et al., 1999). Both proteinases are expressed in the mouse decidua (Hurskainen et al., 1999; Shindo et al., 2000; Mittaz et al., 2004), and preferentially cleave members of the gene family of chondroitin sulphate glycoproteins known as the hyalectins (Apte, 2004; Porter et al., 2005). Furthermore, mice null-mutant for the ADAMTS-5 gene are viable and fertile (Stanton et al., 2005). To our knowledge, any reciprocal and compensatory changes in the expression levels of distinct ADAMTS subtypes in the endometrium of either ADAMTS-1 or ADAMTS-5 gene knockout mice have not been examined.
ADAMTS-1 is a secreted, multidomain, multifunctional protein composed of an amino terminal prodomain, a proteolytic domain, a disintegrin-like domain and an ECM binding domain [which is composed of a central thrombospondin (TSP) type 1 motif, a spacer region and 3 TSP-like motifs] (Kaushal and Shah, 2000; Tang, 2001; Apte, 2004; Porter et al., 2005). In addition to its proteolytic activity, ADAMTS-1 has been shown to have both angioinhibitory and angiogenic properties in vitro and in vivo (Vazquez et al., 1999; Carpizo and Iruela-Arispe, 2000; Shindo et al., 2000). Thus, ADAMTS-1 has the potential to contribute to the development of an uterine environment capable of supporting a pregnancy via the regulated degradation of the decidual ECM and/or the extensive vascular changes that occur in this dynamic tissue during implantation and placentation, another cytokine-mediated developmental event (Zygmunt et al., 2003).
TGF-1 and IL-1 have been shown to have differential effects on the decidualization of human endometrial stromal cells in vitro (Frank et al., 1995; Godkin and Dore, 1998). In agreement with our observations, TGF-1 has been shown to decrease ADAMTS-1 mRNA levels in human prostate cells in vitro (Cross et al., 2005b). Similarly, IL-1 increased ADAMTS-1 mRNA levels in mouse colon carcinoma cells (Kuno et al., 1997b) and in rat motor neurons (Sasaki et al., 2001), but decreased the levels of the ADAMTS-1 transcript present in human articular chondrocytes (Wachsmuth et al., 2004). Collectively, these observations suggest that the regulatory effects of TGF-1 and IL-1 on ADAMTS-1 expression are indirect and dependent upon the cellular context.
P4, a key regulator of decidualization (Graham and Lala, 1992; Salamonsen et al., 2000, 2003; Fazleabas et al., 2004) has also been shown to regulate ADAMTS-1 mRNA and protein expression levels in the rodent ovary (Espey et al., 2000; Robker et al., 2000; Russell et al., 2003). However, computer-based searches of the nucleotide sequence and functional assays have failed to identify a P4 receptor response element in the promoter region of the murine ADAMTS-1 gene (Kuno et al., 1997a; Doyle et al., 2004). Instead, P4 appears to regulate ADAMTS-1 gene expression, at least in the mouse, through an indirect mechanism(s) that involves the DNA binding transcription factors Sp1/Sp3, C/EBPb and/or NF-1 (Doyle et al., 2004). Interestingly, IL-1 and TGF-1 have been shown to mediate many of the biological actions of progesterone on the human endometrium (Graham and Lala, 1992; Godkin and Dore, 1998; Salamonsen et al., 2000, 2003; Fazleabas et al., 2004), and to regulate gene expression in other human cell types via the Sp1/Sp3 complex (Chadjichristos et al., 2002, 2003).
Initial biochemical studies predicted that the ADAMTS-1 zymogen (110 kDa) undergoes two consecutive post-translational cleavage steps that generate two distinct bioactive fragments (87 kDa and 67 kDa) of this protein (Rodriguez-Manzaneque et al., 2000). All three of the ADAMTS-1 protein species have subsequently been detected in cellular extracts prepared from mouse ovaries (Russell et al., 2003). However, similar to our findings, only the ADAMTS-1 zymogen was detected in primary cultures of human chondrocytes (Wachsmuth et al., 2004). These differences are likely attributable to the loss of endogenous proteolytic factors capable of cleaving ADAMTS-1 (Rodriguez-Manzaneque et al., 2000) following the isolation and culture of these enriched populations of cells. Of these proteolytic factors, only MMP-2 has been detected in the human endometrium (Fata et al., 2000). However, MMP-2 activity in primary cultures of endometrial stromal cells is dependent upon the presence of soluble factor(s) derived from the glandular epithelium (Goffin et al., 2002). Furthermore, the ADAMTS-1 mRNA transcript, zymogen and distinct bioactive forms of this protein have been shown to be either coordinately or differentially regulated in tissues under normal and pathological conditions (Russell et al., 2003; Shimada et al., 2004; Cross et al., 2005a; Richards et al., 2005). To date, the biological significance of these complex mRNA and protein expression patterns remains to be elucidated.
In summary, we have determined that ADAMTS-1 is expressed in human decidual stromal cells in vivo and in vitro. Furthermore, we have demonstrated that IL-1 and TGF-1, two key regulators of the proteolytic mechanisms operative at the maternal-fetal interface, are capable of differentially regulating ADAMTS-1 mRNA and protein expression levels in primary cultures of decidual stromal cell cultures. Collectively, these findings serve as the basis for future studies into the expression and function(s) of ADAMTS-1 in the human endometrium under normal and pathological conditions.
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These studies were supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to CDM and PCKL. PCKL is the recipient of a senior investigatorship from the Michael Smith Foundation for Health Research. CDM and CJP are career investigators of the BC Research Institute for Children’s and Women’s Health.
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Submitted on October 27, 2005; resubmitted on February 9, 2006; resubmitted on March 10, 2006; accepted on March 22, 2006.
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