Hum. Reprod. Advance Access originally published online on October 26, 2006
Human Reproduction 2007 22(1):63-74; doi:10.1093/humrep/del356
Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada
1 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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BACKGROUND: The restricted expression of ADAMTS-5 (A Disintegrin And Metalloproteinase with ThromboSpondin repeats-5) to the maternal–fetal interface in mice has led to this novel metalloproteinase being assigned the trivial name ‘implantin’. METHODS: As a first step in determining whether ADAMTS-5 also contributes to the implantation process in humans, we have examined the spatiotemporal expression of this ADAMTS subtype in the endometrium during the menstrual cycle and pregnancy by immunohistochemical analysis. A quantitative competitive PCR (QC-PCR) strategy and western blotting were subsequently used to determine whether interleukin (IL)-1
and transforming growth factor (TGF)-1, two cytokines involved in the formation of the maternal–fetal interface, were capable of regulating ADAMTS-5 messenger RNA (mRNA) and protein levels in primary cultures of stromal cells isolated from first trimester decidual tissues. RESULTS: ADAMTS-5 expression in the stroma of the human endometrium correlates with decidualization of this cellular compartment in vivo. IL-1 was found to increase (P < 0.05) whereas TGF-1 decreased (P < 0.05) ADAMTS-5 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 directed against either cytokine. CONCLUSIONS: ADAMTS-5 expression is restricted to decidualized stromal cells of the human endometrium in vivo and is subject to regulation by cytokines in vitro.
Key words: ADAMTS-5/cytokines/decidua/human/implantin
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Introduction |
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Proteolysis of the extracellular matrix (ECM) of the human endometrium is a critical step in establishing the architecture of this dynamic tissue during the menstrual cycle and pregnancy (Aplin et al., 1988
; Iwahashi et al., 1996). The proteolytic enzymes responsible for this highly regulated series of tissue remodelling events include urokinase plasminogen activator (uPA) and the matrix metalloproteinases (MMPs) which work in concert or in cascades to degrade or process specific components of the ECM (Schatz et al., 1999; Fata et al., 2000; Curry and Osteen, 2001). However, as aberrant expression or distribution of ECM components in the endometrial stroma has been associated with infertility and recurrent pregnancy loss (Bilalis et al., 1996; Jokimaa et al., 2002), it is important to define the full repertoire of proteinases expressed by these cells, their regulation and, ultimately, their individual contribution(s) to the development of a uterine environment that is capable of supporting pregnancy.
The ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) are a novel family of secreted metalloproteinases that are characterized by four structural and functional domains: an amino terminal prodomain, a catalytic domain, a disintegrin-like domain and an ECM-binding domain (composed of a central thrombospondin type 1 motif, a spacer region and a variable number of thrombospondin-like motifs) at the carboxyl terminal of the mature protein species (Tang, 2001; Apte, 2004; Porter et al., 2005). To date, more than 20 members of this rapidly expanding gene family have been characterized in mammals. Although the biological functions of the majority of the distinct ADAMTS subtypes remain poorly characterized, there is increasing evidence to suggest that these novel proteinases play important roles in embryonic growth and development (Shindo et al., 2000; Mittaz et al., 2004), the onset and progression of cancer (Porter et al., 2004), arthritis (Nagase and Kashiwaga, 2003), several thrombotic and inflammatory conditions (Kuno et al., 1997; Tsai, 2002) and the cyclic remodelling events that occur in adult reproductive tissues (Shindo et al., 2000; Li et al., 2001; Mittaz et al., 2004; Richards et al., 2005; Shozu et al., 2005).
Multiple ADAMTS subtypes have been detected, many albeit at low levels, in total RNA lysates prepared from human term placenta or uterine tissues suggesting that these metalloproteinases may play important roles in implantation and placentation (Abbaszade et al., 1999; Vazquez et al., 1999). Furthermore, ADAMTS-5, also known as aggrecanase-2 and ADAMTS-11 (Abbaszade et al., 1999; Hurskainen et al., 1999), is expressed primarily in the murine placenta and corresponding maternal decidua during the peri-implantation period (day 7.5 of gestation) but not at later stages of gestation (Abbaszade et al., 1999). This restricted expression pattern has led to the proposal that ADAMTS-5 contributes to the ECM-remodelling events underlying the establishment of pregnancy, at least in the mouse. Consequently, ADAMTS-5 has been assigned the trivial name ‘implantin’ (Abbaszade et al., 1999). To date, the expression and regulation of ADAMTS-5 in the human endometrium have not been determined.
Cytokines are potent regulators of the proteolytic mechanisms operative at the maternal–fetal interface (Godkin and Dore, 1998; Salamonsen et al., 2000; Karmakar and Das, 2002; Salamonsen et al., 2003). In particular, interleukin (IL)-1 and transforming growth factor (TGF)-1 have been shown to have differential effects on the localized activity of the MMP and uPA systems in the human placenta and endometrium via their ability to coordinately regulate the expression levels of these proteinases and/or their cognate inhibitors in both tissues (Huang et al., 1998; Chung et al., 2001; Karmakar and Das, 2002). In these studies, we have examined the ability of IL-1 and TGF-1 to regulate ADAMTS-5 expression levels in primary cultures of stromal cells isolated from first trimester decidual tissues in a concentration- and time-dependent manner. We have also determined whether ADAMTS-5 is spatiotemporally expressed in the human endometrium during the menstrual cycle and pregnancy.
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Tissues
For the isolation of enriched cultures of stromal cells, tissue samples of first trimester decidua parietalis (gestational ages ranging from 6 to 12 weeks) 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). All patients provided informed written consent.
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; Ng et al., 2006). 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 was 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, resuspended and plated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 25 mM glucose, L-glutamine and antibiotics (100 U/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 culture medium was subsequently replaced 30 min after plating to reduce epithelial cell contamination. 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 decidual 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 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 as described above, 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 phosphate-buffered saline (PBS) and cultured under serum-free conditions in DMEM supplemented with antibiotics, E2 and P4 for the duration of these studies.
Twenty-four hours after removal of serum from the culture medium, the cells were again washed with PBS and cultured in the presence of TGF-1 (0.001, 0.01, 0.1, 1, 5 or 10 ng/ml; Sigma Aldrich) or IL-1 (1, 10, 100 or 1000 IU/ml; Sigma Aldrich) for 24 h or TGF-1 (5ng/ml) or IL-1 (100 IU/ml) for 0,12, 24 or 48 h. Decidual stromal cells treated with vehicle (0.1% v/v ethanol) alone served as controls for these experiments.
To inhibit the regulatory effects of TGF-1 and IL-1 on ADAMTS-5 messenger RNA (mRNA) and protein expression levels in these primary cell cultures, we cultured decidual stromal cells 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 (5 or 10 µg/ml; clone 9016.2; 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; clone 8516.311; Sigma Aldrich) for 24 h.
The concentrations of cytokines and corresponding function-perturbing antibodies and the time points 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
Total RNA was prepared from the decidual stromal cell cultures using a RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada) and a protocol outlined by the manufacturer. 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, we electrophoresed aliquots of the total RNA in a 1% (w/v) denaturing agarose gel containing 3.7% (v/v) formaldehyde and the 28S and 18S ribosomal RNA subunits visualized by ethidium bromide staining. The purity and concentration of total RNA present in each of the extracts were quantified by absorbance (260/280 nm) using a Du-64 UV-spectrophotometer (Beckman Coulter, Mississauga, ON, Canada).
Aliquots (1 µg) of the total RNA extracts prepared from each of the decidual stromal cell cultures were then reverse-transcribed into complementary DNA (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-5 were identified in the mRNA sequence deposited in GenBank (Accession No. NM_007038
[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, BC, Canada. To generate a competitive cDNA fragment, we attached a stretch of nucleotides corresponding to a sequence present within the target ADAMTS-5 PCR product to the 3'-end of the initial forward primer (Figure 1). We have recently used a similar approach to examine the effects of IL-1 and TGF-1 on ADAMTS-1 mRNA levels in these primary cell cultures (Ng et al., 2006).
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Quantitative competitive PCR
The quantitative competitive PCR (QC-PCR) strategy employed in these studies is based upon the competitive co-amplification of a known amount of competitive ADAMTS-5 PCR product added to aliquots of first-strand cDNA prepared from the primary cultures of decidual stromal cells. PCR was performed using these cDNA mixtures as templates and our ADAMTS-5 primers under the following optimized conditions: 1 min at 94°C, 1 min at 59°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 and competitive ADAMTS-5 PCR products were separated using gel electrophoresis and visualized by ethidium bromide staining (data not shown). An aliquot of these PCR products were then subcloned into the PCR II vector (Invitrogen, Carlsbad, CA, USA) and subjected to DNA sequence analysis to confirm the specificity of the primers.
To determine the optimal amount of competitive cDNA to be added to each reaction mixture, we performed PCR using a fixed amount of template cDNA combined with decreasing concentrations of competitive cDNA. Aliquots (10 µl) of the subsequent PCR products were subjected to electrophoresis in a 1% (w/v) 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 the Scion Image computer software (Scion Image Co., Frederick, MD, USA). The volume counts obtained for both PCR products were plotted against the corresponding concentration of competitive cDNA in a line graph. The point of interception of the two lines indicated that 9.77 x 10–3 pg of competitive cDNA was the optimal amount to be added to each QC-PCR mixture.
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QC-PCR was performed under the optimized conditions for the ADAMTS-5 primers described above. The number of cycles were repeated between 22 and 28 times allowing for any differences in the levels of target ADAMTS-5 initially detected in the aliquots of first-strand cDNA prepared from each of the primary cultures of decidual stromal cells without altering the optimal amount of competitive cDNA to be added to each reaction mixture. The ratio of target : competitive ADAMTS-5 PCR products in each reaction mixture was subsequently determined by UV densitometry.
Western blot analysis
Decidual stromal cell cultures (grown to 80–100% conflueny) 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 proteinase-inhibitor cocktail for 30 min on a rocking platform. The cell lysates were centrifuged at 10 000 x g for 10 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 (15–30 µg) of the cell lysates were prepared and immunoblotted as previously described (MacCalman et al., 1996) using a commercially available polyclonal antibody directed against the C-terminal region of human ADAMTS-5 (K39050R; Biodesign International, Saco, ME, USA). Western blot analysis revealed the presence of four distinct ADAMTS-5 protein species of 120, 73, 50 and 40 kDa in human decidual stromal cell cultures (Figure 3). The Mr of these protein species corresponds to the predicted zymogen, mature active form and two post-translational cleavage products (with unknown biological activity) of human ADAMTS-5, respectively (Abbaszade et al., 1999).
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The specificity of this ADAMTS-5 antibody was confirmed by pre-adsorption with a neutralizing peptide supplied by the manufacturer (A39050H; Biodesign International). The antibody was pre-incubated with the peptide at a ratio of 1:20 for 1 h at room temperature on a shaking platform before being used for western blot analysis. Pre-adsorption of the ADAMTS-5 antibody with this neutralizing peptide inhibited its ability to bind to antigen in the protein lysates prepared from the decidual stromal cell cultures (Figure 3).
To standardize the amounts of protein loaded in each lane, we reprobed the western blots with a polyclonal antibody directed against human -actin (Sigma Aldrich). The Amersham enhanced chemiluminescence (ECL) system was used to detect the amount of each antibody bound to antigen. The resultant autoradiograms were analysed by densitometry. The absorbance value obtained for each of the ADAMTS-5 protein species was normalized relative to the corresponding -actin absorbance value. The data are presented as total ADAMTS-5 protein expression and as levels of the mature, active form (73 kDa) of this ADAMTS subtype detected in each of the decidual stromal cell cultures.
Immunohistochemistry
Tissue sections were prepared from archived permanent paraffin blocks containing proliferative (n = 3) or mid-secretory endometrium (n = 3) obtained from women of reproductive age with proven fertility (Getsios et al., 1998) or decidual tissues obtained during the first trimester of pregnancy (n = 3) (Chou et al., 2004). These endometrial tissue sections were immunostained with polyclonal antibodies directed against either human ADAMTS-5 or -actin (Sigma Aldrich). Decidual tissue sections probed with each of these antibodies pre-adsorbed with the ADAMTS-5 neutralizing peptide 1:1000 (w/w) served as controls for these studies.
Sequential incubations were performed according to the methods of Cartun and Pedersen (1989) and included 10% normal goat serum for 30 min, primary antibody at 37°C for 1 h, secondary biotinylated antibody at 37°C for 45 min, streptavidin–biotinylated horseradish peroxidase complex reagent at 37°C for 30 min and three 5-min washes in 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.
Statistical analysis
The absorbance values obtained from the ethidium bromide-stained gels containing the ADAMTS-5 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 cell cultures generated from three or more different tissue samples on independent occasions.
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Time-dependent effects of TGF-
1 and IL-1 on ADAMTS-5 mRNA and protein levels in human decidual stromal cellsADAMTS-5 mRNA transcripts were detected in all of the decidual stromal cell cultures (Figures A1–
A3). The addition of vehicle to the culture medium had no significant effect on ADAMTS-5 mRNA levels in these cells at any of the time points examined in these studies (data not shown).
A significant decrease in ADAMTS-5 mRNA levels was first detected in decidual stromal cells cultured in the presence of TGF-1 for 24 h (Figure A1, panel A). The levels of the ADAMTS-5 mRNA transcript present in these primary cell cultures continued to decrease until the termination of these studies at 48 h (Figure A1, panel A). In contrast, the addition of IL-1 to the culture medium of these primary cells caused a significant increase in ADAMTS-5 mRNA levels after 24 h (Figure A1, panel B). Levels of the ADAMTS-5 mRNA transcript remained elevated after 48 h of culture in the presence of this cytokine (Figure A1, panel B).
There was a significant decrease in total ADAMTS-5 protein expression levels in decidual stromal cells cultured in the presence of TGF-1 for 24 h with levels continuing to decline until the termination of these studies at 48 h (Figure A1, panel C). In contrast, IL-1 induced a significant increase in total ADAMTS-5 protein expression in these primary cells after 24 h with levels remaining elevated at 48 h of culture in the presence of this cytokine (Figure A1, panel D). A similar expression pattern was observed for the mature, active form (73 kDa) of ADAMTS-5 in cells cultured in the presence of these two cytokines (Figure A1, panels C and D).
Concentration-dependent effects of TGF-1 and IL-1 on ADAMTS-5 mRNA and protein levels in human decidual stromal cells
ADAMTS-5 mRNA and total protein expression levels were significantly decreased in decidual stromal cells cultured in the presence of 0.1 ng/ml TGF-1 for 24 h but not at the lower concentrations of cytokine examined in these studies (Figure A2, panels A and C, respectively). The addition of higher concentrations of TGF-1 (1–10 ng/ml) resulted in a continuous and significant decrease in the levels of the ADAMTS-5 mRNA transcript, total protein and 73-kDa protein species present in these cells (Figure A2, panels A and C).
A significant increase in ADAMTS-5 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 A2, panels B and D, respectively).
Attenuation of cytokine-modulated ADAMTS-5 mRNA levels in human decidual stromal cells using monoclonal antibodies directed against either TGF-1 or IL-1
Function-perturbing monoclonal antibodies directed against either TGF-1 or IL-1 had no significant effect on ADAMTS-5 mRNA or protein expression levels in our human decidual stromal cells after 24 h of culture (Figure A3). However, the monoclonal antibody directed against TGF-1 abolished the decrease in ADAMTS-5 mRNA and protein levels observed in decidual stromal cells cultured in the presence of this cytokine (Figure A3, panels A and C). Similarly, addition of the anti-IL-1 antibody to the culture medium of these primary cells was capable of inhibiting the IL-1-mediated increase in ADAMTS-5 mRNA and protein expression levels (Figure A3, panels B and D).
Immunolocalization of ADAMTS-5 in the human endometrium during the menstrual cycle and pregnancy
ADAMTS-5 expression was readily detectable in the glandular epithelium of both the proliferative and the secretory endometria (Figure A4, panels A and B). In the stroma, significant levels of ADAMTS-5 were not detected in the proliferative endometrium (Figure A4, panel A). Instead, ADAMTS-5 was first detected in predecidual cells surrounding the spiral arterioles present in this cellular compartment during the secretory phase of the menstrual cycle (Figure A4, panel B). Intense ADAMTS-5 expression was also detected in the large polyhedral stromal cells of first trimester decidua (Figure A4, panel C). This immunostaining pattern was abolished by pre-adsorption of the ADAMTS-5 antibody with a neutralizing peptide (Figure A4, panel D). In contrast, immunostaining was detected in the stromal cells of decidual tissue sections probed with a polyclonal antibody directed against -actin (Figure A4, panel E) or with this antibody pre-adsorbed with the ADAMTS-5 neutralizing peptide (Figure A4, panel F).
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Here we report that ADAMTS5 mRNA transcripts and four distinct ADAMTS-5 protein species are present in stromal cells isolated from human decidual tissues obtained during the first trimester of pregnancy. IL-1
was found to have a stimulatory effect on ADAMTS-5 mRNA and protein levels, whereas TGF-1 decreases the expression of this ADAMTS subtype in these primary cultures. In addition, we have demonstrated that similar to the mouse (Abbaszade et al., 1999), ADAMTS-5 expression in the human endometrium is associated with decidualization in vivo.
ADAMTS-5, together with ADAMTS-1, -4, -8, -9 and –15, forms a subfamily of proteinases, known as the aggrecanases owing to their ability to cleave the large chondroitin sulphate proteoglycans, aggrecan, brevican, neurocan and versican (Tang, 2001; Nagase and Kashiwaga, 2003; Apte, 2004; Glasson et al., 2005; Porter et al., 2005). Of these, only versican has been found to be present in the decidua of the mouse endometrium (San Martin et al., 2003) and to be over-expressed in human endometrial carcinomas (Hanekamp et al., 2003). Versican is a multidomain, multifunctional protein that plays a central role in the assembly of the ECM via interactions with hyaluronan, tenascin, fibulin-1, fibrillin and fibronectin (Wight, 2002). An indirect correlation between ADAMTS-1/ADAMTS-5 and versican expression levels has been described in human prostate cells in vivo and in vitro (Cross et al., 2005). In contrast, ADAMTS-1 and ADAMTS-4 are co-expressed with versican in the rodent ovary during folliculogenesis and appear responsible for the generation of cleavage products of this chondroitin sulphate proteoglycan that have distinct bioactivities (Russell et al., 2003; Richards et al., 2005). Collectively, these observations suggest that aggrecanases are capable of regulating both the expression and function(s) of versican in mammalian tissues. To date, the role(s) of ADAMTS-5 and other members of the aggrecanase subfamily of proteinases in the formation of high-ordered, multimolecular complex structures present in the endometrial ECM under normal and pathological conditions remain to be elucidated. However, decidualization in the human endometrium has been associated with an increase in the expression levels of several versican-binding proteins including hyaluronan (Salamonsen et al., 2001), fibulin (Haendler et al., 2004) and fibrillin (Fleming and Bell, 1997).
ADAMTS-5 gene-knockout mice are viable and fertile (Stanton et al., 2005). These observations suggest that embryonic implantation is not dependent upon the regulated expression of ADAMTS-5 in the endometrium and that other endometrial ADAMTS subtypes may have overlapping/compensatory functions in this multi-step reproductive process. Such redundancy in gene families is common (Fata et al., 2000; Curry and Osteen, 2001; Madan et al., 2003). Indeed, there is increasing evidence of both redundant and non-redundant roles for distinct ADAMTS subtypes in follicular growth and ovulation and in the development and regression of the corpus luteum in the rodent and bovine ovary (Espey et al., 2000; Madan et al., 2003; Russell et al., 2003; Richards et al., 2005). In addition to ADAMTS-5, only ADAMTS-1 has been detected in mouse and human decidua (Kim et al., 2005; Ng et al., 2006). However, the role(s) of ADAMTS-1 in the morphological and functional differentiation of the endometrium also remains unclear as mice null-mutant for this ADAMTS subtype are capable of undergoing normal decidualization (Mittaz et al., 2004) or proceed to develop large endometrial cysts during the reproductive cycle (Shindo et al., 2000). Both uterine environments are, however, capable of supporting pregnancy. To our knowledge, any reciprocal and compensatory changes in the expression levels of ADAMTS subtypes in the endometrium of either ADAMTS-1 or ADAMTS-5 gene-knockout mice have not been examined.
The factors capable of regulating ADAMTS-5 expression remain poorly characterized. Thyroid-stimulating hormone has been shown to specifically regulate ADAMTS-5 mRNA levels in rabbit chondrocytes (Makihira et al., 2003). In addition, IL-1
and TNF- increased ADAMTS-5 mRNA levels in bovine cartilage tissue–explants with IL-6 and its soluble receptor (sIL-6R), potentiating the stimulatory effects of these cytokines on ADAMTS-5 expression and activity in this model system (Flannery et al., 2000). TGF-1 and IL-1 have also been shown to differentially regulate ADAMTS-5 mRNA in human glioblastoma cells (Held-Feindt et al., 2006) but have no significant effect on the constitutive expression of this ADAMTS subtype in human chondrocytes isolated from normal or osteoarthritic cartilage (Bau et al., 2002; Moulharat et al., 2004). Collectively, these observations suggest that the regulatory effects of TGF-1 and IL-1 on ADAMTS-5 mRNA and protein expression levels are dependent upon the cellular context and are likely mediated indirectly via altered expression levels of other cytokines. For example, IL-1 has been shown to increase whereas TGF-1 has been shown to decrease the expression of IL-6 in human endometrial cells in vitro (Fazleabas et al., 2004; Perrier d’Hauterive et al., 2005). As IL-6 is capable of regulating both MMP and ADAMTS-5 activity (Flannery et al., 2000; Salamonsen et al., 2000), these observations suggest that TGF-1 and IL-1 are upstream components of a proinflammatory cascade that regulates the proteolytic mechanisms operative at the maternal–fetal interface.
ADAMTS-5 is synthesized in an inactive proform which is activated upon secretion by cleavage of its prodomain (Abbaszade et al., 1999; Hurskainen et al., 1999). This activated form is subsequently subjected to a second round of post-translational cleavage that results in the generation of two distinct fragments. In agreement with these observations, four protein species of ADAMTS-5 were detected in our primary cultures of human decidual stromal cells. The varying contributions of each of the four distinct protein species to the total ADAMTS-5 protein expression levels in response to IL-1 and TGF-1 suggest that the proteolytic factors capable of cleaving ADAMTS-5 in human decidual stromal cells are also differentially regulated by these cytokines in a concentration- and time-dependent manner. To date, the biological significance of these different forms of ADAMTS-5 in mammalian tissues and cells is not clear. Similarly, the mature forms of ADAMTS-1 and ADAMTS-4 are also subjected to a second independent processing step culminating in the removal of thrombospondin-like repeat(s) from the carboxyl-terminal ends of these proteins (Rodriguez-Manzaneque et al., 2000; Tortorella et al., 2000). The resultant protein fragments of these ADAMTS subtypes have been shown to have reduced proteolytic activities and ECM-binding properties, which in turn alter cell proliferation, adhesion and invasion in vitro and in vivo (Rodriguez-Manzaneque et al., 2000; Tortorella et al., 2000; Kuno et al., 2004; Liu et al., 2005).
In summary, we have determined that ADAMTS-5 is expressed in human decidual stromal cells in vivo and in vitro. IL-1 and TGF-1, two key regulators of the proteolytic mechanisms operative at the maternal–fetal interface, were also found to be capable of regulating ADAMTS-5 mRNA and protein expression levels in these cells in vitro. Collectively, these observations support our hypothesis that the regulated expression of members of the ADAMTS gene family of MMPs contributes to the cytokine-mediated degradation of decidual ECM during pregnancy in humans.
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4) in the bar graphs (a, P < 0.05 versus 0 h control).
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This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to C.D.M. and P.C.K.L. P.C.K.L. is the recipient of a Distinguished Scientist Award from the Michael Smith Foundation for Health Research. C.D.M. is a career investigator of the Child and Family Research Institute.
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References |
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Submitted on March 29, 2006; resubmitted on July 10, 2006; resubmitted on August 9, 2006; accepted on August 14, 2006.
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