RESEARCH ARTICLE Molecular Reproduction & Development 82:387–396 (2015)

Compensatory Fetal Membrane Mechanisms Between Biglycan and Decorin in Inflammation LUCIANA BATALHA DE MIRANDA DE ARAUJO,1 CASIE E. HORGAN,1 ABRAHAM ARON,1 RENATO V. IOZZO2, AND BEATRICE E. LECHNER1* 1

Departmentof Pediatrics, Women and Infants’ Hospital of Rhode Island, The Warren Alpert Medical School of Brown University, Providence, Rhode Island 2 Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania

SUMMARY Preterm prematureruptureof fetalmembranes(PPROM)isassociatedwithinfection,and is one of the most common causes of preterm birth. Abnormal expression of biglycan and decorin, two extracellular matrix proteoglycans, leads to preterm birth and aberrant fetal membrane morphology and signaling in the mouse. In humans and mice, decorin dysregulation is associated with inflammation in PPROM. We therefore investigated the link between biglycan and decorin and inflammation in fetal membranes using mouse models of intraperitoneal Escherichia coli injections superimposed on genetic biglycan and decorin deficiencies. We assessed outcomes in vivo as well as in vitro using quantitative PCR, Western blotting, and enzyme-linked immunosorbent assays. Our results suggest that biglycan and decorin compensate for each other in the fetal membranes, but lose the ability to do so under inflammation, leading to decreased latency to preterm birth. Furthermore, our findings suggest that biglycan and decorin play discrete roles in fetalmembranesignalingpathways during inflammation, leading tochanges in the abundance of MMP8 and collagen a1VI, two components of the fetal membrane extracellular matrix that influence the pathophysiology of PPROM. In summary, these findingsunderlinetheimportanceof biglycananddecorinastargetsforthemanipulationof fetal membrane extracellular matrix stability in the context of inflammation.

Mol. Reprod. Dev. 82: 387396, 2015. ß 2015 Wiley Periodicals, Inc. Received 5 January 2015; Accepted 1 April 2015

INTRODUCTION Preterm birth is the leading cause of newborn morbidity and mortality in the United States (Murphy et al., 2012). About a third of preterm births are caused by preterm premature rupture of fetal membranes (PPROM) (Steer, 2005). While the pathophysiology of PPROM is unclear and

ß 2015 WILEY PERIODICALS, INC.



Corresponding author: Department of Pediatrics Women and Infants’ Hospital of Rhode Island The Warren Alpert Medical School of Brown University 101 Dudley Street Providence, RI 02905. E-mail: [email protected]

Grant sponsor: National Institute of Child Health and Human Development; Grant number: 1K08HD054676; Grant sponsor: National Institute of General Medical Sciences of the National Institutes of Health; Grant number: P20GM12345; Grant sponsor: National Institutes of Health grants; Grant numbers: R01 CA39481, R01 CA47282

Published online 23 April 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22488

no therapies currently exist (Ananth and Vintzileos, 2006; Miyazaki et al., 2012), infection as well as genetic Abbreviations: Bgn, biglycan; Dcn, decorin; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PPROM, preterm premature rupture of fetal membranes; TGFb, transforming growth factor b; TIMP, tissue inhibitor of matrix metalloproteases

Molecular Reproduction & Development

susceptibility are thought to play a role in its onset (Parry and Strauss, 1998; Shen et al., 2008). Mice deficient in biglycan and decorin, two highly homologous extracellular matrix proteoglycans, deliver their pups prematurely and display abnormalities in fetal membrane morphology and signaling (Calmus et al., 2011; Wu et al., 2014). Biglycan and decorin, members of the small leucine-rich proteoglycan family (Iozzo 1999, 2011; Iozzo and Murdoch, 1996), are the most abundant proteoglycans expressed in human fetal membranes (Meinert et al., 2001; Gogiel et al., 2003; Valiyaveettil et al., 2004). Both proteoglycans are involved in a number of biological processes, including tumor angiogenesis and fibrosis (Neill et al., 2012a, 2012b, 2013); cancer growth (Iozzo and Cohen 1993; Reed et al., 2005; Sofeu Feugaing et al., 2013); stem cell biology (Bi et al., 2005; Berendsen et al., 2011; Ichii et al., 2012); myogenesis (Brandan and Gutierrez, 2013); and osteoarthritis and osteoporosis (Ameye et al., 2002; Ameye and Young 2002; Nikitovic et al., 2012). They are involved in collagen fibrillogenesis and contribute to the mechanical properties of connective tissues (Reed and Iozzo, 2002; Zhang et al., 2009; Chen et al., 2011) as the absence of these proteoglycans reduce connectivetissue tensile strength (Corsi et al., 2002): Humans with abnormal post-translational processing of biglycan and decorin display a subtype of EhlersDanlos syndrome, a pathology associated with a decrease in tensile strength of connective tissues as well as an increased risk of PPROM (Barabas 1966; Quentin et al., 1990). Indeed, we have also shown that decorin is dysregulated in human fetal membranes with PPROM (Horgan et al., 2014). Both biglycan and decorin have also been implicated in a number of signaling pathways, including the TGFb pathway. TGFb signals via Smads (Liu et al., 1996), transcription factors that play a role in the modulation of the extracellular matrix. Biglycan and decorin are both regulated by TGFb (Bassols and Massague, 1988), and decorin modulates TGFb activity via negative-feedback mechanisms (Yamaguchi et al., 1990). Smad2 and Smad3 regulate downstream gene expression of tissue inhibitors of matrix metalloproteinases (TIMPs) and collagens (Verrecchia et al., 2001), proteins that in turn modulate the mechanical stability of the fetal membrane extracellular matrix. Matrix metalloproteinases (MMPs) are linked to the pathogenesis of PPROM, both by their ability to degrade fetal membrane extracellular matrix proteoglycans and collagens (Ferrand et al., 2002) and genetically, as mutations in MMP (Fujimoto et al., 2002) as well as collagen genes (Anum et al., 2009) can be predisposing factors. The link is further supported by the observed interaction between proteoglycans and MMPs. For example, decorin induces the expression of MMP1 (Huttenlocher et al., 1996), and both biglycan and decorin modulate the TGF-b-SMADMMP-TIMP-collagen pathway in fetal membranes in a gestational-age-dependent manner (Wu et al., 2014). Substantial clinical data suggest that infection is a significant risk factor for PPROM (Torricelli et al., 2013). When exposed to an infectious insult, term human fetal membranes display increased MMP expression (Estrada-

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Gutierrez et al., 2010). A link also exists between infection and decorin expression in human fetal membranes with PPROM (Horgan et al., 2014). Coupled with studies indicating that biglycan and decorin both play a role in inflammatory cascades (Babelova et al., 2009; Merline et al., 2011), these observations suggest that infection may significantly impact the biglycan and decorin-dependent signaling that modulate fetal membrane mechanical properties. While we know that mutations leading to abnormal processing of biglycan and decorin (Barabas 1966; Quentin et al., 1990) as well as infection (Torricelli et al., 2013) play a role in the pathogenesis of PPROM, the mechanism by which infection modulates the function of biglycan and decorin and their signaling activity in the fetal membrane is not known.

RESULTS Latency to Preterm Birth as Well as Live Birth is Decreased in the Absence of Biglycan and Decorin During Inflammation In order to assess the role that biglycan and decorin play in the maintenance of gestation to term under inflammation, we first examined the natural history of mouse gestation after the intraperitoneal injection of Escherichia coli into pregnant mice at embryonic day 15 (E15). For initial comparison with the wild-type mice, we chose genotypes in which one of the four possible small leucine-rich proteoglycan genes (two biglycan and two decorin) were functional since these mice are more affected than the biglycan (Bgn) or decorin (Dcn) single-null mutant, but are less severely affected than the double nulls (Bgn/;Dcn/). More specifically, Bgn/; Dcn/ mice delivered their pups extremely prematurely evenwithouttheadditionalenvironmentalinsult ofE.coli, and were thus not suitable for this experimental design. We found that the period between injection of live E. coli and preterm birth is significantly decreased in Bgn+/;Dcn/ and Bgn/;Dcn+/ females compared to E. coli-injected wild-type mice as well as compared to the phosphatebuffered saline (PBS)-injected Bgn+/;Dcn/ and Bgn/; Dcn+/ mice (Fig 1A) (P¼0.001). Whereas the incidence of live-pup birth was significantly decreased in the E. coliinjected compared to the PBS-injected wild-type dams, no live pups were delivered from the E. coli-injected Bgn+/; Dcn/ and Bgn/;Dcn+/ mice (Fig 1B) (P ¼ 0.004). While there was no statistically significant difference in the latency between the E. coli- and the PBS-exposed wildtype mice, the standard deviation was larger in the E. coli-exposed cohort, demonstrating a greater range of latencies  from decreased latency to increased latency with dystociacompared to the PBS-injected control group.

Biglycan and Decorin Compensate for Each Other in the Fetal Membrane in the Absence of Inflammation We next investigated if biglycan and decorin could mediate compensatory changes in gene expression in fetal

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Figure 1. A: Days to delivery are decreased in Bgn+/;Dcn-/- and Bgn-/-;Dcn+/ dams injected with E. coli compared to both the same genotype injected with PBS and the wild-type, regardless of injection category (P ¼ 0.001 via ANOVA). Error bars represent the standard deviation. B: The percentage of live pups born from wild-type dams injected with E. coli compared to PBS decreased to zero in the Bgn+/;Dcn-/- and Bgn-/-;Dcn+/ dams injected with E. coli (P ¼ 0.004 via x2). Gestational age of injections was embryonic day 15 (n ¼ 38 per condition).

membranes. In the absence of inflammation (PBS-injected mice), we observed an up-regulation of biglycan transcript in decorin-null fetal membranes (P ¼ 0.05) as well as a complementary up-regulation of decorin transcript in biglycan-null fetal membranes (P ¼ 0.025) (Fig 2). Interestingly, the presence of inflammation modified these compensatory expression mechanisms between biglycan and decorin in the fetal membranes. Following E. coli injection, biglycan transcription remained unchanged in the decorin-null compared to the wild-type fetal membranes, thus departing from the compensatory increase observed in the absence of inflammation. Decorin transcription also displayed no compensatory upregulation in biglycan-null dams under inflammation. Furthermore, decorin transcription decreased in E. coli-injected mice

compared to the PBS-injected cohort in the absence of biglycan (P ¼ 0.01) (Fig 2A).

MMP8 decreases in Wild-Type and Biglycan-Null Fetal Membranes Under Inflammation, But Remains Unchanged in Decorin-Null Dams To understand how MMPs contribute to fetal membrane rupture and PPROM, we assessed their expression within our mouse model. We found that inflammation leads to a decrease in MMP8 abundance in both wild-type (P ¼ 0.03) and the biglycan-null (P ¼ 0.05), but not in decorin-null mice, following E. coli or PBS injection (Fig 3). MMP9 expression, on the other hand, was unchanged in all groups (data not shown).

Figure 2. Biglycan and decorin compensate for each other in fetal membranes in the absence but not in the presence of inflammation. A: Biglycan

transcript abundance is increased in the PBS-injected, decorin-null fetal membranes compared to wild-type (P ¼ 0.02); this compensatory increase does not occur during inflammation. B: Decorin transcription is increased in the PBS-injected, biglycan-null fetal membranes compared to wild-type fetal membranes (P ¼ 0.006); this compensatory increase does not occur during inflammation, but decorin transcript levels decrease in the E. coliinjected, biglycan-null fetal membranes compared to the PBS-injected, biglycan-null fetal membranes (P ¼ 0.009). Four dams were assessed per genotype and condition. Error bars represent standard deviation. Student’s t-test was performed for each set of data. BgnKO, biglycan null; DcnKO, decorin null; WT, wild-type.

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Figure 3. MMP8 expression is decreased in fetal membranes of E. coli-injected mice in wild-type as well as the biglycan-null, but not in the decorin-null mice. Four dams per genotype and condition were evaluated (P ¼ 0.03 and 0.028). Error bars represent standard deviation. Student’s t-test was performed for each set of data. BgnKO, biglycan null; DcnKO, decorin null; WT, wild-type.

Collagen a1VI Transcription is Unchanged Under Inflammation in the Absence of Biglycan or Decorin, Whereas its Protein Level Increased Given the role that collagens play in the maintenance of connective tissue mechanical strength and integrity, we examined the biglycan- and decorin-dependent expression of collagen a1VI, a biglycan and decorin binding protein, in inflammation. In the absence or presence of both biglycan or decorin, there was no change in collagen a1VI transcript levels (Fig 4A), although protein abundance was increased under E. coli-mediated inflammatory conditions compared to PBS injection for both biglycan (P ¼ 0.05) or decorin (P ¼ 0.03) (Fig 4BC).

DISCUSSION Inflammation leads to preterm birth in the mouse (Reznikov et al., 1999; Hirsch and Wang 2005) and is associated with PPROM in humans (Bendon et al., 1999). Biglycan and decorin are necessary for the maintenance of gestation to full term in a gene-dose-dependent and -compensation manner (Calmus et al., 2011). Further, biglycan/decorin-null fetal membranes posses abnormal morphology (Wu et al., 2014). Consistent with these previous observations, our study demonstrated that the superimposition of an environmental insultin this case inflammationon a genetic defect worsens the pretermbirth phenotype compared the same insult in animals without a genetic deficiency. Furthermore, our study provided important mechanistic insight into the role of fetal membrane biglycan and decorin in the pathophysiology of PPROM in the context of bacteria-induced inflammation. Specifically, inflammation exacerbates the gestational phenotype of the biglycan/decorin-null mouse, leading to abnormalities of transcriptional compensation between biglycan and decorin as well as translational and transcriptional changes in downstream targets that play a role in fetal membrane stability. Our data indicate that biglycan and decorin regulate each other in the unchallenged fetal membrane by

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Figure 4. Collagen a1VI protein expression is increased in the biglycan- and decorin-null, but not in wild-type fetal membranes, during inflammation. A: Collagen a1VI transcript abundance in biglycan- and decorin-null mice with or without inflammation, compared to the wildtype. B: Representative Western blots and C: summary data demonstrating increased collagen a1VI protein abundance in biglycan- and decorin-null, but not in the wild-type fetal membranes, during inflammation (P ¼ 0.05 and 0.03). Four mice were assessed per genotype and condition. Error bars represent standard deviation. Student’s t-test was performed for each set of data. BgnKO, biglycan null; DcnKO, decorin null; WT, wild-type.

compensatory increases in transcription when one gene is non-functional. Such compensatory upregulation between biglycan and decorin reflects their relationship in other tissues, including the cornea, kidney, skin, bone, muscle (Ameye and Young 2002; Corsi et al., 2002; Zhang et al., 2009), and the placenta during gestation (Calmus et al., 2011). In contrast to the transcriptional changes, we did not observe a rebalancing of biglycan protein expression in

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decorin-null fetal membranes or vice versa (Calmus et al., 2011). This may be due to an absence of protein compensation in the fetal membranes or to changes that are not readily apparent according to the techniques we utilized, given the abundance of both small leucine-rich proteoglycans in fetal membranes. Inflammation, however, abolishes the transcriptional compensation observed between these two proteoglycans. Furthermore, when inflammation is present in a wild-type genetic background, biglycan, or decorin expression remains unchanged. Therefore, the etiology for the decreased latency to preterm birth in the Bgn+/;Dcn/ and Bgn/;Dcn+/ mice could follow a two-hit model in which genetic predisposition is aggravated by an environmental factor. Specifically, genetic predispositions can be compensated for, but this compensation is tempered by an environmental challenge. Such a model mirrors clinical observations in patients with EhlersDanlos syndrome, which significantly elevates the incidence of preterm birth from PPROM compared to their unaffected siblings (Barabas 1966; Yen et al., 2006). The progeroid variant of Ehlers-Danlos syndrome, for example, carries a mutation that results in the secretion of abnormal, non-glycosylated biglycan and decorin (Quentin et al., 1990). The link between connective-tissue genetic abnormalities and inflammation-sensitive PPROM is further bolstered by reports that women with a history of multiple PPROM have undiagnosed connective-tissue anomalies similar to EhlersDanlos syndrome (Hermanns-Le et al., 2005). One caveat to these observations is that the mice used for the in vivo E. coli-injection experiments are heterozygous/ homozygous and homozygous/heterozygous biglycan/decorin nulls (Bgn+/;Dcn/ and Bgn/;Dcn+/). Based on prior data (Calmus et al., 2011), it is likely that our observations reflect a similar but tempered response compared to the homozygous double nulls (Bgn/;Dcn/), which have too severe a phenotype to lend themselves to these types of experiments. On the other hand, these observations are likely an exaggerated response compared to the single nulls (Bgn/ or Dcn/), on which we have performed the in vitro component of this study. When both biglycan and decorin are present in fetal membranes, inflammation leads to a decrease in MMP8 proteinwhich may be a protective mechanism to counteract destruction/remodeling effects on the membrane. Mmp8 could be transcribed at typical rates, but inflammation could increase the rate of protein or transcript turnover as MMP8 degrades the fetal membrane. In the absence of biglycan, MMP8 protein abundance decreases in a manner similar to the wild-type genotype; on the other hand, MMP8 protein levels are unchanged in decorin-null mice. Since the expected decrease in MMP8 under inflammation does not occur in the absence of decorin, but does occur in the absence of biglycan, decorin likely contributes to MMP8 regulation during inflammation. Indeed, decorin gain-offunction experiments confirm that decorin regulates components of this metalloproteinase pathway since Mmp8 and Mmp9 gene and protein expression as well as Mmp13 transcription increases in fetal membranes close to term

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(embryonic day 18) in the absence of decorin, but not in the absence of biglycan (Wu et al., 2014). Furthermore, decorin suppresses the abundance and activity of MMP2 and MMP9, which are associated with PPROM (Neill et al., 2012a), yet it also induces the synthesis of a number of MMPs, including MMP1, MMP2 and MMP14 (Schonherr et al., 2001). During early gestation (embryonic day 12), the absence of decorin leads to a decrease in MMP8 and 9 protein expression in fetal membranes. Decorin levels also decrease prior to parturition as membrane tensile strength decreases, whereas biglycan increases (Meinert et al., 2007). Together, these observations suggest that decorin is a fetal membrane-regulating factor that contributes to extracellular matrix remodeling in early gestation, but transitions to a membrane-stabilizing role in late gestation and during inflammation. Our data indicate that biglycan and decorin are both necessary for the maintenance of appropriate collagen a1VI levels during inflammation. Transcription of the collagen a1VI gene is unchanged during inflammation, independent of the presence or absence of biglycan or decorin. Collagen a1VI protein, however, accumulates in the absence of biglycan or decorin during inflammation. One possible explanation for this increase could be to structurally compensate for the absent proteoglycans. Within the context of the two-hit model a genetic defect followed by inflammation leading to diseasethis increase in collagen a1VI expression may be a protective strategy that stabilizes the fetal membrane. Such a feedback system is consistent with the observation that decreased decorin in the kidney leads to increased accumulation of extracellular matrix with increased biglycan expression (Merline et al., 2009) while the absence of decorin leads to hepatic fibrosis (Baghy et al., 2011). An alternative interpretation could be that an increase in collagen a1VI in the absence of decorin and/or biglycan reflects the anti-fibrotic role of these proteoglycans. The reciprocal MMP8 and collagen a1VI profiles in the absence of biglycan or decorin suggest that these two proteoglycans signal via discrete pathways. If they signaled via one common pathway, as their ability to compensate for one another suggests, one would expect no change in MMP8 and collagen a1VI levels between wild-type and single-null mice. Instead, it is likely that compensation is not complete, perhaps because each proteoglycan possesses distinct functions as well. Indeed, biglycan and decorin have separate roles in TGFb signaling in the mouse fetal membrane, leading to the differential expression of MMP8, MMP9, and MMP13; TIMP1, TIMP2, TIMP3, and TIMP4; and collagen a1VI (Wu et al., 2014). Such differences in expression and function may also be related to the midpregnancy gestational age studied here, since biglycan and decorin signaling activities change in a gestational-agedependent manner (Wu et al., 2014). The role of biglycan and decorin in the maintenance of fetal membrane integrity during inflammation likely reflects their complex interaction as connective tissue-stabilizing proteins and as proinflammatory signaling molecules. For example, biglycan also functions as an inflammatory-

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cascade activator via its role as a ligand for Toll-like receptors 2 and 4 (Babelova et al., 2009; Moreth et al., 2014; Zeng-Brouwers et al., 2014). Septic inflammation can evoke decorin expression, while excess decorin feeds back and boosts the inflammatory response during both sepsis and cancer (Merline et al., 2011). Considering these distinct functions during inflammation, our findings that biglycan and decorin normally compensate for each other in the fetal membranes until inflammation is present suggest that biglycan and decorin play discrete roles in fetalmembrane signaling that ultimately lead to changes in MMP8 and collagen a1VI expression; modifications in the abundance of these two fetal membrane extracellular matrix components can ultimately accelerate the pathophysiology of PPROM

MATERIALS AND METHODS Mouse Husbandry and Genotyping Wild-type mice (C3H) were purchased from Jackson Laboratories (Bar Harbor, ME). A Bgn/ breeding pair (C3H) (generated by Marian Young (Xu et al., 1998)) was a gift from Justin Fallon. A Dcn+/ breeding pair (C57BL/6) was used to establish the Dcn/ colony. A Bgn/ female was crossed with a Dcn/ male to establish double-heterozygous breeding pairs (Bgn+/;Dcn+/ and Bgn/0;Dcn+/). These pairs were mated to obtain Bgn+/; Dcn/ and Bgn/;Dcn+/ heterozygous/homozygousnull pups. Breeding pairs were set up for mating at 57 weeks of age. Plugs were checked each morning, and the day the plug was found was defined as embryonic day 0. Dams selected for embryonic-day-15 tissue harvesting were sacrificed between 4 and 5 hr after E. coli or PBS injection. Protocol approval was obtained from the Lifespan Institutional Animal Care and Use Committee. Genomic DNA was extracted from each 3-mm tail biopsy using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) for genotyping. Polymerase chain reaction (PCR) was performed using the Taq DNA Polymerase kit (New England Biolabs, Ipswich, MA) and a PTC200 thermal cycler. The PCR product was run on a 1.8% (w/ v) agarose gel to visualize the following diagnostic bands. The decorin PCR produced bands of 161 bp for the wildtype allele and 238 bp for the null allele. The biglycan PCR produced bands of 212 bp for the wild-type allele and 310 bp for the null allele.

Bacteria Preparation Previously frozen E. coli was inoculated on an agar plate overnight. The next morning, one colony from the plate was incubated overnight in 10 ml of Luria-Bertani (LB) broth (MP Biomedicals, Santa Ana, CA). This seed culture was diluted 1:50 in fresh LB broth, and incubated until an optical density of 0.50.8 was detected at 600 nm, indicating the bacteria was in log phase growth. A more accurate bacterial concentration was determined post-hoc by plating a 106-serial dilution series in triplicate. Colony counts were performed

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on these plates the next morning. Colony forming units ranged from 3  106 to 1.4  107 units per ml.

Injection Preparation Three hundred microliters of a log-phase bacterial culture were concentrated by centrifugation with the addition of 700 ml fresh LB broth. The resulting pellet was washed with 1 ml of sterile, room temperature PBS, and was concentrated again via centrifugation. The resultant pellet was re-suspended in 0.5 ml of sterile, room temperature PBS. The bacterial suspensions were drawn into 1-ml tuberculin syringes and injected intraperitoneally into pregnant, embryonic-day-15 mice of the following genotypes: Bgn/; Dcn+/, Bgn+/;Dcn/, and wild-type (Bgn+/+;Dcn+/+). Vehicle controls of 0.5 ml sterile, room temperature PBS were also used. In order to assess preterm delivery rates, mice were observed twice daily while health status and preterm deliveries were recorded. Health status was determined using an adaptation of the Mouse Sepsis Index scale (Biswas et al., 2002). The scale ranges from five for normal, healthy clinical status to zero for death. Four is defined as slightly ill with ruffled fur and lethargy; three is moderate illness with severe lethargy, ruffled fur and hunched back; two is severe illness with all signs of three plus exudative accumulation around the eyes; and one is a moribund state. Mice of both single-null genotypes injected with E. coli did not differ significantly from wild-type E. coli-injected mice with respect to their illness index. Single-nulls of both genotypes injected with E. coli had a mean score of 3.75  0.58 and wild-type mice injected with E. coli a mean score of 3.87  0.73 (standard deviations) (P ¼ 0.7). Mice that were designated for tissue harvest were sacrificed 45 hr after E. coli or PBS injection.

Antibodies The following antibodies were used: rabbit polyclonal antiCOLa1VI (sc-20649, dilution 1:800) and mouse monoclonal anti-GAPDH (sc-137179, dilution 1:1000) (Santa Cruz Biotech, Santa Cruz, CA). Secondary antibody labeling was performed with anti-mouse and anti-rabbit IgG horseradish peroxidase conjugate (Cell Signaling, Danvers, MA).

Western Blotting Fetal membranes from each genotype and condition were dissected at embryonic day 15, between 4 and 5 hr after E. coli or PBS injection, and frozen at 808C. Tissue samples were cut into small pieces and placed in 1.0 ml of T-PER tissue protein extraction buffer (Pierce, Rockford, IL) with one tablet of proteinase inhibitor cocktail per 10 ml of buffer (Roche, Basel, Switzerland). Tissue samples were then homogenized for 310 sec in an ice bath, and kept on ice for 30 min. The homogenate was centrifuged at 10,000g at 48C for 8 min, and the supernatant was collected and stored at 808C until needed. Total protein content was determined using the BCA assay (Pierce, Rockford, IL). Thirty micrograms of total

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protein were loaded on duplicate 10% SDSpolyacrylamide gels, and subsequently blotted to a polyvinylidene difluoride membrane. Following blocking in 5% milk in PBST buffer (PBS with 0.2% Tween 20) for 30 min, each blot was incubated overnight at 48C with primary antibody. The blot was then briefly washed with PBST, and then incubated with secondary antibody for 40 min. The Super-Signal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL) was used to develop the membrane. Immunoreactive bands were compared with protein markers of known molecular size run in parallel on the same SDSpolyacrylamide gel (Bio-Rad, Hercules, CA). The membrane was then stripped using Restore Western Blot stripping buffer (Thermo Scientific, Waltham, MA) for 10 min, blocked with 5% milk PBST for 20 min, then re-probed with anti-GAPDH antibody (Santa Cruz Biotech, Santa Cruz, CA). Immunoblots were digitally scanned and densitometrically analyzed using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD). The relative optical density (specific protein band to GAPDH, as a loading control) was normalized to the wild-type-mice-with-PBSinjection band. The experiment was repeated with three individual sets of samples.

Enzyme-Linked Immunosorbent Assay (ELISA)

Wild-type, Bgn/, and Dcn/ dams injected with PBS or E. coli were dissected at embryonic day 15 between 4 and 5 hr after injection. Fetal membranes were collected, flash frozen in liquid nitrogen and isopentane, and stored at 808C. Four whole fetal membranes from each condition were used. Each fetal membrane sample was homogenized in 1 ml of protein lysate buffer (2.5 ml Triton X-100 and one tablet of Roche Complete EDTA-free Protease Inhibitor per 50 ml volume), flash frozen in liquid nitrogen, and stored at 808C until needed. Commercially available ELISA kits (MMP8 from Anaspec, Fremont, CA; MMP9 from Abnova, Taipei City, Taiwan) were run in triplicate according to the

manufacturer’s instructions. Plates were read at 450 nm on a Bio-Rad Laboratories Microplate Reader.

RNA, cDNA Preparation, and Quantitative Reverse-Transcriptase PCR

Fetal membranes from wild-type, Bgn/, and Dcn/ dams injected with PBS or E. coli were dissected at embryonic day 15 between 4 and 5 hr after injection in 0.1 mol l1 PBS [pH 7.4], snap-frozen in liquid nitrogen, and stored at 808C. RNA extraction, genomic DNA removal, and conversion to cDNA was performed as described previously (Wu et al., 2014). Quantitative PCR (qPCR) reactions were performed on the 7500 Fast Real-Time PCR System thermocycler (Applied Biosystems, Foster City, CA) using the SYBR-Green method (Invitrogen, Carlsbad, CA). Primers were designed using Primer-Blast software (National Library of Medicine, Bethesda, MD); Gapdh was used as a normalizer (see Table 1). All primers were synthesized by Invitrogen (Carlsbad, CA). Melting-curve analysis of the product was performed to ensure the absence of alternative products or primer dimers. Data analysis was performed via the comparative Ct method with a validation experiment. A standard sample of RNA pooled from samples of wild-type as well as biglycan- and decorin-null fetal membranes were used in each qPCR experiment as a calibrator whose relative transcript level was defined as one. qPCRanalysis wasperformed in triplicate, using 34 dams per genotype and condition.

Statistical Analysis Analysis of differences in gene and protein expression between genotypes was performed using the Student’s ttest or, in the case of non-normally distributed data, the nonparametric MannWhitney Rank Sum test. ANOVA and x2 analysis were performed for the in vivo experiments. Where appropriate, Bonferroni correction was

TABLE 1. Primer List qPCR collagen a1VI

forward: 5’-CTGGTGAAGGAGAACTATGCAG reverse: 5’-GTCTAGCAGGATGGTGATGTC

biglycan (Bgn)

forward: 5’-ATTGCCCTACCCAGAACTTGAC reverse: 5’-GCAGAGTATGAACCCTTTCCTG

decorin (Dcn)

forward: 5’-TTCCTACTCGGCTGTGAGTC, reverse: 5’-AAGTTGAATGGCAGAACGC

Gapdh

forward: 5’-CTCACAATTTCCATCCCAGAC reverse: 5’-TTTTTGGGTGCAGCGAAC

Genotyping biglycan (Bgn), wild-type

forward: 5’-tgatgaggaggcttcaggtt reverse: 5’-gcagtgtggtgtcaggtgag;

biglycan (Bgn), null

forward: 5’-tgtggctactcaccttgctg reverse: 5’-gccagaggccacttgtgtag;

decorin (Dcn), shared decorin (Dcn), wild-type decorin (Dcn), null

forward: 5’-ccttctggcacaagtctcttgg, reverse: 5’-tcgaagatgacactggcatcgg reverse: 5’-tggatgtggaatgtgtgcgag

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performed for multiple comparisons using the Student’s t-test. Analysis was performed using SigmaPlot 11.0 (San Jose, CA).

ACKNOWLEDGMENTS This work was supported by National Institute of Child Health and Human Development grant 1K08HD054676 (to BEL), an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM12345 (to BEL) and National Institutes of Health grants R01 CA39481 and R01 CA47282 (to RVI).

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Compensatory fetal membrane mechanisms between biglycan and decorin in inflammation.

Preterm premature rupture of fetal membranes (PPROM) is associated with infection, and is one of the most common causes of preterm birth. Abnormal exp...
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