Biomedical subjects

Martin Knöfler

Publications and source records attributed to Martin Knöfler.

16 recordsLinked to original sources

PubMed2006

Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast.

The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-beta-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated beta-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with beta-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and up-regulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating in-hibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear beta-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the path-way could contribute to trophoblastic hyperplasia and local invasion.

Cell Differentiation↗

PubMed2004

Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts.

Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migration-related genes. In this 'invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca(2+) levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identified Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Calcium↗

PubMed2004

Transcriptional regulation of the human chorionic gonadotropin beta gene during villous trophoblast differentiation.

Expression of the trophoblast-specific subunit of human chorionic gonadotropin, CGbeta, is associated with fusion of cytotrophoblasts into a multinuclear syncytium. Here, we studied regulation of the CGbeta5 gene in trophoblasts undergoing in vitro syncytialization. Transfection of luciferase reporters harboring different lengths of the CGbeta5 upstream sequence revealed that the proximal promoter region (-345 to +114) is sufficient to govern differentiation-dependent induction. Mutational analyses suggested that two selective promoter factor (Sp) and three activating protein 2 (AP-2) recognition sequences are necessary for full activity of the promoter. During syncytialization these elements interacted with increasing amounts of the transcription factors Sp1, Sp3, and AP-2alpha in electrophoretic mobility shift assay, but only AP-2alpha binding rose upon elevation of cAMP levels with forskolin. Increasing expression of different isoforms of Sp1 and Sp3 could also be detected by Western blot analyses. Sp1/Sp3 localized to syncytial nuclei both in differentiated cultures and in term placental tissue, suggesting assembly of functional transcriptional complexes. Costaining of the transcription factors with E-cadherin on term placental sections revealed that 47 and 33% of cytotrophoblast nuclei were negative for Sp1 and Sp3, respectively. In contrast, immunohistochemistry of early tissue demonstrated expression of Sp1 in the majority of cyto- and syncytiotrophoblasts, whereas Sp3 was absent from the syncytium. Sp1 and Sp3 induced wild-type/mutant promoter constructs upon transfection in Sp-deficient SL-2 cells, indicating that the Sp elements function as activating sequences. The data suggest that increasing concentrations of Sp1, Sp3, and AP-2alpha enhance transcription of CGbeta in differentiating term trophoblasts, whereas a different combination of factors may control expression in early placentas.

Base Sequence↗

PubMed2004

Decreased plasma adiponectin concentrations in women with gestational diabetes mellitus.

OBJECTIVE: Adiponectin is an adipocyte-specific protein that has been found to be associated with insulin sensitivity and obesity. Because gestational diabetes mellitus is associated with obesity and decreased insulin sensitivity, we have analyzed plasma adiponectin levels in women with gestational diabetes mellitus. STUDY DESIGN: Twenty women with gestational diabetes mellitus and 21 unaffected women were included in the study. Plasma adiponectin levels were analyzed with the use of enzyme-linked immunosorbent assay. RESULTS: Women with gestational diabetes mellitus were significantly older (34.3 years vs 29.4 years; P < .001) than unaffected women. Adiponectin plasma levels were significantly lower in women with gestational diabetes mellitus when compared with women without gestational diabetes mellitus (5827 +/- 1988 ng/mL vs 8085 +/- 3816 ng/mL; P = .02). Adiponectin plasma levels were correlated negatively with plasma glucose concentrations of the oral glucose tolerance test ( r > -0.38; P < .04) and correlated positively with gestational age ( r = 0.36; P = .03). CONCLUSION: Our data show that decreased plasma adiponectin levels were found in women with gestational diabetes mellitus compared with unaffected women.

Adiponectin↗

PubMed2004

Calpain-10 haplotype combination and association with gestational diabetes mellitus.

OBJECTIVE: Gestational diabetes mellitus (GDM) is a frequent complication of pregnancy. Epidemiologic and pathophysiologic data suggest a close link of this disease to non-insulin-dependent diabetes mellitus. Within the calpain-10 gene various single-nucleotide polymorphisms have been identified that increased the risk for non-insulin-dependent diabetes mellitus. Therefore, we examined single-nucleotide exchanges of this gene in women with GDM. METHODS: A total of 875 unselected women were prospectively screened for GDM. Eighty women of this population, 40 patients with an abnormal oral glucose tolerance test and 40 normal controls, were randomly selected. DNA samples isolated from sera of the control and study groups were analyzed with respect to single-nucleotide polymorphisms of the calpain-10 gene at positions 43, 19, and 63 using polymerase chain reaction amplification and restriction analysis. RESULTS: Women with GDM were more likely to be homozygous for the allele 1 of single-nucleotide polymorphism 63 (P =.02 by chi(2) test). With respect to single-nucleotide polymorphisms 19 and 43, no significant differences in allele distribution were detected between controls and women with GDM. When comparing the different haplotypes for calpain-10 (single-nucleotide polymorphisms 43, 19, and 63), all women with the haplotype combination 121/221 (n = 8) had gestational diabetes (P =.005 by Fisher exact test). CONCLUSION: Our results indicate that the haplotype 121/221 of the calpain-10 gene may be associated with disturbances of glucose metabolism during pregnancy. LEVEL OF EVIDENCE: II-1

Adult↗

PubMed2004

Tumor necrosis factor-alpha inhibits trophoblast migration through elevation of plasminogen activator inhibitor-1 in first-trimester villous explant cultures.

We have tested the hypothesis that elevated concentrations of TNF alpha could impair trophoblast invasion. Using first-trimester placental explant cultures, we have demonstrated that the cytokine inhibits in vitro migration of extravillous trophoblasts (EVT) on collagen I, and invasion through Matrigel. To elucidate the underlying mechanism, proliferation and differentiation of EVT in vitro were analyzed by immunohistochemistry of serial sections, Western blotting, zymography, ELISA, and RT-PCR from RNA pools of mechanically separated cell populations. At 24 h of cultivation in the presence or absence of TNF alpha, anchorage and proliferation of trophoblasts had occurred to generate cell columns containing viable, post-mitotic, differentiated EVT [positive for integrins alpha 1 and alpha 5, matrix metalloproteinase (MMP)-2, and human leukocyte antigen-G1; negative for proliferating cellular nuclear antigen, cytokeratin 18 neoepitope, and in 5-Bromo-2-deoxy-uridine labeling]. At 72 h, control cells had broken away from the column to migrate through the extracellular matrix; whereas, in contrast, TNF alpha-treated EVT remained as contiguous cell columns, despite increased MMP-9 expression. Thus, in vitro MMP9 activity appears not to be essential for trophoblast migration. Expression of plasminogen activator inhibitor (PAI)-1 was elevated in TNF alpha-treated EVT, and adding antibodies that inhibit PAI-1 activity restored migration, whereas tissue-inhibitor-of-metalloproteinases-1-blocking antibodies were ineffective. Induction of PAI-1 by TNF alpha could be related to restricted trophoblast invasion in preeclampsia.

Cell Cycle↗

PubMed2003

Detection of corticotropin-releasing hormone receptors R1 and R2 (CRH-R1, CRH-R2) using fluorescence immunohistochemistry in the myometrium of women delivering preterm or at term.

OBJECTIVE: To determine differences in expression of corticotropin-releasing hormone receptors R1 and R2 in the myometrium of women delivering preterm or at term, with or without labor. MATERIAL AND METHODS: Small pieces of myometrial smooth muscle were taken from forty patients undergoing caesarian section. One sample each was taken from all preterm and term patients, with and without labor. Antibodies against CRH-R1/2 and CRH-R2 were used for localization by conventional fluorescence immunohistochemistry. The evaluation of staining was based on examination of the entire histologic section by three independent observers. RESULTS: In women at term, CRH-R2 levels were elevated before labor and decreased with the onset of labor. In eight of 10 term samples from women in labor, no staining was detected. In preterm patients we found no difference in CRH-R2 staining between women with and without labor. The intensity of staining for CRH-R1/2 was generally uniform in all four groups. CONCLUSION: The high level of CRH-R2 expression in term patients without labor, together with the subsequent decrease of CRH-R2 in the myometrium during progression of labor, is consistent with the possibility that CRH may have autocrine/paracrine effects on myometrial contractility in the lower segment of the uterus.

Cesarean Section↗

PubMed2003

Elevated serum concentrations of androgens in women with pregnancy-induced hypertension.

Alterations of steroid hormone profiles have been suggested to be involved in the pathophysiology of pregnancy-induced hypertension (PIH). The aim of our study was first to investigate serum concentrations of testosterone, dihydrotestosterone, androstenedione and dehydroepiandrostenedione sulfate in women with PIH and normotensive pregnant women and secondly to evaluate an association between elevated serum concentrations of androgens and the development of severe disease. Serum concentrations of androgens were measured in 40 patients with PIH and 40 normotensive pregnant women, matched for gestational age, determined by enzyme linked immunosorbent assay. Multivariate logistic regression models were used to analyze the influence of elevated serum concentrations of androgens on the occurrence of PIH and the development of severe disease. The median serum concentrations of androstenedione and testosterone were significantly elevated in women with PIH compared to controls (6.3 and 5.0 ng/ml, 1.8 and 1.1 ng/ml, p = 0.005 and p = 0.04, respectively). The difference between the median serum concentrations of dihydrotestosterone and dehydroepiandrostenedione sulfate in women with PIH and controls was not significant. Elevated serum concentrations of androstenedione revealed a significant influence on the odds of presenting with PIH (p = 0.043) and were significantly associated with the development of severe disease (p = 0.014). Women with PIH have elevated serum concentrations of androstenedione and testosterone. Moreover, elevated serum concentrations of androstenedione are associated with development of severe disease.

Adult↗

PubMed2003

Expression of the human Hand1 gene in trophoblastic cells is transcriptionally regulated by activating and repressing specificity protein (Sp)-elements.

The tissue-specific basic helix-loop-helix protein Hand1 is essential for the formation of trophoblast giant cells of the murine placenta. In humans, Hand1 is detectable in trophoblastic tumour cells suggesting an equivalent role in trophoblast differentiation. To understand its mode of expression we have cloned and characterized the human Hand1 gene promoter. Primer extension analyses suggest that transcription initiates 19 nucleotides downstream of the TATA element of the proximal 5' flanking region. Expression of luciferase reporter constructs harboring deletions of the 9.5 kb Hand1 5' flanking sequence defines a promoter region within 274 bp upstream of the transcriptional start site. Compared to a reporter bearing only the TATA box, the proximal promoter activates transcription up to 30-fold. However, transcriptional activity of the region was observed in both Hand1-expressing and non-expressing cell lines. Sequencing, DNAseI footprint analyses and electrophoretic mobility shift assays reveal the presence of four GC-rich sequences, which show different affinities to the endogenous specificity proteins (Sp), and a CCAAT box. In vitro, the Sp-elements mainly interact with Sp1 and Sp3 while the CCAAT element is recognized by the alpha CAAT binding factor protein. Mutant luciferase reporters bearing single active or inactive recognition sites demonstrate that two of the four Sp-binding sites (I and IV) contribute little to the overall transcription rate. The two other Sp-cognate sequences, II and III, downregulate and activate reporter expression 2.3- and 2.6-fold, respectively. Co-transfections of Sp1/Sp3 expression vectors and mutated reporter constructs in Sp-deficient SL2 cells indicate that the Sp-binding site II and III indeed function as repressing and activating enhancer sequences. In summary, the data suggest that constitutive expression of the Hand1 gene in cultured cells is regulated by a complex interplay of Sp-proteins interacting with activator and repressor elements.

5' Flanking Region↗

PubMed2003

Elevated serum concentrations of the angiogenesis inhibitor endostatin in preeclamptic women.

OBJECTIVE: Changes in serum levels of angiogenic factors such as vascular endothelial growth factor or placental growth factor were associated with preeclampsia. The aim of our study was to investigate the concentrations of endostatin, a potent endogenous inhibitor of angiogenesis and endothelial or tumor cell growth, in preeclamptic patients. METHODS: Levels of soluble endostatin were determined by enzyme-linked immunosorbent assay and Western blot analysis in sera of nonpregnant women, healthy pregnant women, and patients with different forms of preeclampsia. RESULTS: Statistical analyses of age-matched study groups revealed elevated medians of endostatin concentration for severely (30.5 ng/mL) and mildly (26.05 ng/mL) preeclamptic patients compared with healthy pregnant (17.2 ng/mL) and nonpregnant women (17.2 ng/mL). Western blot analysis confirmed the up-regulation of soluble endostatin molecules (24 kD) in sera of severely preeclamptic patients. CONCLUSION: Elevated concentrations of endostatin could play a role in the pathophysiology of preeclampsia by counteracting the effects of vascular endothelial growth factor. The inhibitor could also be responsible for the observed growth-inhibitory effects of preeclamptic plasma on endothelial cells.

Adult↗

PubMed2002

Redistribution of corticotropin-releasing hormone receptor R2 using fluorescence immunohistochemistry in fetal membranes of women delivering preterm or at term.

OBJECTIVE: The aim of our study was to determine whether a difference exists in expression of corticotropin-releasing hormone receptor-R1 (CRH-R1) and corticotropin-releasing hormone receptor-R2 (CRH-R2) in fetal membranes of preterm and term women with or without labor. MATERIAL AND METHODS: Small pleces of fetal membranes were obtained from the placenta of each of forty patients undergoing cesarean section. Ten samples each were taken from preterm and term patients, with and without labor. Antibodies against CRH-R1/2 and CRH-R2 were used for localization by conventional fluorescence immunohistochemistry. The evaluation of staining was based on examination of the entire histologic section by three independent observers. RESULTS: In women at term without labor, CRH-R2 receptor was predominantly expressed in the amniotic epithelium and the amniotic mesenchyme. In laboring women at term, the expression of CRH-R2 receptor was shown in the chorionic mesenchyme and the cytotrophoblast cells, but no specific staining could be detected in the amniotic membranes. Changes in CRH-R2 receptor expression could not be demonstrated during preterm labor of early pregnancies. In preterm women, the antibody against CRH-R1/2 receptor detected additional signals in the amniotic mesenchyme and epithelium, suggesting expression of CRH-R1 in these tissues. In women at term, the overlapping pattern of CRH-R1/2 was recognized in both the chorionic and amniotic mesenchyme, in contrast to the specific CRH-R2 staining, suggesting expression of CRH-R1 in the mesodermal cell compartments. CONCLUSION: At term, changes in CRH-R2 expression are directly related to the progression of normal labor; such changes were not observed during preterm labor of early pregnancies. The increased CRH-R2 expression in the chorionic mesenchyme may possibly provoke rupture of the membranes or at least play a role in some key regulatory events in the initiation of normal labor. The fact that this mechanism does not occur in preterm labor strengthens the hypothesis that onset of labor could be controlled by distinct mechanisms in preterm and term pregnancies.

Amnion↗

PubMed2002

Serum levels of heat shock protein 70 in patients with preeclampsia: a pilot-study.

OBJECTIVE: To evaluate the serum levels of heat shock protein (Hsp) 70 in patients with severe preeclampsia (PE) in comparison to controls. The pathophysiology of PE can be explained, in part, by alterations of endothelial function caused by endothelial cell activation and injury. HSP 70 is essential for cellular recovery, survival and maintenance of homeostasis. STUDY DESIGN: In a matched pair study, serum levels of Hsp 70 were measured in 55 patients with late (group A, n = 24) and early (group B, n = 31) onset of severe PE, and in 55 normotensive controls (group C, n = 24 and group D, n = 31) matched for gestational age. Early onset of severe PE was defined as onset of disease at less than 34 weeks of gestation (34 + 0). Serum levels were determined using a sandwich enzyme-linked immunosorbent assay. RESULTS: The overall median serum levels of Hsp 70 were 2.82 ng/mL (SD +/- 8.33) in preeclamptic women, and 1.01 (SD +/- 1.38) ng/mL in controls (P = 0.08). The median serum levels of Hsp 70 were 0.52 ng/mL (SD +/- 1.14) in group A and 0.86 (SD +/- 1.29) ng/mL in group C (P = 0.15). The median serum levels of Hsp 70 were 4.94 ng/mL (SD +/- 10.46) in group B and 1.33 (SD +/- 2.28) ng/mL (P = 0.04) in group D. The difference between group A and B was also statistically significant (P = 0.01). CONCLUSION: Our study revealed higher serum levels of Hsp 70 in patients with early onset of severe PE. Further studies are recommended in order to elucidate the possible role of Hsp 70 in the pathophysiology of PE.

Adult↗

PubMed2002

Concentrations of estrogens in patients with preeclampsia.

The role of estrogens in the pathophysiology of preeclampsia remains to be determined. The aim of our study was to compare serum concentrations of 17 beta-estradiol and estriol in women with preeclampsia to normotensive pregnant controls. Serum concentrations of estrogens were measured in women with mild (n = 24) and severe (n = 24) preeclampsia as well as is normotensive pregnant controls (n = 24). Patients were matched for gestational age. Pregnancies complicated by early onset severe preeclampsia are associated with increased rates of maternal and fetal morbidity. Subsequently, we created further subgroups before and after 34 weeks of gestation (34 + 0). Serum estrogen concentrations were determined by standard ELISA technique. Compared to normotensive controls, the differences between the overall median serum concentrations of 17 beta-estradiol in women with mild (3811 v. 3730 pg/ml, P = 0.9) and severe (3811 v. 3630 pg/ml, P = 0.1) preeclampsia were statistically not significant. The differences between the overall median serum concentrations of estroil in controls and in patients with mild (121 v. 76 ng/ml, P = 0.6) and severe (121 v. 79 ng/ml, P = 0.4) preeclampsia were similar. The differences between the median concentrations of 17 beta-estradiol in patient with early onset severe preeclampsia compared to patients with mild preeclampsia (3061 v. 3715 pg/ml, P = 0.004) and controls (3061 v. 3807 pg/ml, P = 0.006) were statistically significant. In addition, the differences between the median concentrations of estriol in women with early onset severe preeclampsia compared to controls were statistically significant (20 v. 92 ng/ml, P = 0.02). The differences between the median concentrations of estrogens in those with late onset severe preeclampsia compared to women with mild preeclampsia were not significant. We found significantly lower concentrations of estrogens in women with early onset severe preeclampsia.

Adult↗

PubMed2002

Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains.

The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.

Basic Helix-Loop-Helix Proteins↗

PubMed2002

Basic fibroblast growth factor and hypertensive disorders in pregnancy.

OBJECTIVE: We examined serum concentrations of basic fibroblast growth factor (bFGF) in women with hypertensive disorders in pregnancy and analyzed whether serum concentrations of bFGF can be used as a discriminator between mild and severe preeclampsia. METHODS: One hundred and twenty pregnant women were included in this prospective cohort study. We evaluated serum concentrations of bFGF in pregnant women with chronic hypertension (n=22), mild preeclampsia (n=40), severe preeclampsia (n=31), and healthy pregnant women (n=27). RESULTS: Median serum concentrations of bFGF in healthy pregnant women, women with chronic hypertension, and women with mild or severe preeclampsia were 0.0 (0-37.2), 0.0 (0-3.0), 1.7 (0-97.2), and 0.0 (0-52.0), respectively. Comparison of the median values of serum bFGF concentrations showed a significant difference between healthy pregnant women and women with mild preeclampsia (P=0.02). In a logistic regression model, we found a significant influence of bFGF serum concentrations on the diagnosis of mild preeclampsia (P=0.01), but not on the diagnosis of chronic hypertension (P=0.19) or severe preeclampsia (P=0.41). CONCLUSIONS: Elevated serum concentrations of bFGF are associated with mild preeclampsia, but are not discriminatory for the distinction between mild and severe preeclampsia.

Analysis of Variance↗

PubMedDate not supplied

Production of oxytocin receptor and cytokines in primary uterine smooth muscle cells cultivated under inflammatory conditions.

OBJECTIVE: We studied the production of the oxytocin receptor and interleukins in human uterine smooth muscle cells cultured in vitro in the presence of cytokines that were shown to be elevated in gestational diseases such as intrauterine infections and chorioamnionitis. METHODS: Human uterine smooth muscle cells were cultured in the absence or presence of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS). Additionally, cells were cultivated under hypoxic conditions (3.5% oxygen). After 6, 12, 24, and 48 hours of incubation, oxytocin receptor mRNA was measured from total RNA using quantitative, competitive reverse transcriptase-polymerase chain reaction. Secreted cytokines (IL-1beta, IL-6, or IL-8) were quantitated from supernatants after 6, 12, 24, and 48 hours of stimulation by commercially available enzyme-linked immunosorbent assay. RESULTS: In nonstimulated cultures basal secretion of IL-1beta, IL-6, and IL-8 was detectable. Supplementation of IL-1beta induced a statistically significant decrease in oxytocin receptor mRNA abundance, whereas IL-6, TNFalpha, LPS, or hypoxia did not significantly affect oxytocin receptor gene expression. The cytokines IL-1 and TNFalpha induced IL-6 and IL-8 release, whereas secretion of the two interleukins was not altered in the presence of LPS or hypoxia. Expression of IL-1beta was not significantly induced under inflammatory or hypoxic culture conditions. CONCLUSION: The constitutive and cytokine-inducible expression of interleukins from uterine smooth muscle cells suggests that the myometrium may contribute to the overall production of inflammatory mediators in the uterus that are thought to govern term- or infection-induced preterm labor. Down-regulation of the oxytocin receptor under IL-1beta in myometrial cells may indicate that initiation and maintenance of labor could be partially limited under severe inflammatory conditions such as chorioamnionitis.

Cells, Cultured↗