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Endocrinology Vol. 139, No. 3 1208-1212
Copyright © 1998 by The Endocrine Society

ARTICLES

Demonstration of a Relaxin Receptor and Relaxin-Stimulated Tyrosine Phosphorylation in Human Lower Uterine Segment Fibroblasts1

Smita Palejwala, Daniel Stein, Andrea Wojtczuk, Gerson Weiss and Laura T. Goldsmith

Department of Obstetrics and Gynecology, New Jersey Medical School, Newark, New Jersey 07103

Address all correspondence and requests for reprints to: Dr. Laura T. Goldsmith, Department of Obstetrics and Gynecology, New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (±SEM) dissociation constant (Kd) of 4.36 ± 1.7 x 10-9 M and a mean of 3220 ± 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MANY mammalian species, the 6-kDa protein hormone relaxin has pronounced effects on the female reproductive tract that are generally associated with the maintenance of pregnancy and successful parturition (1). Relaxin is obligatory for normal delivery in several species, largely due to its marked rearrangement of reproductive tract connective tissue (1, 2). In addition to its important and well-established role in female reproductive function, results of various recent studies implicate relaxin in sperm motility (3) and in a number of nonreproductive functions such as regulation of blood pressure (4, 5, 6), control of heart rate (5, 6, 7, 8, 9), and the release of oxytocin and vasopressin (10, 11, 12, 13). In spite of the importance of this hormone in normal reproductive function and its potential roles in other nonreproductive tract tissues, our understanding of the mechanism of action of relaxin, including the nature of the relaxin receptor and its signal transduction mechanisms, is extremely limited.

To elucidate the mechanism of relaxin action, we used a model system of human cervix, human lower uterine segment fibroblasts to study the binding characteristics of human relaxin and its effect on intracellular concentrations of cAMP. Although relaxin does not bind to the insulin receptor nor does it have insulin-like activity, relaxin is strikingly similar in tertiary configuration to insulin and is considered a member of the insulin-like growth factor family (14). Because insulin and other members of the insulin-like growth factor family bind to tyrosine kinase receptors, we questioned whether relaxin would affect tyrosine phosphorylation of any cellular protein in a target organ. Our results demonstrate the existence of a relaxin receptor in human lower uterine segment fibroblasts, and that relaxin markedly increases tyrosine phosphorylation of a protein with an apparent Mr approximately 220 kDa in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Lower uterine segment tissue was obtained at term pregnancy, from a nonlaboring patient at the time of cesarean section delivery following informed consent. The protocol was approved by the University of Medicine and Dentistry of New Jersey-New Jersey Medical School Institutional Review Board. Tissue (~1 g) was minced with an iris scissors into 1- to 2-mm3 pieces and incubated in 0.2% collagenase (Type 1; Worthington, Freehold, NJ; CLS-1, 130 U/mg) with 0.05% DNase (Sigma Chemical Co., St. Louis, MO) in Earle’s balanced salt solution (EBSS) at 37 C for 30 min in a shaking water bath. The enzyme-tissue mixture was then triturated for 15 min and incubated again at 37 C for 30 min. After repeated trituration for an additional 10 min, the cell suspension was centrifuged for 10 min at 150 x g. The cell pellet was resuspended in DMEM, cells were counted, and viability was assessed by trypan blue exclusion. Viability of the dispersed cells was greater than 85%. Lower uterine segment cells were maintained as monolayer cultures in DMEM containing 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere of 5% CO2-95% air. EBSS, DMEM, and penicillin/streptomycin were obtained from Life Technologies (Grand Island, NY). For experimental studies, cells were plated in complete DMEM at 1 x 105 cells per well in 24-well tissue culture plates.

To assess purity, cell cultures were evaluated for the distribution of markers for epithelial and fibroblast cells. Previous studies have demonstrated that keratin-positive filaments are uniquely found in epithelial cells, and vimentin-positive filaments are uniquely found in cells of mesenchymal origin (i.e. fibroblasts and endothelial cells) (15, 16). Therefore, cultures were evaluated for the presence of vimentin and cytokeratin by immunohistochemistry as described previously (17). Cells were plated on Permanox Chamber Slides (Nunc, Naperville, IL) and fixed in ice-cold acetone for 10 min at 4 C. Mouse anti-vimentin monoclonal (clone V9) and mouse antihuman epithelial keratin (AE1/AE3) antibodies (Boehringer Mannheim, Indianapolis, IN) were used as the primary antibodies at concentrations of 5 µg/ml. The specificity of these antibodies has been previously demonstrated (18, 19, 20). Additional reagents were components of the Histostain-SP kit (Zymed, South San Francisco, CA), and the protocol was that supplied with the kit. Five hundred cells were tested, and all cells stained positive for vimentin. Only a few cells (<5%) stained positive for cytokeratin, indicating that the cultures of lower uterine segment fibroblasts have little, if any, contamination with epithelial cells.

Preparation of labeled relaxin
Human H2 relaxin, obtained from Genentech (South San Francisco, CA) was iodinated by a modification of the chloramine-T procedure (21). 125I-Labeled relaxin was separated from free 125I by gel chromatography using 0.7 x 20 cm columns of Bio-Gel P-6 (Bio-Rad Labs., Hercules, CA). Specific activity of the 125I-relaxin used in these studies ranged from 102–185 µCi/µg.

Binding studies
To test the binding of human relaxin, cells (~2 x 105 cells/well) were washed in binding buffer (HBSS containing 10 mM HEPES, pH 7.2, 0.1 mg/ml BSA, 1 µg/ml leupeptin, and 0.05 mM phenylmethylsulfonylfluoride) for 2 h at 4 C and then incubated with 10 nM 125I-labeled relaxin at 4 C for 45 min. Additional replicate wells of cells had either unlabeled relaxin at various amounts (0.1 µM-10 µM) or insulin (10 µM) added. Cells were then washed in binding buffer (500 µl/well) at 4 C for 10 min followed by two similar washes with PBS, and the cells were lysed by addition of 100 µl/well boiling, dye-free 2x SDS-PAGE buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol). Cell lysates were collected into tubes, and the radioactivity was determined in a Micromedic Gamma 200 {gamma}-counter (ICN Pharmaceuticals, Inc., Costa Mesa, CA). Binding data were analyzed by the method of Munson and Rodbard (22). Scatchard analysis of the binding data were performed as a function of tracer concentration (23). Cells in multiwell tissue culture plates were incubated in binding buffer containing 5 nM 125I-labeled relaxin in the absence or presence of different concentrations (0–50 nM) of unlabeled relaxin. The amounts of bound and free relaxin were calculated based on radioactivitity detected.

Effect of relaxin on intracellular cAMP
Cells were plated and incubated in complete DMEM for 4 days, media were removed and replaced with serum-free DMEM containing 0.2% lactalbumin hydrolysate (Life Technologies) without or with human relaxin at various concentrations and cells were incubated at 37 C in a humidified atmosphere of 5% CO2-95% air for 15 min. Additional replicate wells of cells were incubated similarly with 20 µM forskolin as a positive control or 3-isobutyl-1-methyl-xanthine (IBMX) (Sigma Chemical Co.) at 0.5 mM with or without human relaxin at various concentrations. Media were removed from the wells, and the monolayered cells were lysed by the addition of 200 µl 0.1% Triton X-100 (Sigma Chemical Co.) in EBSS, followed by shaking on a plate shaker for 1 min. Cell lysates were extracted with ice-cold ethanol, and the dried-down extracts were reconstituted in assay buffer (0.05 M acetate buffer, pH 5.8, 0.02% BSA, 0.005% thimerosal) and assessed for cAMP content using the BIO-TRAK cAMP enzyme immunoassay system (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions.

Phosphotyrosine Western blots
Cells were plated and incubated in complete DMEM for 3 days, and media were removed and replaced with serum-free media and incubated for an additional 2 days. Media were then replaced with serum-free DMEM containing the tyrosine phosphatase inhibitor sodium vanadate (0.5 mM), and cells were incubated in the absence or presence of various concentrations of human relaxin for specific time periods. At the end of the incubation periods, media were removed, and cells were washed with EBSS and lysed with boiling SDS-PAGE buffer containing 0.5 mM sodium vanadate. Cell lysates were then boiled for 2 min, cooled on ice, and then passed four times through a 25-gauge needle to shear the DNA. An aliquot was removed for assessment of protein content using the DC Protein Assay kit (Bio-Rad Labs.) and BSA as standard. To the remaining total cellular protein extracts from each group of cells, 2-mercaptoethanol and bromophenol blue were added to a final concentration of 5% and 0.001%, respectively. Extracts were then boiled for 2 min, electrophoresed (15 µg protein per lane) on 8% resolving, 4% stacking SDS-PAGE gels, and Western transferred onto polyvinylidine difluoride membranes (Sigma Chemical Co.). Membranes were immunoblotted using horseradish peroxidase-conjugated recombinant anti-phosphotyrosine antibody PY20 (RC20H; Transduction Labs., Lexington, KY) at a 1:500 dilution. The specificity of this antibody for phosphorylated tyrosine has been previously demonstrated (24). Blots were developed by the enhanced chemiluminescent method using reagents from Amersham, and the intensities of the signals on the films exposed to the blots were determined using a Molecular Dynamics 300B computing densitometer (Sunnyvale, CA) as we have described previously (25, 26).

Statistical analyses
Data were assessed to determine whether they were normally distributed using the Shapiro-Wilk test. P values greater than 0.05 were required for demonstration of a normal distribution. Variances were evaluated using the Brown Forsythe test. Data from the cAMP studies showed unequal variances and were thus evaluated by the Kruskal Wallis test. Data from the phosphotyrosine blots were demonstrated to be normally distributed with equal variances, and they were thus assessed using two tailed Student’s t tests. All data analyses used JMP statistical software (SAS Institute, Cary NC) written for the Macintosh Computer (Apple Computers, Cupertino, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding of 125I-labeled relaxin to human lower uterine segment fibroblasts
Human relaxin labeled with 125I bound specifically to human lower uterine segment fibroblasts. Table 1Go shows the displacement of 125I-labeled relaxin (10 nM) binding to human lower uterine segment fibroblasts by increasing concentrations (0.1 µM-10 µM) of unlabeled relaxin. A 1000-fold molar excess (10.0 µM) of relaxin displaced 78.3% of the binding of 125I-labeled relaxin. In contrast, a 1000-fold molar excess (10.0 µM) of insulin did not cause any displacement of 125I-labeled relaxin from the cells, indicating that the binding is specific to relaxin, and that the binding site is distinct from the insulin receptor. The binding of 125I-labeled relaxin to lower uterine segment fibroblasts appeared to increase slightly in the presence of a 1000-fold molar excess of insulin. Saturation analyses showed that, although the total relaxin binding was generally low, the specific binding of 125I-labeled relaxin to lower uterine segment fibroblasts was 67–71% of the total binding (n = 4 experiments). Figure 1Go shows the Scatchard analysis of the binding displacement data from three independent experiments. These data indicated a single class of high-affinity relaxin binding sites with a mean (±SEM) dissociation constant (Kd) of 4.36 ± 1.7 x 10-9 M and a mean of 3220 ± 557 binding sites per cell.


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  		Table 1. Displacement of 125I-labeled relaxin binding to human lower uterine segment fibroblasts

 


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  		Figure 1. Scatchard plot of receptor binding in human lower human segment fibroblasts. Relaxin binding consists of a single class of high-affinity binding sites (Kd = 4.36 nM) with 3220 ± 557 binding sites per cell. Analysis is based on data from three independent experiments, each conducted in triplicate.

 
Effect of relaxin on intracellular cAMP concentrations
Because relaxin has been shown to increase intracellular levels of cAMP and/or affect other aspects of the cAMP/protein kinase A (PKA) pathway in several other cell types (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), we examined the effect of relaxin on intracellular concentrations of cAMP in human lower uterine segment fibroblasts. Cells were incubated with relaxin or forskolin (a positive control) for 15 min, a time period in which relaxin elicited a significant increase in intracellular cAMP concentrations in other cell types. The results of these experiments are shown in Fig. 2Go. Incubation of the human lower uterine segment fibroblasts with 20 µM forskolin for 15 min caused an impressive, statistically significant (P < 0.05, for all experiments) increase in intracellular levels of cAMP. In contrast, relaxin at 1, 10, 100, or 500 ng/ml, quantities sufficient to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP. Incubation of the cells in the presence of IBMX increased the intracellular concentrations of cAMP, yet relaxin did not affect levels of intracellular cAMP, even in the presence of IBMX.



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  		Figure 2. Relaxin does not stimulate intracellular cAMP concentrations in human lower uterine segment fibroblasts. Data shown is from three experiments (plus one experiment conducted with phosphodiesterase inhibitor IBMX), each performed in duplicate. F, Forskolin, a positive control.

 
Effect of relaxin on tyrosine phosphorylation of cellular proteins
Because relaxin is so strongly similar in tertiary configuration to insulin, and because the insulin receptor is a tyrosine kinase, we determined the effect of relaxin on tyrosine phosphorylation of cellular proteins of lower uterine segment fibroblasts. The results of these studies are shown in Figs. 3Go and 4Go. Figure 3Go shows a representative Western blot of total cellular proteins from cells that were treated with 1, 10, or 100 nM relaxin and assessed at 5, 20, or 30 min of relaxin exposure. Increased tyrosine phosphorylation of a protein with an apparent Mr approximately 220 kDa was observed. Figure 4Go shows the reproducibility and consistency of this finding.



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  		Figure 3. Relaxin stimulates tyrosine phosphorylation of an approximately 220 kDa protein in human lower uterine segment fibroblasts. Cells were treated with various concentrations of relaxin for 5, 20, and 30 min, and cellular protein extracts were electrophoresed, Western transferred, and blotted with an anti-phosphotyrosine antibody. Blots were developed using enhanced chemiluminescence. Film exposed to a blot from one representative experiment is shown.

 


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  		Figure 4. Relaxin stimulates tyrosine phosphorylation of an approximately 220 kDa protein in human lower uterine segment fibroblasts. Cells were treated with various concentrations of human relaxin for 15 (open bar), 30 (hatched bar), and 45 (solid bar) min, and cellular protein extracts were electrophoresed, Western transferred, and blotted with an antiphosphotyrosine antibody. Blots were developed using enhanced chemiluminescence, and intensities of signals on film exposed to blots were determined using computing densitometry. Densitometric values obtained for control, untreated cells were set at 100, and values for relaxin treated cells are expressed as percent of control. Values presented are means ± SEM of four independent experiments, each conducted in triplicate. *, Significant increase above that of control, untreated cells at same time points (P <= 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although there is ample evidence to demonstrate that relaxin modulates cervical connective tissue in several species (1, 2, 40), the mechanisms responsible for relaxin’s activity on the mammalian cervix are unknown. The definitive mechanism of action of relaxin, including identification of the structure of the relaxin receptor, has yet to be identified in any target organ. The present data demonstrate that fibroblasts isolated from human lower uterine segment tissue at term pregnancy express receptors for relaxin, and that relaxin stimulates tyrosine phosphorylation of a high Mr protein, greater than apparent Mr of 208 kDa, in this cell type. This is the first evidence that relaxin stimulates tyrosine phosphorylation of any protein in any cell type. In concert with results of recent studies that demonstrate that the Mr of the relaxin receptor is approximately 220 kDa (41, 47), our data suggest that the phosphorylated protein is likely to be the relaxin receptor. Ligand-stimulated tyrosine autophosphorylation of the relaxin receptor has not been previously demonstrated in any cell type.

The binding of relaxin to specific binding sites has been studied in several target organs. Although early studies by Mercado-Simmen et al. demonstrated that two classes of binding sites exist in rat and pig uterus (42, 43), more recent studies all demonstrated a single class of high-affinity binding sites in mouse (44), rat (45, 46), and human (41, 47, 48, 49) cells. All studies reported to date demonstrate a binding site with similar affinity to each other and to the data presented here.

A good deal of evidence has been presented that demonstrates that relaxin action in several cell types is associated with the cAMP/adenylate cyclase/PKA pathway (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39). However, no data have been presented that demonstrate coupling of a relaxin-receptor complex to a heterotrimeric G protein. Thus, increased intracellular cAMP concentrations that occur under the influence of relaxin may result from cross-talk between signal transduction pathways. Results of some studies demonstrate that relaxin does not appear to influence intracellular cAMP concentrations (50, 51). The data presented here do not support a direct role for the cAMP/PKA pathway in relaxin action, but rather raise the possibility that the relaxin receptor is a tyrosine kinase or is associated with a high molecular weight tyrosine kinase. Our supposition is supported by results of Osheroff et al. (41), which suggest that binding of relaxin to human uterine cells is not associated with a G protein-coupled mechanism. Studies by Zhang and Bagnell (52) demonstrate that the action of relaxin on proliferation of porcine granulosa cells is inhibited by a tyrosine kinase inhibitor.

Relaxin has been shown to modulate various biochemical components of cervical connective tissue in a variety of species (1, 2, 40). However, studies of the role of relaxin on human cervical collagenolytic activity have been seriously hampered by the extremely limited ability to obtain normal cervical tissue from pregnant women. Studies on relaxin action require cervix from pregnant women to enable determination of relaxin action in tissue that has been exposed to the proper endocrinological milieu to allow for relaxin activity. Due to the ethical limitations preventing cervical biopsies from pregnant women, biopsies from the lower uterine segment adjacent to the cervix taken from women undergoing cesarean delivery have been used as a representative model to investigate the biochemical changes that occur in the cervix during delivery in women (53, 54, 55, 56). Rationale for use of the lower uterine segment as a model to study the changes that occur in the cervix is based on several facts. During labor, the lower uterine segment undergoes significant changes that are quite similar to the changes that occur in the cervix, including thinning and dilatation (57). The lower uterine segment and cervix differ structurally and biochemically from the uterine fundus; both contain fewer smooth muscle cells and lower concentrations of oxytocin receptors than the fundus (57, 58, 59, 60). The lower uterine segment and the cervix contain similar amounts of extractable collagenase during pregnancy in women not in labor at full-term gestation (54). During labor in women at full-term gestation, interstitial collagenase, the rate-limiting enzyme in degradation of type 1 collagen in the matrix, increases 13- to 23-fold in the lower uterine segment (54, 55). An earlier study by Wiqvist et al. demonstrated that the relaxin inhibition of [3H]proline incorporation in lower uterine segment tissue was similar in magnitude to that in cervical biopsy tissue taken in early pregnancy (56). Thus, lower uterine segment appears to be a highly useful model for the study of the effects of relaxin.

Although the specific function of relaxin in normal human parturition is not clear, elevated relaxin levels may play a pathological role during human pregnancy. Results of recent studies demonstrate a significant association between elevated circulating maternal relaxin concentrations and premature birth (61, 62). Because this effect is likely mediated by an effect of relaxin on the cervix, knowledge of the complete signal transduction pathway for relaxin, including the structural determination of the receptor, should prove useful in alleviating this type of prematurity.


   Footnotes
 
1 This work was supported by NIH Grant HD-22338. Back

Received September 5, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sherwood, OD 1994 Relaxin. In: Knobil E, Neill J (eds) The physiology of reproduction, ed 2. Raven Press, New York, pp 861–1009
  2. Steinetz BG, O’Byrne EM, Kroc RL 1980 The role of relaxin in cervical softening in mammals. In: Naftolin F, Stubblefield PG (eds) Dilation of the uterine cervix. Raven Press, New York, pp 157–177
  3. Weiss G 1989 Relaxin in the male. Biol Reprod 40:197–200[Abstract]
  4. St-Louis J, Massicotte G 1985 Chronic decrease of blood pressure by rat relaxin in spontaneously hypertensive rats. Life Sci 37:1351–1357[CrossRef][Medline]
  5. Mumford AD, Parry LJ, Summerlee AJS 1989 Lesion of subfornical organ affects the haemotensive response to centrally administered relaxin in anaesthetized rats. J Endocrinol 122:7747–755
  6. Yang R-H, Bunting S, Wyss JM, Berecek KH, Zhang L, Jin H 1995 Pressor and bradycardic effects of centrally administered relaxin in conscious rats. Am J Hypertens 8:375–381[CrossRef][Medline]
  7. Kakouris H, Eddie LW, Summers RJ 1992 Cardiac effects of relaxin in rats. Lancet 339:1076–1078[CrossRef][Medline]
  8. Han X, Habuchi Y, Giles WR 1994 Relaxin increases heart rate by modulating calcium current in cardiac pacemaker cells. Circ Res 74:537–541[Abstract/Free Full Text]
  9. Ward DG, Thomas R, Cronin MJ 1992 Relaxin increases rat heart rate by a direct action on the cardiac atrium. Biochem Biophys Res Commun 186:999–1005[CrossRef][Medline]
  10. Dayanithi G, Cazalis M, Nordmann JJ 1987 Relaxin affects the release of oxytocin and vasopressin from the neurohypothesis. Nature 325:813–816[CrossRef][Medline]
  11. Wilson BC, Summerlee AJ 1994 Effects of exogenous relaxin on oxytocin and vasopressin release and the intramammary pressure response to central hyperosmotic challenge. J Endocrinol 141:75–80[Abstract/Free Full Text]
  12. Summerlee AJS, O’Byrne KT, Paisley AC, Breeze MF, Porter DG 1984 Relaxin affects the central control of oxytocin release. Nature 325:372–374[CrossRef]
  13. Way SA, Leng G 1992 Relaxin increases the firing rate of supraoptic neurones and increases oxytocin secretion in the rat. J Endocrinol 132:149–158[Abstract/Free Full Text]
  14. Schwabe C, Bullesbach EE 1994 Relaxin: structures, functions, promises and nonevolution. FASEB J 8:1152–1160[Abstract]
  15. Moll T, Franke WW, Schiller DL, Geiger B, Krepler R 1982 The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24[CrossRef][Medline]
  16. Franke WW, Schmid E, Osborn M, Weber K 1978 Different intermediate-sized filaments distinguished by immunofluorescent microscopy. Proc Natl Acad Sci USA 75:5034–5038[Abstract/Free Full Text]
  17. Richards RG, Brar AK, Frank GR, Hartman SM, Jikihara H 1995 Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression. Biol Reprod 52:609–615[Abstract]
  18. Osborn M, Debus E, Weber K 1984 Monoclonal antibodies specific for vimentin. Eur J Cell Biol 34:137–143[Medline]
  19. Woodcock-Mitchell J, Eichner R, Nelson WG, Sun TT 1982 Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol 95:580–588[Abstract/Free Full Text]
  20. Tseng SC, Jarvinen MJ, Nelson WG, Huang JW, Woodcock-Mitchell J, Sun TT 1982 Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies. Cell 30:361–372[CrossRef][Medline]
  21. Greenwood FC, Hunter WM, Glover JS 1963 The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 89:114–123[Medline]
  22. Munson P, Rodbard D 1980 Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[CrossRef][Medline]
  23. Scatchard G 1949 The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672[CrossRef]
  24. Ruff-Jamison S, Campos-Gonzalez R, Glenney JR 1991 Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features. J Biol Chem 266:6607–6613[Abstract/Free Full Text]
  25. Palejwala S, Goldsmith LT 1991 Detection of Ki-ras proto-oncogene protein by a specific monoclonal antibody. Biotechniques 11:606–608[Medline]
  26. Palejwala S, Goldsmith LT 1992 Ovarian expression of cellular Ki-ras p21 varies with physiological status. Proc Natl Acad Sci USA 89:4202–4206[Abstract/Free Full Text]
  27. Kramer SM, Gibson UEM, Fendley BM, Mohler M, Drolet DW, Johnston PD 1990 Increase in cyclic AMP levels by relaxin in newborn rhesus kidney monkey uterus cell culture. In Vitro Cell Dev Biol 26:647–656[Medline]
  28. Braddon SA 1978 Relaxin-dependent adenosine 3':5'-monophosphate concentration changes in the mouse pubic symphysis. Endocrinology 102:1292–1299[Abstract]
  29. Sanborn BM, Kuo HS, Weisbrot NW, Sherwood OD 1980 The interaction of relaxin with the rat uterus. I. Effect on cyclic nucleotide levels and spontaneous contractile activity. Endocrinology 106:1210–1215[Abstract]
  30. Hsu CJ, McCormack SM, Sanborn BM 1985 The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in rat myometrial cells in culture. Endocrinology 116:2029–2035[Abstract]
  31. Meera P, Anwer K, Monga M, Oberti C, Stefani E, Toro L, Sanborn BM 1995 Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A. Am J Physiol 269:C312–C317
  32. Sanborn BM, Anwer K, Monga M, Wen Y, Singh SP, Meera P, Oberti C, Toro L, Stefani E 1995 Mechanisms controlling the acute effects of relaxin on the myometrium. In: MacLennan AH, Tregear GW, Bryant-Greenwood GD (eds) Progress in relaxin research, chapt 19. World Scientific Publishing Co., Singapore, pp 289–297
  33. Osa T, Inoue H, Okabe K 1991 Effects of porcine relaxin on contraction, membrane response and cyclic AMP content in rat myometrium in comparison with the effects of isoprenaline and forskolin. Br J Pharmacol 104:950–960[Medline]
  34. Hughes SJ, Hollingsworth M, Elliott KRF 1997 The role of a cAMP-dependent pathway in the uterine relaxant action of relaxin in rats. J Reprod Fertil 109:289–296[Abstract/Free Full Text]
  35. Fei DTW, Gross MC, Lofgren JL, Mora-Worms M, Chen AB 1990 Cyclic AMP responses to recombinant human relaxin by cultured human endometrial cells-a specific and high throughput in vitro bioassay. Biochem Biophys Res Commun 170:214–222[CrossRef][Medline]
  36. Bigazzi M, Brandi ML, Bani G, BaniSacchi T 1992 Relaxin influences the growth of MCF-7 breast cancer cells. Cancer 70:639–643[CrossRef][Medline]
  37. Chen G, Huang JR, Tseng L 1988 The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells. Biol Reprod 39:519–525[Abstract]
  38. Tseng L, Lane B 1995 Role of relaxin in the decidualization of human endometrial cells. In: MacLennan AH, Tregear GW, Bryant-Greenwood GD (eds) Progress in relaxin research, chap 23. World Scientific Publ. Co., Singapore, pp 325–338
  39. Cronin MJ, Malaska T, Bakhit C 1987 Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells. Biochem Biophys Res Commun 148:1246–1251[CrossRef][Medline]
  40. Sherwood OD 1995 The role of relaxin in animals: an overview. In: MacLennan AH, Tregear GW, Bryant-Greenwood GD (eds) Progress in relaxin research, chap 27. World Scientific Publ. Co., Singapore, pp 391–417
  41. Osheroff PL, King KL 1995 Binding and cross-linking of 32P-Labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes. Endocrinology 136:4377–4381[Abstract]
  42. Mercado-Simmen RC, Bryant-Greenwood GD, Greenwood FC 1980 Characterization of the binding of 125I-relaxin to rat uterus. J Biol Chem 255:3617–3623[Free Full Text]
  43. Mercado-Simmen RC, Goodwin B, Ueno MS, Yamamoto SY, Bryant-Greenwood GD 1982 Relaxin receptors in the myometrium and cervix of the pig. Biol Reprod 26:120–128[CrossRef][Medline]
  44. Yang S, Rembiesa B, Bullesbach EE, Schwabe, C 1992 Relaxin receptors in mice: demonstration of ligand binding in symphyseal tissues and uterine membrane fragments. Endocrinology 130:179–185[Abstract]
  45. Osheroff PL, Cronin MJ, Lofgren JA 1992 Relaxin binding in the rat heart atrium. Proc Natl Acad Sci USA 89:2384–2388[Abstract/Free Full Text]
  46. Osheroff PL, Phillips HS 1991 Autoradiographic localization of relaxin binding sites in rat brain. Proc Natl Acad Sci USA 88:6413–6417[Abstract/Free Full Text]
  47. Parsell DA, Mak JY, Amento EP, Unemori EN 1996 Relaxin binds to and elicits a response from cells of the human monocyte cell line, THP-1. J Biol Chem 271:27936–27941[Abstract/Free Full Text]
  48. Carrell DT, Peterson CM, Urry RL 1995 The binding of recombinant human relaxin to human spermatozoa. Endocr Res 21:697–707[Medline]
  49. Garibay-Tupas JL, Maaskant RA, Greenwood FC, Bryant-Greenwood GD 1995 Characteristics of the binding of 32P-labelled human relaxins to the human fetal membranes. J Endocrinol 145:441–448[Abstract/Free Full Text]
  50. Summers RJ, Tan YY, Kakouris H, Eddie LW 1995 Cardiac actions of relaxin. In: MacLennan AH, Tregear GW, Bryant-Greenwood GD (eds) Progress in Relaxin Research, chap 34. World Scientific Publ. Co., Singapore, pp 487–498
  51. Colon J, Goldsmith L, Lessing J, Brenner S, Ginsburg F, Schoenfeld C, Amelar R, Dubin L, Weiss G Effect of relaxin on the cAMP content of human sperm. Program of the 33rd Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, 1986, p 151 (Abstract)
  52. Zhang Q, Bagnell CA, Effect of tyrosine kinase inhibitors on relaxin-induced deoxyribonucleic acid (DNA) synthesis in cultured procine granulosa cells. Program of the 26th Annual Meeting of the Society for the Study of Reproduction, 1993, Fort Collins, CO, p 182 (Abstract)
  53. Granstrom LM, Ekman GE, Malmstrom A, Ulmsten U, Woessner JF 1992 Serum collagenase levels in relation to the state of the human cervix during pregnancy and labor. Am J Obstet Gynecol 167:1284–1288[Medline]
  54. Rajabi MR, Dean DD, Beydoun SN, Woessner JF 1988 Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition. Am J Obstet Gynecol 159:971–976[Medline]
  55. Rechberger T, Woessner JF 1993 Collagenase, its inhibitors, and decorin in the lower uterine segment in pregnant women. Am J Obstet Gynecol 168:1598–1603[Medline]
  56. Wiqvist I, Norstrom A, O’Byrne E, Wiqvist N 1984 Regulatory influence of relaxin on human cervical and uterine connective tissue. Acta Endocrinol (Copenh) 106:127–132[CrossRef]
  57. Danforth DN 1980 Early studies of the anatomy and physiology of the human cervix and implications for the future. In: Naftolin F, Stubblefield PG (eds) Dilatation of the uterine cervix. Raven Press, New York, pp 3–15
  58. Danforth DN, Veis A, Breen M, Weinstein HG, Buckingham JC, Manalo P 1974 The effect of pregnancy and labor on the human cervix: changes in collagen, glycoproteins and glycosaminoglycans. Am J Obstet Gynecol 120:641–649[Medline]
  59. Blandau RJ, Moghissi K 1973 The biology of the cervix. The University of Chicago Press, Chicago
  60. Singer A, Jordan JA 1976 The cervix. W.B. Saunders, Philadelphia, PA
  61. Petersen LK, Skajaa K, Uldbjerg N 1992 Serum relaxin as a potential marker for preterm labor. Br J Obstet Gynecol 99:292–295[Medline]
  62. Weiss G, Goldsmith LT, Sachdev R, VonHagen S, Lederer K 1993 Elevated first-trimester serum relaxin concentrations in pregnant women following ovarian stimulation predict prematurity risk and preterm delivery. Obstet Gynecol 82:821–828[Abstract/Free Full Text]



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