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Pregnancy and labor increase the capacity of human myometrial cells to secrete parathyroid hormone-related protein
Life Sciences 68 (2001) 1557–1566
Pregnancy and labor increase the capacity of human myometrial cells to secrete parathyroid hormone-related protein Jeffrey S. Shenbergera,*, Patricia S. Dixonb, Jerome Choateb, Kenneth Helalc, Ronald L. Shewd, William Barthc a
Department of Pediatrics, University of Kentucky, 800 Rose Street, Lexington KY 40536-0084, USA b Clinical Investigations, 2200 Bergquist Drive, Wilford Hall USAF Medical Center, Lackland AFB, TX 78236-5300, USA c Department of Obstetrics and Gynecology, 2200 Bergquist Drive, Wilford Hall USAF Medical Center, Lackland AFB, TX 78236-5300, USA d Department of Anatomy and Cell Biology, Department of Anatomy and Cell Biology Medical Sciences Building 5035, Indiana University Medical Center, 635 Barnhill Drive, Indianapolis, IN 46202-5120, USA Received 25 May 2000; accepted 9 October 2000
Abstract Parathyroid hormone-related protein (PTHrP), a oncofetal gene product possessing smooth muscle relaxant properties, has been found in rat and human uterine smooth muscle cells (USMC) where it is postulated to regulate myometrial tone and/or blood flow. Studies investigating the gestational regulation of PTHrP in human USMC have not been performed. This study was conducted to determine if pregnancy alters the capacity of USMC to secrete or respond to PTHrP. USMC cultures were established from 8 hysterectomy specimens (H) and 7 non-laboring (NP) and 5 laboring term pregnant uterine biopsies (LP). PTHrP secretion was measured at baseline and in response to TGF-b1 using a immunoradiometric assay. The USMC response to PTHrP was assessed by incubating cultures with human (1-34)PTHrP and measuring cellular cAMP by radioimmunoassay. We found that cultures from the groups did not differ with respect to basal PTHrP secretion. TGF-b1, on the other hand, produced dose-dependent increases in secreted PTHrP in each group such that LP.NP.H at 12 hrs and LP.NP and H 24 hrs. Maximal responses were found at 24 hrs in cells treated with 10 ng/ml TGF-b1 (LP: 20346366 vs NP: 14856427; H: 12506202 fmol/mg). Incubation of cultures with PTHrP produced dose-dependent increases in cAMP production, with 1027M increasing levels by 64%. Neither pregnancy nor labor significantly affected the cAMP response. These findings indicate that the human myometrium has the capacity to increase PTHrP secretion during pregnancy and labor through a TGF-b-dependent pathway. Such findings are con* Corresponding author: Department of Pediatrics/Neonatology, MS-473 University of Kentucky Chandler Medical Center 800 Rose Street Lexington, KY 40536-0084. Tel.: 859-323-5530; fax: 859-257-4384. E-mail address: [email protected] (J.S. Shenberger) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 0 9 4 9 -3
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sistent with a role of PTHrP in enhancing uterine blood flow. rights reserved.
© 2001 Elsevier Science Inc. All
Keywords: Parathyroid hormone-related protein; Uterus; Smooth muscle; Pregnancy
Introduction Parathyroid hormone-related protein (PTHrP) is an oncofetal gene product originally identified as the source of humoral hypercalcemia of malignancy [1]. Although many tumor cells overexpress PTHrP, the peptide is normally produced by keratinocytes, epithelial cells, osteoblasts, and smooth muscle cells where it often functions in a “classical” endocrine fashion [2]. During pregnancy, PTHrP and PTH/PTHrP receptor protein and mRNA are found throughout the uterus, with an abundance located within the amnion, chorion, luminal epithelium, and myometrium [3–5]. In each uterine tissue location, the peptide is postulated to perform a unique physiologic function including the facilitation of calcium transport across the placenta, preparation and maintenance of the conceptus implantation site, and regulation of myometrial tone and blood flow [6–9]. In the rat uterus for instance, PTHrP mRNA increases in the longitudinal and circular smooth muscle layers late in pregnancy where its expression is enhanced by both uterine occupancy and the administration of 17b-estradiol [9,10]. Recently, PTHrP gene expression has also been documented in sheep, though the message is present in greater quantities preterm than term, suggesting significant inter-species variation [7]. In vitro, human myometrial cells secrete PTHrP, and when stimulated with TGF-b1, increase PTHrP gene expression and peptide secretion [11,12]. Data collected from isolated smooth muscle strip preparations indicate that PTHrP functions as a smooth muscle relaxant by decreasing resting myometrial tone and inhibiting spontaneous and oxytocin-induced uterine contractions [13,14]. Despite the animal studies detailing the gestational regulation of PTHrP, no human investigations have been performed to examine either the presence or regulation of PTHrP during pregnancy. Therefore, we conducted the present study to investigate PTHrP regulatory mechanisms in myometrial cell cultures established from non-pregnant, term pregnant non-laboring, and term pregnant laboring women. Our goals were to determine whether pregnancy and/or labor alter the capacity of human myometrial cells to 1) secrete PTHrP basally and in response to TGF-b1; 2) to determine if estrogen increases PTHrP protein secretion from nonpregnant cells; and 3) to assess the ability of myometrial cells to synthesize cAMP following PTHrP stimulation. We speculated that PTHrP secretion would be greater in the latter stages of pregnancy but that the cellular response to the peptide would be unchanged. Methods Tissue collection and establishment of cell cultures Myometrial specimens were obtained from 8 laboring pregnant (LP) and 8 non-laboring pregnant (NP) women at 37 weeks gestation or greater undergoing cesarean section at Wilford Hall Medical Center (WHMC) and from 8 non-pregnant (H), premenapausal women un-
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dergoing hysterectomy at Brook Army Medical Center (BAMC). Criteria for cesarean section and hysterectomy were at the discretion of the attending physician. Informed consent was obtained prior to surgery. Patients were excluded if they had clinical or laboratory evidence of infection, fetal distress, pre-eclampsia, placental pathology, or infiltrative myometrial carcinoma. The WHMC/BAMC Institutional Review Board approved the protocol. Full-thickness myometrial samples (130.530.5–1 cm) were obtained from the upper margin of the hysterotomy incision. Samples were immediately placed into cold Hank’s balanced saline solution and placed at 48C. Specimens were then placed in 10 ml of red cell lysis buffer [NH4CL (0.2M), KHCO3 (10mM), EDTA (0.13 mM), pH 7.4] for 5 min. Serosal tissue was removed and specimens minced and placed in DMEM supplemented with 10% fetal bovine serum containing 200 U/ml collagenase for 4 hrs at 378C. Dispersed cells were centrifuged and the resulting pellet resuspended in M3 media containing 10% FBS, HEPES buffer (25nM, pH 7.4), penicillin (200 U/ml), streptomycin (200 mg/ml), and kanamycin sulfate (200 mg/ml) and incubated in room air 1 5%CO2 at 378C. Purity of cultures Confluent uterine smooth muscle cells (USMC) grown on slides were incubated in M3 1 0.5% bovine serum albumin for 24 hrs. Slides were fixed for 5 min in 100% methanol at 2208C, treated with 0.1% Tween-20, rinsed, and blocked with 2% normal goat serum. Monolayers were incubated with mouse, monoclonal, anti-a-actin (1:100) and stained with goat, anti-mouse IgG-FITC (1:50). Slides were then examined under a fluorescent microscope (Nikon Optiphot, Tokyo, Japan). PTHrP secretion and TGF-b1 stimulation Cells were permitted to grow to 90% confluence, growth-arrested in M3 1 0.5% BSA for 24 hrs, and incubated with TGF-b1 (0.1–10 ng/ml) for 24 hrs. Following incubation, media was removed and stored at 2708C for PTHrP analysis. The remaining monolayer was dissolved in 0.5N NaOH, centrifuged, and assayed for total well protein using the bicinchoninic acid assay. PTHrP determinations were performed in duplicate using a immunoradiometric assay (IRMA) containing 125I-labelled goat anti-PTHrP(57-80) antibody and goat antiPTHrP(1-40) antibody bound to polystyrene beads. Samples were counted on a gamma counter and converted to PTHrP (pmol/L) using a standard curve derived from known amounts of (1-84)PTHrP. The sensitivity of the assay is 0.2 pmol/liter. Values were corrected to total protein from the corresponding well and expressed as the average from triplicate wells. Pilot studies revealed that 1 ng/ml of TGF-b1 did not substantially increase PTHrP secretion until 12 hrs, therefore the incubation times of 12 and 24 hrs were utilized. Effect of 17b-estradiol on PTHrP secretion To study the impact of 17b-estradiol on PTHrP secretion, 6 NP cultures were treated with 17b-estradiol (1–100 nM) after growth-arrest. In a second experiment, cultures were treated with 10 nM 17b-estradiol for 24 hrs. Following incubation, media was changed to fresh M3 1 10 nM 17b-estradiol 1 TGF-b1 (0.1–10 ng/ml). Secreted PTHrP was determined as previously described and corrected to total protein. Both experiments were performed using triplicate wells.
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cAMP determinations Cells in triplicate wells were growth-arrested in M3 1 0.5% BSA. Media was replaced with fresh M3 1 100mM iso-butyl-methylxanthine (IBMX) for 1 hr. Human (1-34)PTHrP (1029, 1028, 1027M) or 1026M isoproterenol was added and plates incubated for 10 min. Media was removed and monolayers extracted with ice-cold 65% ethanol. Extracts were added to the media and monolayers rinsed an additional time with ethanol. Combined extracts were then centrifuged at 20003g for 15 min, supernatants transferred to glass tubes, and evaporated under nitrogen. Dried extracts were dissolved in PBS and assayed in duplicate for cAMP using radioimmunoassay. Values were averaged and normalized to cell number from duplicate wells. Materials Human (1-34)PTHrP was attained from Calbiochem (La Jolla, CA). Immunoradiometric PTHrP assay kits were purchased from DiaSorin (Stillwater, MN) while cAMP RIA kits were obtained from Amersham (Arlington Hts, IL). Protein assay kits were purchased from Pierce (Rockford, IL) and all other chemicals and antibodies were obtained from Sigma (St. Louis, MO). Statistics Trends in PTHrP secretion, cAMP production, and 17b-estradiol were tested with repeated measures ANOVA and Newman-Keuls multiple comparison testing. The doseresponse of 17b-estradiol on basal PTHrP secretion was tested by one-way ANOVA. Maximal cAMP responses to isoproterenol were compared using Students’ t test. Data is listed as mean 6 standard error and the level of significance set at p,0.05. Results Cell cultures All H cultures grew avidly while only 7 of 8 NP and 5 of 8 LP grew well enough to utilize for experiments. In addition, a single NP culture did not grow during TGF-b1 assay and was omitted from analysis. All remaining monolayers attained the typical “hill and valley” appearance of smooth muscle cells when confluent and all stained positively for a-smooth muscle actin in greater than 90% of the cells. PTHrP secretion and TGF-b1 stimulation Basal secretion of PTHrP did not differ between groups at either 12 or 24 hrs. As shown in figure 1, however, increasing concentrations of TGF-b1 did increase PTHrP secretion (p,0.0001) and this effect was greater at 24 than 12 hrs (p,0.0001). Furthermore, the three groups differed in their responses to TGF-b1 (p,0.005) with LP cultures secreting more PTHrP than either H or NP following incubation with 1 and 10 ng/ml of TGF-b1 for 12 and 24 hrs. Maximal differences were noted with 10 ng/ml TGF-b1 at 24 hrs (LP: 20346366 vs NP: 14856427; H: 12506202 fmol/mg protein, p,0.01). Control studies revealed that TGF-b1
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Fig. 1. Effect of TGF-b1 on PTHrP secretion. Media from cells treated for 24 hrs with TGF-b1 was assayed for PTHrP by IRMA and values corrected to total protein. Increasing TGF-b1 increased PTHrP secretion in a dosedependent fashion (p,0.0001). Secretion was greater at 12 and 24 hrs in cultures from term laboring pregnant women compared to term, non-laboring women and hysterectomy patients (* p,0.05). At 24 hrs, 1 ng/ml TGF-b1 increased PTHrP secretion from both LP and NP compared to H (** p,0.05). Bars 5 standard error.
had neither significant effect on cell number nor did it generate a dose-dependent increase in total cellular protein (data not shown). Effect of 17b-estradiol on PTHrP secretion The addition of 17b-estradiol to NP cultures for 24 hrs did not significantly alter the secretion of PTHrP as shown in figure 3. Pre-treatment of NP cultures with 10 nM 17b-estradiol also failed to alter the dose-response to TGF-b1. Effect of pregnancy on cAMP production Isoproterenol increased cAMP production in cultures by 4–5-fold but the maximal response was similar between the groups (LP: 24396467; NP: 22926315; H: 24426290 fmol/ 105 cells, NS). PTHrP also caused a dose-dependent increase in cAMP production across the groups (Fig. 2). Treatment of each group with 1027M (1-34)PTHrP increased cAMP production by an average of 64% over basal values (p,0.01) and although there was a trend toward higher cAMP levels in H, the differences did not reach statistical significance (LP: 704643; NP: 665692; H: 9086105 fmol/105 cells, NS at 1027M). Discussion Thiede and colleagues first reported the presence of PTHrP in the rat uterus a decade ago observing increased PTHrP mRNA in the myometrium during late gestation with vastly
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Fig. 2. Effect of 17b-estradiol on PTHrP secretion. Hysterectomy specimens were treated for 24 hrs with varying concentrations of 17b-estradiol and media assayed for PTHrP using IRMA and corrected to total protein. There was no difference in the amount of secreted PTHrP. Bars 5 standard error.
greater message in uterine horns containing fetuses [3]. The authors postulated that PTHrP was under the control of a local stimulus and that the peptide functioned in a paracrine or autocrine fashion to alter myometrial tone and/or blood flow [3,9]. Subsequent reports revealed that changes in PTHrP expression could be mimicked by the inflation of an intra-uterine balloon and by the administration of 17b-estradiol, implicating uterine stretch and steroid hormones as stimuli capable of altering myometrial PTHrP expression [15,16]. Physiologic studies solidified PTHrP as a smooth muscle relaxant demonstrating that the peptide prevents spontaneous and oxytocin-induced uterine contractions and induces vasodilation of the fetalplacental vessels [13,17,18]. The findings of the present study demonstrate that, in human USMC, basal PTHrP secretion and cAMP-stimulating capacity are unaltered by pregnancy and labor. Furthermore, the greater secretion of PTHrP from pregnant and pregnant laboring cells stimulated with TGF-b1 suggests that TGF-b1, in addition to stretch and steroid hormones, may be involved in altering PTHrP expression during pregnancy.
Fig. 3. Effect of TGF-b1 on 17b-estradiol primed USMC. USMC from hysterectomy patients were treated for 24 hrs with 10 nM 17b-estradiol and then incubated with varying concentrations of TGF-b1. Media derived from the cells was assayed for PTHrP using IRMA and values corrected to total monolayer protein. Increasing TGF-b1 increased PTHrP secretion in a dose-dependent fashion both in cells without 17b-estradiol (Control) and those with 17b-estradiol (p,0.0001). There was no effect, however, of 17b-estradiol on PTHrP secretion. Bars 5 standard error.
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Fig. 4. Effect of PTHrP on cAMP production. USMC from term laboring pregnant women, non-laboring women, and hysterectomy patients were incubated for 10 min with human (1-34)PTHrP. Cellular extracts were analyzed for cAMP via RIA and corrected to cell number. PTHrP increased cAMP production dose-dependently (p,0.0001), however there was no difference between groups. Bars 5 standard error.
PTHrP has been extracted from normal human uterine smooth muscle tissue and leiomyomas while PTHrP mRNA and secreted immunoreactive PTHrP have been observed in primary passage USMC cultures [11,12]. This study sought to confirm the ability of early passage USMC to secrete PTHrP and to test the effect of pregnancy and labor on this ability. Cell culture was chosen to evaluate PTHrP instead of whole tissue extraction because of the ability to study multiple pathways using the same specimen and to reduce the quantity of tissue needed. Using this protocol, we were unable to delineate a difference in basal PTHrP secretion between pregnant and non-pregnant or laboring and non-laboring tissues. Although this finding does not preclude the possibility that PTHrP mRNA is upregulated and not translated or secreted, it nonetheless signifies that additional factors are likely to be necessary to induce changes in PTHrP. One stimulus known to influence myometrial PTHrP expression is TGF-b1, a multifunctional cytokine which enhances PTHrP mRNA in keratinocytes, squamous carcinoma cells, and epidermal carcinoma cells [19–21]. Both TGF-b1 mRNA and protein have been documented within myometrial tissue using in-situ hybridization and immunohistochemistry [22]. Casey et al reported that treatment of myometrial cultures with TGF-b1 increased PTHrP mRNA and secreted PTHrP in a dose-dependent manner with maximal effects occurring following treatment with 1 ng/ml [11]. We also witnessed a dose- and time-dependent increase in secreted PTHrP following TGF-b1 treatment. Furthermore, TGF-b1-treated LP cells secreted more PTHrP than those from NP cells, which in turn, secreted more PTHrP than H cells. As such, the previously reported alterations in the rat myometrial PTHrP during pregnancy might reflect changes in TGF-b1. This scenario is supported by observations in both rats and humans that myometrial levels of TGF-b1 are gestationally regulated [23–25]. In fact, myometrial TGF-b1 levels are elevated during pregnancy and increased further in spontaneously laboring tissue [24]. Likewise, the expression of TGF-b receptor types I and II increase during pregnancy but are down-regulated at the onset of labor [24]. Ultimately then, TGF-b1 may enhance PTHrP mRNA transcription and/or mRNA stability as has been documented in W256 tumor cells [24,27]. Steroid hormones represent an additional pathway whereby PTHrP gene expression may be regulated. In humans, levels of estrogen increase progressively throughout gestation and
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remain high until delivery. Treatment of non-cycling rats treated with 17b-estradiol increases myometrial PTHrP mRNA and protein [9,15]. Estrogen is also known to increase the synthesis of TGF-b1 in human USMC [25]. Thus estrogen might increase PTHrP through either transcriptional upregulation of PTHrP mRNA or through increases in TGF-b1. To assess the involvement of these two pathways, we treated H cultures with 17b-estradiol for 24 h and studied basal and TGF-b1-stimulated PTHrP secretion. We found that incubation of H cell cultures with 17b-estradiol did not alter basal nor TGF-b1-stimulated secretion of immunoreactive PTHrP. While it is conceivable that the dose of 17b-estradiol used in the present study may have been ineffective at increasing PTHrP transcription, this same range of 17b-estradiol concentrations has been shown to increase PTHrP mRNA and protein secretion in MCF-7 breast cancer cells [28]. It is also possible that the upregulation of PTHrP mRNA is transient and that elevations in PTHrP occurred prior to 24 hrs. This would agree with findings that 17b-estradiol increases PTHrP mRNA maximally for only 4 hrs [9]. Alternatively, the lack of effect might relate to changes in TGF-b receptor density. In human myometrial cells, 17bestradiol has been found to decrease TGF-b receptor mRNA [25]. As a consequence, TGFb-stimulated PTHrP secretion would be attenuated, a trend which we observed, but one that did not reach statistical significance. Although the precise physiologic role of myometrial PTHrP is unclear, it is well known that the smooth muscle relaxant properties of PTHrP are linked to the generation of cAMP. In human uterine and rat aortic smooth muscle cells, (1-34)PTHrP stimulates a 2–3-fold increase in cAMP [12, 29]. Studies using isolated pig tracheal smooth muscle strips indicate that the relaxant effect of PTHrP is driven by cAMP via Ca21-activated potassium channels [30]. In the current study, PTHrP also increased cAMP production dose-dependently. Pregnancy and labor, on the other hand, tended to decrease the cAMP-stimulating ability of (1-34)PTHrP, although the trend did not reach statistical significance. Just after mid-gestation in the rat, the effects of PTHrP on myometrial motility also diminish, suggesting that the role of PTHrP within the myometrium is unlikely to promote uterine quiescence in the presence of the growing fetus [14]. Given that uterine artery blood flow increases 2.5-fold through the 38th week of gestation and that blood flow is interrupted by contractions, the finding of increased PTHrP secretion during late gestation and labor is more consistent with a role of PTHrP in the paracrine augmentation of uterine blood flow [31]. Conclusion In conclusion, our findings show that PTHrP is gestationally regulated in human myometrial cells. The mechanism of regulation is complex, involving secondary interactions with TGF-b, steroid hormones, and perhaps other undefined variables. Until studies are performed which directly measure myometrial PTHrP protein and mRNA in the human myometrium, however, the exact role of PTHrP during pregnancy will remain speculative. Acknowledgments The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other Departments of the U.S. Government.
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Pregnancy and labor increase the capacity of human myometrial cells to secrete parathyroid hormone-related protein Jeffrey S. Shenbergera,*, Patricia S. Dixonb, Jerome Choateb, Kenneth Helalc, Ronald L. Shewd, William Barthc a
Department of Pediatrics, University of Kentucky, 800 Rose Street, Lexington KY 40536-0084, USA b Clinical Investigations, 2200 Bergquist Drive, Wilford Hall USAF Medical Center, Lackland AFB, TX 78236-5300, USA c Department of Obstetrics and Gynecology, 2200 Bergquist Drive, Wilford Hall USAF Medical Center, Lackland AFB, TX 78236-5300, USA d Department of Anatomy and Cell Biology, Department of Anatomy and Cell Biology Medical Sciences Building 5035, Indiana University Medical Center, 635 Barnhill Drive, Indianapolis, IN 46202-5120, USA Received 25 May 2000; accepted 9 October 2000
Abstract Parathyroid hormone-related protein (PTHrP), a oncofetal gene product possessing smooth muscle relaxant properties, has been found in rat and human uterine smooth muscle cells (USMC) where it is postulated to regulate myometrial tone and/or blood flow. Studies investigating the gestational regulation of PTHrP in human USMC have not been performed. This study was conducted to determine if pregnancy alters the capacity of USMC to secrete or respond to PTHrP. USMC cultures were established from 8 hysterectomy specimens (H) and 7 non-laboring (NP) and 5 laboring term pregnant uterine biopsies (LP). PTHrP secretion was measured at baseline and in response to TGF-b1 using a immunoradiometric assay. The USMC response to PTHrP was assessed by incubating cultures with human (1-34)PTHrP and measuring cellular cAMP by radioimmunoassay. We found that cultures from the groups did not differ with respect to basal PTHrP secretion. TGF-b1, on the other hand, produced dose-dependent increases in secreted PTHrP in each group such that LP.NP.H at 12 hrs and LP.NP and H 24 hrs. Maximal responses were found at 24 hrs in cells treated with 10 ng/ml TGF-b1 (LP: 20346366 vs NP: 14856427; H: 12506202 fmol/mg). Incubation of cultures with PTHrP produced dose-dependent increases in cAMP production, with 1027M increasing levels by 64%. Neither pregnancy nor labor significantly affected the cAMP response. These findings indicate that the human myometrium has the capacity to increase PTHrP secretion during pregnancy and labor through a TGF-b-dependent pathway. Such findings are con* Corresponding author: Department of Pediatrics/Neonatology, MS-473 University of Kentucky Chandler Medical Center 800 Rose Street Lexington, KY 40536-0084. Tel.: 859-323-5530; fax: 859-257-4384. E-mail address: [email protected] (J.S. Shenberger) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 0 9 4 9 -3
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sistent with a role of PTHrP in enhancing uterine blood flow. rights reserved.
© 2001 Elsevier Science Inc. All
Keywords: Parathyroid hormone-related protein; Uterus; Smooth muscle; Pregnancy
Introduction Parathyroid hormone-related protein (PTHrP) is an oncofetal gene product originally identified as the source of humoral hypercalcemia of malignancy [1]. Although many tumor cells overexpress PTHrP, the peptide is normally produced by keratinocytes, epithelial cells, osteoblasts, and smooth muscle cells where it often functions in a “classical” endocrine fashion [2]. During pregnancy, PTHrP and PTH/PTHrP receptor protein and mRNA are found throughout the uterus, with an abundance located within the amnion, chorion, luminal epithelium, and myometrium [3–5]. In each uterine tissue location, the peptide is postulated to perform a unique physiologic function including the facilitation of calcium transport across the placenta, preparation and maintenance of the conceptus implantation site, and regulation of myometrial tone and blood flow [6–9]. In the rat uterus for instance, PTHrP mRNA increases in the longitudinal and circular smooth muscle layers late in pregnancy where its expression is enhanced by both uterine occupancy and the administration of 17b-estradiol [9,10]. Recently, PTHrP gene expression has also been documented in sheep, though the message is present in greater quantities preterm than term, suggesting significant inter-species variation [7]. In vitro, human myometrial cells secrete PTHrP, and when stimulated with TGF-b1, increase PTHrP gene expression and peptide secretion [11,12]. Data collected from isolated smooth muscle strip preparations indicate that PTHrP functions as a smooth muscle relaxant by decreasing resting myometrial tone and inhibiting spontaneous and oxytocin-induced uterine contractions [13,14]. Despite the animal studies detailing the gestational regulation of PTHrP, no human investigations have been performed to examine either the presence or regulation of PTHrP during pregnancy. Therefore, we conducted the present study to investigate PTHrP regulatory mechanisms in myometrial cell cultures established from non-pregnant, term pregnant non-laboring, and term pregnant laboring women. Our goals were to determine whether pregnancy and/or labor alter the capacity of human myometrial cells to 1) secrete PTHrP basally and in response to TGF-b1; 2) to determine if estrogen increases PTHrP protein secretion from nonpregnant cells; and 3) to assess the ability of myometrial cells to synthesize cAMP following PTHrP stimulation. We speculated that PTHrP secretion would be greater in the latter stages of pregnancy but that the cellular response to the peptide would be unchanged. Methods Tissue collection and establishment of cell cultures Myometrial specimens were obtained from 8 laboring pregnant (LP) and 8 non-laboring pregnant (NP) women at 37 weeks gestation or greater undergoing cesarean section at Wilford Hall Medical Center (WHMC) and from 8 non-pregnant (H), premenapausal women un-
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dergoing hysterectomy at Brook Army Medical Center (BAMC). Criteria for cesarean section and hysterectomy were at the discretion of the attending physician. Informed consent was obtained prior to surgery. Patients were excluded if they had clinical or laboratory evidence of infection, fetal distress, pre-eclampsia, placental pathology, or infiltrative myometrial carcinoma. The WHMC/BAMC Institutional Review Board approved the protocol. Full-thickness myometrial samples (130.530.5–1 cm) were obtained from the upper margin of the hysterotomy incision. Samples were immediately placed into cold Hank’s balanced saline solution and placed at 48C. Specimens were then placed in 10 ml of red cell lysis buffer [NH4CL (0.2M), KHCO3 (10mM), EDTA (0.13 mM), pH 7.4] for 5 min. Serosal tissue was removed and specimens minced and placed in DMEM supplemented with 10% fetal bovine serum containing 200 U/ml collagenase for 4 hrs at 378C. Dispersed cells were centrifuged and the resulting pellet resuspended in M3 media containing 10% FBS, HEPES buffer (25nM, pH 7.4), penicillin (200 U/ml), streptomycin (200 mg/ml), and kanamycin sulfate (200 mg/ml) and incubated in room air 1 5%CO2 at 378C. Purity of cultures Confluent uterine smooth muscle cells (USMC) grown on slides were incubated in M3 1 0.5% bovine serum albumin for 24 hrs. Slides were fixed for 5 min in 100% methanol at 2208C, treated with 0.1% Tween-20, rinsed, and blocked with 2% normal goat serum. Monolayers were incubated with mouse, monoclonal, anti-a-actin (1:100) and stained with goat, anti-mouse IgG-FITC (1:50). Slides were then examined under a fluorescent microscope (Nikon Optiphot, Tokyo, Japan). PTHrP secretion and TGF-b1 stimulation Cells were permitted to grow to 90% confluence, growth-arrested in M3 1 0.5% BSA for 24 hrs, and incubated with TGF-b1 (0.1–10 ng/ml) for 24 hrs. Following incubation, media was removed and stored at 2708C for PTHrP analysis. The remaining monolayer was dissolved in 0.5N NaOH, centrifuged, and assayed for total well protein using the bicinchoninic acid assay. PTHrP determinations were performed in duplicate using a immunoradiometric assay (IRMA) containing 125I-labelled goat anti-PTHrP(57-80) antibody and goat antiPTHrP(1-40) antibody bound to polystyrene beads. Samples were counted on a gamma counter and converted to PTHrP (pmol/L) using a standard curve derived from known amounts of (1-84)PTHrP. The sensitivity of the assay is 0.2 pmol/liter. Values were corrected to total protein from the corresponding well and expressed as the average from triplicate wells. Pilot studies revealed that 1 ng/ml of TGF-b1 did not substantially increase PTHrP secretion until 12 hrs, therefore the incubation times of 12 and 24 hrs were utilized. Effect of 17b-estradiol on PTHrP secretion To study the impact of 17b-estradiol on PTHrP secretion, 6 NP cultures were treated with 17b-estradiol (1–100 nM) after growth-arrest. In a second experiment, cultures were treated with 10 nM 17b-estradiol for 24 hrs. Following incubation, media was changed to fresh M3 1 10 nM 17b-estradiol 1 TGF-b1 (0.1–10 ng/ml). Secreted PTHrP was determined as previously described and corrected to total protein. Both experiments were performed using triplicate wells.
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cAMP determinations Cells in triplicate wells were growth-arrested in M3 1 0.5% BSA. Media was replaced with fresh M3 1 100mM iso-butyl-methylxanthine (IBMX) for 1 hr. Human (1-34)PTHrP (1029, 1028, 1027M) or 1026M isoproterenol was added and plates incubated for 10 min. Media was removed and monolayers extracted with ice-cold 65% ethanol. Extracts were added to the media and monolayers rinsed an additional time with ethanol. Combined extracts were then centrifuged at 20003g for 15 min, supernatants transferred to glass tubes, and evaporated under nitrogen. Dried extracts were dissolved in PBS and assayed in duplicate for cAMP using radioimmunoassay. Values were averaged and normalized to cell number from duplicate wells. Materials Human (1-34)PTHrP was attained from Calbiochem (La Jolla, CA). Immunoradiometric PTHrP assay kits were purchased from DiaSorin (Stillwater, MN) while cAMP RIA kits were obtained from Amersham (Arlington Hts, IL). Protein assay kits were purchased from Pierce (Rockford, IL) and all other chemicals and antibodies were obtained from Sigma (St. Louis, MO). Statistics Trends in PTHrP secretion, cAMP production, and 17b-estradiol were tested with repeated measures ANOVA and Newman-Keuls multiple comparison testing. The doseresponse of 17b-estradiol on basal PTHrP secretion was tested by one-way ANOVA. Maximal cAMP responses to isoproterenol were compared using Students’ t test. Data is listed as mean 6 standard error and the level of significance set at p,0.05. Results Cell cultures All H cultures grew avidly while only 7 of 8 NP and 5 of 8 LP grew well enough to utilize for experiments. In addition, a single NP culture did not grow during TGF-b1 assay and was omitted from analysis. All remaining monolayers attained the typical “hill and valley” appearance of smooth muscle cells when confluent and all stained positively for a-smooth muscle actin in greater than 90% of the cells. PTHrP secretion and TGF-b1 stimulation Basal secretion of PTHrP did not differ between groups at either 12 or 24 hrs. As shown in figure 1, however, increasing concentrations of TGF-b1 did increase PTHrP secretion (p,0.0001) and this effect was greater at 24 than 12 hrs (p,0.0001). Furthermore, the three groups differed in their responses to TGF-b1 (p,0.005) with LP cultures secreting more PTHrP than either H or NP following incubation with 1 and 10 ng/ml of TGF-b1 for 12 and 24 hrs. Maximal differences were noted with 10 ng/ml TGF-b1 at 24 hrs (LP: 20346366 vs NP: 14856427; H: 12506202 fmol/mg protein, p,0.01). Control studies revealed that TGF-b1
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Fig. 1. Effect of TGF-b1 on PTHrP secretion. Media from cells treated for 24 hrs with TGF-b1 was assayed for PTHrP by IRMA and values corrected to total protein. Increasing TGF-b1 increased PTHrP secretion in a dosedependent fashion (p,0.0001). Secretion was greater at 12 and 24 hrs in cultures from term laboring pregnant women compared to term, non-laboring women and hysterectomy patients (* p,0.05). At 24 hrs, 1 ng/ml TGF-b1 increased PTHrP secretion from both LP and NP compared to H (** p,0.05). Bars 5 standard error.
had neither significant effect on cell number nor did it generate a dose-dependent increase in total cellular protein (data not shown). Effect of 17b-estradiol on PTHrP secretion The addition of 17b-estradiol to NP cultures for 24 hrs did not significantly alter the secretion of PTHrP as shown in figure 3. Pre-treatment of NP cultures with 10 nM 17b-estradiol also failed to alter the dose-response to TGF-b1. Effect of pregnancy on cAMP production Isoproterenol increased cAMP production in cultures by 4–5-fold but the maximal response was similar between the groups (LP: 24396467; NP: 22926315; H: 24426290 fmol/ 105 cells, NS). PTHrP also caused a dose-dependent increase in cAMP production across the groups (Fig. 2). Treatment of each group with 1027M (1-34)PTHrP increased cAMP production by an average of 64% over basal values (p,0.01) and although there was a trend toward higher cAMP levels in H, the differences did not reach statistical significance (LP: 704643; NP: 665692; H: 9086105 fmol/105 cells, NS at 1027M). Discussion Thiede and colleagues first reported the presence of PTHrP in the rat uterus a decade ago observing increased PTHrP mRNA in the myometrium during late gestation with vastly
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Fig. 2. Effect of 17b-estradiol on PTHrP secretion. Hysterectomy specimens were treated for 24 hrs with varying concentrations of 17b-estradiol and media assayed for PTHrP using IRMA and corrected to total protein. There was no difference in the amount of secreted PTHrP. Bars 5 standard error.
greater message in uterine horns containing fetuses [3]. The authors postulated that PTHrP was under the control of a local stimulus and that the peptide functioned in a paracrine or autocrine fashion to alter myometrial tone and/or blood flow [3,9]. Subsequent reports revealed that changes in PTHrP expression could be mimicked by the inflation of an intra-uterine balloon and by the administration of 17b-estradiol, implicating uterine stretch and steroid hormones as stimuli capable of altering myometrial PTHrP expression [15,16]. Physiologic studies solidified PTHrP as a smooth muscle relaxant demonstrating that the peptide prevents spontaneous and oxytocin-induced uterine contractions and induces vasodilation of the fetalplacental vessels [13,17,18]. The findings of the present study demonstrate that, in human USMC, basal PTHrP secretion and cAMP-stimulating capacity are unaltered by pregnancy and labor. Furthermore, the greater secretion of PTHrP from pregnant and pregnant laboring cells stimulated with TGF-b1 suggests that TGF-b1, in addition to stretch and steroid hormones, may be involved in altering PTHrP expression during pregnancy.
Fig. 3. Effect of TGF-b1 on 17b-estradiol primed USMC. USMC from hysterectomy patients were treated for 24 hrs with 10 nM 17b-estradiol and then incubated with varying concentrations of TGF-b1. Media derived from the cells was assayed for PTHrP using IRMA and values corrected to total monolayer protein. Increasing TGF-b1 increased PTHrP secretion in a dose-dependent fashion both in cells without 17b-estradiol (Control) and those with 17b-estradiol (p,0.0001). There was no effect, however, of 17b-estradiol on PTHrP secretion. Bars 5 standard error.
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Fig. 4. Effect of PTHrP on cAMP production. USMC from term laboring pregnant women, non-laboring women, and hysterectomy patients were incubated for 10 min with human (1-34)PTHrP. Cellular extracts were analyzed for cAMP via RIA and corrected to cell number. PTHrP increased cAMP production dose-dependently (p,0.0001), however there was no difference between groups. Bars 5 standard error.
PTHrP has been extracted from normal human uterine smooth muscle tissue and leiomyomas while PTHrP mRNA and secreted immunoreactive PTHrP have been observed in primary passage USMC cultures [11,12]. This study sought to confirm the ability of early passage USMC to secrete PTHrP and to test the effect of pregnancy and labor on this ability. Cell culture was chosen to evaluate PTHrP instead of whole tissue extraction because of the ability to study multiple pathways using the same specimen and to reduce the quantity of tissue needed. Using this protocol, we were unable to delineate a difference in basal PTHrP secretion between pregnant and non-pregnant or laboring and non-laboring tissues. Although this finding does not preclude the possibility that PTHrP mRNA is upregulated and not translated or secreted, it nonetheless signifies that additional factors are likely to be necessary to induce changes in PTHrP. One stimulus known to influence myometrial PTHrP expression is TGF-b1, a multifunctional cytokine which enhances PTHrP mRNA in keratinocytes, squamous carcinoma cells, and epidermal carcinoma cells [19–21]. Both TGF-b1 mRNA and protein have been documented within myometrial tissue using in-situ hybridization and immunohistochemistry [22]. Casey et al reported that treatment of myometrial cultures with TGF-b1 increased PTHrP mRNA and secreted PTHrP in a dose-dependent manner with maximal effects occurring following treatment with 1 ng/ml [11]. We also witnessed a dose- and time-dependent increase in secreted PTHrP following TGF-b1 treatment. Furthermore, TGF-b1-treated LP cells secreted more PTHrP than those from NP cells, which in turn, secreted more PTHrP than H cells. As such, the previously reported alterations in the rat myometrial PTHrP during pregnancy might reflect changes in TGF-b1. This scenario is supported by observations in both rats and humans that myometrial levels of TGF-b1 are gestationally regulated [23–25]. In fact, myometrial TGF-b1 levels are elevated during pregnancy and increased further in spontaneously laboring tissue [24]. Likewise, the expression of TGF-b receptor types I and II increase during pregnancy but are down-regulated at the onset of labor [24]. Ultimately then, TGF-b1 may enhance PTHrP mRNA transcription and/or mRNA stability as has been documented in W256 tumor cells [24,27]. Steroid hormones represent an additional pathway whereby PTHrP gene expression may be regulated. In humans, levels of estrogen increase progressively throughout gestation and
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remain high until delivery. Treatment of non-cycling rats treated with 17b-estradiol increases myometrial PTHrP mRNA and protein [9,15]. Estrogen is also known to increase the synthesis of TGF-b1 in human USMC [25]. Thus estrogen might increase PTHrP through either transcriptional upregulation of PTHrP mRNA or through increases in TGF-b1. To assess the involvement of these two pathways, we treated H cultures with 17b-estradiol for 24 h and studied basal and TGF-b1-stimulated PTHrP secretion. We found that incubation of H cell cultures with 17b-estradiol did not alter basal nor TGF-b1-stimulated secretion of immunoreactive PTHrP. While it is conceivable that the dose of 17b-estradiol used in the present study may have been ineffective at increasing PTHrP transcription, this same range of 17b-estradiol concentrations has been shown to increase PTHrP mRNA and protein secretion in MCF-7 breast cancer cells [28]. It is also possible that the upregulation of PTHrP mRNA is transient and that elevations in PTHrP occurred prior to 24 hrs. This would agree with findings that 17b-estradiol increases PTHrP mRNA maximally for only 4 hrs [9]. Alternatively, the lack of effect might relate to changes in TGF-b receptor density. In human myometrial cells, 17bestradiol has been found to decrease TGF-b receptor mRNA [25]. As a consequence, TGFb-stimulated PTHrP secretion would be attenuated, a trend which we observed, but one that did not reach statistical significance. Although the precise physiologic role of myometrial PTHrP is unclear, it is well known that the smooth muscle relaxant properties of PTHrP are linked to the generation of cAMP. In human uterine and rat aortic smooth muscle cells, (1-34)PTHrP stimulates a 2–3-fold increase in cAMP [12, 29]. Studies using isolated pig tracheal smooth muscle strips indicate that the relaxant effect of PTHrP is driven by cAMP via Ca21-activated potassium channels [30]. In the current study, PTHrP also increased cAMP production dose-dependently. Pregnancy and labor, on the other hand, tended to decrease the cAMP-stimulating ability of (1-34)PTHrP, although the trend did not reach statistical significance. Just after mid-gestation in the rat, the effects of PTHrP on myometrial motility also diminish, suggesting that the role of PTHrP within the myometrium is unlikely to promote uterine quiescence in the presence of the growing fetus [14]. Given that uterine artery blood flow increases 2.5-fold through the 38th week of gestation and that blood flow is interrupted by contractions, the finding of increased PTHrP secretion during late gestation and labor is more consistent with a role of PTHrP in the paracrine augmentation of uterine blood flow [31]. Conclusion In conclusion, our findings show that PTHrP is gestationally regulated in human myometrial cells. The mechanism of regulation is complex, involving secondary interactions with TGF-b, steroid hormones, and perhaps other undefined variables. Until studies are performed which directly measure myometrial PTHrP protein and mRNA in the human myometrium, however, the exact role of PTHrP during pregnancy will remain speculative. Acknowledgments The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other Departments of the U.S. Government.
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