0021-972x/92/7404-0950$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
TRANSFORMING HUMAN UTERINE
GROWTH FACTOR-& CELLS IN CULTURE:
Casey, Masato Mibe, Ahmet
STIMULATION OF PARATHYROID HORMONE-RELATED mRNA LEVELS AND PROTEIN SECRETION’
PROTEIN
EXPRESSION
IN
Erk, and Paul C. MacDonald
The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Deparmtents University of Texas Southwestern Medical Center, Dallas, Texas USA.
of Obstetrics-Gynecology
and Biochemis0y,
The
ABSTRACT In this study, we found that human myometrial smooth muscle cells and endometrial stromal cells in monolayer culture express parathyroid hormone-related protein (PTH-rP) mRNA and secrete immunoreactive and bioactive PTH-rP into the culture medium. The levels of PTH-rP mRNA in both of these cell types increased in response to treatment with transforming growth factor-/3 as did the amounts of immunoreactive and bioactive PTH-rP secreted into the culture medium. INTRODUCTION We evaluated the potential for transforming growth factor-p (TGF;B) stimulation of parathyroid hormone-related protein (PTHrP) gene expression and protein secretion by human uterine cells in monolayer culture, viz., myometrial smooth muscle cells and endometrial stromal cells. We were prompted to conduct these studies by the intriguing findings of Thiede and colleagues (l-3) that PTH-rP is expressed in the uterus of estrogen-treated rats and in the avian oviduct. The levels of PTH-rP mRNA during rat pregnancy were much greater in uterine horns occupied by fetuses. We found that both human myometrial smooth muscle and endometrial stromal cells express PTH-rP mRNA and secrete PTH-rP into the medium. The level of mRNA for PTH-rP in both of these cell types increased in response to treatment with TGF;B. The amount of immunoreactive and bioactive PTH-rP secreted into the culture medium of these uterine cells also increased in response to TGF-P. MATERIALS AND METHODS Human PTH-rP l-34 was from Peptides Int. (Louisville, KY) or Peninsula Laboratories (Belmont, CA). TGF-P (from human platelets) was from R & D Systems (Minneapolis, MN); isobutylmethyLvanthine (IBMX) and tetradecanoylphorbol acetate (TPA) were from Sigma Chem. Co. (St. Louis, MO). Cells in culture. Tissues were obtained from uteri of nonpregnant women after hysterectomy. The consent form and protocol for use of human tissues were approved by the IRB of this University. Myometrial smooth muscle cells (4) and endometrial stromal cells (5) were isolated by enzymatic digestion as described and cultured in Ham F12:Dulbecco minimal essential medium (l:l, v/v) that contained fetal bovine serum (FBS, lo%, v/v), Hepes buffer (25 mM, pH 7.4) penicillin (200 U/ml), streptomycin (200 &ml), and kanamycin sulfate (200 &ml) (“Fl2:DMEM with serum”). Confluent, first passage cells were used for all experiments. Human sex skin (foreskin) fibroblasts, provided to us by Dr. James E. Griffin of this University, were maintained in F12:DMEM with serum. MCF-7 cells, from the American Type Culture Collection, were maintained in Eagle MEM that contained FBS (lo%, v/v), insulin (10 @ml), and antibiotics-antimycotics; 24 h prior to the commencement of a given treatment paradigm, the culture medium was changed to serum-free medium (SFM). The cells were treated with TGF-P or TPA, in various concentrations, for 2 to 48 h. Thereafter, the culture medium was stored at -80 C prior to RIA and bioassay of PTH-rP or isolation of RNA, data are expressed as fmol PTH-rP 1-34 and are normalized to the amount of total cell protein. Preparation of RNA and northern analyses. Total RNA was prepared by the acid guanidium thiocyanate-phenol-chlororform extraction method of Chomczynski and Sacchi (6) with modifications (7), size-fractionated by electrophoresis on 1% formaldehyde-agarose gels, transferred electrophoretically to nylon membrane, and crosslinked to the membrane by ultraviolet irradiation. Prehybridization was conducted for 16 h at 65 C in buffer [formamide (50%, v/v), NaCI (0.25 M), NasHPOd (0.25 M, pH 7.2) EDTA (1 mM), and SDS (7%, w/v)]. Hybridization was conducted for 16 h at 43 C in this buffer with a human PTH-rP cDNA probe (537 bp corresponding to coding region, kindly provided to us by Dr. Geoffrey N. Hendy, McGill University, Montreal, Quebec, Canada) radiolabeled with [32P]dCTP by random hexamer priming. Blots were washed with 2x SSC and SDS (O.l%, w/v) for 15 min at 25 C, twice with 0.1x SSC and SDS (O.l%, w/v) Submitted November
18,199l
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for 10 min at 25 C and once for 15 min at 43 C. Autoradiography was performed at -80 C with Kodak X-Omat AR film. The presence of .similar amounts of total RNA in each lane was verified by visualization of 28s and 18s rRNA subunits or by analysis of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA using a specific 24-mer oligonucleotide probe (5’-TCT AGA CGG CAG GTC AGG TCC ACC-3’) end-labeled with [+P]ATP. Radioimmunoassay of PTH-rP. Immunoreactive PTH-rP (irPTH-rP) in the culture medium of myometrial smooth muscle cells and endometrial stromal cells was quantified by RIA (Peninsula Laboratories, Inc., Belmont, CA) using culture medium as the diluent and human PTH-rP 1-34 as the standard. The lower limit of the RIA was 0.5-l fmol PTH-rP per assay tube. Aliquots of 50 or 100 ~1 of test media were used. The intraassay coefficient of variation was 9.6%. Linearity of the RIA was demonstrated using The crossvarious amounts of conditioned culture medium. reactivity of human PTH 1-34 is < 1%. Quan@cation of biaactive PTH-rP. Bioactive PTH-rP in culture medium of myometrial and endometrial stromal cells in culture was assessed by evaluating the effectiveness of these conditioned media in stimulating CAMP production by UMR-106 cells (a rat osteoblastlike cell line that is responsive lo PTH-rP and PTH). Confluent UMR-106 cells [obtained from The American Type Culture Collection (Rockville Pike, MR) and maintained in F12:DMEM with serum] in 24-well plastic plates were treated with SFM that contained bovine serum albumin (O.l%, w/v) for 18-24 h prior to use. For bioassay, UMR-106 cells were treated for 15 min at 37 C with conditioned culture medium from treated cells to which IBMX (100 FM) was added; in each assay, some cells were treated with authentic PTH-rP 1-34 (plus IBMX). Thereafter, the cells were sonicated in ice-cold aqueous ethanol (65%, v/v). Cell protein in an aliquot of the sonicate (after vacuum evaporation of the solvent) was quantified by the method of Lowry et al. (8) The sonicates were centrifuged at 14,000 xg for 15 min at 4 C. After acetylation, CAMP was quantified in aliquots of the supernate by use of RIA kits from Amersham Corp. (Arlington Heights, IL) or Advanced Magnetics (Cambridge, MA). The lower limit of the RIA was approximately 2 fmol CAMP/tube; the intraassay coefficient of variation was < 10%. RESULTS PTH-rP mRNA was demonstrable in human myometrial smooth muscle cells and endometrial stromal cells in monolayer culture; and, irPTH-rP was produced by both types of cells. Treatment of cells of either type with TGF-P (1 ngiml) led to an increase in PTHrP mRNA levels (Fig. 1).
CT
CT
Figure 1. PTH-rP mRNA in human myometrial smooth muscle cells (M) and endometrial stromal cells (E) with (T) or without (C) TGFp treatment (1 “g/ml; 8 h); 2Opg total RNA/lane.
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/950/3004864 by Medizinische Lesehalle user on 03 February 2019
M. Linette
Vol. 74, No. 4 Printed in U.S.A.
RAPID
COMMUNICATION
951
The predominant mRNA species in both cell types was - 1.8 kb in length; other minor species also were demonstrable (data not shown). The level of irPTH-rP in the culture medium was increased by treatment with TGF-P (Fig. 2).
CTL x-+6
Figure 2. Production of irPTH-rP by human myometrial cells and endometrial stromal cells in monolayer culture. Confluent cells in SFM were treated with TGF-P (in various concentrations) for 24 h. PTH-rP in the culture medium was quantified by RIA. The level of PTH-rP mRNA was increased somewhat after 2-4 h of TGF-P treatment; much greater levels of PTH-rP mRNA were apparent after 8-24 h of TGF-p treatment (Fig. 3).
3. Time course of induction of PTH-rP mRNA in endometrial stromal cells by TGF-/3 (1 @ml); total RNA (15 pg/lane); control (C) and TGF-P-treatment (T); bars (left) denote location of 28s and 1% rRNA subunits. Figure
In myometrial cells treated with TGF-P, irPTH-rP accumulated in the culture medium to levels that were maximal at - 12 h and maintained to 48 h (Fig. 4). Treatment of myometrial cells with TPA (lo-lo-10.7M) caused an increase in irPTH-rP and PTH-rP mRNA levels (Fig. 5).
0 1 --
1 10 TPA (nM)---
100
Figure 5. Left panel: Accumulation of irPTH-rP in culture medium of myometrial cells treated with TPA (in various concentrations) or TGF-P (1 &ml) for 24 h. Right panel: Effect of TPA on PTH-rP mRNA (top panel) or G3PDH mRNA (bottom panel) levels in myometrial cells. Confluent cells were treated with vehicle alone (lane A) or TPA (lo-sM, lane B or IO-sM, lane C) for 24 h; 20 pg total RNA per lane. The PTH-rP-like bioactivity produced by these cells was assessed using activation of adenylate cyclase in UMR-106 cells as the bioassay. Conditioned medium from myometrial cells treated with TGF-P (1 r&ml) for 24 h was more effective in stimulating cyclic AMP production by UMR-106 cells (216 f 51 pmol cAMP/mg UMR cell protein) than was conditioned medium from nontreated cells (69 f 7, ~~0.04, n=4); likewise, conditioned medium from TGF-p-treated endometrial stromal cells was more effective (217 + 36 pmol/mg protein) than conditioned medium from controls (57 + 6 pmol/mg protein; p
TRANSFORMING HUMAN UTERINE
GROWTH FACTOR-& CELLS IN CULTURE:
Casey, Masato Mibe, Ahmet
STIMULATION OF PARATHYROID HORMONE-RELATED mRNA LEVELS AND PROTEIN SECRETION’
PROTEIN
EXPRESSION
IN
Erk, and Paul C. MacDonald
The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Deparmtents University of Texas Southwestern Medical Center, Dallas, Texas USA.
of Obstetrics-Gynecology
and Biochemis0y,
The
ABSTRACT In this study, we found that human myometrial smooth muscle cells and endometrial stromal cells in monolayer culture express parathyroid hormone-related protein (PTH-rP) mRNA and secrete immunoreactive and bioactive PTH-rP into the culture medium. The levels of PTH-rP mRNA in both of these cell types increased in response to treatment with transforming growth factor-/3 as did the amounts of immunoreactive and bioactive PTH-rP secreted into the culture medium. INTRODUCTION We evaluated the potential for transforming growth factor-p (TGF;B) stimulation of parathyroid hormone-related protein (PTHrP) gene expression and protein secretion by human uterine cells in monolayer culture, viz., myometrial smooth muscle cells and endometrial stromal cells. We were prompted to conduct these studies by the intriguing findings of Thiede and colleagues (l-3) that PTH-rP is expressed in the uterus of estrogen-treated rats and in the avian oviduct. The levels of PTH-rP mRNA during rat pregnancy were much greater in uterine horns occupied by fetuses. We found that both human myometrial smooth muscle and endometrial stromal cells express PTH-rP mRNA and secrete PTH-rP into the medium. The level of mRNA for PTH-rP in both of these cell types increased in response to treatment with TGF;B. The amount of immunoreactive and bioactive PTH-rP secreted into the culture medium of these uterine cells also increased in response to TGF-P. MATERIALS AND METHODS Human PTH-rP l-34 was from Peptides Int. (Louisville, KY) or Peninsula Laboratories (Belmont, CA). TGF-P (from human platelets) was from R & D Systems (Minneapolis, MN); isobutylmethyLvanthine (IBMX) and tetradecanoylphorbol acetate (TPA) were from Sigma Chem. Co. (St. Louis, MO). Cells in culture. Tissues were obtained from uteri of nonpregnant women after hysterectomy. The consent form and protocol for use of human tissues were approved by the IRB of this University. Myometrial smooth muscle cells (4) and endometrial stromal cells (5) were isolated by enzymatic digestion as described and cultured in Ham F12:Dulbecco minimal essential medium (l:l, v/v) that contained fetal bovine serum (FBS, lo%, v/v), Hepes buffer (25 mM, pH 7.4) penicillin (200 U/ml), streptomycin (200 &ml), and kanamycin sulfate (200 &ml) (“Fl2:DMEM with serum”). Confluent, first passage cells were used for all experiments. Human sex skin (foreskin) fibroblasts, provided to us by Dr. James E. Griffin of this University, were maintained in F12:DMEM with serum. MCF-7 cells, from the American Type Culture Collection, were maintained in Eagle MEM that contained FBS (lo%, v/v), insulin (10 @ml), and antibiotics-antimycotics; 24 h prior to the commencement of a given treatment paradigm, the culture medium was changed to serum-free medium (SFM). The cells were treated with TGF-P or TPA, in various concentrations, for 2 to 48 h. Thereafter, the culture medium was stored at -80 C prior to RIA and bioassay of PTH-rP or isolation of RNA, data are expressed as fmol PTH-rP 1-34 and are normalized to the amount of total cell protein. Preparation of RNA and northern analyses. Total RNA was prepared by the acid guanidium thiocyanate-phenol-chlororform extraction method of Chomczynski and Sacchi (6) with modifications (7), size-fractionated by electrophoresis on 1% formaldehyde-agarose gels, transferred electrophoretically to nylon membrane, and crosslinked to the membrane by ultraviolet irradiation. Prehybridization was conducted for 16 h at 65 C in buffer [formamide (50%, v/v), NaCI (0.25 M), NasHPOd (0.25 M, pH 7.2) EDTA (1 mM), and SDS (7%, w/v)]. Hybridization was conducted for 16 h at 43 C in this buffer with a human PTH-rP cDNA probe (537 bp corresponding to coding region, kindly provided to us by Dr. Geoffrey N. Hendy, McGill University, Montreal, Quebec, Canada) radiolabeled with [32P]dCTP by random hexamer priming. Blots were washed with 2x SSC and SDS (O.l%, w/v) for 15 min at 25 C, twice with 0.1x SSC and SDS (O.l%, w/v) Submitted November
18,199l
950
for 10 min at 25 C and once for 15 min at 43 C. Autoradiography was performed at -80 C with Kodak X-Omat AR film. The presence of .similar amounts of total RNA in each lane was verified by visualization of 28s and 18s rRNA subunits or by analysis of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA using a specific 24-mer oligonucleotide probe (5’-TCT AGA CGG CAG GTC AGG TCC ACC-3’) end-labeled with [+P]ATP. Radioimmunoassay of PTH-rP. Immunoreactive PTH-rP (irPTH-rP) in the culture medium of myometrial smooth muscle cells and endometrial stromal cells was quantified by RIA (Peninsula Laboratories, Inc., Belmont, CA) using culture medium as the diluent and human PTH-rP 1-34 as the standard. The lower limit of the RIA was 0.5-l fmol PTH-rP per assay tube. Aliquots of 50 or 100 ~1 of test media were used. The intraassay coefficient of variation was 9.6%. Linearity of the RIA was demonstrated using The crossvarious amounts of conditioned culture medium. reactivity of human PTH 1-34 is < 1%. Quan@cation of biaactive PTH-rP. Bioactive PTH-rP in culture medium of myometrial and endometrial stromal cells in culture was assessed by evaluating the effectiveness of these conditioned media in stimulating CAMP production by UMR-106 cells (a rat osteoblastlike cell line that is responsive lo PTH-rP and PTH). Confluent UMR-106 cells [obtained from The American Type Culture Collection (Rockville Pike, MR) and maintained in F12:DMEM with serum] in 24-well plastic plates were treated with SFM that contained bovine serum albumin (O.l%, w/v) for 18-24 h prior to use. For bioassay, UMR-106 cells were treated for 15 min at 37 C with conditioned culture medium from treated cells to which IBMX (100 FM) was added; in each assay, some cells were treated with authentic PTH-rP 1-34 (plus IBMX). Thereafter, the cells were sonicated in ice-cold aqueous ethanol (65%, v/v). Cell protein in an aliquot of the sonicate (after vacuum evaporation of the solvent) was quantified by the method of Lowry et al. (8) The sonicates were centrifuged at 14,000 xg for 15 min at 4 C. After acetylation, CAMP was quantified in aliquots of the supernate by use of RIA kits from Amersham Corp. (Arlington Heights, IL) or Advanced Magnetics (Cambridge, MA). The lower limit of the RIA was approximately 2 fmol CAMP/tube; the intraassay coefficient of variation was < 10%. RESULTS PTH-rP mRNA was demonstrable in human myometrial smooth muscle cells and endometrial stromal cells in monolayer culture; and, irPTH-rP was produced by both types of cells. Treatment of cells of either type with TGF-P (1 ngiml) led to an increase in PTHrP mRNA levels (Fig. 1).
CT
CT
Figure 1. PTH-rP mRNA in human myometrial smooth muscle cells (M) and endometrial stromal cells (E) with (T) or without (C) TGFp treatment (1 “g/ml; 8 h); 2Opg total RNA/lane.
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/950/3004864 by Medizinische Lesehalle user on 03 February 2019
M. Linette
Vol. 74, No. 4 Printed in U.S.A.
RAPID
COMMUNICATION
951
The predominant mRNA species in both cell types was - 1.8 kb in length; other minor species also were demonstrable (data not shown). The level of irPTH-rP in the culture medium was increased by treatment with TGF-P (Fig. 2).
CTL x-+6
Figure 2. Production of irPTH-rP by human myometrial cells and endometrial stromal cells in monolayer culture. Confluent cells in SFM were treated with TGF-P (in various concentrations) for 24 h. PTH-rP in the culture medium was quantified by RIA. The level of PTH-rP mRNA was increased somewhat after 2-4 h of TGF-P treatment; much greater levels of PTH-rP mRNA were apparent after 8-24 h of TGF-p treatment (Fig. 3).
3. Time course of induction of PTH-rP mRNA in endometrial stromal cells by TGF-/3 (1 @ml); total RNA (15 pg/lane); control (C) and TGF-P-treatment (T); bars (left) denote location of 28s and 1% rRNA subunits. Figure
In myometrial cells treated with TGF-P, irPTH-rP accumulated in the culture medium to levels that were maximal at - 12 h and maintained to 48 h (Fig. 4). Treatment of myometrial cells with TPA (lo-lo-10.7M) caused an increase in irPTH-rP and PTH-rP mRNA levels (Fig. 5).
0 1 --
1 10 TPA (nM)---
100
Figure 5. Left panel: Accumulation of irPTH-rP in culture medium of myometrial cells treated with TPA (in various concentrations) or TGF-P (1 &ml) for 24 h. Right panel: Effect of TPA on PTH-rP mRNA (top panel) or G3PDH mRNA (bottom panel) levels in myometrial cells. Confluent cells were treated with vehicle alone (lane A) or TPA (lo-sM, lane B or IO-sM, lane C) for 24 h; 20 pg total RNA per lane. The PTH-rP-like bioactivity produced by these cells was assessed using activation of adenylate cyclase in UMR-106 cells as the bioassay. Conditioned medium from myometrial cells treated with TGF-P (1 r&ml) for 24 h was more effective in stimulating cyclic AMP production by UMR-106 cells (216 f 51 pmol cAMP/mg UMR cell protein) than was conditioned medium from nontreated cells (69 f 7, ~~0.04, n=4); likewise, conditioned medium from TGF-p-treated endometrial stromal cells was more effective (217 + 36 pmol/mg protein) than conditioned medium from controls (57 + 6 pmol/mg protein; p
