Human Myometrial Smooth Muscle Cells Are Novel Targets of Direct ...

Download PDF
16 downloads 17 Views 922KB Size Report
Comment
Department of Obstetrics and Gynecology, University of Louisville, School of Medicine. Louisville, Kentucky 40292. ABSTRACT. Human myometrium contains ...
BIOLOGY OF REPRODUCTION 49, 1149-1157 (1993)

Human Myometrial Smooth Muscle Cells Are Novel Targets of Direct Regulation by Human Chorionic Gonadotropin' J.L. KORNYEI, 3 Z.M. LEI, and Ch.V. RAO 2 Department of Obstetrics and Gynecology, University of Louisville, School of Medicine Louisville, Kentucky 40292 ABSTRACT Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind ' 51-hCG in a ligand-specific manner. The presence ofhCG during culture resulted in a significant increase ofmyometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density. In summary, our results demonstrate that hCG can directly regulate myometrial smooth muscle cells, causing hyperplasia as well as hypertrophy.

INTRODUCTION

MATERIALS AND METHODS

Human CG and human LH (hLH) belong to a family of glycoprotein hormones that contain noncovalently bound dissimilar at and [3 subunits [1]. Among this family, hCG and hLH bear a significant structural and functional homology [1]. These two hormones bind to the same receptors, which have recently been cloned [2-5]. For a long time, these receptors have been thought to be present only in gonadal tissues. It is now known that several nongonadal human reproductive tissues, including brain, also contain these receptors [6-14]. Recent studies have shown that hCG can directly regulate the functions of several nongonadal reproductive tissues, indicating that the receptors are functional in these tissues [13-19]. Human myometrium is one of the nongonadal reproductive tissues containing hCG/LH receptors [6]. However, it has not been known whether hCG could directly regulate human myometrial functions. We investigated in the present studies whether hCG can directly regulate in vitro proliferation of human myometrial smooth muscle cells. The results demonstrate that hCG can indeed increase myometrial smooth muscle cell numbers as well as the size of a subpopulation of cells. Accepted July 28, 1993. Received May 27, 1993. 'This work was supported by NIH grant HD26173. 2Correspondence: Dr. Ch.V. Rao, Department of Obstetrics and Gynecology, 438 MDR Bldg., University of Louisville, Louisville, KY 40292. FAX: (502) 588-0881. c urrent address: Institute of Physiology, University Medical School of Pecs, Pcs, H-7643, Hungary.

Materials

Type XI collagenase, type I DNase, HEPES, fetal bovine serum (FBS), minimum essential medium (MEM) amino acids, MEM nonessential amino acids, MEM vitamins, kanamycin, indomethacin, nordihydroguaiaretic acid (NDGA), monoclonal anti-a smooth muscle actin antibody, anti-hCG and antichicken IgG rabbit antisera, estradiol, progesterone, and cell dissociation sieves (60 mesh, 0.23- mm opening) were purchased from Sigma Chemical Co. (St. Louis, MO); antibioticantimycotic solution and trypsin-EDTA solution from Gibco (Grand Island, NY); basic Waymouth's medium (WM) from ICN (Costa Mesa, CA); monoclonal antibody specific to type I collagen from Southern Biotech (Birmingham, AL); monoclonal antibody to macrophage CD68 antigen from Dako Corp. (Carpenteria, CA); monoclonal antibody to endothelial cell CD34 antigen from Serotec (Kidlington, UK). The commercial sources of other reagents are the same as previously described [5, 6, 12]. The following items were obtained as gifts: highly purified hCG (CR-123, 12 780 IU/mg; CR-127, 14 900 IU/mg), hCGa (CR-123, 27 IU/mg), and hCG-3 (CR-123, 13 IU/mg) subunits; human LH (AFP-0624B; 4015 IU/mg); human FSH (hFSH) (AFP-8792B, 1683 IU/mg); human thyroid stimulating hormone (TSH) (AFP-4314C, 15 IU/mg); human prolactin (PRL) (AFP-8982C, 40 IU/mg); and human growth hormone (GH) (AFP-9755A, 2.4 IU/mg) from NIDDK, NHPP, University of Maryland School of Medicine (Baltimore, MD).

1149

1150

KORNYEI ET AL.

Highly purified hCG (1M22[33], 12 000 IU/mg) with a very low degree of peptide bond nicking (< 3%) was a gift from Professor Wolfgang Merz, University of Heidelberg (Heidelberg, Germany); the polyclonal hCG/LH receptor antibodies raised against synthetic N-terminal rat receptor sequences of 1-11 and 15-38 was from Dr. Patrick Roche of the Mayo Clinic (Rochester, MN); full-length porcine hCG/LH receptor cDNA in pBluescript (SK+) was from Dr. Hugues Loosfelt from Hormones and Reproduction, Hopital de Bicetre (Bicetre, France); and recombinant human epidermal growth factor (EGF) was from Dr. Pablo Valenzuela of Chiron Corp. (Emeryville, CA). Tissue Collection Myometrial tissue from premenopausal women undergoing hysterectomy (for uterine prolapse, fibroids, etc.), with no history of hormonal treatment or any other uterine disease prior to surgery, were obtained from the University of Louisville Affiliated Hospitals. The use of the tissues was approved by our institutional Human Studies Committee. Immediately after collection, the tissues were placed in ice-cold Hanks' balanced salt solution (pH 7.4) containing 2% HEPES and 2% antibiotic-antimycotic solution (referred to hereafter as Hanks' medium) and transported immediately to the laboratory. Sterile conditions were maintained from the time of tissue collection until the end of all the experiments. Cell Dispersion The method of Casey et al. [20] with modifications was used for dispersion of myometrial smooth muscle cells. Up to 1 g of myometrial smooth muscle was cut from the interior of myometrium and rinsed for 30 sec with 70% alcohol, washed with Hanks' medium, and minced into fragments of approximately 2-3 mm 3 with the use of two scalpels. About 50 tissue fragments were digested in two 150-cm 2 Corning (Dow Corning, Midland, MI) cell culture flasks for 18 h at 37°C under 5% CO2 in 50 ml of Hanks' medium containing 2 mg/ml type XI collagenase and 0.01 mg/ml type I DNase. At the end of digestion, the tissue fragments were pressed, pestled, filtered through cell-dissociation sieves, and washed with Hanks' medium. The filtrate was centrifuged for 5 min at 300 x g; the cell pellets were washed three times with Hanks' medium and suspended in 100 ml of basic WM containing 10% FBS, 2% antibiotic-antimycotic solution, 1% sodium pyruvate, 2% MEM amino acids, 1% MEM nonessential amino acids, 1% MEM vitamins, 0.2 mg/ml kanamycin, 2% HEPES, and 0.2 nM estradiol (referred to as culture medium). The culture medium containing cells was equally divided and placed into two 150cm2 plastic culture flasks, then incubated in a CO 2 incubator. Immediately after cell dissociation, the cell viability was only 1-5%. This low viability was expected because of the harshness of the digestion procedure. During culture, the cell viability was greater than 95% because all the dead cells were removed during an initial 24-h period that was allowed for

attachment of cells. The cells that survived and grew were myometrial smooth muscle cells (99%) as determined by immunocytochemistry. Cell viability was determined by Trypan Blue exclusion. The yield of viable cells after 10-14 days of culture was 30-40 million. Several monolayer smooth muscle cell cultures from different tissue specimens were successfully established and kept for more than 6 mo without any significant change in morphology or growth rate. Determination of cell viability during the culture period required detachment of subconfluent cells by decanting the medium and washing the cells three times with 10 ml of Hanks' balanced salt solution containing no Ca 2+ or Mg2 +. Then 7 ml of ice-cold trypsin-EDTA solution was added and the flasks were rolled around gently to cover the whole monolayer area. The enzyme solution was then quickly removed and the culture flasks were placed in the incubator for 3 min. Cell detachment, which sometimes was aided by sharp raps on the edges of the culture flasks, was monitored under a microscope. After complete detachment, trypsin action was blocked by addition of 5 ml of ice-cold FBS. Then the flasks were sequentially rinsed with 30 ml and 10 ml of ice-cold Hanks' balanced salt solution. This cell suspension was centrifuged for 5 min at 300 x g and the pellet was washed three more times. Determinationof Purity of Cells Purity was determined by immunocytochemistry using the anti-oa smooth muscle actin antibody for myometrial smooth muscle cells, anti-type I collagen antibody for fibroblasts, antiCD68 antibody for macrophages, and anti-CD34 antibody for endothelial cells. Whole myometrial tissues were concurrently immunostained for comparison with cultured cells. While the cultured cells did not immunostain with antibodies specific for fibroblasts, macrophages, or endothelial cells, greater than 99% of them immunostained with an antibody specific for smooth muscle cells (data not shown). Immunocytochemistry The cells were scraped, washed, and centrifuged; cell pellets were fixed in Bouin's solution, embedded in paraffin, and processed for immunocytochemistry by an avidinbiotin immunoperoxidase method as previously described [6]. The whole myometrial tissue was similarly processed. The following dilutions of primary antibodies were used: 1:300 for the receptor antibody (anti-LHR, 15-38); 1:300 for the anti-a smooth muscle actin antibody; 1:50 for the antibody to type I collagen; 1:100 for the CD68 antibody; and 1:50 for the CD34 antibody. For the controls, either the primary antibody was preabsorbed with excess antigen prior to immunostaining (receptor antibody) or unabsorbed primary antibodies were omitted during the immunostaining procedure (all the other antibodies). The control sections were counterstained with hematoxylin.

DIRECT hCG REGULATION OF HUMAN MYOMETRIAL CELLS

Northern Blot Analysis Northern blotting was performed as previously described [7, 21]. Briefly, the mRNA was isolated using Micro-FastTrack mRNA isolation kits from Invitrogen Corp. (San Diego, CA). The kits contain lysing buffer, prepackaged oligo(dT) columns, and instructions for rapid and efficient isolation of mRNA from the cells. The hybridizations were performed for 18 h at 65°C in a solution containing 50% deionized formamide, 58 mg/ml NaCI, 5% dextran, and 1 x 105 cpm/ml of porcine receptor cDNA labeled with 32 p by the random priming method through use of commercial kits. The blots were washed twice at 65°C with double-strength SSC (single-strength SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) containing 1% SDS and twice more at 42°C with double-strength SSC containing no SDS. The washed blots were exposed to x-ray film for 5 days at -80°C with intensifying screens. The molecular size of transcripts was determined by running an RNA ladder in an adjacent lane. Immunoblotting The myometrial cells were homogenized at 4C with a Polytron homogenizer at a speed setting of 6 for 1 min in 10 mM PBS (pH 7.4) containing 5 mM N-ethylmaleimide and 0.2 mM phenylmethanesulfonyl fluoride. The homogenates were centrifuged at 4C for 20 min at 120 x g. The protein in supernates was determined by Bradford's method through use of a commercial kit. The proteins were separated on discontinuous 7.5% SDS-PAGE under reducing conditions [22] and electroblotted [23] to Immobilon-P membranes; receptors were detected by 1:1000 dilution of a polyclonal hCG/LH receptor antibody (anti-LHR, 15-38) and an enhanced chemiluminescence detection system. The molecular size of proteins was determined by running the standard marker proteins in an adjacent lane. Radioiodinationof hCG Unlabeled hCG was radioiodinated by the lactoperoxidase technique [24, 25]. The specific activity of 25I-hCG was 80.2 ptCi/Klg, and approximately 58.7% of the added hormone bound to excess bovine CL plasma membrane protein. Ligand Blotting The ligand blotting procedure was performed as previously described by Keinanen et al. [26]. Briefly, the myometrial cells were homogenized; the proteins in 120 x g supernates of homogenates were solubilized in 1% Triton X-100, separated on discontinuous 7.5% SDS-PAGE under nonreducing conditions, and electroblotted to Immobilon-P membranes. The receptors were detected by 125I-hCG (1 x 106 cpm/ml) binding in the presence and absence of various excess unlabeled hormones. The membranes were then extensively washed and exposed to x-ray film for 7 days at -80°C with intensifying screens. The molecular size of receptor

1151

protein was determined by running molecular weight standards in an adjacent lane. Cell Culture and Treatments At the end of cell dispersion, 1000 viable cells/cm2 were plated in 25-cm 2 Corning cell culture flasks (Corning, Inc., Corning, NY) or 9-cm 2 Nunc slide flasks (Nunc Inc., Naperville, IL). This number was chosen so that confluency could be reached in about 7-10 days with an average population doubling time of 1.0 to 1.8 days. Hormones and other treatments were added in the culture medium to the flasks on the second day when the cell attachment was complete. The protein kinase inhibitors were added at the highest concentrations that did not affect the basal growth or morphology of cells. All the cells were continuously primed with 0.2 nM 1713estradiol (E2) during the entire duration of culture. The culture medium was changed every 48 h, and the treatments were present during the entire duration of culture. The cultures were stopped just before confluency was reached on about Day 7 to avoid bias from contact inhibition. The cell density in a subconfluent phase varied up to about 100 000 cells/cm2 . At the end of experiments, culture medium was replaced with basic WM and the flasks were kept at 4°C until all treatment groups could be counted one at a time. As much as 8 h was required to finish all the counting, and there was no change in cell numbers or general morphology during this period. Cell Counting Immediately before counting, cells were detached by trypsin treatment, as described above, with volumes adjusted to 25-cm2 flasks. The final pellets from each flask were suspended in 12.5 ml ISOTON-II electrolyte solution, and cells in two 500-1Il aliquots were counted in a Coulter Counter-ZM (Coulter Electronic, Hialeah, FL) equipped with a 100-jimdiameter aperture tube. The diameter range, determined by prior measurement of cell size distribution, was set to count everything at 8 Im and above. A micro counting method was developed for determining the effects of antibodies to hCG and its receptors on cell growth. In this method, cells were cultured in 9-cm 2 slide flasks that take only 3-ml volume. At the end of the experiments, the upper flask parts were removed and the cell monolayers were fixed and stained with hematoxylin-eosin. The stained cells were counted under a microscope through use of a calibrated grid. On each slide, 256 000-pLm 2 strips were counted ten times on a crossing line marked at the 1.5 cm position. StatisticalAnalysis All experiments were repeated at least three times on cells from different tissue specimens. The results, which are expressed as 103 cells/cm 2 unit, are the means and their standard errors. The number of data points used in the calcula-

1152

KORNYEI ET AL. 2 TABLE 1. Effect of various treatments on myometrial smooth muscle cell density (103 cells/cm ). The concentrations of hormones were 30 nM for hCG, 1 nM for EGF, 2.2 nM for 17)-estradiol (E2), and 40 nM for progesterone (P 4). The cells were treated for about 7 days.

Treatment

Control

Alone

40

1

EGF

hCG b

51 + 2

111

c

4 d 86 + 3

+hCG +EGF +E2 a-eThe values with different superscript letters are significantly different at p < 0.01.

tion of these means ranged from 6 to 10. Analysis of variance with Student-Newman-Keuls multiple range test was used to examine data in Figures 5, 7, 8, 9, 10, and Table 1 [27]. Twotailed t-test was used for data in Figure 6 [27]. RESULTS Human CG/LH Receptor mRNA in Myometrial Smooth Muscle Cells Figure 1 shows that human myometrial smooth muscle cells contained a 4.3-kb receptor transcript (lane 3). The relative abundance of this transcript was lower than in rat testis (lane 1), a classical target of hCG/LH action. Rat testis also contained other receptor transcripts; the 4.3 kb was the primary transcript. Rat liver, a non-target tissue of hCG/LH action, showed no detectable receptor transcripts (lane 2). Human CG/LH Receptor Protein in Myometrial Smooth Muscle Cells Myometrial smooth muscle cells also contained an immunoreactive receptor protein of 50 kDa (Fig. 2, lane 2),

FIG. 1. Northern blotting for hCG/LH receptors. Lane 1 is rat testis as a positive control; lane 2 is rat liver as a negative control; lane 3 is human myometrial smooth muscle cells. Two micrograms of mRNA of each type of tissue or cells was used.

E2

P4

34 -t 1 2b 50 78 + 3d

41 +1 b 2 49 110 + 4c

33 + le

which became nondetectable when the receptor antibody preabsorbed with excess receptor peptide was used (lane 4). As expected, rat testis also contained an immunoreactive receptor protein, but its 80-kDa molecular size was higher than in myometrial cells (lane 1). Rat liver showed no detectable receptor protein (lane 3). The immunocytochemistry showed that the receptor protein was present in myometrial smooth muscle cells (Fig. 3B) as in intact myometrium (Fig. 3A). The receptor immunostaining both in the cells and in the tissue appeared to be perinuclear. The immunostaining in the tissue and cells was absent when the receptor antibody preabsorbed with receptor peptide was used (Fig. 3, C and D). Ligand Binding of hCG/LHReceptors in Myometrial Smooth Muscle Cells Figure 4 shows that the 50-kDa protein in myometrial smooth muscle cells (lane 3) and the 80-kDa protein in rat testis (lane 1) bound '2 5I-hCG. The rat liver showed no de25 tectable 125I-hCG binding (lane 2). The 1 I-hCG binding to myometrial smooth muscle cells was inhibited by excess unlabeled hCG (lane 4) and hLH (lane 5) but not by hTSH.

FIG. 2. Western immunoblotting for hCG/LH receptors. Lane 1 is rat testis; lanes 2 and 4 are human myometrial smooth muscle cells; lane 3 is rat liver. Unabsorbed receptor antibody was used in lanes 1 to 3, and receptor antibody preabsorbed with excess receptor peptide was used in lane 4. Fifteen micrograms of protein of each type of tissue or cells was used.

DIRECT hCG REGULATION OF HUMAN MYOMETRIAL CELLS

1153

FIG. 3. Immunocytochemistry for hCG/LH receptors in human myometrial tissue (A) and myometrial smooth muscle cells (B). C and D are immunostaining controls for myometrial tissue and myometrial smooth muscle cells, respectively. Magnification A and B = x150; C and D = x300.

Effects of hCG on Myometrial Smooth Muscle CellDensity Culturing myometrial smooth muscle cells with lower than 1 nM hCG had no effect on cell density (Fig. 5), whereas 1 nM hCG caused a small but significant increase in cell density. The response increased with increasing hCG concentrations, reaching a maximum at about 30 nM, followed by a decline. The decreased response at 100 nM hCG was still significantly higher than in the control. Figure 6 shows that the hCG effect on myometrial smooth muscle cell density had a time lag of about 4 h. Subsequently, the hCG effect persisted during the remainder of the culture period. Hormone Specificity of hCG Effect Figure 7 shows that as before, 30 nM hCG increased myometrial smooth muscle cell density whereas isolated hCG-a

and hCG-3 subunits had no effect. Human LH, which is structurally and functionally similar to hCG, mimicked hCG in increasing the myometrial smooth muscle cell density. FSH and TSH, which are in the same glycoprotein hormone family as hCG and hLH, had no effect. On the other hand, PRL and GH, which belong to a different hormone family, were able to increase myometrial smooth muscle cell density. Blocking of hCG Effect by hCG/LH Receptor andhCG Antibodies Culturing myometrial smooth muscle cells with each of two different receptor antibodies alone had no effect on cell density (Fig. 8). But when these were combined with hCG, they were able to block the hCG effect. Similarly, hCG antibody alone had no effect, but it blocked hCG response when

1154

KORNYEI ET AL. 120 * 100

*

80

!a

*

60 40

*,

20 0 0 FIG. 4. Ligand blotting for hCG/LH receptors. Lane 1 is rat testis; lane 2 is rat liver; lanes 3 to 6 are human myometrial smooth muscle cells. Only 1251-hCG was present in lanes 1 to 3; 10 g/ml unlabeled hCG, human LH, and human TSH was also present in lanes 4, 5, and 6, respectively. Fifteen micrograms protein of each type of tissue or cells was used.

it was present with hCG (Fig. 8). The ability of these antibodies to block hCG action is specific, as an anti-chicken IgG raised in rabbits had no effect either by itself or when present with hCG (Fig. 8). SignalingMechanism in hCG Action The signaling mechanisms were investigated by use of various kinase inhibitors. H-89 (a protein kinase A inhibitor), lavendustin-A (a tyrosine-kinase inhibitor), and calphostin (a protein kinase-C inhibitor) had no effect on basal growth rate of myometrial smooth muscle cells (Fig. 9). Among these, only protein kinase A inhibitor was able to block the hCG effect on myometrial smooth muscle cell density. Lack of Involvement of Eicosanoidsin hCG Action Eicosanoids mediate the actions of hCG in several tissues including nongonadal human reproductive tissues [13, 14, 16, 17, 19, 28, 29]. This led us to examine the possi-

2

4

6

8

10

12

days FIG. 6. Time dependency of hCG effect on myometrial smooth muscle cell density. Broken line is control; solid line is treatment with 30 nM hCG. Asterisks indicate significant differences as compared to the corresponding controls at p < 0.01.

bility that eicosanoids from cyclooxygenase or lipoxygenase pathways of arachidonic acid metabolism might play a mediatory role in the hCG effect on myometrial smooth muscle cell density. Figure 10 shows that neither indomethacin, a cyclooxygenase inhibitor, nor NDGA, a lipoxygenase inhibitor, had any effect on basal or hCG-stimulated myometrial smooth muscle cell growth. Interactionof hCG With OtherRegulators EGF, estradiol, and progesterone are some of the other regulators of human myometrial smooth muscle cell functions. We investigated whether these regulators could mimic or modify hCG's action in myometrial cells. Table 1 shows that EGF not only mimicked hCG, but in fact was more effective than hCG in increasing myometrial smooth muscle cell density. When both were present, the increase in smooth muscle cell density was intermediate between that caused by hCG *

--

25

* *

-a

20

*

6

3G z

a

*

.98

lb

15

10 0

0.01

0.03

0.1

0.3

1

3

10

30

100

nM hCG FIG. 5. Dose dependency of hCG effect on myometrial smooth muscle cell density. The cells were cultured for 7 days. Asterisks indicate significant differences as compared to the control at p < 0.01.

cont

hC(:

hC(;-a hCG-h

I.H

PSH

TSH

PRI.

(G:

30nM FIG. 7. Hormone specificity of hCG effect on myometrial smooth muscle cell density. The asterisks indicate significant differences as compared to the control at p < 0.01. The cells shown in this and the remaining figures were treated for about 7 days.

1155

DIRECT hCG REGULATION OF HUMAN MYOMETRIAL CELLS *

BRA

/

--

25 -

i'I !

a

*

*___

* -I--

20-

lb 15- I

hCG sad IHhC-R l

+

+

-

.+.+

-

+ +

amlhCG Ab M dU Ab

+

+ +

4-

FIG. 8. Reversal of hCG effect on myometrial smooth muscle cell den1 sity by receptor and hCG antibodies. Receptor antibody R was raised against the synthetic N-terminal sequence of 1-11; R2 was raised against the synthetic N-terminal sequences of 15-38. Nonspecific antibody used was antichicken IgG raised in rabbits. All the antibodies were used at 1:50 dilution; hCG was used at 30 nM. Asterisks indicate significant differences as compared to the control at p < 0.01.

or EGF alone. Estradiol alone inhibited the smooth muscle cell density; when it was combined, it had no effect on hCG response, but it inhibited EGF response. Progesterone had no effect on its own nor was it able to modify responses to hCG, EGF, or even estradiol. Effect of hCG on Cell Size The size of myometrium increases during pregnancy. Cellular hypertrophy has been considered to be partly responsible for this increase. Since hCG is present during pregnancy, we investigated whether hCG could increase the size of myometrial smooth muscle cells. Diameters of trypsinized rounded myometrial smooth muscle cells, as mea--A 5u

10hCG INDM NDGA

-

.

+

.

.4

+

-

+

+

+

FIG. 10. Effect of indomethacin (INDM) and NDGA on hCG effect on myometrial smooth muscle cell density. Human CG was used at 30 nM, INDM at 1 jiM, and NDGA at 0.1 M. Asterisks indicate significant differences as compared to the control at p < 0.01.

sured on photographs taken in hemacytometers, showed a normal size distribution with an average cell size of about 20 [m (Fig. 11). Treatment with hCG resulted in the appearance of a major peak corresponding to the one in the control cells and a second minor peak of very large cells with an average cell diameter of about 42 pim. Similar cell size profiles were obtained when Coulter Counter-ZM apparatus was used for measurement of cell sizes. Since cell size differences of this magnitude could be caused by alterations in the genome, the cells' ploidy was determined in normal and largesize cells. The results showed that both groups of cells had a normal female karyotype 46,XX. DISCUSSION We were led to the present studies because human myometrium contains hCG/LH receptors, indicating that this tis-

*

*

7 +

_

--

0.55 .S0

* 0.45 e 0.40 : 0.3S 0.35 , 0.30 a 0.25

40'

a

.

30-

I J _f 1v

Ia

hCG H9 LAY CAL

A 5.

_L

+

+ +

+

+

0.20 0.15

0.10 0.05 0.00 5-9

10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 microns

+ +

+ +

+

FIG. 9. Effect of various kinase inhibitors on hCG's effect on myometrial smooth muscle cell density. Human CG was used at 30 nM, H-89 at 10 FiM, lavendustin (LAV) at 1 IM, and calphostin (CAL) at 0.1 nM. Asterisks indicate significant differences as compared to the control at p < 0.01.

FIG. 11. Effect of hCG on the size distribution of myometrial smooth muscle cells. Broken line represents control and solid line represents treatment with 30 nM hCG. Cells released by mild trypsin digestion, which subsequently became rounded, were photographed in hemacytometers. Measurements were taken of the diameters of 514 cells for control and 685 cells for hCG treatment.

1156

KORNYEI ET AL.

sue is also a direct target of gonadotropin regulation. In the present studies, we focused on investigating whether hCG and LH could regulate myometrial smooth muscle cell growth in terms of cell number and cell size, both of which increase during pregnancy when hCG is present in the circulation. Human CG stimulated myometrial smooth muscle cell density in a concentration-dependent manner. Concentrations below 1 nM hCG were ineffective, whereas those above 1 nM were effective. The maximal effect was seen at about 30 nM hCG; this was followed by a decline that was probably due to a receptor desensitization phenomenon. The maximal increase in smooth muscle cell density ranged from 30% to 60% depending on the tissue specimen. The myometrial smooth muscle cells prepared from proliferative-phase specimens did not usually respond, whereas those prepared from secretory phase usually, but not always, did respond. The reasons for these findings are not known. The results were not due to the absence of receptors, however, because tissues from the same or different reproductive states contained different levels of receptors. The hCG response was generally lost during the second subculture even though cells contained receptors. This again suggests the possibility that one or more postreceptor mechanisms required for hCG action are lost during the second subculture. The cells that lost hCG response, however, continued to respond to EGF [30], suggesting that the loss was specific to an hCG receptor mechanism. The hCG action on myometrial smooth muscle cells required the conformation of an hCG dimer, as isolated hCG subunits were ineffective. Human LH, which binds to the same receptors as hCG [31], mimicked hCG. FSH and TSH, which are in the same glycoprotein hormone family, could not mimic hCG or hLH because they do not bind to the same receptors [31]. Prolactin and GH, which belong to a different hormone family [32], increased myometrial smooth muscle cell density. Since these two hormones are not known to bind to gonadal or nongonadal hCG/LH receptors [31], they may be acting via their own separate receptors. To our knowledge, the presence of GH or its receptors has never been demonstrated in uterus. However, it is known that human myometrium synthesizes decidual-type prolactin [33,34]; prolactin can regulate a number of uterine functions including endometrial proliferation in animals [35, 36], and prolactin receptors that can also bind human GH are present in animal uteri [36-39]. Therefore, the present findings are suggestive of a possible autocrine role for prolactin in human myometrial growth. Myometrial smooth muscle cell response to hCG was blocked by hCG/LH receptor antibodies, suggesting that free receptor is required for hCG action. Human CG response was also blocked by hCG antibody; this suggests that free hCG that can bind to its receptors is required for hCG action. In addition, the latter finding rules out the action of an impurity in the hCG preparation (rather than hCG per se) on myometrial smooth muscle cells. This view is further supported by the findings that two different highly purified hCG prepara-

tions similarly increased myometrial smooth muscle cell density. These two hCG preparations had different levels of peptide bond nicking (CR-127 hCG, 20%; IM22 [33], 3%). This finding of similar action despite a difference in peptide bond nicking suggests that at least up to 20% of nicking may not interfere with hCG action on myometrial smooth muscle cell density. It now appears that dual signaling systems are involved in hCG/LH actions, i.e., cAMP and protein kinase A and inositol phospholipid turnover and protein kinase C [5, 40]. In studies performed to investigate whether one or both of these or tyrosine kinase signaling is involved in hCG's action, it was found that protein kinase A signaling is required for hCG action. Eicosanoids from cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism do not appear to be involved. EGF is also mitogenic to human myometrial smooth muscle cells [30], but its mode of action is different from that of hCG [30]. For example, EGF decreased average cell size while it increased cell density. EGF's action is mediated by tyrosine kinase and protein kinase C signaling systems. Finally, the synthesis of eicosanoids from the lipoxygenase pathway of arachidonic acid metabolism appeared to be required for full expression of the EGF action. Progesterone, which is abundant during pregnancy and is generally presumed to cause myometrial changes, did not mimic or modify hCG action. Progesterone also could not modify the inhibitory action of estradiol or the stimulatory action of EGF. These findings are in agreement with those of Rossi et al. [41]. Estradiol alone inhibited myometrial smooth muscle cell growth. It could not modify hCG action, but it did inhibit responsiveness to EGF. The inhibitory effects of estradiol in the presence and absence of EGF are also in agreement with Rossi et al. [41]. Contrary to these findings, Chen et al. [42] have demonstrated that estradiol stimulates the growth of myometrial smooth muscle cells that have been in culture for a long time. Therefore, it is possible that estradiol may have stimulatory effects in myometrial smooth muscle cells aged in culture. Human myometrial growth during pregnancy has been considered to be due to hypertrophy and/or hyperplasia, but there are no definitive data. Nevertheless, it is reasonable to presume that myometrial growth seen during pregnancy, at least in part, is caused by hCG. Human CG may also have other roles in pregnant myometrium. Whether hLH can also similarly act on nonpregnant myometrium during the periovulatory period is not known. In summary, hCG can directly act on human myometrial smooth muscle cells to increase their number as well as their size in a subpopulation of cells. This action of hCG is hormone specific, uses a protein kinase A signaling system, and does not require mediation by eicosanoids from cyclooxygenase or lipoxygenase pathways of arachidonic acid metabolism.

DIRECT hCG REGULATION OF HUMAN MYOMETRIAL CELLS

ACKNOWLEDGMENTS We thank Dr. Frank Yen of the Child Evaluation Center at the University of Louisville for determination of chromosomal ploidy, Dr. Xian Li for performing some of the experiments, and numerous Ob/Gyn physicians and other staff at the University of Louisville Affiliated hospitals for their cooperation in collecting tissue specimens

REFERENCES 1. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981; 50:466-495. 2. McFarland KC, Sprengel R, Phillips HS, KOhler M,Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH. Lutropin-choriogonadotropin receptor: an unusual member of the G-protein coupled receptor family. Science 1989; 245:494-499. 3. Loosfelt H, Misrahi M, Atger M,Salesse R, Thi MTVH-L,JolivetA, Guiochon-Mantel A, Sar S,Jallal B, GarnierJ, Milgrom E. Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 1989; 245:525-528. 4. Minegish T, Nakamura K,Takakura Y,Miyamoto K,Hasegawa Y, Ibuki Y, Igarashi M. Cloning and sequencing of human LH/hCG receptor cDNA. Biochem Biophys Res Commun 1990; 172:1049-1054. 5. Guderman T, Birnbaumer M, Birnbaumer L.Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide 2+ breakdown and Ca mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.J Biol Chem 1992; 267:4479-4488. 6. Reshef E, Lei ZM, Rao ChV, Pridham DD, Chegini N, LuborskyJL. The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua.J Clin Endocrinol &Metab 1990; 70:421-430. 7. Lei ZM, Reshef E, Rao ChV. The expression of human chorionic gonadotropin/luteinizing hormone receptors in human endometrial and myometrial blood vessels. J Clin Endocrinol &Metab 1992; 75:651-659. 8. Lei ZM, Rao ChV. Gonadotropin receptors in human fetoplacental unit: implications for hCG as an intracrine, paracrine and endocrine regulator of human fetoplacental function. Trophoblast Res 1992; 6:213-224. 9. Lei ZM, Rao ChV, Ackerman DM, Day TG. The expression of human chorionic gonadotropin/human luteinizing hormone receptors in human gestational trophoblastic neoplasms.J Clin Endocrinol &Metab 1992; 74:1236-1241. 10. Lincoln SR, Lei ZM, Rao ChV, Yussman MA. The expression of human chorionic gonadotropin/human luteinizing hormone receptors in ectopic human endometrial implants.J Clin Endocrinol &Metab 1992; 75:1140-1144. 11. Lei ZM, Rao ChV, Lincoln SR, Ackermann DM. Increased expression of human chorionic gonadotropin/human luteinizing hormone receptors in adenomyosis. J Clin Endocrinol &Metab 1993; 76:763-768. 12. Lei ZM, Rao ChV, KornyeiJL, Licht P, Hiatt ES. Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain. Endocrinology 1993; 132:2262-2270. 13. Lei, ZM, Toth P, Rao ChV, Pridham, D. Novel co-expression of human chorionic gonadotropin (hCG)/human luteinizing hormone receptors and their ligand, hCG, in human fallopian tubes.J Clin Endocrinol &Metab 1993; 77:863 872. 14. Rao ChV, Li X, Toth P, Lei ZM, Cook VD. Novel expression of functional human cho rionic gonadotropin/luteinizing hormone receptors gene in human umbilical cords. J Clin Endocrinol &Metab 1993; (in press). Lei ZM, Rao ChV, LinJ. Novel role of human chorionic gonadotropin in dif15. Shi QOJ, ferentiation of human cytotrophoblasts. Endocrinology 1993; 132:1387-1395. 16. Toth P, Rao ChV. Direct novel regulation of cyclooxygenase and prostacyclin synthase by human chorionic gonadotropin in the human amnion. Trophoblast Res 1993; (in press). 17. Toth P, Rao ChV, Lei ZM, Lincoln SR, Spinnato IIJA, SanfilippoJ. Expression of functional hCG/LH receptors in human uterine blood vessels. In: Program of the 40th annual meeting of the Society for Gynecologic Investigation; 1993; Toronto, Canada. Abstract S46. 18. Licht P, Cao, H, Lei ZM, Rao ChV, Merz WE. Novel self-regulation of human chorionic gonadotropin biosynthesis in term pregnancy human placenta. Endocrinology 1993; (in press).

1157

19. Toth P, LiX, Rao ChV. Expression of hCG/LH receptor gene and its functional coupling to the regulation of cyclooxygenase-1 and -2 enzymes in human fetal membranes. Placenta 1993; 14:A78. 20. Casey ML, MacDonald PC, Mitchell MD, Snyder IM. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In Vitro 1984; 20:396-403. 21. SambrookJ, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, ed. 2, Book 2. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1989: 7.12-7.52, 10.2710.36. 22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 .Nature 1970; 227:680-685. 23. Dunn SD. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem 1986; 157:144-153. 24. Miyachi Y,VaitukaitisJL, Nieschlag E, Lipsett MB. Enzymatic radioiodination of gonadotropins.J Clin Endocrinol &Metab 1972; 34:23-28. 25. Rao ChV, Griffin LP, Carman FRJr. Gonadotropin receptors in human corpora lutea of the menstrual cycle and pregnancy. AmJ Obstet Gynecol 1977; 128:146-153. 26. Keinanen KP, Kellokumpu S, Rajaniemi H. Visualization of the rat ovarian lutropin receptor by ligand blotting. Mol Cell Endocrinol 1987; 49:33-38. 27. Steel RG, TorrieJA. Principles and Procedures of Statistics. New York: McGraw-Hill; 1960. 28. Armstrong DT, Grinwich DL. Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1972; 1:21-28. 29. North RA, Whitehead R, Larkins RG. Stimulation by human chorionic gonadotropin of prostaglandin synthesis by early human placental tissue. J Clin Endocrinol &Metab 1991; 73:60-70. 30. KornyeiJ, Rao ChV. Molecular analysis of epidermal growth factor action in human myometrial smooth muscle cells. In: Program of the 39th annual meeting of the Society for Gynecologic Investigation; 1992; San Antonio, TX. Abstract 581. 31. Rao ChV. Properties of gonadotropin receptors in the cell membrane of bovine corpus luteum.J Biol Chem 1974; 249:2864-2872. 32. Linzer DIH, Nathans D. Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family. Proc Natl Acad Sci USA 1984; 81:4255-4259. 33. Walters CA, Daly DC, ChapitisJ, Kuslis ST, PriorJC, Kusmik WF, Riddick DH. Human myometrium: a new potential source ofprolactin. AmJ Obstet Gynecol 1983; 147:639644. 34. Gellersen B, Bonhoff A, Hunt N, Bohnet HG. Decidual-type prolactin expression by the human myometrium. Endocrinology 1991; 129:158-168. 35. Chilton BS, Daniels JC. Influence of prolactin on DNA synthesis and glandular differentiation in rabbit uterine endometrium. In: MacLeod RM, Thorner MO, Scapagini U (eds.), Prolactin, Basic and Clinical Correlates. Padova, Italy: Fidia Research Series; 1985: 351-359. 36. Young KH, Bazer FW. Porcine endometrial prolactin receptors detected by homologus radioreceptor assay. Mol Cell Endocrinol 1989; 64:145-154. 37. Grissom FE, Littleton GK. Evolution of lactogen receptors in selected rabbit tissues during pregnancy. Endocr Res 1988; 14:1-19. 38. Galosy SS, Gertler A,Elberg G, Laird DM. Distinct placental lactogen and prolactin (lactogen) receptors in bovine endometrium. Mol Cell Endocrinol 1991; 78:229-236. 39. Dusantier-Furt I, Gaye P, Belair L,Petridou B, Kelly PA,DjianeJ. Prolactin receptor gene expression in the rabbit: identification, characterization and tissue distribution of several prolactin receptor messenger RNA encoding a unique precursor. Mol Cell Endocrinol 1991; 77:181-192. 40. Davis JS, West LA, Farese RV. Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells.J Biol Chem 1984; 259:15028-15034. 41. Rossi MJ, Chegini N, Masterson BJ. Presence of epidermal growth factor, plateletderived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro. Endocrinology 1992; 130:1716-1727. 42. Chen L, Lindner HR, Lancet M. Mitogenic action of oestradiol-173 on human myometrial and endometrial cells in long term tissue cultures. J Endocrinol 1973; 59:8797.