Itp.w3.kanazawa-u.ac.jp

Progesterone and Mifepristone Regulate L-Type Amino Acid Transporter 2 and
4F2 Heavy Chain Expression in Uterine Leiomyoma Cells
Xia Luo, Ping Yin, Scott Reierstad, Hiroshi Ishikawa, Zhihong Lin, Mary Ellen Pavone, Hong Zhao, Erica E. Marsh and Serdar E. Bulun J. Clin. Endocrinol. Metab. 2009 94:4533-4539 originally published online Oct 6, 2009; , doi: 10.1210/jc.2009-1286 To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine
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Copyright The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online Progesterone and Mifepristone Regulate L-Type
Amino Acid Transporter 2 and 4F2 Heavy Chain
Expression in Uterine Leiomyoma Cells
Xia Luo, Ping Yin, Scott Reierstad, Hiroshi Ishikawa, Zhihong Lin,Mary Ellen Pavone, Hong Zhao, Erica E. Marsh, and Serdar E. Bulun Division of Reproductive Biology Research (X.L., P.Y., S.R., H.I., Z.L., M.E.P., H.Z., E.E.M., S.E.B.),Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University,Chicago, Illinois 60611; and Department of Obstetrics and Gynecology (X.L.), Qilu Hospital, ShandongUniversity, Jinan, Shandong 250012, People's Republic of China Context: Progesterone and its receptor (PR) play key roles in uterine leiomyoma growth. Previously,
using chromatin immunoprecipitation-based cloning, we uncovered L-type amino acid transporter
2 (LAT2) as a novel PR target gene. LAT2 forms heterodimeric complexes with 4F2 heavy chain
(4F2hc), a single transmembrane domain protein essential for LAT2 to exert its function in the
plasma membrane. Until now, little is known about the roles of LAT2/4F2hc in the regulation of the
growth of human uterine leiomyoma.
Objective: The aim of the study is to investigate the regulation of LAT2 and 4F2hc by progesterone
and the antiprogestin mifepristone and their functions in primary human uterine leiomyoma
smooth muscle (LSM) cells and tissues from 39 premenopausal women.
Results: In primary LSM cells, progesterone significantly induced LAT2 mRNA levels, and this was
blocked by cotreatment with mifepristone. Progesterone did not alter 4F2hc mRNA levels, whereas
mifepristone significantly induced 4F2hc mRNA expression. Small interfering RNA knockdown of
LAT2 or 4F2hc markedly increased LSM cell proliferation. LAT2, PR-B, and PR-A levels were signif-
icantly higher in freshly isolated LSM cells vs. adjacent myometrial cells. In vivo, mRNA levels of LAT2
and PR but not 4F2hc were significantly higher in leiomyoma tissues compared with matched
myometrial tissues.
Conclusion: We present evidence that progesterone and its antagonist mifepristone regulate the
amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells. Our findings suggest that
products of the LAT2/4F2hc genes may play important roles in leiomyoma cell proliferation. We
speculate that critical ratios of LAT2 to 4F2hc regulate leiomyoma growth. (J Clin Endocrinol Metab
94: 4533– 4539, 2009)
Uterine leiomyomata (fibroids) are the most common leastonethirdofapproximately600,000hysterectomies
benign tumors in women, originating from uterine performed annually in the United States (3).
smooth muscle cells. Symptomatic leiomyoma occurs in as Although the etiology of uterine fibroids is unknown, many as 30% of women over 35 yr of age (1). They are a the growth of these tumors has been known to be depen- frequent cause of abnormal uterine bleeding and are in- dent on ovarian steroid hormones (4). Accumulating data volved in recurrent pregnancy loss. Uterine leiomyomata support the concept that progesterone plays an important are the single most common underlying indication for hys- role in regulating the growth of uterine leiomyoma (4, 5).
terectomy (2). Uterine leiomyomata are responsible for at The role of progesterone and its receptor (PR) in the patho- ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: Ct, Cycle threshold; 4F2hc, 4F2 heavy chain; GAPDH, glyceraldehyde-3- Printed in U.S.A.
phosphate dehydrogenase; LAT2, L-type amino acid transporter 2; LSM, leiomyoma Copyright 2009 by The Endocrine Society smooth muscle; PCNA, proliferation cell nuclear antigen; PR, progesterone receptor; doi: 10.1210/jc.2009-1286 Received June 18, 2009. Accepted August 26, 2009.
siRNA, small interfering RNA.
First Published Online October 6, 2009 J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 Luo et al.
Progesterone Action, LAT2 and 4F2hc J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 genesis of fibroids is highlighted by the following set of find- leiomyoma, following a protocol approved by the Institutional ings. First, PR has been observed to be higher in fibroid tissue Review Board for Human Research of Northwestern University compared with the myometrial tissue by several groups (6, (Chicago, IL). The size of the tumors ranged from 3.5 to 15 cmin diameter. The subjects had not received any hormonal treat- 7). Secondly, the presence of PR in fibroid tissue was also ment for at least three menstrual cycles before surgery. Each shown to be positively correlated with their growth (8). Most specimen was evaluated histologically by a pathologist. The cycle importantly, progestins are able to reverse the reduction of phase was estimated by the last menstrual period; this was con- fibroid size that is induced by GnRH agonist therapy when firmed by endometrial histology. Twenty-two samples were ob- administered as add-back treatment (9, 10).
tained during the follicular phase, 12 during the luteal phase, and The in vivo effects of progesterone on leiomyoma growth, five during menstruation. Of these 39 samples, 16 were used forcell culture experiments, whereas 29 were used for the tissue however, are still not clearly understood. For example, the experiments. We used tissues from six women to prepare cells average leiomyoma size during the first trimester of preg- and also for in vivo studies. We isolated LSM cells from the nancy is significantly higher compared with that before the peripheral portions approximately 1 cm from the outer capsule onset of pregnancy (11). On the other hand, leiomyoma size of the leiomyoma, and cultured them as previously described remains stable or can decrease slightly when the first and with minor modifications (21). Immunocytochemistry using an third trimesters of pregnancy are compared (12–14). Thus, antibody against smooth muscle ␣-actin confirmed purity of the progesterone may play dual roles regarding leiomyoma cells (data not shown). Primary cells were used only up to thesecond passage to avoid changes in phenotype and gene expres- growth under various in vivo conditions.
sion. LSM cells were cultured in DMEM/F12 1:1 (GIBCO/BRL, Previously, we and others have described functions of pro- Grand Island, NY) containing 10% fetal bovine serum (Invitro- gesterone-responsive genes that promote fibroid growth (5, gen, Carlsbad, CA). The monolayer cultures at about 70% con- 15, 16). We recently performed a genome-wide chromatin fluency were starved in serum-free medium overnight and treated immunoprecipitation-cloning procedure to identify novel with vehicle (ethyl alcohol 1:1000; Sigma-Aldrich, St. Louis, binding sites of PR in chromatin isolated from leiomyoma MO), progesterone (3 ⫻ 10⫺7 M; Sigma-Aldrich), or mifepris-tone (10⫺8-10⫺4 M; Sigma-Aldrich).
smooth muscle (LSM) cells. We noted that PR was recruited All cell culture-based experiments were repeated using cells to intron 12 of the L-type amino acid transporter 2 (LAT2) from at least four subjects. One representative experiment was gene (our unpublished observations). Here, we define novel illustrated. Each cell culture-based experiment was carried out in roles of LAT2 and its functional partner, a heavy chain of 4F2 triplicate replicates using cells in first or second passage.
antigen (4F2hc) in LSM cell fate.
LAT2 has a 12-membrane-spanning domain that medi- RNA preparation and real-time quantitative PCR
ates Na⫹-independent amino acid exchange. It requires Total RNA from LSM cells was extracted using Tri-reagent an additional single-membrane-spanning domain pro- (Sigma-Aldrich). cDNA was prepared with qScript cDNA Super- tein, 4F2hc, to exert its function in the plasma membrane.
Mix (Quanta BioSciences, Inc., Gaithersburg, MD) from 2 ␮g of LAT2 and 4F2hc form a heterodimeric complex via a disul- RNA. Primers against LAT2, 4F2hc, and the constitutively ex- fide bond (17, 18). The mRNAs of LAT2 and 4F2hc are pressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressed in most embryonic and adult tissues (18, 19).
were used as described in previous reports (22, 23). For LAT2, theforward and reverse primers were 5⬘-AATGCATTTGAGAATT- LAT2 transports large neutral amino acids, as well as small TCCAGGA-3⬘ and 5⬘-GAGCCCTGAAGGAAAGCCA-3⬘, re- neutral amino acids (18, 19). LAT2 is involved mostly in the spectively (22). For 4F2hc, the forward and reverse primers were basolateral efflux step of transepithelial amino acid transport 5⬘-CTCAGGCAAGGCTCCTGACT-3⬘ and 5⬘-GGCAGGGT- in the kidney and intestine (19). However, the exact local- GAAGAGCATCA-3⬘, respectively (22). For GAPDH, the forward ization of LAT2 on cell membranes is not fully known, and and the reverse primers were 5⬘-GAAGGTGAAGGTCG- the tissue distribution data are sometimes conflicting (20).
GAGTC-3⬘ and 5⬘-GAAGATGGTGATGGGATTTC-3⬘, respec-tively (23). Primer specificity was confirmed by the demonstration The expression and functional properties of amino acid of single peaks using dissociation curves after amplification of transporters for supplying organic nutrition to cells have not cDNA and a lack of amplification of genomic DNA.
been entirely clarified. Furthermore, nothing is known re- Real-time quantitative PCR was performed to determine the garding the expression and function of LAT2/4F2hc in hu- relative amounts of each transcript using the DNA-binding dye man uterine leiomyoma.
SYBR green (Applied Biosystems, Foster City, CA) and the ABIPrism 7900HT Detection System (Applied Biosystems). Cyclingconditions started at 50 C for 2 min, followed by 95 C for 10 min,then 40 cycles of 95 C for 15 sec and 60 C for 1 min. The cycle Materials and Methods
threshold (C ) was placed at a set level where the exponential increase in PCR amplification was approximately parallel be- Tissue collection and primary cell culture
tween all samples. Relative fold change was calculated by com- Human uterine leiomyoma and matched myometrial tis- paring C values between the target gene and GAPDH as the sues were obtained at surgery from 39 women (mean age, 40 reference guide. The 2⫺⌬⌬Ct method was used to analyze these yr; range, 33– 48) undergoing hysterectomy for symptomatic relative changes in gene expression (24).

J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 medium without antibiotics and incu- bated for 24 h for mRNA and 72 h for Western blot.
Cell viability assay
The effects of LAT2 siRNA or 4F2hc siRNA on the viability of LSM cells was also (fold change) 0.5 evaluated by the 3-(4, 5-dimethylthiazolyl- 2)-2, 5-diphenyltetrazolium bromide (MTT) assay (Invitrogen). LSM cells were plated at a density of 4.0 ⫻ 104 cells per well in 24- well culture plate 1 d before transfection toachieve approximately 50% confluence at the time of transfection. The transfection was done as previously described and incu- bated for 72 h. The MTT assay was per- formed as described by the manufacturer'sprotocol with minor modifications. Briefly, at 72 h from start of transfection, 40 ␮l MTT solution was added to each well, and cells were incubated at 37 C for 4 h. Then 400 ␮l of the sodium dodecyl sulfate-HCl solution were added to each well and mixed vehicle 10-8M 10-7M 10-6M 10-5M 10-4M thoroughly. The plate was then incubated at 37 C for another 16 –18 h, and samples weretransferred to a 96-well plate and read at an absorbance of 570 nm.
Protein isolation and
Cultured LSM and myometrial cells were lysed using Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL) and 1 ⫻ protease inhibitor (Sigma-Aldrich). Pro- tein concentrations were determined by col- orimetric BCA protein Assay (Pierce), and equal concentrations of total protein were FIG. 1. Regulation of LAT2 and 4F2hc expression by progesterone or mifepristone. Primary
loaded in each well. Samples were subjected to cultured LSM cells were starved in phenol red-free and serum-free DMEM/F12 (1:1) medium PAGE (Bio-Rad, Hercules, CA) and trans- overnight and then treated with vehicle (ethanol), progesterone, or mifepristone for different ferred onto nitrocellulose membranes (In- times. LAT2 (A, D, E) and 4F2hc (B, D, F) mRNA levels were measured by real-time PCR and vitrogen). Membranes were probed using normalized to GAPDH mRNA levels. LAT2 protein levels were evaluated by Western blot using antibodies against ␤-actin (Sigma-Aldrich), a LAT2 peptide antibody (C). Blots were reprobed with a ␤-actin antibody for loading control.
proliferation cell nuclear antigen (PCNA; Results for A, B, D, E and F were reported as a fold change compared with cells treated with Millipore, Burlington, MA), LAT2 (Santa vehicle only and represented as the mean ⫾ SEM. All graphs were derived from one Cruz Biotechnology, Santa Cruz, CA) or PR representative experiment, and all experiments were repeated in triplicate in four subjects.
(PGR; courtesy of Dr. Dean Edwards, Baylor *, P ⬍ 0.05, compared with vehicle treatment.
College of Medicine, Houston, TX). Anti-mouse and antigoat IgG conjugated to horse- Small interfering RNA (siRNA)
radish peroxidase (Cell Signaling, Danvers, RNA oligonucleotides directed against LAT2, 4F2hc, and a MA) were used as secondary antibodies. Immunoreactive bands mismatch negative control siRNA were purchased from Dhar- were visualized using the ECL-detection system (GE Healthcare, macon (Lafayette, CO). LSM cells were plated at a density of Piscataway, NJ). Quantification of chemiluminescence signal in- 1.0 ⫻ 106 cells per 10-cm dish in phenol free DMEM/F12 con- tensity was performed after completion of all autoradiographic taining 10% charcoal stripped fetal bovine serum, but lacking studies with ImageJ software (National Institutes of Health, Be- gentamycin/amphotericin B, 1 d before transfection to achieve thesda, MD).
approximately 50% confluence at the time of transfection. Onthe day of transfection, the RNAiMAX lipofectamine-based re-agent was combined in conjunction with 200 nM siRNA du- plexes diluted in Opti-Mem I (Invitrogen) and applied to the cells Statistical significance was determined by Student's t test and according to the manufacturer's instructions. Five hours after the one-way ANOVA followed by Fisher's protected least signifi- start of the transfection, the medium was changed to growth cance difference test. Significance was accepted at P ⬍ 0.05.

Luo et al.
Progesterone Action, LAT2 and 4F2hc J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 but it did not influence LAT2 mRNA level at any concentration (Fig. 1D).
To elucidate whether mifepristone could block the action of progesterone, we treated LSM cells with progester- one alone, mifepristone alone, or their (fold change) 0.2 combination for 4 h. Consistently, mifepristone alone did not affect LAT2 expression, but it abolished progester- one-induced LAT2 mRNA level (Fig.
1E). On the other hand, progesterone did not influence 4F2hc mRNA levels, whereas mifepristone significantly in- duced them (Fig. 1F).
Relative intensity Knockdown of LAT2 is associated
with increased LSM cell
FIG. 2. The effects of knocking down LAT2 on LSM cell proliferation. LSM cells were
We performed knockdown of en- transfected with control siRNA or LAT2 siRNA for 72 h. Knockdown efficiency and specificity dogenous LAT2 to investigate how it of the LAT2 gene were examined by both real-time PCR (A) and Western blot (B). Cell may be involved in the regulation of proliferation was analyzed by measuring PCNA protein level with mouse antihuman PCNA LSM cell proliferation by examining antibody (B). Blots were reprobed with a ␤-actin antibody for loading control. Western blotdensities were quantified with ImageJ software (C). The effect of knocking down LAT2 on PCNA protein levels and MTT assays.
LSM cell proliferation was confirmed with MTT assay (D). Data for A (represented as mean ⫾ Real-time PCR demonstrated that SEM) and B were from one representative experiment and were repeated in four subjects. Data siRNA against LAT2 significantly re- for C and D show the mean ⫾ SEM from four subjects. Each experiment was done in triplicate.
*, P ⬍ 0.05, compared with control siRNA.
duced its endogenous mean mRNAlevel by over 90% (Fig. 2A). Westernblot analysis confirmed that LAT2 pro- tein level was also markedly decreased by LAT2 siRNA(Fig. 2, B and C). Knockdown of LAT2 increased the pro- Regulation of LAT2 and 4F2hc mRNA levels by
liferation marker PCNA (Fig. 2, B and C), suggesting that progesterone or mifepristone in LSM cells
LAT2 inhibited proliferation of LSM cells. The effect of Our preliminary dose-response (10⫺10-10⫺6 M) exper- siRNA LAT2 knockdown on the proliferation of LSM iment showed that progesterone regulated LAT2 expres- cells was confirmed by MTT assay, which reflects the total sion in LSM cells at 10⫺8, 10⫺7, and 10⫺6 M concentra- number of viable cells. As shown in Fig. 2D, knockdown tions; peak regulation occurred at 10⫺6 M dose after 4 h of of LAT2 significantly increased the number of viable cells.
treatment (data not shown). We chose to use the 3 ⫻ 10⫺7 M Knockdown of LAT2 also increased the proliferation of concentration of progesterone to further examine its ef- myometrial cells from matched tissues (data not shown).
fects on LAT2 and 4F2hc gene expression.
Real-time quantitative PCR analyses were performed Knockdown of 4F2hc is also involved in increased
after LSM cells were incubated with progesterone (3 ⫻ LSM cell proliferation
10⫺7 M) for 2 and 4 h. LAT2 mRNA levels were signifi- We performed knockdown of endogenous 4F2hc to cantly induced by progesterone at both 2 and 4 h (Fig. 1A).
investigate its effects on proliferation of LSM cells. Real- However, progesterone did not affect 4F2hc mRNA lev- time PCR analysis showed that 4F2hc siRNA reduced en- els (Fig. 1B). Treatment at the same concentration for dogenous 4F2hc mRNA expression by nearly 80% (Fig.
72 h also significantly up-regulated LAT2 protein levels 3A). 4F2hc protein expression could not be demonstrated because a commercial antibody was not available. 4F2hc To determine whether progesterone antagonist mife- knockdown by siRNA significantly increased PCNA level pristone affects LAT2 or 4F2hc expression differentially, (Fig. 3, B and C). Alterations in LSM cell proliferation we treated LSM cells with various concentrations (10⫺8- were also verified by the MTT assay. Figure 3D illustrates 10⫺4 M) of mifepristone for 4 h. In contrast to the regu- that 4F2hc knockdown by siRNA modestly but signifi- lation of these two genes by progesterone, mifepristone cantly increased the number of viable cells. These results induced 4F2hc mRNA levels in a dose-dependent manner, suggest that 4F2hc may also be involved in inhibition of

J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 4F2hc, and PR were normalized to GAPDH, and then the mRNA levels in leiomyoma tissues were expressed as a multiple of that value in matched myo- metrial tissues, allowing the detection of relative mRNA differences between (fold change) 0.2 the two tissues.
As shown in Fig. 5, mean LAT2 and PR mRNA levels were significantly higher in leiomyoma than myometrial tissues. However, there was no signifi- cant difference in mRNA levels of 4F2hc between leiomyoma and matched myo- (fold change) 0.6 metrial tissues.
PCNA relative intensity FIG. 3. The effects of knocking down 4F2hc on LSM cell proliferation. LSM cells were
In this study, we have demonstrated transfected with control siRNA or 4F2hc siRNA for 72 h. The cells were then harvested for that progesterone and its antagonist analysis. Knockdown efficiency and specificity of the 4F2hc gene were examined by real-timePCR (A). LSM cells were harvested for determination of cell proliferation by measuring of mifepristone regulated LAT2 and 4F2hc PCNA protein level with mouse antihuman PCNA antibody (B). Western blot densities were expression. LAT2 knockdown studies quantified with ImageJ software (C). The effect of knockdown 4F2hc on LSM cell proliferation suggested that LAT2 inhibits LSM cell was confirmed with MTT assay (D). Blots were reprobed with a ␤-actin antibody, which wasused as a control. 4F2hc mRNA measured by real-time PCR was normalized to GAPDH mRNA.
growth, although its product is consid- Data for A (represented as mean ⫾ SEM) and B were from one representative experiment and ered to be an amino acid transporter.
were repeated in four subjects. Data for C and D are shown as the mean ⫾ SEM from four Thus, the role of LAT2 in leiomyoma subjects. Each experiment was done in triplicate. *, P ⬍ 0.05, compared with control siRNA.
pathology may seem paradoxical atfirst sight. The functional properties of LSM cell growth. Similar to its effects on LSM cell pro- LAT2 for supplying organic nutrition to cells have not liferation, knockdown of 4F2hc stimulated myometrial been entirely clarified. It has been reported that LAT2 cell proliferation (data not shown).
operates as an amino acid exchanger (19, 25). There is alsoevidence to suggest that amino acid efflux via LAT2 is not Expression of LAT2 and PR in cultured LSM cells
dependent upon extracellular amino acids (18, 26). The and matched myometrial cells
net direction of the transport of large neutral amino acids The effects of progesterone on target tissues are medi- by LAT2 is believed to depend on the unidirectional trans- ated by PR. Previous studies have demonstrated that PR porters that are coexpressed in the cells. Thus, the role of levels are higher in LSM cells than in myometrial cells (6).
LAT2 is most likely an equilibration of the amino acid To determine whether cell-specific differences in LAT2 distribution across the two membranes, whereas other levels are accompanied by comparable differences in PR, transporters determine the actual net amino acid flux (27).
LAT2 and PR protein expression in LSM cells and Knockdown of LAT2 in LSM cells might have changed the matched myometrial cells was examined using Western equilibration of the amino acid distribution across the cell blot analysis. Compared with myometrial cells, both PR membrane, which could then alter the net flux of amino and LAT2 protein expression was higher in cultured LSMcells (Fig. 4, A and B).
acid determined by other transporters. The final outcome,therefore, may be the increase of cell growth.
Gene expression of LAT2, 4F2hc, and PR in
We also found that knockdown of 4F2hc increased the leiomyoma and matched myometrial tissues
proliferation of LSM cells, suggesting that this gene may To understand the in vivo relevance of progesterone- inhibit leiomyoma growth. Our result is different from regulated LAT2 and 4F2hc expression in leiomyoma, we what was demonstrated in a previous study (28), which analyzed mRNA levels of PR, LAT2, and 4F2hc genes in showed that a monoclonal antibody against 4F2hc anti- human leiomyoma and matched myometrial tissues from gen inhibited lymphocyte activation and cell proliferation.
29 subjects. To allow comparisons of data obtained from This difference may be due to the different cell systems and samples of different subjects, the mRNA levels of LAT2, methods used. To our knowledge, the present study is the

Luo et al.
Progesterone Action, LAT2 and 4F2hc J Clin Endocrinol Metab, November 2009, 94(11):4533– 4539 to 4F2hc ratio in leiomyoma tissue mayfavor its growth. Further studies areneeded to clearly understand this com-plex mechanism.
We find that progesterone or mife- pristone regulates LAT2 or 4F2hc ex-pression via PR. PR exists as two iso-forms, PR-A and PR-B (30). In humans,PR-B and PR-A may have distinct or FIG. 4. Comparison of LAT2 and PR protein levels in cultured LSM cells with matched
similar functions depending on the pro- myometrial cells. Cell lysates were prepared for immunoblotting with anti-LAT2, anti-PR and moter context and cell type (31, 32).
␤-actin (loading control) antibodies (A) and quantified with ImageJ software (B). Data in A Thus, it seems justified to further from one representative result were reproduced in cells from four patients. Data in B evaluate the effects of each isoform on represent the mean ⫾ SEM from four subjects. *, P ⬍ 0.05, compared with myometrial cells.
LAT2 or 4F2hc expression in LSM first to demonstrate the function of LAT2/4F2hc through knockdown of these genes, and the first to report the ex- Information on the hormonal mechanisms regulating pression and function of LAT2/4F2hc in leiomyoma.
system-L transporter activity is still scarce (33–35). Ste- It should be noted that LAT2 requires 4F2hc to func- roid hormones may act via multiple mechanisms. They tion normally in the plasma membrane (17, 18). Knocking affect not only the transcriptional modulation of target down 4F2hc could lead to a loss of function of LAT2.
genes via intracellular receptors within minutes to hours Thus, our observation that knockdown of 4F2hc stimu- but also elicit rapid nongenomic effects within seconds to lated leiomyoma cell proliferation is consistent with the minutes. Steroid hormones have been shown to enhance or effect of LAT2 knockdown on the same cell type. In vivo, diminish amino acid transport depending on the nature of however, LAT2 but not 4F2hc expression in leiomyoma the hormone. The stimulation may involve either in- tissue was higher. It is likely that critical ratios of LAT2 to creased carrier synthesis or rapid nongenomic effects at 4F2hc determine the net in vivo effects of this L-type the cell membrane (36, 37), whereas inhibition may in- amino acid transporter system in leiomyoma tissue. To volve synthesis of a labile protein that either decreases the add a further twist, treatment with progesterone increases, rate of synthesis or increases the rate of degradation of a whereas mifepristone decreases, the LAT2 to 4F2hc ratio.
component of the transport system (38).
Evidence from a number of laboratories suggests that pro- In summary, we demonstrated the functions of two gesterone stimulates, whereas mifepristone inhibits genes involved in amino acid transport through the cell leiomyoma growth (4, 5, 29). The sum of these observa- membrane in leiomyoma cell growth. The products of tions is consistent with a model that a higher in vivo LAT2 these genes heterodimerize to regulate amino acid trans-port and also regulate leiomyoma proliferation; more-over, progesterone or an antiprogestin regulates their ex- pression. We speculate that critical ratios of LAT2 to 4F2hc may regulate leiomyoma growth.
We thank Dr. Dean Edwards (Baylor College of Medicine, Hous-ton, TX) for the PR antibody.
(fold difference) Tissue mRNA levels Address all correspondence and requests for reprints to: Serdar E. Bulun, Division of Reproductive Biology Research, FeinbergSchool of Medicine, Northwestern University, 303 East Superior Street, Suite 4-123, Chicago, Illinois 60611. E-mail: s-bulun@ FIG. 5. LAT2, 4F2hc, and PR mRNA levels in human leiomyoma and
This work was supported by National Institutes of Health matched myometrial tissues. Overall, 58 samples from 29 patients Grant HD46260.
were analyzed; 29 samples were obtained from leiomyoma and 29 Disclosure Summary: X.L., P.Y., S.R., H.I., Z.L., M.E.P., from adjacent myometrial tissues. To allow comparisons of data H.Z., and E.E.M. have nothing to declare. S.E.B. serves as a obtained from samples from different patients, mRNA levels in themyometrial tissues were normalized to 1. The data were shown as the consultant for the pharmaceutical companies Repros, Medit- mean ⫾ SEM. *, P ⬍ 0.01, compared with myometrial tissues.
rina, and Novartis.
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