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. 2009 Nov;94(11):4533-9.
doi: 10.1210/jc.2009-1286. Epub 2009 Oct 6.

Progesterone and mifepristone regulate L-type amino acid transporter 2 and 4F2 heavy chain expression in uterine leiomyoma cells

Xia Luo  1 , Ping Yin, Scott Reierstad, Hiroshi Ishikawa, Zhihong Lin, Mary Ellen Pavone, Hong Zhao, Erica E Marsh, Serdar E Bulun
Affiliations

Progesterone and mifepristone regulate L-type amino acid transporter 2 and 4F2 heavy chain expression in uterine leiomyoma cells

Xia Luo et al. J Clin Endocrinol Metab. 2009 Nov.

Abstract

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 significantly 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.

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Figures

Figure 1
Figure 1
Regulation of LAT2 and 4F2hc expression by progesterone or mifepristone. Primary cultured LSM cells were starved in phenol red-free and serum-free DMEM/F12 (1:1) medium overnight and then treated with vehicle (ethanol), progesterone, or mifepristone for different times. LAT2 (A, D, E) and 4F2hc (B, D, F) mRNA levels were measured by real-time PCR and normalized to GAPDH mRNA levels. LAT2 protein levels were evaluated by Western blot using a LAT2 peptide antibody (C). Blots were reprobed with a β-actin antibody for loading control. Results for A, B, D, E and F were reported as a fold change compared with cells treated with vehicle only and represented as the mean ± sem. All graphs were derived from one representative experiment, and all experiments were repeated in triplicate in four subjects. *, P < 0.05, compared with vehicle treatment.
Figure 2
Figure 2
The effects of knocking down LAT2 on LSM cell proliferation. LSM cells were transfected with control siRNA or LAT2 siRNA for 72 h. Knockdown efficiency and specificity of the LAT2 gene were examined by both real-time PCR (A) and Western blot (B). Cell proliferation was analyzed by measuring PCNA protein level with mouse antihuman PCNA antibody (B). Blots were reprobed with a β-actin antibody for loading control. Western blot densities were quantified with ImageJ software (C). The effect of knocking down LAT2 on LSM cell proliferation was confirmed with MTT assay (D). Data for A (represented as mean ± sem) and B were from one representative experiment and were repeated in four subjects. Data for C and D show the mean ± sem from four subjects. Each experiment was done in triplicate. *, P < 0.05, compared with control siRNA.
Figure 3
Figure 3
The effects of knocking down 4F2hc on LSM cell proliferation. LSM cells were transfected with control siRNA or 4F2hc siRNA for 72 h. The cells were then harvested for analysis. Knockdown efficiency and specificity of the 4F2hc gene were examined by real-time PCR (A). LSM cells were harvested for determination of cell proliferation by measuring of PCNA protein level with mouse antihuman PCNA antibody (B). Western blot densities were quantified with ImageJ software (C). The effect of knockdown 4F2hc on LSM cell proliferation was confirmed with MTT assay (D). Blots were reprobed with a β-actin antibody, which was used as a control. 4F2hc mRNA measured by real-time PCR was normalized to GAPDH mRNA. Data for A (represented as mean ± sem) and B were from one representative experiment and were repeated in four subjects. Data for C and D are shown as the mean ± sem from four subjects. Each experiment was done in triplicate. *, P < 0.05, compared with control siRNA.
Figure 4
Figure 4
Comparison of LAT2 and PR protein levels in cultured LSM cells with matched myometrial cells. Cell lysates were prepared for immunoblotting with anti-LAT2, anti-PR and β-actin (loading control) antibodies (A) and quantified with ImageJ software (B). Data in A from one representative result were reproduced in cells from four patients. Data in B represent the mean ± sem from four subjects. *, P < 0.05, compared with myometrial cells.
Figure 5
Figure 5
LAT2, 4F2hc, and PR mRNA levels in human leiomyoma and matched myometrial tissues. Overall, 58 samples from 29 patients were analyzed; 29 samples were obtained from leiomyoma and 29 from adjacent myometrial tissues. To allow comparisons of data obtained from samples from different patients, mRNA levels in the myometrial tissues were normalized to 1. The data were shown as the mean ± sem. *, P < 0.01, compared with myometrial tissues.
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