Early growth response-2 expression in uterine leiomyoma cells: regulation and function

Abstract

Objective: To investigate the regulation of early growth response-2 (Egr-2) by transforming growth factor β3 (TGF-β3) and its functions in cultured human uterine leiomyoma smooth muscle cells.

Design: Laboratory research.

Setting: Academic medical center.

Patient(s): Primary leiomyoma cells from patients with symptomatic leiomyomata.

Intervention(s): Tissue culture followed by RNA and protein analysis.

Main outcome measure(s): Cell proliferation, alteration in extracellular matrix component expression.

Result(s): In vivo mRNA levels of Egr-2 were statistically significantly higher in leiomyoma tissues compared with matched myometrial tissues, and showed a statistically significant correlation with TGF-β3 messenger RNA (mRNA) levels in leiomyoma tissues. In primary leiomyoma smooth muscle cells, TGF-β3 statistically significantly induced Egr-2 gene expression in a dose-dependent and time-dependent manner. Small interfering RNA (siRNA) knockdown of Egr-2 markedly increased the level of the proliferation marker proliferating cell nuclear antigen and the expression of proto-oncogene c-myc. On the other hand, ablation of Egr-2 stimulated collagen-1A1 and collagen-3A1 transcription and inhibited dermatopontin gene expression. However, the mRNA levels of α-smooth muscle actin and fibronectin were not affected by Egr-2 knockdown.

Conclusion(s): We demonstrated that TGF-β3 regulated Egr-2 gene expression and presented evidence that Egr-2 decreases collagen production and stimulates dermatopontin gene expression.

Figure 1

Correlation between mRNA levels of Egr-2 and TGF-β3 in human leiomyoma and myometrial tissues in vivo. (A) Egr-2 and TGF-β3 mRNA levels in human leiomyoma and matched myometrial tissues in vivo. To allow comparisons of data obtained from samples from different subjects, mRNA levels in the myometrial tissues were normalized to 1. The data are shown as the mean ± SEM. (B) Regression analysis indicated Egr-2 mRNA levels correlated significantly and positively with TGF-β3 mRNA levels (R=0.805, P=0.016). *P<0.05 versus myometrium tissues.

Figure 2

Effect of TGF-β3 treatment on the expression of Egr-2 in LSM cells. Serum-starved LSM cells were stimulated with (A) variable concentrations of TGF-β3 (ranging from 0.01 to 5 ng/ml) or vehicle (4 mM HCL containing 1 mg/ml BSA) for 3 hours; (B) TGF-β3 (1 ng/ml) or vehicle for 1, 3, 6, 24 hours; or (C) TGF-β3 (0.5, 1, or 5 ng/ml) or vehicle for 72 hours; Messenger RNA levels of Egr-2 at each dose (A) and time point (B) were measured by real-time PCR and normalized by GAPDH. Egr-2 protein levels were measured by immunoblot (C). β-actin was used as a loading control. Results are reported as fold change compared with cells treated with vehicle only and represented as the mean ± SEM of 3 independent experiments. *P<0.05 as compared with vehicle treatment.

Figure 3

The effects of siRNA knockdown of Egr-2 on LSM cell proliferation. A, Knockdown efficiency and specificity of the Egr-2 gene was examined by real-time PCR. B, Cell proliferation was determined by measuring PCNA protein levels using western blot. Blots were reprobed with a β-actin antibody for loading control. A representative experiment of 3 using LSM cells from 3 different subjects is shown. Western blot densities (n=3) were quantified using ImageJ software. C, Effect of Egr-2 knockdown on c-myc mRNA levels. Results are reported as fold change compared with control siRNA transfected cells and represented as the mean ± SEM of 3 indepent experiments. *P<0.05 as compared with control siRNA transfected cells.

Figure 4

The effects of Egr-2 knockdown on the expression of ECM genes in LSM cells. A. Knockdown of Egr-2 increased the mRNA levels of collagen1A1 (upper panel) and collagen3A1 (lower panel) in LSM cells. B. Egr-2 knockdown decreased dermatopontin expression in LSM cells. C. Egr-2 silencing down-regulated TGF-β3 gene expression. D. The mRNA levels of α-SMA (upper panel) and fibronectin (lower panel) were not affected by Egr-2 siRNA in LSM cells. Results are reported as fold change compared with control siRNA transfected cells and represented as the mean ± SEM of 3 independent experiments. *P<0.05 as compared with control siRNA transfected cells.