Aberrant expression of leptin in human endometriotic stromal cells is induced by elevated levels of hypoxia inducible factor-1alpha

Abstract

Elevated expression of leptin in endometriotic tissue results in an increase in stromal cell proliferation and may contribute to the development of endometriosis. However, the underlying mechanism responsible for aberrant expression of leptin is not known. We hypothesize that aberrant expression of leptin in endometriotic stroma may be regulated by increased levels of hypoxia-inducible factor-1alpha (HIF-1alpha), the master transcription factor that controls gene expression in response to hypoxia. Herein we show that the mRNA and protein levels of HIF-1alpha were greater in ectopic endometriotic tissue compared with its eutopic counterpart. Exposure of eutopic endometrial stromal cells under hypoxic conditions or treated with desferrioxamine (DFO, chemical hypoxia) resulted in a time-dependent increase in leptin gene expression. A promoter activity assay demonstrated that HIF-1alpha induced leptin promoter activity after DFO treatment. Chromatin immunoprecipitation assay further demonstrated that binding of HIF-1alpha to leptin promoter was evident after DFO treatment. Finally, depletion of HIF-1alpha by short interference RNA abolished leptin expression in ectopic endometriotic stromal cells. Taken together, our data demonstrate that aberrant expression of leptin in ectopic endometriotic stromal cells is induced, at least in part, by an elevated level of HIF-1alpha in these cells, providing new insights into the etiology of endometriosis.

Figure 1

Expression of HIF-1α mRNA in stromal cells obtained from eutopic endometrium and ectopic endometriotic lesions. A: A representative RT-PCR gel picture shows HIF-1α mRNA levels in stromal cells isolated from normal endometria and endometriotic tissues. GAPDH was used as an internal control. N1–N3, eutopic endometria from disease-free patients (normal control); E1–E3, ectopic endometriotic lesions; NC, negative control. B: Mean HIF-1α to GAPDH mRNA ratios in stromal cells isolated from eutopic endometria in disease-free patients (Normal; n = 9) and ectopic endometria in patients with endometriosis (Endo; n = 9). Asterisk indicates significant difference between these two groups (P < 0.05 with Student’s t-test analysis).

Figure 2

Expression of HIF-1α protein in samples obtained from eutopic endometria and ectopic endometriotic lesions. A: A representative Western blot shows expression of HIF-1α protein in nuclear extracts of stromal cells isolated from normal endometrium (N1 and N2) and ectopic endometriotic lesion (E1 and E2). B: A representative Western blot shows levels of HIF-1α protein in nuclear extracts of stromal cells isolated from paired eutopic (Eu) and ectopic (Ec) endometria of two patients with endometriosis. C: Means of HIF-1α to β-actin protein ratio in eutopic and ectopic stromal cells (n = 5) similar to that shown in B. Asterisk indicates significant difference at P < 0.05 with Student’s t-test.

Figure 3

Induction of leptin mRNA by true and chemically mimicked hypoxia. A: A representative Western blot shows expression of HIF-1α and HIF-1β protein in stromal cells cultured under normoxia (N) or hypoxia (H). N4, 4 hours normoxia; H4, 4 hours hypoxia; N24, 24 hours normoxia; H24, 24 hours hypoxia. This experiment was repeated four times with different batches of cells, and the results were similar. B: A representative RT-PCR gel picture shows expression of leptin mRNA in endometrial stromal cells cultured under normoxia or hypoxia for 24 hours. Norm, normoxia; Hypo, hypoxia. This experiment was repeated four times with different batches of cells, and the results were similar. C: A representative Western blot shows expression of HIF-1α and HIF-1β protein in stromal cells cultured under normoxia treated with or without 10 mmol/L DFO for indicated time. This experiment was repeated four times with different batches of cells and the results were similar. D: A representative RT-PCR gel picture shows expression of leptin (leptin) and 18S ribosomal RNA (18S) mRNA in normal endometrial stromal cells treated with or without DFO for the indicated time. Means and standard errors from five independent experiments using different batches of cells were combined and shown in the bottom panel. Asterisks indicate significant differences between control (Con; without DFO treatment) and DFO-treated (DFO) groups at P < 0.05 using Student’s t-test at each time point.

Figure 4

Levels of leptin protein expression in cell-free supernatant of culture media measured by ELISA method in the paired eutopic and ectopic endometrial stromal cells from the same patients (n = 3). The leptin was undetectable in eutopic stromal cells under normoxia conditions. However, increased leptin expression was noted in both eutopic and ectopic stromal cells under hypoxic (1% O₂) conditions. Asterisk indicates significant difference (P < 0.05) within the ectopic endometriotic stromal cells between normoxia and hypoxia conditions using Student’s t-test.

Figure 5

DFO-induced leptin promoter activity is mediated by proximal hypoxia response element (HRE). A: A schematic drawing of the 5′-regulatory region of human leptin promoter. Two putative HREs (HRE1 and HRE2) analyzed by bioinformatic methods were indicated in boxes with mutated sequences shown under the HRE. Arrows indicate the direction of HRE. B: Human endometrial stromal cells were transiently transfected with reporter plasmid containing empty vector (pGL3), human leptin promoter construct with wild-type HRE (a fragment corresponding to human leptin gene promoter −490/−1), or constructs with site-directed mutated HREs. Transfected cells were then treated with or without DFO (10 mmol/L) for 16 hours and luciferase activity was determined. WT, wild type; m, mutated; dm, double mutated; HRE, hypoxia response element; RLU, relative luciferase activity. Asterisks indicate significant differences (P < 0.05) in luciferase activity between normoxia and hypoxia under different transfected conditions using analysis of variance.

Figure 6

HIF-1α binds to human leptin promoter and regulates its activity. A: A representative PCR gel from eutopic stromal cells using the ChIP assay demonstrates a single band amplified by PCR using HIF-1α antibody precipitated genomic DNA obtained from DFO-treated cells as template (n = 5). C, control (without DFO treatment). D, DFO-treated group. Ig, normal rabbit IgG. This experiment was repeated four times using different batches of cells and the results were identical. B: A representative gel picture shows transfection of siRNA targeted at HIF-1α (SiHIF-1) abolished DFO-induced leptin mRNA expression in ectopic endometriotic stromal cells. The sequence-scrambled siRNA (SrHIF-1) was not effective. This experiment was repeated three times using different batches of cells, and the results were similar.