Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma Yuanyuan Ren, M.Sc., Hao Yin, Ph.D.,

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Presentation transcript:

Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma Yuanyuan Ren, M.Sc., Hao Yin, Ph.D., Ruijuan Tian, M.Sc., Lihua Cui, M.Sc., Yingjun Zhu, M.D., Ph.D., Wanjun Lin, M.D., Xiang D. Tang, M.D., Ph.D., Yu Gui, M.D., Ph.D., Xi-Long Zheng, M.D., Ph.D. Fertility and Sterility Volume 96, Issue 4, Pages 1015-1020.e1 (October 2011) DOI: 10.1016/j.fertnstert.2011.07.004 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Epidermal growth factor (EGF) induces DNA synthesis and polyploidization in human leiomyomal smooth muscle cells (HL-SMCs). Serum-starved human myometrial (HM) and HL-SMCs were treated with 100 ng/mL EGF for 24 hours with and without pretreatment of 5 μmol/L AG1478 or 20 μmol/L PD98059, followed by pulse-labeling with bromodeoxyuridine (BrdU) and laser scanning cytometry (LSC) analysis as described in Materials and Methods. (A) BrdU incorporation rates of HL- and HM-SMCs with various treatments (n = 4). Data represent three independent experiments. (B) Representative Western blots of three independent experiments showing phospho-p44/42 MAPK in HM- and HL-SMCs in response to 100 ng/mL EGF treatment for 15 minutes with and without pretreatment of 5 μmol/L AG1478 or 20 μmol/L PD98059. (C) Polyploid cells, as detected by LSC, were defined by >4N DNA content. ∗P<.05 compared with control group; ∗∗P<.01 compared with EGF-treated group (n = 4). (D) Representative LSC scattergrams (n = 4) of three independent experiments. The y-axis is the total fluorescence of BrdU staining in each cell, and the x-axis is the DNA content in each cell. Cells in the upper quadrants were BrdU positive, and those in the right quadrants were polyploid. Inserts: the relocation images of scanned cells with 2–4N DNA content and >4N DNA content. Fertility and Sterility 2011 96, 1015-1020.e1DOI: (10.1016/j.fertnstert.2011.07.004) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Differential effects of EGF on phosphorylation of EGF receptor (EGFR) and Akt. Cultured HL- and HM-SMCs were treated with 100 ng/mL EGF for different time intervals, followed by Western blot analysis. Representative Western blots of three independent experiments show time-dependent phosphorylation of (A) EGFR and (B) Akt in HM- and HL-SMCs in response to 100 ng/mL EGF treatment. Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 1015-1020.e1DOI: (10.1016/j.fertnstert.2011.07.004) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Different effects of EGF on p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in HM- and HL-SMCs. Cultured HL- and HM-SMCs were treated with 100 ng/mL EGF for different time intervals, followed by Western blot analysis. (A) Representative Western blots of four independent experiments showing time-dependent phosphorylation of p44/42 MAPK in response to EGF treatment in HM- and HL-SMCs. (B) Accumulative data. The x-axis represents different time points. The y-axis represents the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK. Phosphorylation levels at different time points were standardized with the levels at 0 minutes (control). ∗P<.05 compared with HM-SMCs at the same time point (n = 3). (C) Representative Western blots of p44/42 MAPK kinase activity assay from four independent experiments. Cultured HM- and HL-SMCs were treated with EGF for 15 minutes, followed by kinase activity assay as described in Materials and Methods. The bar figure shows cumulative data of kinase activity assay. p44/42 MAPK activities are is indicated by the levels of ELK1 phosphorylation. ∗P<.05 (n = 4) compared with control group. #P<.05 (n = 4) between both cell types treated with EGF for 15 minutes. Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 1015-1020.e1DOI: (10.1016/j.fertnstert.2011.07.004) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 EGF treatment reduces the expression of p27 in cultured HL-SMCs. Cultured HL- and HM-SMCs were treated with 100 ng/mL EGF for different time intervals, followed by Western blot analysis. Representative Western blots from three independent experiments show that 100 ng/mL EGF treatment time-dependently increased and decreased p27 in HM- and HL-SMCs, respectively. Abbreviations as in Figure 1. Fertility and Sterility 2011 96, 1015-1020.e1DOI: (10.1016/j.fertnstert.2011.07.004) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Characterization of primarily cultured smooth muscle cells (SMCs) isolated from human leiomyoma (HL) and matched myometrial tissues (HM). (A) SMC marker proteins, such as SM α-actin, calponin, and SM22α, were detected in cultured HL- and HM-SMCs with the use of Western blot analysis. Data represent 3 independent experiments. (B) The cell numbers (y-axis) of cultured HM- and HL-SMCs were measured with the use of a hematocytometer for up to 7 days (x-axis). ∗P<.01 (n = 4). Fertility and Sterility 2011 96, 1015-1020.e1DOI: (10.1016/j.fertnstert.2011.07.004) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions