All-trans-retinoic acid mediates changes in PI3K and retinoic acid signaling proteins of leiomyomas  Hadas Ben-Sasson, M.Sc., Assaf Ben-Meir, M.D., Asher Shushan, M.D., Laila Karra, M.Sc., Nathan Rojansky, M.D., Benjamin Y. Klein, M.D., Rubina Levitzki, B.Sc., Hannah Ben-Bassat, Ph.D.  Fertility and Sterility  Volume 95, Issue 6, Pages 2080-2086 (May 2011) DOI: 10.1016/j.fertnstert.2011.01.155 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Schematic representation of the retinol signaling pathway. It indicates two principal enzymes: alcohol dehydrogenase (oxidizes retinol to retinal) and aldehyde dehydrogenase (oxidizes retinal to ATRA). CRBP = cellular retinol-binding protein; CYP26 = cytochrome P450 family 26 enzymes; RA = retinoic acid; RAR = retinoic acid receptor; RARE = retinoic acid response element; RXR = retinoid X receptors. (B) Schematic representation of selected phosphatidylinositol 3-kinase (PI3K)/Akt proteins controlling proliferation and/or apoptosis and their activation. The PI3K pathway, activated by many survival factors, leads to activation of PIP3, PDK1, and Akt, important players of survival signaling. PTEN negatively regulates the PI3K pathway. Active Akt inhibits by phosphorylation many downstream proapoptotic proteins: GSK3, FoxO1 (member of the FKHR family), and Bax (a member of the Bcl2 family). Active Akt is also involved in suppressing the activity (by phosphorylation) of tuberin and hamartin, critical regulators for cell growth and proliferation. Akt regulates cell growth also through its effect on the cell cycle and on the level of D-type cyclins (CD1/2). Regulation of D-type cyclins also involves GSK3. It should be noted that cell lineage specificity of individual signaling molecules has to be considered. ↓ = direct stimulatory modification; ⊥ = direct inhibitory modification. Colors indicate protein function: red) kinase; green) phosphatase; blue) transcription factor. Fertility and Sterility 2011 95, 2080-2086DOI: (10.1016/j.fertnstert.2011.01.155) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Densitometric immunoblot analysis (Western blot) showing the effect of all-trans-retinoic acid (ATRA) on alcohol dehydrogenase 1 (ADH1) and aldehyde dehydrogenase 1 (ALDH1) protein expressions of leiomyoma and myometrial cultured cells at 24 hours’ treatment (n = 6). (A) ATRA 10−9 mol/L; (B) ATRA 10−7 mol/L. Data are expressed as mean ± SE. ∗P<.05 (Wilcoxon signed rank test, Student t test). Fertility and Sterility 2011 95, 2080-2086DOI: (10.1016/j.fertnstert.2011.01.155) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Densitometric analysis of Western blot (A) showing the effect of all-trans-retinoic acid (ATRA) at (B) 10−9 mol/L and (C) 10−7 mol/L on PI3K/Akt protein expressions of leiomyoma and myometrial cultured cells at 24 hours’ treatment (n = 6). Data are expressed as mean ± SE. ∗P<.05 (Wilcoxon signed rank test, Student t test). Fertility and Sterility 2011 95, 2080-2086DOI: (10.1016/j.fertnstert.2011.01.155) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 ATRA affects morphology, proliferation, and cell cycle distribution of smooth muscle cultured cells in a dose- and time-dependent manner. (A) Representative phase-contrast micrographs of leiomyoma cultured cells treated with ATRA at 10−9 mol/L, 10−7 mol/L, or 10−6 mol/L for 48 hours compared with untreated control sample. Morphologic changes observed with adjacent myometrial cells were generally similar (data not shown). (B) ATRA at 10−7 mol/L was already effective on day 3 and cell proliferation remained reduced as monitored on day 12. (C) The inhibitory effect of ATRA was evident and significant on day 7 of treatment (P≤.05). Analysis of 6 matched paired cultures. Data are expressed as mean ± SE. ∗P<.05 (Student t test). (D) Representative cell cycle analysis of matched leiomyoma and myometrial cells treated with 10−7 mol/L ATRA for 24 hours (red) and 48 hours (black). Cell cycle distribution and apoptosis were determined by fluorescence-activated cell sorter analysis. ATRA altered the cell cycle distribution, with increased subG1/G0 (apoptotic) fraction, slightly decreased G1 fraction, and no effect on G2/M fraction compared with the untreated control subjects. The response of the paired leiomyoma and myometrial cells to treatment with ATRA was similar. Fertility and Sterility 2011 95, 2080-2086DOI: (10.1016/j.fertnstert.2011.01.155) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions