Endocannabinoid system regulates migration of endometrial stromal cells via cannabinoid receptor 1 through the activation of PI3K and ERK1/2 pathways

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Endocannabinoid system regulates migration of endometrial stromal cells via cannabinoid receptor 1 through the activation of PI3K and ERK1/2 pathways Davide Gentilini, Ph.D., Alessandra Besana, Ph.D., Paola Vigano, Ph.D., Paolo Dalino, M.D., Michele Vignali, M.D., Michela Melandri, M.D., Mauro Busacca, M.D., Anna Maria Di Blasio, M.D. Fertility and Sterility Volume 93, Issue 8, Pages 2588-2593 (May 2010) DOI: 10.1016/j.fertnstert.2010.02.006 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of methanandamide on chemotaxis (A) and chemokinesis (B) of endometrial stromal cells (ESCs). All of the experiments were performed in a Boyden chamber using collagen IV to coat the filters. The results are expressed for both assays as mean chemotactic index ± SD and represent the number of cells migrated in response to treatments compared with basal values (BAS). ∗P<.05 vs. corresponding unstimulated cells. (C) Effect of treatment with cannabinoid receptor 1 (CNR1)–selective agonist ACEA as chemoattractant on ESC migration. (D) Effect of pretreatment with the CNR1-selective antagonist AM251 before methanandamide stimulation on ESC migration. Fertility and Sterility 2010 93, 2588-2593DOI: (10.1016/j.fertnstert.2010.02.006) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Effect of the ERK1/2 inhibitor PD98059 (10−6 mol/L) and of the PI3K-selective inhibitor wortmannin (WORT; 10−7 mol/L) on methanadamide-stimulated migration of ESCs. Results are indicated as mean chemotactic index ± SD and represents the ratio between number of cells migrated under stimulation and in basal condition. ∗P<.05 vs. corresponding unstimulated cells. (B) Effect of methanadamide on Akt and ERK1/2 phosphorylation. ESCs were treated with and without methanandamide at different concentrations (from 10−5 to 10−8M) for 15 minutes. Lysates were immunoblotted with the indicated antibodies. The experiments were repeated five times with similar results. Abbreviations as in Figure 1. Fertility and Sterility 2010 93, 2588-2593DOI: (10.1016/j.fertnstert.2010.02.006) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Typical electric signal recorded from a single ESC after blocking ICa and INa currents. (A) Arrows indicate the interval considered to measure the average steady-state current at each potential. The perfusion of methanandamide (10−5 mol/L) increased the basic current (B, typical cell), and the methanandamide-sensitive current had the characteristics of a K+ current (C, average data; n = 8). Perfusion with AM251 (10−4 mol/L) slightly but not significantly inhibited the current and prevented the stimulating effect of methanandamide (D, typical cell). Abbreviations as in Figure 1. Fertility and Sterility 2010 93, 2588-2593DOI: (10.1016/j.fertnstert.2010.02.006) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of methanandamide (10−5 mol/L) on actin cytoskeleton pattern of ESCs. (A, left) Untreated cells showed a classic static phenotype. (A, right) Treatment with methanandamide (10−5 mol/L) induced a migratory phenotype. Groups of cells were analyzed and cells showing a static (B, left) or a migratory phenotype (B, right) were counted. (C) Results are expressed as mean ± SEM percentage of cells which manifested a migratory phenotype ∗P<.05 vs. corresponding unstimulated cells. Abbreviations as in Figure 1. Fertility and Sterility 2010 93, 2588-2593DOI: (10.1016/j.fertnstert.2010.02.006) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions