Subhradip Karmakar1, Chandana Das. 1. Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Abstract
OBJECTIVES: The present study examines the effects of IL-1beta and TGF-beta1 in modulation of ezrin, E-cadherin, CD44 and beta-catenin expression in human trophoblast cells which may lead to their altered cytoskeleton dynamics during cell-to-cell and cell-to-matrix interactions. METHODS: Trophoblast (extravillous and villous) cells isolated and purified from early and term placentae and human choriocarcinoma cell line JEG-3 used in this study were challenged with either IL-1beta or TGF-beta1 (10 ng/ml) for 12 h following which RT-PCR was performed for ezrin, E-cadherin, CD44 and beta-catenin. Immunolocalization of these proteins was carried out in the chorionic villi as well as in the cultured cells stimulated by the cytokines. Western Blot was also performed to study the regulation of ezrin and E-cadherin in primary extravillous, villous and term trophoblast by these cytokines. Scanning electron microscopy (SEM) and Matrigel Invasion Assay was used to study the effect of these cytokines on cellular morphology and invasion. RESULTS: IL-1beta induced a down regulation in the expression of ezrin, E-cadherin and beta-catenin while upregulation of CD44 message in both primary trophoblast and JEG-3 cells. On the contrary, TGF-beta1 exhibited just an opposite effect, i.e. up regulation of ezrin, E-cadherin, beta-catenin, and down regulation of CD44. These observations were further corroborated with the immunolocalization findings of the above proteins in first trimester and term villous tissue, the former having predominance of IL-1beta and the latter of TGF-beta1 [Am. J. Reprod. Immunol. 48 (2002) 210]. Cellular morphology as observed through SEM revealed an enhanced cell-to-matrix adhesion with poor cell-cell interaction following IL-1beta challenge and a strong intercellular adhesion with weak cell-to-matrix interaction in presence of TGF-beta1. Crystal violet staining and Matrigel invasion revealed a higher invasion index following IL-1beta challenge and a low invasion index following TGF-beta1 challenge. CONCLUSION: IL-1beta mediated increased cell-to-matrix interaction with reduced cell-to-cell adhesion along with reduced ezrin and E-cadherin expression is associated with enhanced invasiveness while TGF-beta1 mediated up regulation of cell-to-cell adhesion with reduced cell-to-matrix interaction along with an increased ezrin and E-cadherin expression, is associated with reduced invasiveness, along with an altered cellular morphology. These facts therefore indicate the possible role of the two cytokines during cell motility and invasion through alteration of cell-matrix and cell-cell interaction.
OBJECTIVES: The present study examines the effects of IL-1beta and TGF-beta1 in modulation of ezrin, E-cadherin, CD44 and beta-catenin expression in human trophoblast cells which may lead to their altered cytoskeleton dynamics during cell-to-cell and cell-to-matrix interactions. METHODS: Trophoblast (extravillous and villous) cells isolated and purified from early and term placentae and humanchoriocarcinoma cell line JEG-3 used in this study were challenged with either IL-1beta or TGF-beta1 (10 ng/ml) for 12 h following which RT-PCR was performed for ezrin, E-cadherin, CD44 and beta-catenin. Immunolocalization of these proteins was carried out in the chorionic villi as well as in the cultured cells stimulated by the cytokines. Western Blot was also performed to study the regulation of ezrin and E-cadherin in primary extravillous, villous and term trophoblast by these cytokines. Scanning electron microscopy (SEM) and Matrigel Invasion Assay was used to study the effect of these cytokines on cellular morphology and invasion. RESULTS:IL-1beta induced a down regulation in the expression of ezrin, E-cadherin and beta-catenin while upregulation of CD44 message in both primary trophoblast and JEG-3 cells. On the contrary, TGF-beta1 exhibited just an opposite effect, i.e. up regulation of ezrin, E-cadherin, beta-catenin, and down regulation of CD44. These observations were further corroborated with the immunolocalization findings of the above proteins in first trimester and term villous tissue, the former having predominance of IL-1beta and the latter of TGF-beta1 [Am. J. Reprod. Immunol. 48 (2002) 210]. Cellular morphology as observed through SEM revealed an enhanced cell-to-matrix adhesion with poor cell-cell interaction following IL-1beta challenge and a strong intercellular adhesion with weak cell-to-matrix interaction in presence of TGF-beta1. Crystal violet staining and Matrigel invasion revealed a higher invasion index following IL-1beta challenge and a low invasion index following TGF-beta1 challenge. CONCLUSION:IL-1beta mediated increased cell-to-matrix interaction with reduced cell-to-cell adhesion along with reduced ezrin and E-cadherin expression is associated with enhanced invasiveness while TGF-beta1 mediated up regulation of cell-to-cell adhesion with reduced cell-to-matrix interaction along with an increased ezrin and E-cadherin expression, is associated with reduced invasiveness, along with an altered cellular morphology. These facts therefore indicate the possible role of the two cytokines during cell motility and invasion through alteration of cell-matrix and cell-cell interaction.
Authors: Jeffrey J Whyte; Ashley E Meyer; Lee D Spate; Joshua A Benne; Raissa Cecil; Melissa S Samuel; Clifton N Murphy; Randall S Prather; Rodney D Geisert Journal: Proc Natl Acad Sci U S A Date: 2017-12-26 Impact factor: 11.205