Tight junctions (TJs) are crucial to both the development and normal functioning of most organs. Studies have shown that TJs play dual roles in the proper physiological functions of tissues by forming a continuous belt-like structure that contributes to cell polarity and regulates paracellular transport (Fanning et al. 1999)
TJs consist of a complex of proteins that can be divided into three general categories: cytoplasmic plaque proteins like zonula occludens; regulatory proteins such as heterotrimeric G-proteins; and four-transmembrane domain proteins, which include occludin and claudins. Via these numerous components, TJs can affect cell signaling and gene expression involved with such processes as cellular proliferation, differentiation, and morphogenesis (Fanning et al, 1999; Gonzalez-Mariscal et al, 2008; Tsukita et al, 2008 and Gonzalez-Mariscal et al, 2007).
Claudins are essential in both maintaining the gate and fence functions of the TJs as well as being involved in multiple normal cellular processes. These proteins are part of a 24-member family and are expressed in a tissue-specific manner. Interestingly, recent research has demonstrated alterations in claudin gene expression in a variety of cancers including breast, prostate, colon, pancreatic and stomach. Claudin-3 and -4, the specific focus of this study, are two notable examples of claudin isoforms that are routinely overexpressed in a variety of cancers, including ovarian, breast, endometrial, and pancreatic. While a greater abundance of a TJ protein would seem to decrease tumor invasiveness, studies have found that this is not always the case. Increased levels of claudin-4 in breast tumor samples correspond with higher tumor grade and lower survival rate (Lanigan et al., 2008). Analysis of claudin-1, -3, and -4 expression in other breast tumor samples revealed that more invasive tumors expressed claudin-3 and -4 over claudin-1 (Blanchard et al., 2009). Similarly, increases in claudin-3 and -4 expression positively correlated with tumor progression in the endometrium, and overexpression of these two proteins corresponded with increased myometrial invasion (Pan et al., 2007). The overexpression of these two proteins can disrupt the TJ, as revealed by electron micrographs of TJs in endometrioid adenocarcinomas. Additionally, clear-cell endometrial cancer and uterine serous papillary carcinoma, two rare and highly aggressive forms of endometrial cancer, showed even greater claudin-3 and -4 expression compared to endometrioid adenocarcinomas (Konecny et al., 2008).
The endometrium and breast tissues are classical hormone-dependent tissues, and the effects of estradiol (E2) on the physiology of both are well known. The breast and endometrium are regularly exposed to varying concentrations of E2 throughout the menstrual cycle, inducing cellular proliferation in both tissues and affecting ductal growth in the breast. Similarly, progesterone (P4) is a key steroid hormone in these tissues, contributing to alveolar development in the breast and discouraging proliferation in the endometrium following ovulation. In addition to controlling reproductive tract structure and function, E2 has been shown to act as a mitogenic factor. The role of E2 as a mitogen affects normal processes, such as the tissue remodeling that occurs during the menstrual cycle and embryo implantation. However, the mitogenic function of E2 can also contribute to tumor initiation and progression, enhancing tumor cell motility and thus invasiveness. Strikingly, many of the specific molecular changes observed in the endometrium during implantation are also seen in tumorigenesis - diminished endometrial cell-cell attachment and TJ closures, expression of metalloproteinases, angiogenesis and differential expression of integrins to name a few.
Despite studies reporting the expression pattern of claudins in a variety of tumors and normal tissues, their roles and the hormonal regulation of their expression in cancer are not well known. Studies have shown that E2 decreases transepithelial resistance in normal human cervical (hECE and hEVEC) and breast cancer (MCF-7) epithelial cells by modulating the expression of occludin isoforms (Zeng et al, 2004; Gorodeski, 2007 and Salazar et al, 2012). Although there are several studies on the role of E2 on cell motility (breast, endometrial, ovarian, aortic endothelial cells and vascular smooth muscle cells), the data is contradictory, showing both a stimulatory and inhibitory effect on migration depending on cell type, suggesting cell-specific and E2-dependent mechanisms of action (Rochefort et al, 1998; Acconcia et al, 2006 and Geraldes et al 2002). Understanding how E2 modulates endometrial TJ proteins could improve our understanding of the biology of the endometrium and provide insights into treating endometrial cancer.
The reason for the upregulation of claudin-3 and -4 in certain endometrial and breast tumors is currently unclear. As demonstrated by the studies reviewed above, a better understanding of claudin-3 and -4 overexpression in these cancers could potentially provide clinically relevant information about tumor progression. The research collaboration of which this study is a part of previously observed in a panel of reproductive cell lines overexpression of claudin-3 and -4 in three estrogen receptor (ER)-positive cell lines - the breast cancer cell lines MDA-MB415 and MCF-7 as well as the endometrial cancer cell line HEC-1A (Todd et al, data unpublished). Strikingly, analysis of normal human tissues revealed expression of claudin-3 in the breast and faint expression of claudin-4 while neither claudin was observed in the uterus. This contrast in expression was further highlighted by the complete lack of claudin-3 and -4 expression in the normal human mammary epithelial cell line HMEC versus MCF-7 and HEC-1A cells. In addition, an initial examination of the subcellular distributions of claudin-3 and -4 in MCF-7 and HEC-1A cells showed the expected expression of both proteins in the membrane. Expression of claudin-3 and -4 was also observed in the cytosolic, nuclear, and cytoskeletal fractions, with the pattern of expression differing between the two cell lines and the two proteins. Together, these findings provided the basis for the present study, which explores the possible connections between these observations and the presence of hormones.
In order to learn more about the regulation of these two proteins, the breast epithelial MCF-7 and endometrial HEC-1A cancer cell lines were exposed to varying concentrations E2 P4 as well as the chemotherapeutic 4-hydroxy-tamoxifen (4-OH-TAM). Using immunoblot analysis, claudin-3 and -4 expression and subcellular localization were subsequently assessed.
We confirmed overexpression of claudin-4 in two endometrial (HEC-1A and RL95-2) cancer cell lines and two breast cancer cell lines (MCF-7 and MDA-MB415). Furthermore, our studies demonstrated that E2 has a biphasic effect on claudin-4 expression in both the breast epithelial MCF-7 and endometrial HEC-1A cancer cells as well as on claudin-3 expression in HEC-1A cells. Changing hormone concentration generally had a greater effect on claudin-4 versus claudin-3 expression, and subcellular distribution of the two proteins varied in a dose-dependent manner for almost all conditions. These differential effects of E2, P4, and 4-OH-TAM on claudin-3 and -4 expression in HEC-1A and MCF-7 cells could thus provide novel information concerning the role of hormones in tumorigenesis.
While this study examined only alterations of claudin-3 and -4 expression by a singular hormone, it is important to consider the potential effects combinations of hormones could have on claudin expression. A recent mathematical model demonstrated that the transcriptional response to 4-OH TAM through the ER in HEK293/ERα cell line is dependent on several factors, such as background concentrations of E2 and expression of ERs (Lebedeva et al., 2012). Findings such as these highlight the complexity of studying cellular processes at the molecular level. Additionally, while this research focused on claudin-3 and -4 expression, further studies are needed to test for the presence of other claudins in these cells. The variety of cis- and trans-interactions that can occur between claudin isoforms results in a TJ with unique and complex properties that can be subsequently regulated by phosphorylation or through a variety of signaling pathways (Gonzalez-Mariscal et al., 2010). Thus, this study contributes toward the understanding of alterations in claudin expression incurred by complex changes within the cell upon hormone exposure.
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