Title Peripheral myelin protein-22 ( PMP 22 ) modulates alpha 6 integrin expression in the human endometrium

Background: PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium. Methods: Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on a6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices. Results: In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and a6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of a6 integrin. Expression of both PMP22 and a6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase a6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed a6 integrin surface expression. Conclusion: These findings suggest a physiologic role for PMP22 on the expression of a6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of a6 integrin expression in the endometrium. Background Peripheral myelin protein 22 (PMP22) is a member of the Growth Arrest Specific 3 (GAS3) family of tetraspan proteins. Expression of the PMP22 gene is driven by two alternate promoters P1 and P2 which drive transcription for two transcripts containing different noncoding exons termed 1A and 1B [1]. Although both transcripts translate into identical proteins, the presence of two promoters is thought to confer tissue specific control of expression [2]. Transcripts arising from promoter 1 (termed 1A) have been shown in the peripheral and central nervous systems and are thought to be important for myelin formation [3,4]. Transcripts originating from promoter 2 (termed 1B) have been identified in neuronal and non-neuronal tissue throughout the body [1]. Within non-neuronal tissue, transcripts of PMP22 1B have been identified in the epithelia of the lungs and uterus, the choroid plexus, and the heart [5,6]. * Correspondence: mwadehra@mednet.ucla. edu † Contributed equally Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA Full list of author information is available at the end of the article Rao et al. Reproductive Biology and Endocrinology 2011, 9:56 http://www.rbej. com/content/9/1/56 © 2011 Rao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Translation of the PMP22 gene gives rise to a 160amino-acid protein, with four predicted transmembrane domains. The highest expression of PMP22 occurs in Schwann cells, and there, PMP22 localizes strictly with compact myelin [7]. Altered expression of PMP22 has grave consequences as it is associated with certain heritable demyelinating peripheral neuropathies. In particular, elevated expression of PMP22 causes CharcotMarie-Tooth disease type 1A (CMT1A), an autosomal dominant condition that is characterized by progressive motor and sensory polyneuropathy [8-10]. Haploinsufficiency of PMP22 results in hereditary neuropathy with liability to pressure palsies (HNPP) [11,12]. Outside of its role in myelin formation, studies have implicated PMP22 in a number of cellular roles including adhesion and the regulation of proliferation [13]. In fact, PMP22 was first discovered as a gene upregulated in growth-arrested fibroblasts in culture [14], and since then, PMP22 protein has been shown to help regulate cell spreading and regulate apoptosis in these cells [15]. Its importance in non-neuronal cells was further expanded when it found that in epithelial cells, PMP22 localized within tight junctions and formed complexes with integrins such as a6b4 and with the integral cation channel P2X7 [16-18]. Several studies suggest a complex mechanism for the regulation of PMP22 expression, and recent studies have implicated steroid hormones in its regulation. Studies have shown that both progesterone and glucocorticosteroids act as positive regulators of expression in Schwann cells [19-21], and anti-progesterone therapy has been shown to reduce PMP22 levels, reducing the CMT1A phenotype [22,23]. However, outside of this cell type, limited information is available as to hormonal control of PMP22 expression. PMP22 has been observed in the uterus, with high PMP22 mRNA levels in proliferative stroma [24], but no studies have examined PMP22 expression in epithelial cells of the endometrium or throughout the estrous cycle. In this study, we characterize the expression of human PMP22 during the proliferative and secretory phases of the female menstrual cycle. As previous studies have suggested that PMP22 associates with integrins, we generated human endometrial cell lines with varying levels of PMP22 expression and characterized their integrin profiles. We report for the first time, the expression of PMP22 protein in the human endometrium, with greater expression in the proliferative phase as compared to the secretory phase. Furthermore, we show that PMP22 colocalizes a6 integrin both in vitro and in normal human tissue samples. The dichotomy of PMP22 and a6 integrin expression in the female menstrual cycle suggests roles for the both proteins in adhesion and state of endometrial differentiation. Methods