Secretory Leukocyte Protease Inhibitor Mediates Proliferation of Human Endometrial Epithelial Cells by Positive and Negative Regulation of Growth-associated Genes

Abstract

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-␤1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-␤1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated. Secretory leukocyte protease inhibitor (SLPI),1 also known as anti-leukoprotease, human mucus proteinase inhibitor, and human seminal plasma inhibitor, is a 12-kDa member of the chelonianin class of serine protease inhibitors, which also includes SKALP/elafin (1, 2). SLPI is composed of two homologous cysteine-rich domains. The carboxyl-terminal domain manifests inhibitory activities against chymotrypsin, trypsin, granulocyte and pancreatic elastases, cathepsin G, and mast cell chymase (3, 4). SLPI is expressed primarily in secretory/glandular epithelial cells of a variety of human tissues including the bronchi, parotid glands, small and large intestines, skin, breast, pancreas, male and female genital tracts, and kidney (5-14). Its relative absence in serum, except in certain pathological conditions (15, 16), and its localization to extracellular matrix and subcellular sites not readily accessible to larger molecular weight protease inhibitors such as ␣ 1 -antitrypsin (17), suggests a primarily autocrine/paracrine mode of action. In addition to its anti-protease activity, SLPI has anti-inflammatory, anti-bacterial, anti-fungal, and anti-retroviral (human immunodeficiency virus) activities that appear to reside in its amino-terminal cysteine-rich domain (18 -23). Furthermore, SLPI has been shown to regulate intracellular enzyme synthesis, suppress matrix metalloproteinase production and activity, mediate normal wound healing, prevent scar formation, and augment fertility (24 -27). The higher uterine endometrial expression of SLPI during pregnancy ...

Identification of new genes associated with breast cancer progression by gene expression analysis of predefined sets of neoplastic tissues

“…60 SLPI also exhibits proliferative effects, although its mechanism remains unknown. 61 Several studies demonstrated that SLPI is altered in cancer; it was found upregulated in ovarian and in lung carcinomas, and its serum level correlated with tumor stage and response to therapy. 62,63 The exact mechanism by which SLPI promotes malignancy is not yet known: in addition to tumor growth support, the enhancement of malignancy could be due to its effects on angiogenesis.…”

Subfertility, Uterine Hypoplasia, and Partial Progesterone Resistance in Mice Lacking the Krüppel-like Factor 9/Basic Transcription Element-binding Protein-1 (Bteb1) Gene

“…To examine whether alterations in expression of specific uterine genes at 6.5 dpc accompany the reduced number of implantation sites noted in Bteb1 Ϫ/Ϫ mice, uteri from WT and Bteb1 Ϫ/Ϫ mice at 6.5 dpc were evaluated by QPCR for expression of progesterone-responsive, Bteb1-regulated, and/or implantation-specific genes (37,38,44,45) (Fig. 7).…”

The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells

“…cross-linking for 90s followed by baking at 80 C for 25 min. The pre-hybridization and hybridization of membranes with 32 P-labeled human SLPI cDNA fragment (399 bp) using ULTRAhyb followed previously described protocols (Zhang et al 2002a). Membranes were washed twice in 2 SSC, 0·1% SDS and in 0·1 SSC, 0·1% SDS at 42 C for 15 min each time to remove non-specifically bound probe.…”

“…Over-expression of SLPI has been noted in human tumors and human carcinoma cell lines of ovarian, endometrial, respiratory tract, and neural origins (Koshikawa et al 1996, Ameshima et al 2000, Hough et al 2001, Shigemasa et al 2001, Westin et al 2002. In endometrial clonal cell lines engineered to under-express SLPI by forced expression of antisense SLPI mRNA, a direct linkage between cellular SLPI production and proliferation has been observed (Zhang et al 2002a). In these cell lines, expression of SLPI was positively correlated with increased expression of the cell cycle progression factors cyclin D1 and proliferating nuclear antigen, and was inversely correlated with expression levels of the epithelial growth inhibitors, transforming growth factor-(TGF-) and insulin-like growth factor-binding protein (IGFBP)-3, and of the tumor suppressor lysyl oxidase (Zhang et al 2002a).…”

“…In endometrial clonal cell lines engineered to under-express SLPI by forced expression of antisense SLPI mRNA, a direct linkage between cellular SLPI production and proliferation has been observed (Zhang et al 2002a). In these cell lines, expression of SLPI was positively correlated with increased expression of the cell cycle progression factors cyclin D1 and proliferating nuclear antigen, and was inversely correlated with expression levels of the epithelial growth inhibitors, transforming growth factor-(TGF-) and insulin-like growth factor-binding protein (IGFBP)-3, and of the tumor suppressor lysyl oxidase (Zhang et al 2002a). A causal role for SLPI in the promotion of malignant behavior was also recently demonstrated in lung carcinoma cells stably transfected with mouse or human SLPI expression constructs (Devoogdt et al 2003).…”