Introduction
In greatly simplified form the process by which cells of the immune system target sites of infection can be described as a two step process. Target cells and nearby immune system cells produce and secrete pro-inflammatory proteins called cytokines. The prototypical cytokine is interleukin 1-beta (IL-1β). IL-1β (and IL-18) are synthesized as inactive precursors that are converted to their active forms by proteolytic cleavage catalyzed by caspase 1 (IL-1β converting enzyme; ICE protease). IL-1β and other cytokines exhibit diverse effects that include stimulating the differentiation of cells of the immune system, and attracting immune system cells to sites of inflammation. Once attracted to the site of cytokine production immune system cells, such as T-cells, use a complex system based on T-cell receptor to recognize foreign antigens on cells infected with viruses and other intracellular pathogens or malignant cells. This recognition process triggers the release of granules, or granzymes, contained in the T-cells and natural killer (NK) cells. These granules contain several proteases, of which the most important is granzyme B. After entry into target cells, granzyme B cleaves the precursor forms of several caspases, and thereby induces apoptosis.
Because of their importance inflammatory processes and the production of pro-inflammatory cytokines is regulatged by a complex set of pro anti-inflammatory agents including orones such as glucocorticoids and estrogens and the numerous proteins involved in control of the nuclear factor kappa b (NF-κB) system. It is now widely accepted that inflammatory processes and inflammatory cytokines play an important role in several chronic disease states, including atherosclerosis, osteoporosis, and cirrhosis of the liver.
Sites of Action of PI-9
The serine proteinase inhibitor, proteinase inhibitor 9 (PI-9) is unique in its ability to inhibit both inflammation and apoptosis. PI-9 is a potent inhibitor of granzyme B and interferes with granzyme B mediated apoptosis when a cell is targeted by the immune system. PI-9 also inhibits caspase 1 and thereby reduces the production of pro-inflammatory cytokines important in maturation and migration of cells of the immune system. Together these effects of PI-9 represent a powerful multi-level system for modulating the interrelated inflammatory and apoptotic functions of caspase 1 and granzyme B.
Sites of PI-9 Action
Regulation of PI-9 Gene Expression
PI-9 was initially identified in our laboratory as a strongly estrogen-inducible mRNA in a line of ER positive human liver cells. Our studies using human liver biopsy specimens demonstrated that PI-9 is estrogen inducible in human liver (Kanamori et al, 2000). PI-9 was subsequently shown by others to be the first cellular inhibitor of caspase 1. Important physiologic roles for PI-9 were suggested by studies in other laboratories demonstrating that PI-9 expression is disregulated and largely absent in early stage atherosclerotic plaques and that PI-9 is overproduced in some cancers and protects them from immune system-mediated apoptosis. Since we found that estrogen induced PI-9 gene transcription, we cloned the human PI-9 gene. However, PI-9 does not contain a classical estrogen response element palidrome upstream of the transcription start site (Kanamori et al., 2000). We used transient trasnfections, gel mobility shift asssays, Dnase I forptinting and ChIP to demonstrate that estrogen induction of PI-9 gene transcrption is mediated through a novel downstream estrogen responsive unit (ERU) approximately 20 nucleotdies downstream of the transcription initiation site. The ERU consists of an imperfect ERE palindrome immediately followed by a direct repeat containing two consensus ERE half sites separated by 13 nucleotides. All four of the ERE half sites are important for efficient transcription and 2 ER dimmers are bound to the ERU. These studies were significant because they showed that a direct repeat of ERE half sites can function in a composite element to confer estrogen inducibility n a native gene (Krieg et al., 2001).
Glucocorticoids exihibit well characterized anti-inflammatory activities and PI-9 in an anti-inflammatory protein. We find that glucocorticoids moderately induce PI-9 through an imperfect glucocorticoids response element upstream of the transcription initiation site. Interestingly, the cellular PI-9 protein and PI-9 mRNA are induced by diverse pro-inflammatory agents including IL-1b, bacterial lipopolysaccharide (LPS), the phorbol ester, and other regulators including the phorbol ester, TPA, and tumor necrosis factor (TNF). These agents induce PI-9 gene transcriton through two functional nuclear factor kappa b (NF-κB) sites and a consensus AP-1 site. The complex regulation of PI-9 gene expression is summarized in the able below.
Regulation of PI-9 Gene Expression. This table summarizes diverse studies of the control of PI-9 gene expression from our laboratory. The data is derived from (Kanamori et al., 2000, Krieg et al., Kannan-Thulasiraman and Shapiro 2002, and Kannan-Thulasiraman and Shapiro (2003), Submitted for publication, and unpublished data). Daudi cells are a line of B-cells. The bone cells were osteoblasts (generously provided by Prof. T. Spelsberg).
Our surprising finding that thje pro-inflammatory cytokine IL-1b induces PI-9 suggests a novel mchanism for regulating inflammation and apoptosis. Since IL-1β is produced by caspase 1-mediated cleavage of its inactive precursor and PI-9 inhibits caspase 1 (and potentially the formation of active caspase 1 from pro-caspase 1), the IL-1β induction of PI-9 provides a potential mechanism to shut off inflammatory processes so they do not become self-sustaining and provides a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory protein and anti-apoptotic protein, PI-9. A possible description of this regulatory system is illustrated below.
Schematic of How Induction of PI-9 Might Interfere with Producton of IL-1β and Inflammatory processes. The several inducers of PI-9 are shown. These fall into three classes: Steroid hormones, estrogens and glucocorticoids. These hormones have known anti-inflammatory activities. Agents that effect cell growth and apoptosis including TPA and TNF. Pro-inflammatory agents LPS and IL-1β. Induction of PI-9 by any combination of these agents has the potential to reduce production of IL-1β.
Functional Studies of PI-9 Action
Analysis of the biological functions of PI-9 involves collaborative work with diverse laboratories. At one end of the spectrum we are working on structural studies of PI-9 and are interested in the mechanism by which it inhibits granzyme B and caspase 1. Studies using our affinity purified polyclonal antibody to PI-9 suggest there may be unidentified intracellular proteases that bind to PI-9. Our preliminary studies suggest an as yet unidentified high molecular weight protease binds to PI-9.
With an understanding of the basic systems regulating PI-9 gene expression, a major focus becomes determining whether these regulatory properties result in biologically significant phenotypes. Among the areas under investigation are functional studies of the role of PI-9 and its regulation in immune surveillance (in collaboration with the laboratory of Prof. David Kranz), fetal implantation and early development (in collaboration with the laboratory of Prof. Asgi Fazleabas), and inflammation.
Some Recent Publications
Kanamori, H., Krieg, S., Mao, C., Di Pippo, V.A., Wang, S., Zajchowski, D.A. and Shapiro, D.J. (2000) Proteinase, Inhibitor 9, an Inhibitor of Granzyme B-mediated Apoptosis is a Primary Estrogen-inducible gene in Human Liver Cells. J. Biol., Chem., 275: 5867-5873.
Krieg, S.A., Krieg, A.J., and Shapiro D.J. (2001) A unique Downstream Estrogen Responsive Unit Mediates Estrogen induction of proteinase Inihibitor-9, A Cellular inhibitor of IL-1beta Converting Enzyme (Caspase 1). Molec. Endocrinol., 15: 1971-1982.
Kanan-Thulasiraman, P., and Shapiro, D.J. (2002) modularors of Inflammation use Nuclear Factor-κB and Activator Protein-1 Sites to Induce the Caspase-1 and Granzyme B Inhibitor, Proteinase Inhibitor 9. J. Biol. Chem., 277: 41230-41239.
Last Updated: 08/12/03