Steven L. Weinstein
An effective immune system is absolutely essential for health and long life, protecting us against a wide variety of infectious disease-causing microorganisms. Macrophages are key cells within the immune system that are part of the first line of defense against infection. These cells “eat” microbes and destroy them internally and they release several toxic molecules that kill microbes in the local area. However, arguably macrophages most important function during an infection is to release mediator proteins called cytokines that recruit immune system cells in great numbers to the site of infection, a watershed event in mounting an effective immune defense. My research seeks to elucidate the molecular mechanisms that regulate the production of cytokines in macrophages. A fuller understanding of these of mechanisms is a critical ingredient in efforts to generate new insight into human disease and to develop more effective therapeutic interventions.
1. ATF3-Mediated Transcriptional Repression of Interferon-ß
Interferon-beta (IFN-ß) is a critical mediator of the early immune response to infection as well as being a complex modulator that potentiates or prevents adverse immune responses which contribute to autoimmunity and allergy. A large body of data from animal experiments illustrate that inappropriate levels of IFN-ß cause severe pathology. Consequently, it is not surprising that cells producing IFN-ß have evolved both inductive and repressive mechanisms to tightly control the levels of this cytokine.
IFN-ß production by macrophages (Mø), a major source of this cytokine, is constitutively quite low, but synthesis is rapidly increased upon exposure to microbial stimuli present during infection. Much of the increased production of this cytokine in stimulated Mø is attributable to transcriptional induction of the IFN-ß gene. The specific transcription factors and their binding sites within the IFN-ß promoter that mediate transactivation are known in extensive detail. In contrast, the mechanisms that mediate transcriptional repression of this gene are not as well understood, although evidence for this type of regulation exists.
Recently, we obtained preliminary data indicating that the transcriptional regulator, Activating Transcription Factor 3 (ATF3) represses IFN-ß transcription. Mø lacking ATF3 or expressing reduced amounts of ATF3 produce higher levels of IFN-ß mRNA after LPS stimulation, compared to wild type Mø. These observations lead us to hypothesize that ATF3 binds to the IFN-ß promoter and represses transcription of this gene in Mø. Further, we hypothesize that regulation of IFN-ß and other genes by ATF3 is immunologically relevant.
The specific aims are designed to test both hypotheses by: 1) determining whether over-expression of ATF3 in Mø represses IFN-ß transcription, 2) determining whether ATF3 binds to the IFN-ß promoter, 3) determining whether ATF3-mediated repression of IFN-ß transcription is caused by histone deacetylation, 4) determining whether ATF3 influences host immune responses, and 5) determining the identity of other ATF3 target genes in Mø.
2. Positive Transcriptional Regulators of CCL4 in LPS-treated Macrophages
Macrophage inflammatory protein-1beta (MIP-1ß) now called CCL4 is a mediator of the inflammatory response that is necessary for effective host defense, but that in its chronic form causes severe tissue damage and disease. CCL4 is produced by macrophages following exposure to lipopolysaccharide (LPS) as part of the immune response to bacterial infection and the expression of CCL4 in these cells is controlled transcriptionally.
The goal of this research is to understand the molecular mechanism for the transcriptional induction of CCL4 in LPS-activated macrophages. Current data indicate that the LPS-responsiveness of the CCL4 gene is governed by an ATF/CRE sequence [TGACATCA] located in the proximal promoter region. Multiple nuclear proteins bind to this sequence, at least in vitro. In preliminary experiments we have identified two ATF/CRE binding proteins as ATF2 and ATF4, making these proteins the leading candidates to transactivate the CCL4 gene. Alternatively, other proteins bound to the ATF/CRE site that are currently not identified may mediate CCL4 transcription. It is known that ATF2 and ATF4 are able to form heterodimers, but each protein also has other binding partners. Thus, ATF2 and ATF3 may bind to the ATF/CRE sequence in the CCL4 promoter as a heterodimer or they may bind partnered with other proteins. As a starting point to investigate these different possibilities, we hypothesize that ATF2/ATF4 heterodimers bind to the ATF/CRE element in the CCL4 promoter and transactivate this gene in LPS-treated macrophages.
This investigation is designed to test this hypothesis by: 1) Determining whether ATF2 / ATF3 heterodimers bind to the ATF/CRE element in the CCL4 promoter; 2) Determining whether ATF2 and ATF3 transactivate CCL4 in LPS-stimulated macrophages; and 3) Determining how ATF2 and ATF3 transactivate CCL4 in LPS treated macrophages.
3. Examination of cyclic AMP-responsive element binding protein (CREB) as a LPS-regulated transcription factor
Many of the immune responses of macrophages to LPS exposure require the synthesis of proteins not present in the cell prior to the appearance of the stimulus. The expression of several of these proteins is regulated at the level of gene transcription. Thus, a detailed understanding of the transcriptional regulatory proteins that control the LPS-modulated genes is of interest.
One of the transcription factors we believe influences LPS-induced genes is the cyclic AMP-responsive element binding protein (CREB). This hypothesis is based on the observations that: 1) LPS rapidly stimulates the addition of a phosphate group on a serine residue in CREB that is known to regulate the function of this transcription factor; and 2) a mutant version of CREB that cannot be phosphorylated on this residue interferes with LPS-induced, CREB-dependent transcription. Thus, CREB has the features of a LPS-activated transcription factor.
To test this hypothesis, we have evaluated whether the Early Growth Response-1 gene (Egr-1) is a LPS-induced CREB-regulated gene. We chose this gene because transcription of Egr-1 is rapidly induced by LPS in macrophages, the Egr-1 promoter contains two binding sites for CREB and Egr-1 has been strongly linked to the cellular responses triggered by LPS. Thus far, we have found that a dominant-negative mutant of CREB blocks (by > 80%) LPS-induction of an Egr-1 promoter-driven reporter. In addition, point mutation of one of the two CREB binding sites within the Egr-1 promoter greatly reduce LPS induction (by 60-70%). We are currently examining the proteins bound to the CREB binding site that appears to mediate LPS-induction of Egr-1. We suspect that CREB or a related transcription factor binds to this site. Our future work on CREB will be directed toward identifying other CREB-dependent, LPS-induced genes.
Publications (* denotes student author)
+Khuu, C.H., *Barroso, R.M., Hai, T. and S.L. Weinstein. Activating transcription factor 3 (ATF3) represses the expression of CCL4 in murine macrophages. Molecular Immunology, 44:1609-1616, 2007.
Weinstein, SL, AJ Finn, SH Davé, F Meng, CA Lowell, JS Sanghera and AL DeFranco. Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-beta. Journal of Leukocyte Biology 67: 405-414, 2000.
DeFranco AL, Crowley MT, Finn A; Hambleton J, SL Weinstein. The role of tyrosine kinases and map kinases in LPS-induced signaling.Progress in Clinical Biology Research 397:119-36, 1998.
DeFranco, A.L., M.T. Crowley, A.J. Finn, J. Hambleton, M.L. MacKichan and S.L. Weinstein. The role of MAP kinases, phosphatidylinositol 3-kinase and ceramide in LPS-induced signaling in macrophages. In Bacterial endotoxin: recognition and effector mechanisms. Proceedings of the Fourth Congress of the International Endotoxin Society, 29:473-482. J. Levin, editor, Elsevier, 1998.
Sanghera, J.S., S.L. Weinstein, M. Aluwalia, J. Girn and S.L. Pelech. Activation of multiple protein kinase pathways by bacterial lipopolysaccharide in murine macrophages. Journal of Immunology 156: 4457-4465, 1996.
Hambleton, J.H., S.L. Weinstein, L. Lem and A.L. DeFranco. Activation of c-Jun N-terminal kinase by bacterial lipopolysaccharide in monocyte/macrophage cells. Proceedings of the National Academy of Sciences USA 93: 2774-2778, 1996.
DeFranco, A.L., J. Hambleton, M. McMahon and S.L. Weinstein. Examination of the role of MAP kinase in the response of macrophages to lipopolysaccharide. Progress in Clinical Biology Research. 392:407-20, 1995.
DeFranco, A.L., S.L.Weinstein, J.S. Sanghera, S.L. Pelech and C.H. June. LPS stimulation of protein tyrosine phosphorylation in macrophages. In Bacterial endotoxin: recognition and effector mechanisms. J. Levin et al., editors, Elsevier Science Publishers, Amsterdam, 1993, pp.255-265.
Weinstein, S.L., C.H. June and A.L. DeFranco. Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14. Journal of Immunology 151:3829-3838, 1993.
Weinstein, S.L., J.S. Sanghera, K. Lemke, A.L. DeFranco and S.L. Pelech. Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages. Journal of Biological Chemistry 267:14955-14962, 1992.
Weinstein, S.L., M.R. Gold and A.L. DeFranco. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proceedings of the National Academy of Sciences USA 88:4148-4152, 1991.
Presentations since 2007 (* denotes student author)
*G. Walton Jr and S.L. Weinstein. Regulation of Interferon-beta Transcription by ATF-3 in RAW 264.7 Macrophages. Annual Biomedical Research Conference for Minority Students (ABRCMS). Phoenix, AZ. 2009
*A. Molina and S.L. Weinstein. Measuring interferon-beta secreted by normal and ATF-3 deficient macrophages. Society for Advancement of Chicanos and Native Americans (SACNAS). Dallas, TX, 2009.
*M.Sandoval, *Ami Antani and S.L. Weinstein.The ATF3 transcription factor is a potential negative regulator of IFN-ß. College of Science and Engineering. Student Project Showcase. SFSU. Spring 2009 (1st place)
*R. Barroso and S.L. Weinstein. College of Science and Engineering. Student Project Showcase. SFSU. Spring 2008.
*B.Olveda II and S.L. Weinstein. Exploring ATF-3 as a Negative Regulator of IFN-Beta. Society for Advancement of Chicanos and Native Americans (SACNAS), Salt Lake City, UT, 2008.
*V. McDaniel and S.L. Weinstein. Is Activating Transcription Factor 4 (ATF4) a Transactivator of the CCL4 gene?. Annual Biomedical Research Conference for Minority Students (ABRCMS), Austin, TX, 2007.
*M. V.Ly and S.L. Weinstein. Effect of RNA Interference-Mediated Knockdow of Activating Transcription Factor 3 (ATF3) on Interferon-ß. Annual Biomedical Research Conference for Minority Students (ABRCMS), Austin, TX, 2007.
*E. Martinez and S.L. Weinstein. Effect of siRNA-mediated ATF3 Knockdown on Interferon-ß Transcription in Macrophages. Society for Advancement of Chicanos and Native Americans (SACNAS), Kansas City, MO, 2007.
*R.Barrozo and S.L. Weinstein. Transcriptional Repressor ATF3 Binds to the IFN-beta Promoter in Macrophages. Society for Advancement of Chicanos and Native Americans (SACNAS), Kansas City MO, 2007.
*R.Barrozo and S.L. Weinstein. Transcriptional Repressor ATF3 Binds to the IFN-beta Promoter in Macrophages. Graduate Student Showcase. SFSU May 2007.
*R.Barrozo and S.L. Weinstein. Transcriptional Repressor ATF3 Binds to the IFN-beta Promoter in Macrophages. College of Science and Engineering. Student Project Showcase. SFSU. May 2007. (Awarded 2nd place)
*K.Braich, Hai, T. and S.L. Weinstein. Effect of Over-Expression of Activating Transcription Factor 3 on IFN-ß Transcription in Macrophages. College of Science and Engineering Student Project Showcase. SFSU. May 2007.
*M. V.Ly and S.L. Weinstein. Effect of RNA Interference-Mediated Knockdown of Activating Transcription Factor 3 (ATF3) on Interferon-ß. College of Science and Engineering Student Project Showcase. SFSU. May 2007.
*R.Barrozo and S.L. Weinstein. Transcriptional Repressor ATF3 Binds to the IFN-Beta Promoter in Macrophages. National Science Foundation GK-12 Annual Meeting. Washington, D.C., 2007.