Erik P. Lillehoj, PhD

1. MUC1 is an airway epithelial cell surface receptor for Pseudomonas aeruginosa. MUC1 was the first mucin gene to be cloned in 1990. By 2000, when I was a Research Associate in the laboratory of Dr. Chul Kim, the mucin gene family had grown to 12 members, most of which encoded for proteins secreted from goblet cells to provide lubrication for mucosal surfaces and protection from microbial pathogens. At this time, however, the function of the membrane-tethered MUC1 mucin was completely unknown. Because Pseudomonas aeruginosa, a Gram-negative bacterium almost invariably associated with airway mucus in cystic fibrosis patients, was known to bind to secreted mucins purified from airway mucus, we hypothesized that P. aeruginosa also would bind to MUC1 expressed on the airway epithelial cell surface. Our published studies demonstrated, for the first time, that (i) P. aeruginosa bound to MUC1 expressed by both transfected CHO cells (1a) as well as MUC1 endogenously expressed by well-differentiated and polarized primary human bronchial epithelial (NHBE) cells (1d), (ii) P. aeruginosa binding to MUC1 was mediated through flagellin (1b), the major structural protein of the bacterial flagellum, and (iii) this receptor-ligand interaction stimulated phosphorylation of the MUC1 intracellular region and activation of an ERK1/2 signaling pathway (1c). These results have provided the first credible evidence at the molecular level of this host-pathogen interaction that is becoming the paradigm for receptor-ligand studies in epithelial cell biology.

1a. Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, Kim KC. Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol. 2001 Jan;280(1):L181-7. PMID: 11133508.

1b. Lillehoj EP, Kim BT, Kim KC. Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol. 2002 Apr;282(4):L751-6. doi: 10.1152/ajplung.00383.2001. PMID: 11880301.

1c. Lillehoj EP, Kim H, Chun EY, Kim KC. Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase. Am J Physiol Lung Cell Mol Physiol. 2004 Oct;287(4):L809-15. doi: 10.1152/ajplung.00385.2003. PMID: 15220114.

1d. Kato K, Lillehoj EP, Kai H, Kim KC. MUC1 expression by human airway epithelial cells mediates Pseudomonas aeruginosa adhesion. Front Biosci (Elite Ed). 2010 Jan 1;2:68-77. PMID: 20036855.

2. MUC1 drives intracellular signaling in airway epithelia. The amino acid sequence of the MUC1 cytoplasmic tail (MUC1-CT) is strikingly similar to that of cytokine and growth factor receptors, suggesting that MUC1 may function as a signaling molecule. In breast and pancreatic carcinomas, the MUC1-CT has been shown to interact with a variety of signaling molecules, including c-Src, ErbB family members, glycogen synthase kinase3β, protein kinase Cδ, β-catenin, p120 catenin, Grb-2, p53, heat shock protein 70, and HSP90. Unlike cytokine/growth factor receptors, however, the MUC1-CT is not capable of autophosphorylation, suggesting a fundamentally different, but as yet unknown, mechanism of signal transduction. Until recently, the ability of the MUC1-CT to interact with these or other signaling molecules in normal human airway epithelial cells was unknown. Our published studies have now confirmed that the MUC1-CT interacts with β-catenin (2a). Further, we have identified three previously unreported, novel MUC1-CT binding partners, calcium-modulating cyclophilin ligand (CAML) (2b), peroxisome proliferator-activated receptor-γ (PPAR-γ) (2c), and TLR5 (2d).

2a. Lillehoj EP, Lu W, Kiser T, Goldblum SE, Kim KC. MUC1 inhibits cell proliferation by a β-catenin-dependent mechanism. Biochim Biophys Acta. 2007 Jul;1773(7):1028-38. doi: 10.1016/j.bbamcr.2007.04.009. PMID: 17524503.

2b. Guang W, Kim KC, Lillehoj EP. MUC1 mucin interacts with calcium-modulating cyclophilin ligand. Int J Biochem Cell Biol. 2009 Jun;41(6):1354-60. doi: 10.1016/j.biocel.2008.12.004. PMID: 19135167.

2c. Park YS, Guang W, Blanchard TG, Kim KC, Lillehoj EP. Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ. Am J Physiol Gastrointest Liver Physiol. 2012 Sep 15;303(6):G765-74. doi: 10.1152/ajpgi.00023.2012. PMID: 22766852.

2d. Kato K, Lillehoj EP, Kim KC. Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation. Inflamm Res. 2016 Mar;65(3):225-33. doi: 10.1007/s00011-015-0908-8. PMID: 26645913.

3. MUC1 expression inhibits airway inflammation. Normal immune homeostasis in the airways is strictly regulated by the opposing activities of proinflammatory and anti-inflammatory pathways. Among the innate immune receptors that drive airway inflammation are the TLRs and NLRs, and their respective signaling pathways have been well-described over the past 10-15 years. The anti-inflammatory mechanisms that counter-regulate airway inflammation, however, are less clear. A major breakthrough in understanding the function of MUC1 in the airways came when we discovered that Muc1 knockout mice exhibit hyper-inflammatory responses in the lungs following experimental infection with P. aeruginosa (3a). Through a series of mechanistic studies, we demonstrated that the anti-inflammatory properties of MUC1/Muc1 were attributed to its ability to suppress TLR signaling in response to microbial PAMPs, through both MyD88-dependent (3c) and MyD88-independent (3d) pathways. We have also extended these findings to Helicobacter pylori infection of the gastric mucosa (3b).

3a. Lu W, Hisatsune A, Koga T, Kato K, Kuwahara I, Lillehoj EP, Chen W, Cross AS, Gendler SJ, Gewirtz AT, Kim KC. Cutting edge: Enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice. J Immunol. 2006 Apr 1;176(7):3890-4. doi: 10.4049/​jimmunol.176.7.3890. PMID: 16547220.

3b. Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. Muc1 cell surface mucin attenuates epithelial inflammation in response to a common mucosal pathogen. J Biol Chem. 2010 Jul 2;285(27):20547-57. doi: 10.1074/jbc.M110.121319. PMID: 20430889.

3c. Kato K, Lillehoj EP, Park YS, Umehara T, Hoffman NE, Madesh M, Kim KC. Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88. J Immunol. 2012 Feb 15;188(4):2014-22. doi: 10.4049/jimmunol.1102405. PMID: 22250084.

3d. Kato K, Lillehoj EP, Kim KC. MUC1 regulates epithelial inflammation and apoptosis by polyI:C through inhibition of Toll/IL-1 receptor-domain-containing adapter-inducing IFN-β (TRIF) recruitment to Toll-like receptor 3. Am J Respir Cell Mol Biol. 2014 Sep;51(3):446-54. doi: 10.1165/rcmb.2014-0018OC. PMID: 24693944.

4. Catalytically-active sialidases are expressed in human airways. In general, glycoprotein receptors for microbial pathogens, including MUC1, contain N- and/or O-linked glycan chains terminating with sialic acid. These terminal sialic acid residues are strategically positioned to influence receptor-ligand, cell-cell, and host-pathogen interactions. The sialylation state of glycoconjugates is dynamically and coordinately regulated through the opposing catalytic activities of sialyltransferases and neuraminidases (NEUs). Although much is known about prokaryotic NEUs as virulence factors, a role for mammalian host NEUs in bacterial pathogenesis has never been considered, and until our studies in 2012, absolutely nothing was known about the expression of NEUs in the airways. Of the four known mammalian NEUs (NEU1, 2, 3, 4), we discovered that NEU1 is the predominant sialidase expressed by human airway epithelial cells (4a). Further, NEU1 expression in airway epithelia regulates the sialylation state of the MUC1 ectodomain (MUC1-ED), MUC1-ED adhesiveness for P. aeruginosa, and flagellin-stimulated, MUC1-dependent ERK1/2 activation (4a, 4c). We also demonstrated, for the first time, that the next most abundant airway epithelial cell sialidase, NEU3, was catalytically-active in these cells and demonstrated a substrate preference for gangliosides (4b). Most recently, we have shown that patients with idiopathic pulmonary fibrosis have increased NEU1 expression in lung tissues, and that overexpression of NEU1 increases pulmonary collagen deposition, lymphocytosis, and fibrosis (4d).

4a. Lillehoj EP, Hyun SW, Feng C, Zhang L, Liu A, Guang W, Nguyen C, Luzina IG, Atamas SP, Passaniti A, Twaddell WS, Puché AC, Wang LX, Cross AS, Goldblum SE. NEU1 sialidase expressed in human airway epithelia regulates epidermal growth factor receptor (EGFR) and MUC1 protein signaling. J Biol Chem. 2012 Mar 9;287(11):8214-31. doi: 10.1074/jbc.M111.292888. PMID: 22247545.

4b. Lillehoj EP, Hyun SW, Feng C, Zhang L, Liu A, Guang W, Nguyen C, Sun W, Luzina IG, Webb TJ, Atamas SP, Passaniti A, Twaddell WS, Puché AC, Wang LX, Cross AS, Goldblum SE. Human airway epithelia express catalytically active NEU3 sialidase. Am J Physiol Lung Cell Mol Physiol. 2014 May 1;306(9):L876-86. doi: 10.1152/ajplung. PMID: 24658138.

4c. Lillehoj EP, Hyun SW, Liu A, Guang W, Verceles AC, Luzina IG, Atamas SP, Kim KC, Goldblum SE. NEU1 sialidase regulates membrane-tethered mucin (MUC1) ectodomain adhesiveness for Pseudomonas aeruginosa and decoy receptor release. J Biol Chem. 2015 Jul 24;290(30):18316-31. doi: 10.1074/jbc.M115.657114. PMID: 25963144.

4d. Luzina IG, Lockatell V, Hyun SW, Kopach P, Kang PH, Noor Z, Liu A, Lillehoj EP, Lee C, Miranda-Ribera A, Todd NW, Goldblum SE, Atamas SP. Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis. Am J Physiol Lung Cell Mol Physiol. 2016 May 15;310(10):L940-54. doi: 10.1152/ajplung.00346.2015. PMID: 26993524.

5. Over 16 years committed to MUC1 mucin research. Since joining the University of Maryland in 2000, I have published 48 papers related to MUC1, focusing on its role as a cell surface receptor for P. aeruginosa, MUC1-driven intracellular signaling, the anti-inflammatory effects of MUC1 activation, and the role of MUC1-ED sialylation in these responses. Two major review articles have summarized these studies (5a, 5d). In addition, we have recently extended these investigations to H. pylori infection of the gastric mucosa where similar results have been described (5b, 5c). Based on these collective studies, it is now clear that MUC1 plays a critical role at multiple mucosal sites in the host response to pathogen infections. We anticipate that our future studies in these and other host-pathogen systems will ultimately identify new molecular and cellular pathways for mucosal immunity that will contribute to the development of novel therapies for life-threatening infectious diseases.

5a. Kim KC, Lillehoj EP. MUC1 mucin: A peacemaker in the lung. Am J Respir Cell Mol Biol. 2008 Dec;39(6):644-7. doi: 10.1165/rcmb.2008-0169TR. PMID: 18617677.

5b. Guang W, Twaddell WS, Lillehoj EP. Molecular interactions between MUC1 epithelial mucin, β-catenin, and CagA proteins. Front Immunol. 2012 May 7;3:105. doi: 10.3389/fimmu.2012.00105. PMID: 22566976.

5c. Lillehoj EP, Guang W, Ding H, Czinn SJ, Blanchard TG. Helicobacter pylori and gastric inflammation: Role of MUC1 mucin. J Pediatr Biochem. 2012 Jul 1;2(3):125-132. doi: 10.3233/JPB-2012-00058. PMID: 23667410.

5d. Lillehoj EP, Kato K, Lu W, Kim KC. Cellular and molecular biology of airway mucins. Int Rev Cell Mol Biol. 2013;303:139-202. doi: 10.1016/B978-0-12-407697-6.00004-0. PMID: 23445810.

Research Support

Ongoing

UM Ventures Seed
Grant Lillehoj (PI)
University of Maryland, Baltimore
06/01/16-05/31/17

MUC1 Ectodomain and Portions Thereof to Treat Pseudomonas aeruginosa Infections.

The goal of this project is to demonstrate that synthetic peptides corresponding to the MUC1 ectodomain block P. aeruginosa adhesion to human airway epithelial cells.

Completed (last 3 years)

07R-1665
Lillehoj (PI)
Stanley Medical Research Institute
07/1/15-06/30/16

Diagnostic Potential of Retrovirus Envelope Proteins in Acute Onset Schizophrenia and Bipolar Disorder

The goal of this project was to determine the diagnostic potential of serological markers of schizophrenia using cloned HERV envelope genes and proteins.

RO1 HL047125
Kim (PI)
NHLBI, NIH
07/01/12-06/30/16

Signaling Mechanisms of MUC1 Mucin

The goal of this project was to characterize the anti-inflammatory role of MUC1 in airway epithelial cells to the host response to Pseudomonas aeruginosa lung infection. The current proposal is a renewal of this prior grant.

Role: Co-investigator

• 1981: American Cancer Society Postdoctoral Fellowship Award
• 2002: American Lung Association Dalsemer Award

Positions and Employment

• 1984 - 1986: Staff Fellow, NIAID, NIH, Bethesda, MD
• 1986 - 1988: Research Scientist, Nucleic Acid and Protein Synthesis Laboratory, Frederick Cancer Research Facility, Frederick, MD
• 1988 - 1992: Assistant Director, Product Technology, Cambridge Biotech, Rockville, MD
• 1992 - 1994: Research Assistant Professor, Department of Biochemistry and Molecular Biology, George Washington University School of Medicine, Washington, D.C.
• 1994 - 2000: Director, Research and Development, Dexall Biomedical Labs, Gaithersburg, MD
• 2000 - 2005: Assistant Professor, Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD
• 2005 - 2008: Assistant Professor, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD
• 2008 - Present: Associate Professor, Department of Pediatrics, University of Maryland School of Medicine

Other Experience and Professional Memberships

• 1981 - Present: Member, American Association for the Advancement of Science
• 1984 - Present: Member, American Association of Immunologists
• 2000 - Present: Member, American Society for Microbiology
• 2006 - 2010: Member, University of Maryland School of Medicine Council
• 2006 - Present: Journal Peer Reviewer (ad hoc), American Journal of Respiratory Cell and Molecular Biology, American Journal of Physiology: Cell Physiology, International Journal of Molecular Sciences
• 2007: NIH Peer Review Committee, NIEHS DISCOVER program, ad hoc reviewer
• 2009: NIH Peer Review Committee, Challenge Grant program, ad hoc reviewer
• 2010 - Present: Member, University of Maryland Institutional Review Board (IRB)