We conduct both laboratory and clinical investigations to understand the molecular mechanisms of complex genetic disorders of inflammation, autoimmunity, and neurodegeneration in order to develop novel and rational therapeutic approaches. To achieve these goals, we focus on the regulation and physiological functions of primarily two genes: uteroglobin (UG), also known as Clara cell 10 kDa protein (CC10), and palmitoyl-protein thioesterase-1 (PPT1); mutations of the latter are the genetic basis of infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disease of childhood. Our investigations have led to ongoing clinical trials. Recently, using PPT1-knockout mice, which recapitulate virtually all clinical and pathological features of INCL, we discovered that deficiency in this enzyme leads to abnormal accumulation of S-acylated proteins in the endoplasmic reticulum (ER), causing ER stress. ER stress mediates activation of the ER cysteine proteinase caspase-12, which leads to caspase-3 activation and apoptosis. The discovery provides insight into a complex mechanism of neurodegeneration in INCL and identifies several potential targets for the development of rational therapeutic approaches for treating this uniformly fatal disease.
Critical homeostatic role of uteroglobin in regulating allergen-induced inflammatory response
Ray, Choi, Zhang, Mandal,1 Mukherjee; in collaboration with Askew, Pattabiraman, Silverman
Asthma is a complex heritable inflammatory disease of the respiratory system. In the United States alone, 6,000 deaths annually are attributable to this disease, which is precipitated by an exaggerated response to allergens, leading to airway inflammation and bronchoconstriction. Uteroglobin (UG), the first-discovered member of the secretoglobin superfamily, is a potent anti-inflammatory protein constitutively expressed at high levels in the airway epithelia of all mammals. We previously reported that the lungs of UG-knockout (UG-KO) mice express elevated levels of Th2 cytokines, such as interleukin (IL)-4 and IL-13; the cytokines are augmented by allergen sensitization and challenge leading to exaggerated airway inflammation. Notably, these responses are suppressed by recombinant UG treatment. Recent reports indicate that human orthologues of murine squamous cell carcinoma antigen-2 (SCCA-2/serpinb3a), a serine protease inhibitor, are overexpressed in the airways of asthmatic patients. We report here that, compared with wild-type littermates, UG-KO mouse lungs express markedly elevated levels of SCCA-2 mRNA and protein, which are augmented by allergen challenge. Most important, recombinant UG treatment abrogates these effects. We further demonstrated that treatment of cultured human bronchial epithelial cells with IL-4 or IL-13 stimulates phosphorylation of STAT-1 and STAT-6, leading to SCCA-1 (SERPINB3) and SCCA-2 (SERPINB4) gene expression. We propose that IL-4– and IL-13–stimulated SCCA gene expression is mediated via STAT-1 and STAT-6 activation and that, by suppressing the production and most likely by interfering with the signaling of these cytokines, UG inhibits the SCCA gene expression associated with airway inflammation in asthma.
Mandal AK, Zhang Z, Ray R, Choi MS, Chowdhury B, Pattabiraman N, Mukherjee AB. Uteroglobin represses allergen-induced inflammation by blocking PGD2 receptor-mediated functions. J Exp Med 2004;199:1317-1330.
Ray R, Choi M, Zhang Z, Silverman GA, Askew D, Mukherjee AB. Uteroglobin suppresses SCCA gene expression associated with allergic asthma. J Biol Chem 2005;280:9761-9764.
Inhibition by uteroglobin of PGF2a receptor–mediated expression of genes critical for the production of pro-inflammatory lipid mediators
Ray, Zhang, Mandal, 1 Chowdhury,2 Mukherjee
Prematurity is one of the leading causes of infant mortality and may result from intrauterine infection, which mediates premature labor by stimulating the production of inflammatory lipid mediators such as prostaglandin F2 a (PGF2 a). The biological effects of PGF2 a are mediated by the G protein–coupled receptor FP; however, the molecular mechanism whereby FP signaling mediates inflammatory lipid mediator production remains unclear. We previously reported that, in the human uterus, a composite organ whose major constituents are fibroblasts and epithelial and smooth muscle cells, an inverse relationship exists between the levels of PGF2 a and the steroid-inducible anti-inflammatory protein uteroglobin. We found that, in NIH 3T3 fibroblasts and in human uterine smooth muscle cells, FP signaling is mediated by multikinase pathways in a cell type–specific manner, thus activating NF-kB, which then stimulates the expression of cyclooxygenase-2. Cyclooxygenase-2 is a critical enzyme for the production of PGs from arachidonic acid, which is released from membrane phospholipids by phospholipase A2 and whose expression is stimulated by PGF2a. Most important, UG inhibits FP-mediated NF-kappaB activation and cyclooxygenase-2 gene expression, thereby suppressing the production of inflammatory lipid mediators. We propose that these various effects of UG play important roles in maintaining homeostasis in organs that are vulnerable to inadvertent stimulation of FP-mediated inflammatory response.
Mandal AK, Ray R, Zhang Z, Chowdhury B, Mukherjee AB. Uteroglobin inhibits PGF2a receptor-mediated expression of genes critical for the production of pro-inflammatory lipid mediators. J Biol Chem 2005;280:32897-32904.
Interaction of uteroglobin with lipocalin-1 receptor suppresses cancer cell motility and invasion
Zhang, Kim, Lee, Choi, Chowdhury,2 Wang,3 Mukherjee
Cellular migration and invasion are precisely regulated processes that play critical roles in chemotaxis, embryonic development, and implantation. Dysregulated migration and invasion of cancer cells lead to metastasis, which accounts for more than 90 percent of cancer deaths. Understanding the molecular mechanism(s) that regulate cancer cell motility and invasion may thus facilitate the development of novel therapeutic approaches. We previously reported that uteroglobin, a multifunctional secreted protein, binds to several cell types, including some cancer cells, and inhibits their migration and invasion. More recently, we reported that HTB-81 adenocarcinoma cells, which do not bind to UG, are refractory to UG-mediated inhibition of migration and invasion. Given that UG shares some biological properties with the protein lipocalin-1, which binds to several cell types via its receptor (Lip-1R), we sought to determine whether UG might also bind to this receptor and mediate inhibitory effects on migration and invasion of HTB-81 adenocarcinoma cells. Using COS-1 cells transfected with a green fluorescent protein–Lip-1R cDNA construct, we demonstrated that induced expression of Lip-1R facilitates specific binding of 125I-humanUG (hUG) to these cells, which do not normally bind to hUG. We further demonstrated that forced expression of Lip-1R in HTB-81 cells promotes binding of 125I-hUG with high affinity and specificity. As a result of Lip-1R expression, the cells become fully responsive to hUG-mediated inhibition of migration and invasion. Our results suggest that Lip-1R is a UG binding protein and that its forced expression in UG-resistant cancer cells renders the cells sensitive to UG-mediated inhibition of migration and invasion.
Zhang Z, Kim S-J, Wang J, Chowdhury B, Lee Y-C, Choi M, Mukherjee AB. Interaction of uteroglobin with lipocalin-1 receptor suppresses cancer cell motility and invasion. Gene (in press).
Regulation of inflammation by prostaglandins with opposing functions
Zhang, Mandal,1 Mukherjee
Cyclooxygenase-2 (COX-2), which is important for the production of lipid mediators of inflammation such as prostaglandins (PGs), has for many years been the favorite target for anti-inflammatory drug development. However, as widely discussed, selective COX-2 inhibitors exhibit adverse effects. While some PGs are pro-inflammatory, others have anti-inflammatory effects, although the molecular mechanism(s) by which the opposing effects are achieved remains unclear. We report here that, via receptor-mediated pathways, inflammatory PGs such as PGD2 and PGF2alpha mediate the activation of nuclear factor kappa B (NF-kappaB), which stimulates the expression of COX-2 essential for inflammatory PG production. Most interestingly, the anti-inflammatory PG (PGA1) counteracts the activation of NF-kappaB and consequently suppresses COX-2 gene expression. Our results suggest that pro- and anti-inflammatory PGs maintain homeostasis by counteracting each other’s effects. We propose that selective COX-2 inhibitors disrupt the balance and thereby manifest the reported adverse effects.
Mandal AK, Zhang Z, Kim SJ, Tsai PC, Mukherjee AB. Yin-Yang: balancing act of prostaglandins with opposing functions to regulate inflammation. J Immunol 2005;175:6271-6273.
A combination therapy with Cystagon® and N-acetylcysteine for INCL patients
Levin, Zhang, Mukherjee; in collaboration with Caruso, Gropman
As a group, neuronal ceroid lipofuscinoses (NCLs) are the most common (1 in 12,500) heritable neurodegenerative disorders of childhood. Mutations in at least seven genes are responsible for various forms of NCL. The infantile form of NCL, or INCL, is the most severe disease, which is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency. PPT catalyzes the hydrolysis of thioester linkages in S-acylated polypeptides, and its deficiency leads to abnormal accumulation in lysosomes of acylated proteins called ceroids. Thus, INCL is now recognized as a lysosomal storage disease. Given that thioester bonds are susceptible to nucleophilic attack, drugs with nucleophilic properties may offer therapeutic potential for INCL. Accordingly, we tested several compounds with these properties (i.e., cysteamine, phosphocysteamine, cysteamine bitartrate, and N-acetylcysteine) and found that they disrupt thioester linkages in the model PPT1-substrate [14C] palmitoylCoA, resulting in the release of [14C] palmitic acid. Among the drugs tested, we characterized phosphocysteamine and cysteamine bitartrate (Cystagon®) in further detail because (1) INCL is a lysosomal storage disease, and Cystagon® concentrates in the lysosomes; (2) we found that the drug functions at a low pH in cleaving thioester linkages; (3) it is reported to cross the blood-brain barrier; (4) the drug prevents apoptosis in INCL lymphoblasts; and (5) the drug is relatively nontoxic. Furthermore, we have shown that phosphocysteamine not only disrupts thioester linkages in S-acylated polypeptides in cultured cells from INCL patients but also mediates the depletion of intracellular ceroid deposits and prevents their reaccumulation (Zhang et al., Nat Med 2002;7:478).
For the past three and a half years, we have been conducting a clinical trial to determine whether Cystagon® is beneficial for INCL patients. Our preliminary results with six Caucasian patients indicate that the drug slows the rapid neurodegeneration characteristic of INCL. Moreover, our patients have not developed epileptic seizures, a common complication of the disease. In fact, before the initiation of Cystagon® treatment, the EEG of one patient revealed epileptic foci that were not detected in EEG tests conducted after six months’ treatment with the drug. The most dramatic effect of the drug is the complete clearance of lysosomal ceroids in white blood cells within six months of therapy. In parallel with these studies, we used a mouse model of INCL to test the effects of Cystagon® alone and of a combination of Cystagon® and N-acetylcysteine (Mucomyst®), which manifests anti-apoptotic and neuroprotective properties. Our preliminary results show that the combination therapy delays the development of neurological symptoms, reduces apoptosis, and maintains the brain volume in the subject mice for a longer period compared with those treated with Cystagon® alone. These preliminary results prompted us to propose a combination therapy with Cystagon® plus Mucomyst® for INCL patients. Given that INCL is a fatal disease and that Mucomyst® has anti-apoptotic and neuroprotective effects and, like Cystagon®, has a proven record of safety, we propose to test a combination of the two drugs in INCL patients.
Zhang Z, Lee YC, Kim SJ, Choi MS, Tsai PC, Xu Y, Xiao YJ, Zhang P, Heffer A, Mukherjee AB. Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL. Hum Mol Genet 2006;15:337-346.
1Asim K. Mandal, PhD, former Postdoctoral Fellow
2Bhabadeb Chowdhury, PhD, former Research Fellow
3Gingya Wang, former Summer Student
Collaborators
David Askew, PhD, University of Pittsburgh School of Medicine, Pittsburgh, PA
Peter Backlund, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
Bruce J. Baum, DMD, Gene Therapy and Therapeutics Branch, NICDR, Bethesda, MD
Rafael Caruso, MD, Ophthalmic and Visual Function Branch, NEI, Bethesda, MD
Eli Eisenstein, PhD, Hadassah Medical Center, Jerusalem, Israel
Andrea Gropman, MD, Georgetown University Medical Center, Washington, DC
Sandra L. Hofmann, MD, PhD, University of Texas Southwestern Medical Center, Dallas, TX
Shau-Ku Huang, PhD, The Johns Hopkins University Medical School, Baltimore, MD
Alan Koretsky, PhD, NMR Center, NINDS, Bethesda, MD
Eric Lee, DVM, Laboratory of Mammalian Genetics and Development, NICHD, Bethesda, MD
Ilona Linnoila, MD, Cancer and Cell Biology Branch, NCI, Rockville, MD
Jeeva Munasinghe, PhD, NMR Center, NINDS, Bethesda, MD
Nagarajan Pattabiraman, PhD, Lombardi Cancer Center, Washington, DC
Gary Silverman, PhD, University of Pittsburgh School of Medicine, Pittsburgh, PA
Heiner Westphal, MD, Laboratory of Mammalian Genetics and Development, NICHD, Bethesda, MD
Krystyna Wisniewski, MD, PhD, Institute for Basic Research, Staten Island, NY
Yi-Jin Xiao, PhD, Cleveland Clinic, Cleveland, OH
Yan Xu, PhD, Cleveland Clinic, Cleveland, OH
Yongping Yang, PhD, Mouse Cancer Genetics Program, NCI, Frederick, MD
Alfred L. Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
Junying Yuan, PhD, Harvard Medical School, Boston, MA
For further information, contact mukherja@mail.nih.gov.