Anil B. Mukherjee, MD, PhD, Head, Section on Developmental Genetics

Zhongjian Zhang, MD, PhD, Staff Scientist
Moonsuk Choi, PhD, Postdoctoral Fellow
Rabindranath Ray, PhD, Postdoctoral Fellow
Bhabadeb Chowdhury, PhD, Research Fellowa
Pei Chen Tsai, MS, Technical Training Fellow
Yi-ching Lee, PhD, Visiting Fellow
Aperna Mital, PhD, Visiting Fellow
Sondra Levin, MD, Guest Researcher
Aprile Pilon-Clayton, PhD, Guest Researcher

We conduct both basic and clinical investigations to understand molecular mechanisms of inflammatory/autoimmune and neurodegenerative diseases and to develop novel approaches for the treatment of these diseases. Our studies focus on understanding the regulation and physiological functions of several genes and their products, including uteroglobin (UG), soluble phospholipases A2 (sPLA2s), palmitoyl-protein thioesterase (PPT), and neutral ceramidase. UG is a multifunctional secreted protein with potent sPLA2-inhibitory and immunomodulatory activities. We have demonstrated that UG-deficient mice suffer from IgA-nephropathy, the most common primary renal glomerular disease, for which there is no effective treatment, and are studying the molecular mechanisms of the disease to develop novel therapeutic approaches. We have also isolated another gene encoding PPT and found that inactivating mutations in this gene lead to infantile neuronal ceroid lipofuscinosis (INCL), the most severe form of childhood Batten disease, for which there is no effective treatment, and are conducting a pilot study to determine whether Cystagon™ may be effective treatment for INCL. We are using a mouse model of INCL to understand the molecular mechanisms of the disease and to develop novel therapeutic approaches using gene transfer and embryonic stem (ES) cell technologies.

Novel role of uteroglobin as an innate repressor of PGD2 receptor–mediated COX-2 gene expression: implications for allergic asthma

Mandal,b Zhang, Chowdhury, Ray, Mukherjee; in collaboration with Pattabiraman

Uteroglobin is secreted by the tracheobronchial epithelia of virtually all mammals. We previously reported that ovalbumin challenge in uteroglobin knock-out mice elicits an exaggerated allergic response characterized by increased expression of Th2 cytokines, eotaxin, and eosinophil infiltration in the lungs. We report here that levels of prostaglandin D2 are markedly elevated in the bronchoalveolar lavage fluid of these mice and that the animals produce high levels of cyclooxygenases-2, a critical enzyme for the production of prostaglandins, which are abrogated by uteroglobin pretreatment. Given that prostaglandin D2 mediates allergic airway inflammation via its receptor DP and that the molecular mechanism of DP signaling is unknown, we studied DP signaling in vitro and determined the role of uteroglobin in this process. We found that DP signaling is mediated via mitogen-activated protein kinases (P38 and p42/44) and protein kinase C in a cell-specific manner leading to the activation of nuclear factor (NF)-kappaB, which stimulates cyclooxygenase-2 expression. Most important, we found that uteroglobin blocks DP signaling, inhibits NF-kappaB activation, and, consequently, suppresses cyclooxygenase-2 gene expression. We propose that uteroglobin is a critical component of an innate homeostatic mechanism against inadvertent stimulation of allergen-induced inflammatory response in the airways. 
 

Chowdhury B, Mantile-Selvaggi G, Miele L, Cordella-Miele E, Zhang Z, Mukherjee AB. Lys 43 and Asp 46 in alpha-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity. Biochem Biophys Res Commun 2002;295:877-883.

Inhibition by uteroglobin of prostaglandin F2alpha receptor–mediated activation of NF-kappaB and COX-2 gene expression

Mandal,b Ray, Zhang, Chowdhury, Mukherjee

Via its F2alpha receptor (FP), prostaglandin F2alpha regulates many important physiological processes such as inflammation, uterine smooth muscle contraction, and parturition. It has been suggested that such effects of PGF2alpha may be mediated by the production of pro-inflammatory lipid mediators. However, the molecular mechanism by which FP signaling regulates the production of these mediators remained unclear until now. We report here that FP signaling is mediated by PKC and that it activates NF-kappaB, which up-regulates cyclooxygenase-2. Cyclooxygenase-2 is a mediator-inducible enzyme that is critical for the production of mediators from arachidonic acid, which is released from cellular phospholipids by cytosolic phospholipase A2, the expression of which is also stimulated by PGF2alpha. Most important, uteroglobin inhibits FP-mediated NF-kappaB activation and cyclooxygenase-2 expression. We propose that uteroglobin is an essential component of a novel innate mechanism of homeostasis against inadvertent activation of FP-mediated stimulation of cyclooxygenases-2 expression and prevents the generation of pro-inflammatory lipid mediators.

Wang CY, Lei HJ, Huang CY, Zhang Z, Mukherjee AB, Yuan CJ. Induction of cyclooxygenase-2 by

staurosporine through the activation of nuclear factor for IL-6 (NF-IL6) and activator protein 2 (AP2) in an osteoblast-like cell line. Biochem Pharmacol 2002;64:177-184.

CC10-null mice are predisposed to lung tumorigenesis by NNK in cigarette smoke

Zhang, Mukherjee; in collaboration with Linnoila, Yang

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with reduced levels of Clara cell 10 kD protein (CC10), a multifunctional, steroid-inducible, secreted protein that is naturally present in the respiratory system of virtually all mammals. CC10 expression is rarely detectable in human non–small cell cancers, despite being abundantly produced by progenitors of normal airway epithelial cells. Its expression is drastically reduced following exposure of animals to the methylnitrosamine NNK, the main carcinogen in tobacco smoke, and during SV40-induced carcinogenesis. Moreover, induced expression of CC10 in cancer cells reverses the transformed phenotype. Here we report that, compared with wild-type controls, CC10-knock-out mice exposed to NNK are highly susceptible (30 percent knockout versus 5 percent wild type) to dramatically increased proliferation of airway epithelial cells and development of adenoma of the lung within five to 12 months of NNK exposure. Interestingly, the hyperproliferation and tumorigenesis in NNK-treated knock-out mice were associated with activation of Ras and the mitogen-activated protein kinase, and 50 percent of the adenomas had the K-Ras mutation. We propose that CC10 deficiency leads to increased susceptibility to carcinogen-induced lung cancers, raising the possibility that this protein possesses tumor suppressor activity.

Interferon-gamma stimulates the expression of a novel secretoglobin gene in lymphoblasts

Choi, Zhang, Mukherjee

Interferons (IFN) are a family of cytokines that alert the immune system to viral infections of host cells. The IFNs (IFN-alpha, IFN-beta, and IFN-gamma) interact with specific cellular receptors and stimulate the production of second messengers, leading to the expression of antiviral and immune-modulatory genes. It has been previously reported that IFN-gamma stimulates the expression of uteroglobin (UG), the founding member of the secretoglobin superfamily, with immunomodulatory properties. We demonstrate here that, in addition to UG, IFN-gamma stimulates a novel secretoglobin gene in lymphoblasts that bears 74 percent sequence identity with lipophilin-B. A BLAST search also revealed that the gene is highly similar to YGB, a lymphocyte-specific secretoglobin. However, unlike YGB, the gene we characterized is expressed not only in lymphoblasts but also in other cell types, including colon carcinoma cells. Moreover, the deduced protein sequence demonstrates that the protein does not contain the C-terminal cysteine residue characteristic of the secretoglobins, including YGB. We named the gene colon carcinoma and lymphoblast-associated secretoglobin (CCLAS). Although the function of the gene is yet to be determined, the stimulation of its expression by IFN-gamma may suggest an important immunological role.

Safety and efficacy of intratracheal recombinant human Clara cell protein in a newborn piglet model of acute lung injury

Pilon-Clayton; in collaboration with Chandra, Chester, Davis, Drexler, Koo, Kowalewska, Levine, Pollack, Welch

Despite the widespread use of exogenous surfactant, acute and chronic lung injury continues to be a major cause of morbidity in preterm infants. CC10 is a protein produced by Clara cells that inhibits phospholipase A2 and has anti-inflammatory and antifibrotic properties. We studied whether intratracheal (IT) recombinant human Clara cell protein (rhCC10) could safely minimize lung injury in a newborn piglet model of acute lung injury. Twenty-nine newborn piglets were given Survanta® and then ventilated for 48 hours receiving the following: room air (group 1); 100 percent O2 (group 2); or 100 percent O2 and 25, 5, or 1 mg/kg (groups 3, 4, and 5, respectively) of IT rhCC10. We performed laboratory studies, oxygen ratios, static pressure-volume curves, bronchoalveolar lavage (for inflammatory markers), and histologic analyses over the 48-hour study period. Pulmonary compliance and oxygenation significantly improved in animals receiving 5 mg/kg IT rhCC10 compared with room air and 100 percent O2 controls. Reductions in inflammatory markers were seen in animals receiving rhCC10, although changes did not reach statistical significance. We noted no significant toxicity. In this newborn piglet model of hyperoxic lung injury, rhCC10 appeared safe and improved pulmonary function and may thus represent a promising therapy for the prevention of lung injury in preterm infants.
 

Chandra S, Davis JM, Drexler S, Kowalewska J, Koo HC, Chester D, Pollack S, Welch R, Pilon

A, Levine CR. Safety and efficacy of intratracheal recombinant human Clara cell protein in a newborn piglet model of acute lung injury. Pediatr Res 2003;54:509-515.

Magnetic resonance imaging of the brain of a mouse model of INCL

Zhang, Mukherjee; in collaboration with Hofmann, Koretsky, Munasinghe

Neuronal ceroid lipofuscinoses (NCL) are the most common (1 in 12,500) heritable progressive encephalopathies of children. Inactivating mutations in the PPT gene cause INCL, a heritable lysosomal storage disease. PPT facilitates the degradation of posttranslationally acylated polypeptides by cleaving thioester linkages that connect the lipids with the polypeptides that are acylated. A deficiency of the enzyme leads to abnormal ceramidase (N-CDase) is virtually identical to that of the human N-CDase. It is expressed at a high level in mouse kidneys and in specific segments of the upper gastrointestinal tract. We also found that neutral ceramidase mRNA expression is stimulated by its substrate, C2-ceramide. Most important, we found that mice fed with C2-ceramide or injected with a specific inhibitor of neutral ceramidase before feeding ceramide show dramatically increased apoptosis in mesangial cells in vitro and in gastrointestinal tract in vivo. Our results raise the possibility that neutral ceramidase is part of a novel homeostatic mechanism to prevent apoptosis in vital organs such as the gastrointestinal tract and the kidneys.

Choi MS, Anderson MA, Zhang Z, Zimonjic DB, Popescu N, Mukherjee AB. Neutral ceramidase

gene: role in regulating ceramide-induced apoptosis. Gene 2003;315:113-122.

COLLABORATORS

Rafael Caruso, MD, Ophthalmic and Visual Function Branch, NEI, Bethesda MD 
Sudhish Chandra, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Darrin Chester, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Jonathan M. Davis, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Steven Drexler, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Kenneth H. Fischbeck, MD, Neurogenetics Branch, NINDS, Bethesda MD

Andrea Gropman, MD, Neurogenetics Branch, NINDS, Bethesda MD

Sandra L. Hofmann, MD, PhD, University of Texas Southwestern Medical Center, Dallas TX

Shau-Ku Huang, PhD, The Johns Hopkins University School of Medicine, Baltimore MD

Hshi-Chi Koo, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Alan Koretsky, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda MD

Jolanta Kowalewska, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Carolyn Robbins Levine, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Ilona Linnoila, MD, Cancer and Cell Biology Branch, NCI, Rockville MD

Jeeva Munasinghe, PhD, Mouse Imaging Facility, NINDS, Bethesda MD

N. Pattabiraman, PhD, Lombardi Cancer Center, Georgetown University, Washington DC

Simcha Pollack, PhD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Nicolae Popescu, PhD, Laboratory of Experimental Carcinogenesis, NCI, Bethesda MD

Richard Welch, MD, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola NY

Yongping Yang, PhD, Cancer and Cell Biology Branch, NCI, Bethesda MD
 

aDeparted in 2002

bAsim K. Mandal, PhD, former Postdoctoral Fellow

cMary A. Anderson, MA, former Technical Training Fellow
 

For further information, contact mukherja@cc1.nichd.nih.gov