Bhabadeb Chowdhury

Uteroglobin Represses Allergen-induced Inflammatory Response by Blocking PGD2 Receptor–mediated Functions

Uteroglobin (UG) is an antiinflammatory protein secreted by the epithelial lining of all organs communicating with the external environment. We reported previously that UG-knockout mice manifest exaggerated inflammatory response to allergen, characterized by increased eotaxin and Th2 cytokine gene expression, and eosinophil infiltration in the lungs. In this study, we uncovered that the airway epithelia of these mice also express high levels of cyclooxygenase (COX)-2, a key enzyme for the production of proinflammatory lipid mediators, and the bronchoalveolar lavage fluid (BALF) contain elevated levels of prostaglandin D2. These effects are abrogated by recombinant UG treatment. Although it has been reported that prostaglandin D2 mediates allergic inflammation via its receptor, DP, neither the molecular mechanism(s) of DP signaling nor the mechanism by which UG suppresses DP-mediated inflammatory response are clearly understood. Here we report that DP signaling is mediated via p38 mitogen–activated protein kinase, p44/42 mitogen–activated protein kinase, and protein kinase C pathways in a cell type–specific manner leading to nuclear factor–κB activation stimulating COX-2 gene expression. Further, we found that recombinant UG blocks DP-mediated nuclear factor–κB activation and suppresses COX-2 gene expression. We propose that UG is an essential component of a novel innate homeostatic mechanism in the mammalian airways to repress allergen-induced inflammatory responses.

Gastrointestinally Distributed UDP-glucuronosyltransferase 1A10, Which Metabolizes Estrogens and Nonsteroidal Anti-inflammatory Drugs, Depends upon Phosphorylation*

Among gastrointestinal distributed isozymes encoded at the UGT1 locus, UDP-glucuronosyltransferase 1A10 (UGT1A10) metabolizes a number of important chemicals. Similar to broad conversion of phytoestrogens (Basu, N. K., Ciotti, M., Hwang, M. S., Kole, L., Mitra, P. S., Cho, J. W., and Owens, I. S. (2004) J. Biol. Chem. 279, 1429–1441), UGT1A10 metabolized estrogens and their derivatives, whereas UGT1A1, -1A3, -1A7, and -1A8 differentially exhibited reduced activity toward the same. UGT1A10 compared with UGT1A7, -1A8, and -1A3 generally exhibited high activity toward acidic nonsteroidal anti-inflammatory drugs and natural benzaldehyde derivatives, while UGT1A3 metabolized most efficiently aromatic transcinnamic acids known to be generated from flavonoid glycosides by microflora in the lower gastrointestinal tract. Finally UGT1A10, -1A7, -1A8, and -1A3 converted plant-based salicylic acids; methylsalicylic acid was transformed at high levels, and acetylsalicylic (aspirin) and salicylic acid were transformed at moderate to low levels. Atypically UGT1A10 transformed estrogens between pH 6 and 8 but acidic structures preferentially at pH 6.4. Furthermore evidence indicates UGT1A10 expressed in COS-1 cells depends upon phosphorylation; UGT1A10 versus its single, double, and triple mutants at three predicted protein kinase C phosphorylation sites incorporated [33P]-orthophosphate and showed a progressive decrease with no detectable label or activity for the triple T73A/T202A/S432G-1A10 mutant. Single and double mutants revealed either null/full activity or null/additive activity, respectively. Additionally UGT1A10-expressing cultures glucuronidated 17β-[14C]estradiol, whereas cultures containing null mutants at protein kinase C sites showed no estrogen conversion. Importantly UGT1A10 in cells supported 10-fold higher glucuronidation of 17β-estradiol than UGT1A1. In summary, our results suggest gastrointestinally distributed UGT1A10 is important for detoxifying estrogens/phytoestrogens and aromatic acids with complementary activity by UGT1A7, -1A8, -1A3, and/or -1A1 evidently dependent upon phosphorylation.

The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION

Background: The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits. Results: The p40 subunit stabilizes p35 and promotes its secretion. Conclusion: Understanding the regulatory steps of IL-12 biosynthesis led to the generation of optimized IL-12 plasmids. Significance: Availability of expression-optimized IL-12 DNA plasmids is important for practical applications as DNA vaccine adjuvants and in cancer immunotherapy. IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

The RNA-stabilizing Protein HuR Regulates the Expression of ζ Chain of the Human T Cell Receptor-associated CD3 Complex

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however, the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3ζ chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3ζ that lacks a 562-bp region in its 3′-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3ζ transcript are critical for the mRNA stability and protein expression of CD3ζ. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3ζ 3′-UTR. Knockdown of HuR resulted in decreased expression of the CD3ζ chain, whereas overexpression led to the increase of CD3ζ chain levels. Additionally, overexpression of HuR in human T cells resulted in increased mRNA stability of CD3ζ. Our results identify the 3′-UTR of CD3ζ as a novel target for the mRNA-stabilizing protein HuR. Thus, the absence of two critical AREs in the alternatively spliced CD3ζ 3′-UTR found in SLE T cells may result in decreased HuR binding, representing a possible molecular mechanism contributing to the reduced stability and expression of CD3ζ in SLE.

Uteroglobin Inhibits Prostaglandin F2α Receptor-mediated Expression of Genes Critical for the Production of Pro-inflammatory Lipid Mediators

Prematurity is one of the leading causes of infant mortality. It may result from intrauterine infection, which mediates premature labor by stimulating the production of inflammatory lipid mediators such as prostaglandin F2α (PGF2α). The biological effects of PGF2α are mediated via the G protein-coupled receptor FP; however, the molecular mechanism(s) of FP signaling that mediates inflammatory lipid mediator production remains unclear. We reported previously that in the human uterus, a composite organ in which fibroblast, epithelial, and smooth muscle cells are the major constituents, an inverse relationship exists between the levels of PGF2α and a steroid-inducible anti-inflammatory protein, uteroglobin. Here we report that, in NIH 3T3 fibroblasts and human uterine smooth muscle cells, FP signaling is mediated via multi-kinase pathways in a cell type-specific manner to activate NF-κB, thus stimulating the expression of cyclooxygenase-2. Cyclooxygenase-2 is a critical enzyme for the production of prostaglandins from arachidonic acid, which is released from membrane phospholipids by phospholipase A2, the expression of which is also stimulated by PGF2α. Most importantly, uteroglobin inhibits FP-mediated NF-κB activation and cyclooxygenase-2 gene expression by binding and most likely by sequestering PGF2α into its central hydrophobic cavity, thereby preventing FP-PGF2α interaction and suppressing the production of inflammatory lipid mediators. We propose that uteroglobin plays important roles in maintaining homeostasis in organs that are vulnerable to inadvertent stimulation of FP-mediated inflammatory response.

Lys 43 and Asp 46 in α-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity ☆

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A2 (sPLA2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices. We previously reported that sPLA2-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca++, essential for sPLA2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA2-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.

Uteroglobin interacts with the heparin-binding site of fibronectin and prevents fibronectin-IgA complex formation found in IgA-nephropathy

Immunoglobulin A (IgA)‐nephropathy (IgAN) is the most common primary renal glomerular disease in the world that has no effective treatment. High levels of circulating IgA–fibronectin (Fn) complexes, characteristically found in IgAN patients, are suggested to cause abnormal deposition of IgA and Fn in the renal glomeruli of these patients causing renal failure. We previously reported that binding of Fn to uteroglobin (UG), a multifunctional anti‐inflammatory protein, inhibits Fn–IgA heteromerization. However, the specific site of Fn–UG interaction until now remained unidentified. We report here that UG interacts with the heparin‐binding site of Fn and propose that small molecules competing for interaction with this site may reduce the level of circulating Fn–IgA complexes in IgAN.

Amino Acid Residues in α‐Helix‐3 of Human Uteroglobin Are Critical for Its Phospholipase A2 Inhibitory Activity

Rabbit blastokinin1 or uteroglobin (UG)2 is a steroid-dependent, multifunctional, secreted protein.3,4 This protein was first discovered in the rabbit uterus during early pregnancy and subsequently found in many other nonreproductive organs. Interestingly, the highest level of UG expression occurs in the rabbit1,2 and human endometrium5 during the progesterone-dominated phase of the ovarian menstrual cycle, when implantation of the embryo in the uterus takes place. This protein is also expressed in many extrauterine tissues, including the thymus, pituitary gland, lungs, gastrointestinal tract, pancreas, mammary gland, prostate, and seminal vesicle.6 UG is also present in the blood7,8 and urine,9 although it is not synthesized in the kidneys. Currently, this protein is known by several names, which are primarily derived from the organ or body fluid in which it is detectable or from the type of xenobiotics with which it interacts. Thus, it is called progesterone-binding protein,10 Clara cell 10-kDa protein,11 urine protein-1,9 polychlorinated biphenyl–binding protein,12 and retinol-binding protein.13 A unified nomenclature is being developed (see Nomenclature Committee Report in this volume). Structurally, UG is a homodimer in which the two identical 70-amino-acid subunits are covalently linked in an antiparallel orientation by two interchain disulfide bonds.14–20 Each monomer consists of four α-helices and one β-turn between α-helix-2 and -3, but there is no β-structure. Recently, we and others have identified a high-affinity binding site (putative receptor) of UG on several cell types.21–23 Through this pathway, UG appears to inhibit cellular motility and invasion of the extracellular matrix,21 suggesting an antichemokinelike property of this protein. Interestingly, human UG (hUG) is encoded by a single copy gene located on chromosome 11q12.3-13.1,24 a region in which a number of candidate disease genes have been mapped by linkage analyses.