Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.
ABSTRACT Background The autoimmune disease lupus erythematosus (lupus) is characterized by photosensitivity, where even ambient ultraviolet radiation (UVR) exposure inflames skin. Beneficial effects of anifrolumab (anti-interferon α/breceptor (anti-IFNAR)) on lupus skin disease support a pathogenic role for IFN-I, but mechanistic understanding is limited. We have shown that Langerhans cell (LC) dysfunction contributes to photosensitivity. Healthy LCs act via a disintegrin and metalloprotease 17 (ADAM17) to release epidermal growth factor receptor (EGFR) ligands that limit UVR-induced keratinocyte apoptosis and photosensitivity. However, LC ADAM17 activity is reduced in non-lesional lupus model skin, and data point to reduced LC-mediated protection in human lupus. Here, we asked about the role of the IFN-rich lupus skin environment in LC dysfunction and the implications of this regulation for photosensitivity. Methods Gene expression patterns in non-lesional skin from human lupus and multiple murine models were examined. We used MRL/lpr, B6.Sle1yaa, and imiquimod models of lupus in in vivo studies to assess the role of IFN-I in LC ADAM17 dysfunction and photosensitivity. Results We show a shared IFN-rich environment in non-lesional skin across human and murine model systems, that IFN-I inhibits LC ADAM17 activity, and that anti-IFNAR in lupus models restores LC ADAM17 function and reduces photosensitivity in EGFR and LC ADAM17-dependent manners. Reactive oxygen species (ROS) can mediate ADAM17 activity, and we show reduced LC ROS expression in lupus models that is restored by anti-IFNAR. Conclusions Our findings suggest that IFN-I promotes photosensitivity by causing LC ADAM17 dysfunction and that anifrolumab ameliorates lupus skin disease at least in part by restoring LC function. This work provides insight into IFN-I-mediated disease mechanisms, LC regulation, and a mechanism of action for anifrolumab in lupus.
Abstract Synthetic biology has the potential to transform cell‐ and gene‐based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease‐related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium “Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics,” which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell‐based therapies.
Eukaryotic ribosomal proteins (rp) are found to be arranged in relatively large (8 to > 20 member) families of unlinked genes. These families are usually composed by a single intron(s)containing expressed gene, and a number of processed, silent genes (1). One of the most studied of these families, L32, has been shown to be highly conserved among the organisms where it has been identified, i.e, mouse, human and flies (2, 3, 4, 5), but no information was available regarding the lower-eukaryote counterparts. While screening for another gene in a cDNA library from the filamentous fungus Trichoderma harzianum, we isolated a clone whose deduced amino acid sequence showed high homology (around 50% identity) with L32 rp from mouse, human, Drosophila melanogaster and D. subobscura (Figure 1). The cDNA is 562 bp in length and codes for a 137-amino-acid polypeptide. This deduced protein, termed TrpL32, is very rich in basic residues (pI = 12.41) and quite hydrophobic (data not shown) which is in agreement with the information related with the other L32 rp studied so far. From Southern analysis (data not shown) we have seen that this protein is encoded by a single gene or by several very highly homologous ones grouped in a 6 kb Hindm genomic fragment. The expression pattern of the mRNA was investigated by Northern blotting of total mRNA and resulted in a constitutive transcription of the TrpL32 gene. To our knowledge, this is the first gene of L32 rp from a lower eukaryote so far reported and shows a striking similarity with those from the higher ones. EMBL accession no. X71914
Abstract Background: The immunosuppressive milieu found within the tumor microenvironment (TME) has long been understood to be a key driver of tumor initiation and progression. More recently it has been appreciated that metabolites derived from biosynthetic pathways are major components in forming this immune-privileged niche. For example, the conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO) or the reduction of adenosine triphosphate to adenosine by the ectoenzymes CD39 and CD73 leads to T cell dysfunction and exhaustion, and a significantly blunted antitumor immune response. At Synlogic we are using synthetic biology in combination with natural probiotics to develop engineered bacteria or “Synthetic Biotic Medicines,” which are programmed with precision to correct disease-causing and -promoting metabolic defects. Here we present results showing the development of two engineered bacterial strains that have been designed to consume either kynurenine or adenosine, two molecules known to play central roles in promoting tumor immune tolerance, with the goal of relieving TME-associated immunosuppression and promoting antitumor immunity. Methods and Results: Synthetic biologic techniques were employed to generate the adenosine-consuming strain (SYN-Ade) or the kynurenine-consuming strain (SYN-Kyn) by introduction of genetic elements that were highly efficient in the metabolism of adenosine or kynurenine, respectively. In in vitro biochemical assays, SYN-Ade and SYN-Kyn were able to deplete test media containing levels of adenosine and kynurenine that are ~100-fold and 20-fold higher than the adenosine or kynurenine levels found in the tumors of cancer patients, (180uM of adenosine or 80uM of kynurenine, respectively) to undetectable levels within 2 hours. For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to robust in vivo pharmacodynamic activity. In mice bearing subcutaneous CT26 tumors, the administration of SYN-Kyn by intratumoral (IT) injection led to significant decreases in tumor kynurenine levels, which was equivalent to small-molecule inhibition of the IDO enzyme. Importantly, the combination of SYN-Kyn with an anti-CTLA4 antibody in the CT26 tumor model or the combination of SYN-Kyn or SYN-Ade with a cocktail of anti-PD1/CTLA4 antibodies in MC38 tumor-bearing mice led to significant antitumor effects over those observed with the antibodies alone. Conclusions: Taken together, these results demonstrate that the application of synthetic biology to engineer nonpathogenic bacteria is a viable approach to deliver profound efficacy in experimental models of cancer, and support the further development of these Synthetic Biotic medicines as potential immuno-oncology therapies. Citation Format: Kip A. West, Adam Fisher, Dan Leventhal, Anna Sokolovska, Ning Li, Chris Plescia, Mary Castillo, Vincent Isabella, Starsha Kolodziej, Paul Miller, Jose M. Lora. Metabolic modulation of the tumor microenvironment using Synthetic Biotic™ Medicines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2920.
A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function. A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function. Cell–cell interactions are crucial for the development and maintenance of multicellular organisms. Protein ectodomain shedding of membrane-anchored signaling molecules and their receptors is considered an important mechanism for regulating cell–cell communications (1Murphy G. The ADAMs: Signalling scissors in the tumour microenvironment.Nat. Rev. Cancer. 2008; 12: 929-941Google Scholar, 2Weber S. Saftig P. Ectodomain shedding and ADAMs in development.Development. 2012; 139: 3693-3709Crossref PubMed Scopus (165) Google Scholar, 3Blobel C.P. ADAMs: Key players in EGFR-signaling, development and disease.Nat. Rev. Mol. Cell. Biol. 2005; 6: 32-43Crossref PubMed Scopus (853) Google Scholar, 4Zunke F. Rose-John S. The shedding protease ADAM17: Physiology and pathophysiology.Biochim. Biophys. Acta Mol. Cell Res. 2017; 1864: 2059-2070Crossref PubMed Scopus (119) Google Scholar). 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The soluble interleukin 6 receptor: Advanced therapeutic options in inflammation.Clin. Pharmacol. Ther. 2017; 102: 591-598Crossref PubMed Scopus (78) Google Scholar, 22Issuree P.D. Maretzky T. McIlwain D.R. Monette S. Qing X. Lang P.A. Swendeman S.L. Park-Min K.H. Binder N. Kalliolias G.D. Yarilina A. Horiuchi K. Ivashkiv L.B. Mak T.W. Salmon J.E. et al.iRHOM2 is a critical pathogenic mediator of inflammatory arthritis.J. Clin. Invest. 2013; 123: 928-932PubMed Google Scholar, 23Qing X. Chinenov Y. Redecha P. Madaio M. Roelofs J.J. Farber G. Issuree P.D. Donlin L. McLlwain D.R. Mak T.W. Blobel C.P. Salmon J.E. iRhom2 promotes lupus nephritis through TNF-alpha and EGFR signaling.J. Clin. Invest. 2018; 128: 1397-1412Crossref PubMed Scopus (39) Google Scholar). 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The goal of this study was to generate mice carrying a mutant form of endogenous ADAM17 that lacks its cytoplasmic domain, including all previously described signaling motifs, in order to learn more about the role of the cytoplasmic domain in regulating the function of ADAM17 in vivo. In order to generate mice that express endogenous ADAM17 with a truncated cytoplasmic domain, we used CRISPR-Cas9 to introduce a targeted knock-in (KI) mutation, in which an HA-tag with a stop codon was attached immediately C-terminal to the transmembrane domain of ADAM17 (following DKKLD699, Fig. 1A, see Experimental procedures and Fig. S1 for details). The resulting mutant ADAM17Δcyto lacks almost all of the cytoplasmic domain of ADAM17 (cytoplasmic amino acid residues 700–827) and corresponds to an ADAM17Δcyto mutant that fully rescues ADAM17-dependent shedding when overexpressed in Adam17−/− mouse embryonic fibroblasts (mEFs) (27Le Gall S.M. Maretzky T. Issuree P.D.A. Niu X.-D. Reiss K. Saftig P. Khokha R. Lundell D. Blobel C.P. ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site.J. Cell Sci. 2010; 123: 3913-3922Crossref PubMed Scopus (143) Google Scholar). Founder mice carrying the Adam17Δcyto KI mutation were generated following standard protocols (see Experimental procedures for details) and bred to homozygosity. The presence of the Adam17Δcyto mutation was verified by sequencing PCR fragments from genomic DNA of mutant mice compared with wild-type controls (Fig. 1, B and C). Offspring of heterozygous matings (Adam17Δcyto/+ x Adam17Δcyto/+) were genotyped by genomic PCR (Fig. 1D) and were born at the expected Mendelian ratio (Fig. 1E). Homozygous mutant Adam17Δcyto animals appeared grossly normal (Fig. 2A) and did not display behavioral abnormalities during routine handling compared with wild-type controls. However, closer inspection revealed that Adam17Δcyto mice had slightly wavy fur (Fig. 2B) and curly whiskers (Fig. 2C, red arrows), which are characteristic phenotypes for mice carrying mutations affecting ADAM17/EGFR signaling (16Chalaris A. Adam N. Sina C. Rosenstiel P. Lehmann-Koch J. Schirmacher P. Hartmann D. Cichy J. Gavrilova O. Schreiber S. Jostock T. Matthews V. Hasler R. Becker C. Neurath M.F. et al.Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice.J. Exp. Med. 2010; 207: 1617-1624Crossref PubMed Scopus (207) Google Scholar, 44Brandl K. Sun L. Neppl C. Siggs O.M. Le Gall S.M. Tomisato W. Li X. Du X. Maennel D.N. Blobel C.P. Beutler B. MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 19967-19972Crossref PubMed Scopus (119) Google Scholar, 45Hassemer E.L. Le Gall S.M. Liegel R. McNally M. Chang B. Zeiss C.J. Dubielzig R.D. Horiuchi K. Kimura T. Okada Y. Blobel C.P. Sidjanin D.J. The waved with open eyelids (woe) locus is a hypomorphic mouse mutation in Adam17.Genetics. 2010; 185: 245-255Crossref PubMed Scopus (34) Google Scholar). Gross and histopathological analysis of newborn mice revealed that Adam17Δcyto animals resemble Adam17−/− animals in that their eyes are open at birth (Fig. 3A, top two rows of panels) (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russel W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. et al.An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Crossref PubMed Google Scholar, 46Horiuchi K. Kimura T. Miyamoto T. Takaishi H. Okada Y. Toyama Y. Blobel C.P. Cutting edge: TNF-{alpha}-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock.J. Immunol. 2007; 179: 2686-2689Crossref PubMed Google Scholar). Moreover, Adam17Δcyto mice show enlarged and thickened pulmonic, aortic, and tricuspid heart valves (Fig. 3A, middle and lower middle panels, tricuspid valve not shown), similar to Adam17−/− mice (12Jackson L.F. Qiu T.H. Sunnarborg S.W. Chang A. Zhang C. Patterson C. Lee D.C. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.EMBO J. 2003; 22: 2704-2716Crossref PubMed Scopus (330) Google Scholar, 46Horiuchi K. Kimura T. Miyamoto T. Takaishi H. Okada Y. Toyama Y. Blobel C.P. Cutting edge: TNF-{alpha}-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock.J. Immunol. 2007; 179: 2686-2689Crossref PubMed Google Scholar). However, unlike Adam17−/− mice (14Hall K.C. Hill D. Otero M. Plumb D.A. Froemel D. Dragomir C.L. Maretzky T. Boskey A. Crawford H.C. Selleri L. Goldring M.B. Blobel C.P. ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.Mol. Cell. Biol. 2013; 33: 3077-3090Crossref PubMed Scopus (34) Google Scholar, 15Saito K. Horiuchi K. Kimura T. Mizuno S. Yoda M. Morioka H. Akiyama H. Threadgill D. Okada Y. Toyama Y. Sato K. Conditional inactivation of TNFalpha-converting enzyme in chondrocytes results in an elongated growth plate and shorter long bones.PLoS One. 2013; 8e54853Crossref PubMed Scopus (17) Google Scholar), Adam17Δcyto mice did not have significantly expanded zones of hypertrophic cells in their long bone growth plate (Fig. 3A, femur shown in lower panel).Figure 3Adam17Δcyto mice closely resemble Adam17−/− mice at birth. A, representative images of the heads of newborn wild-type and Adam17Δcyto (littermates) and Adam17−/− mice show open eyes at birth (OEB) in Adam17Δcyto mutants that are similar to the OEB in Adam17−/− mice (top row, white arrows). Corresponding H&E-stained sections of the eye are shown in row 2, with an asterisk marking the open eyelid. Sections of a representative pulmonic valve (middle panel/row 3) and aortic valve (row 4) show similarly enlarged and misshapen tricuspid valves in Adam17Δcyto mutants compared with Adam17−/− mice. However, the growth plate in the Adam17Δcyto mutant appeared normal in size and comparable to the wild-type control and did not display the enlarged zone of hypertrophic cells seen in Adam17−/− mice (bottom row). Scale bars: sections of eyes and femurs, 100 μm, sections of heart valves, 50 μm. B, representative images of the aortic and pulmonic valves in adult Adam17Δcyto mutants are indistinguishable from wild-type controls (left panels). The hair follicles and skin of Adam17Δcyto mutants showed pyogranulomatous inflammation, which also affected the meibomian glands in the eyelids and the zymbal glands near the ear canal (Fig. 3B, right panels). Scale bars: sections of adult heart valves, 100 μm, sections of skin and glands, 50 μm. All images are representative for sections from at least three mice per genotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of adult Adam17Δcyto animals showed no evident abnormalities in heart weight and gross and histologic morphology of the heart, including the heart valves (Fig. 3B, left panels), suggesting that the heart valve defects are remodeled and return to normal as these animals grow into adults. In addition, we found pyogranulomatous inflammation of hair follicles in the skin and in the meibomian glands in eyelids and zymbal glands near the ear canal, which are both specialized sebaceous glands (Fig. 3B, right panels). There were no other major evident pathological phenotypes in the adult Adam17Δcyto mice compared with their littermate controls. The hypomorphic phenotype of Adam17Δcyto mice raised questions about the underlying cause for the apparently reduced activity of ADAM17. We therefore performed western blots on Concanavalin A-enriched glycoproteins isolated from wild-type, Adam17Δcyto, and Adam17−/− mEFs with rabbit polyclonal antibodies raised against the extracellular domain of mouse ADAM17 (anti-A17-ecto). The samples were run under denaturing, but nonreducing conditions, since the anti-A17-ecto antibodies only recognize nonreduced ADAM17 in western blots (see Fig. S2 and Experimental procedures for details). Under these conditions, the anti-A17-ecto antibody detected the wild-type proform of ADAM17 (Fig. 4A, indicated by an open arrowhead) as well as the mature form (indicted by a black arrowhead) in mEFs. By comparison, the levels of pro-ADAM17Δcyto (open arrowhead) and mature ADAM17Δcyto (black arrowhead) were strongly reduced. When wild-type cells were lysed in the absence of metalloprotease inhibitors (marimastat and 1,10 Phenanthroline, see Experimental procedures for details), the mature form of wild-type ADAM17 underwent postlysis autocatalytic removal of the cytoplasmic domain, as reported previously (47Schlöndorff J. Becherer J.D. Blobel C.P. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE).Biochem. J. 2000; 347 Pt 1: 131-138Crossref PubMed Google Scholar, 48McIlwain D.R. Lang P.A. Maretzky T. Hamada K. Ohishi K. Maney S.K. Berger T. Murthy A. Duncan G. Xu H.C. Lang K.S. Haussinger D. Wakeham A. Itie-Youten A. Khokha R. et al.iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS.Science. 2012; 335: 229-232Crossref PubMed Scopus (219) Google Scholar), which generates a faster migrating form of mature ADAM17 (Fig. 4A, indicated by an asterisk), whereas pro-ADAM17 was not affected. A similar postlysis autocatalytic removal of the cytoplasmic domain was also observed in the related ADAM10 and is thought to have no biological significance in ADAM17 or ADAM10 (49Brummer T. Pigoni M. Rossello A. Wang H. Noy P.J. Tomlinson M.G. Blobel C.P. Lichtenthaler S.F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.FASEB J. 2018; 32: 3560-3573Crossref PubMed Scopus (14) Google Scholar). The proform of ADAM17Δcyto (open arrowhead in Fig. 4A, darker exposure shown on the right) migrated faster than the proform of wild-type ADAM17, consistent with the predicted reduction in molecular weight caused by deletion of the cytoplasmic domain. The mature form of ADAM17Δcyto (black arrowhead in Fig. 4A) comigrated approximately with the autodegraded form of mature wild-type ADAM17 (asterisk). However, the presence or absence of the metalloprotease inhibitors during cell lysis did not affect the migration of mature ADAM17Δcyto. This suggests that ADAM17Δcyto was not detectably subjected to postlysis processing
IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.
We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.
The extracellular matrix (ECM) promotes the differentiation of many cell types, and ECM remodeling in the liver has been implicated in embryonic development, tissue injury, and oncogenesis. Integrins are heterodimeric ECM receptors that play critical roles in transducing the composition of the ECM in the cell environment. We previously showed that mouse H2.35 cells, a conditionally transformed, liver-derived cell line, assume a more differentiated hepatocyte morphology and enhanced liver-specific gene expression when the cells are cultured on gelatinous ECM substrata. Here we show that H2. 35 cells express relatively high levels of alpha3beta1-integrins, similar to that previously shown for immature hepatocytes, transformed hepatocytes, and biliary cells. However, the cell morphological responses that depend on alpha3beta1-integrin have not been defined. We found that transfecting H2.35 cells with antisense RNA construct directed to alpha3-subunit messenger RNA perturbs the initial cell attachment to laminin and collagen, and strongly inhibits cell morphological, proliferative, and gene expression responses to a collagen gel substratum. In situ hybridization to mouse embryo tissues demonstrates the presence of alpha3-subunit messenger RNAs in newly formed hepatocytes. We suggest that alpha3beta1-integrins are important for immature and transformed hepatocytes to respond morphologically to the extracellular matrix.
